Cyclodextrin containing Nano- and Microgels for Uptake and ...

Cyclodextrin containing Nano- and Microgels

for Uptake and Release of Chemical Agents

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften

der RWTH Aachen University zur Erlangung des akademischen Grades

eines Doktors der Naturwissenschaften genehmigte Dissertation

vorgelegt von

Diplom-Chemiker

Markus J. Kettel

aus Mönchengladbach, Deutschland

Berichter: Universitätsprofessor Dr. Martin Möller

Universitätsprofessor Dr. Andrij Pich

Tag der mündlichen Prüfung: 07.11.2013

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.

Für meine Familie

„Man muss immer etwas Neues machen, um etwas Neues zu sehen.“

Georg Christoph Lichtenberg (1742-1799)

I

Table of contents

List of abbreviations ................................................................................................................... III

Acknowledgment .................................................................................................................... V

Publications ..................................................................................................................VII

Summary ................................................................................................................ XIII

Zusammenfassung ................................................................................................................. XV

Chapter 1 Introduction .................................................................................................. 1

Chapter 2 Literature Reviews ....................................................................................... 7

Chapter 3 Functional PMMA nanogels by cross-linking with cyclodextrin

methacrylate ............................................................................................... 31

Chapter 4 Chlorhexidine loaded cyclodextrin containing PMMA nanogels as

antimicrobial coating and delivery system ................................................ 67

Chapter 5 Ibuprofen loaded cyclodextrin containing poly(2-hydroxyethyl

methacrylate) nano/microgels .................................................................. 101

Chapter 6 Thermosensitive aqueous nanogels modified with cyclodextrin ............. 125

Chapter 7 Thermochromic cyclodextrin containing nanogels by complexation

of solvatochromic dye Nile Red .............................................................. 151

Chapter 8 Surfactant-free preparation of nanogels with high functional β-

cyclodextrin content ................................................................................. 187

Chapter 9 Application of β-cyclodextrin containing nanogels as carriers of

permethrin on keratin fibers ..................................................................... 205

Chapter 10 β-Cyclodextrin based host-guest systems for safe application and

release of insecticides (Appendix to Chapter 9) ...................................... 239

III

List of abbreviations

AAEM Acetoacetoxyethyl methacrylate

AIBN 2,2-Azobisisobutyronitrile

AMPA 2,2-Azobis(2-methylpropyonamidine)

dihydrochloride

BIS N,N-methylene bisacrylamide

CD Cyclodextrin

CD-A Cyclodextrin acrylate

CD-MA Cyclodextrin methacrylate

CHX Chlorhexidine

d days

Da Dalton

DLS Dynamic light scattering

DMF Dimethyl formamide

DMSO Dimethylsulfoxide

DS Degree of substitution

DSC Differential scanning calorimetry

FESEM Field-Emission Scanning Electron Microscopy

FTIR spectroscopy Fourier transform infrared spectroscopy

GC Gas chromatography

h hour

HCl Hydrochloric acid

HEMA 2-Hydroxyethyl methacrylate

HPLC High pressure liquid chromatography

LCST Lower critical solution temperature

MALDI-TOF MS spectrometry Matrix assisted laser desorption ionization time

of flight mass spectrometry

MeOH Methanol

MMA Methyl methacrylate

MWCO Molecular weight cut-off

IV

NCO Isocyanate

NG Nanogel

NMR Nuclear magnetic resonance

NR Nile Red

PBS Phosphate-buffered saline

PDI Polydispersityindex

PEO Poly(ethylene oxide)

PHEMA Poly(hydroxyethyl methacrylate)

PMMA Poly(methyl methacrylate)

PNIPAM Poly(N-isopropylacrylamide)

ppm Parts per million

PPO Poly(propylene oxide)

PSD Particle size distribution

PVCL Poly(vinylcaprolactam)

Rh Hydrodynamic radius

rpm Rounds per minute

RT Room temperature

SFPP Surfactant-Free Precipitation Polymerization

sP(EO-stat-PO) Star shaped Poly(propylene oxide-stat-

propylene oxide)

STEP- technology STEP™-Technology

TCM Trichloromethane

TEA Triethylamine

TEM Transmission electron microscopy

TGA Thermogravimetric analysis

THF Tetrahydrofuran

TICT Twisted intermolecular charge transfer

UV Ultraviolet

VCL N-Vinylcaprolactam

VPTT Volume phase transition temperature

V

Acknowledgment

My special thanks goes to Prof. Dr. Martin Möller for the warm welcome as a doctoral

candidate in his research group, for the selection of this interesting topic and the generously

granted freedom in carrying out this work. His scientific expertise and his ideas guided and

coordinated this work in the right direction.

I want to thank Prof. Dr. Andrij Pich for his great interest and the friendly takeover as co-

reporter. I would also like to thank him for the excellent scientific supervision of the research in

the field of responsive nano-and microgels and for the diverse interesting discussions.

I am grateful to Prof. Dr. Jürgen Groll for his great interest, the excellent scientific

support and for technical discussions with regard to nano/hydrogels based on the cross-linking of

pre-polymers.

Dr. Karola Schäfer, I want to thank you for your interest and the friendly support of the

work in terms of the various applications, especially in the field of textile application. In addition,

I thank you for your strong commitment and confidence for this work and in my person.

I want to thank Prof. Dr. Uwe Beginn for introducing me into his research group at the

University of Osnabrück and the technical assistance of computer-based complexation studies

using molecular modelling.

Dr. Hans-Jürgen Buschmann from DTNW Nordwest e.V. in Krefeld, I want to thank you

for the great knowledge in the chemistry of cyclodextrins and for the help with the

characterization of cyclodextrin complexes.

I want to thank Prof. Dr. Rainer Haag for welcoming me in his work group at Freie

Universität Berlin and furthermore, Dr. Marcelo Calderón and Mohiuddin Abdul Quadir for the

nice working atmosphere and for the help with ITC measurements.

My thanks go to Dr. Walter Tillmann for the countless measurements of qualitative and

quantitative IR/Raman spectra and the great interest in my work. I am grateful to Dr. Phan Ho

and Mr. Stefan Rütten for the help with the electron microscope equipment, especially for the

great confidence regarding late evening measurements on FESEM. Thank you, Dr. Andrea

Körner, for the measurement of the MALDI TOF spectra and the incorporation into the

headspace GC-MS spectroscopy. Dr. Elisabeth Heine and Rita Gartzen, I want to thank you for

VI

the implementation of the antimicrobial tests. I want to thank Haika Hildebrandt and Michaela

Meurath for the optimization and supply with the NCO-functionalized sP(EO-stat-EO) pre-

polymers.

I also want to thank the employees who have directly helped with great interest and high

motivation and with practical activities to individual chapters: the research students Dominik

Schmitz, Fiete Dierks, Tanja Peters, Server Uzungelis, Mohammed Mbarki, Benjamin Grimme,

Lennart Sandbrink, Lars Müller the Bachelor students Markus Lansing and Miriam Fleischhack.

For practical support I would also like to thank my technical students Klaus Krolls and Yesmin

Yazici. Special thanks go to the laboratory technicians Marion Arndt, Ramona Kloss and

Alexandra Kopp for the nice working atmosphere, the organizational support in the laboratory

and the technical support for the textile finishing.

In addition, I thank the staff of DWI, ITMC and the Department of Functional and

Interactive Materials for the daily helpfulness, suggestions, discussions and the excellent working

atmosphere. In particular, I would like to thank my laboratory colleagues Hailin Wang, Nikolai

Belov, Ibrahim Hassouna, Karla Dörmbach for the pleasant time and the countless discussions,

day and night in the lab. Jörg Meyer, Wiktor Steinhauer, Thorsten Anders, Christian Herbert,

Janis Lejnieks, Christian Vaida, Metodi Bozukov, Nebia Greving, Björn Schulte, Firat Özdemir,

Dragos Popescu, Michael Erbrich, Ying Chung He, Marco Backes, Subrata Chattopadhyay, Emin

Hrsic, Christian Hahn, Stefan Theiler, Marc Hans, Sascha Pargen, Jens Köhler, Michael Scharpf

and Angela Plum from ITMC building; Mar Diez, Vera Schulte, Meike Beer, Smirti Singh,

Kristina Bruellhoff, Heidrun Keul, Christina Klaus, Ramona Ronge, Miran Yu, Nina Keusken,

Juliana Kurniadi, Tsoomoo Narangerel, Bea Vo-Van, Claudia Formen, Thomas Weyand, Robert

Lösel, Fuat Topuz, Dr. Artur Henke, Dr. Nicolae Goga from (old) DWI building; Dr. Cheng

Cheng, Dr. Dilek Akcakayiran, Andreea Balaceanu, Ricarda Schröder, Garima Agrawal, Philipp

Nachev, Florian Störman, Christian Willms, Dominik Kehren, Yaodong Wu, Sebastian Kühl,

Jason Zografou, Christan Bergs, Richard Meurer from AK Pich and many more, thank you for

the countless hours at the institute and the varied recreational activities outside science.

I especially thank my parents and my brother for the great support and help throughout

my years of study and promotion. Thank you for your faith in me.

I especially want to thank Stefanie Spira for a lot of patience, great support and the

deflection in my spare time. Thank you that you are here.

VII

Publications

Parts of this thesis are published in:

Articles in scientific journals

(3) Markus J. Kettel, Haika Hildebrandt, Karola Schaefer, Martin Moeller, Juergen Groll;

Tenside Free Preparation of Nanogels with High Functional β-Cyclodextrin Content;

ACS Nano, 2012, 6(9), 8087-8093

DOI: 10.1021/nn302694q.

(2) Markus J. Kettel, Fiete Dierkes, Karola Schaefer, Martin Moeller, Andrij Pich;

Aqueous Nanogels Modified with Cyclodextrin;

Polymer, 2011, 52, 1917-1924

DOI: 10.1016/j.polymer.2011.02.037. ISSN: 0032-3861.


Synthese von reaktionsfähigen Cyclodextrin-Mikrogelen durch Batch-

Fällungspolymerisation;

Chemie Ingenieur Technik, 2010, 82 (9), 1342

DOI: 10.1002/cite.201050570.

Articles in conference proceedings

(7) Markus J. Kettel, Karola Schaefer, Andrea Koerner, Uwe Beginn, Martin Moeller;

Cyclodextrin based host-guest systems for safe application and controlled release of

insecticides from textile materials;

Proceedings of the 5th

Aachen-Dresden International Textile Conference, Aachen/D, November

25-26, 2011, ed. Küppers, B., DWI an der RWTH Aachen e.V./Germany

(ISSN 1867-6405), P73.

(6) Markus J. Kettel, Mohammed Mbarki, Elisabeth Heine, Karola Schaefer, Martin Moeller;

Chlorhexidine loaded Cyclodextrin based Microgels as Antimicrobial Systems on Textiles;


Aachen-Dresden International Textile Conference, Dresden/D, November

25-26, 2010, ed. Doerfel, A., ITB/TU Dresden/Germany (ISSN 1867-6405), P56.

(5) Karola Schaefer, Markus J. Kettel, Juergen Groll, Martin Moeller;

Cyclodextrin based Microgels as Carriers of Insecticides for Wool Textiles;


Int. Wool Res. Conference (IWRC 2010), Shanghai/PR of China, October

19-22, 2010, ed. College of Chemistry Engineering and Biotechnology,

Donghua University/China, paper C-007, Vol. I, pp 485-488.

VIII


Complexation of Synthetic and Natural Insecticides in Cyclodextrins for Safely Application

and Controlled Release;


Int. Wool Res. Conference (IWRC 2010), Shanghai/PR of China, October

19-22, 2010, ed. College of Chemistry Engineering and Biotechnology,

Donghua University/ China, paper C-007, Vol. I, pp 198-202.

(3) Markus J. Kettel, Juergen Groll, Karola Schaefer, Martin Moeller;

Complexation of Permethrin in Cyclodextrin based Nanogels and Application on Textiles

(AiF 15123N);

Proceedings of the 3rd

Aachen-Dresden International Textile Conference, Aachen/D, November

26-27, 2009, ed. Küppers, B., DWI an der RWTH Aachen e.V./Germany

(ISSN 1867-6405), P41.


Cyclodextrin based Gels as Carriers of Sensitive Additives for Keratin Fibres;


International Hair-Science (Hair’S09), Weimar/D, September 9-11, 2009,

ed. Küppers, B., DWI an der RWTH Aachen e.V./Germany, P43.

(1) Karola Schäfer, Markus Kettel, Martin Möller;

New Concepts for the Insect Proofing of Textile Materials (AiF 15123 N);


Aachen-Dresden International Textile Conference, Dresden/D, December

4-5, 2008, ed. Doerfel, A., ITB/TU Dresden/Germany (ISSN 1867-6405), P56.

Abstracts in conference proceedings

(6) Karola Schäfer, Markus J. Kettel, Andrij Pich, Martin Möller;

Soft Nanotechnologie zur Funktionalisierung von Textilien;

8th

Textilveredlertag des VDTF,

Münster/D, June 4, 2011.

(5) Markus J. Kettel, Karola Schaefer, Juergen Groll, Andrij Pich, Martin Moeller;

Cyclodextrin based Nano and Microgels - Multifunctional Colloidal Networks -;

3rd

EuCheMS Chemistry Congress, Chemistry-the Creative Force;

Nürnberg/D; August 28- September 03, 2010.


Synthesis and Applications of Urethan Cross linked Cyclodextrin Nanogels;

First European Cyclodextrin Conference;

Aalborg/DK; October 11-13, 2009.

IX


Synthesis of Cross linked Cyclodextrin Polymers and the Creation of Nano- and Microgels;

8th

International Conference on Advanced Polymers via Macromolecular Engineering (APME);

Dresden/D, October 04-07, 2009.

(2) Markus J. Kettel, Karola Schäfer, Martin Möller;

Synthesis of Cyclodextrin based Nano and Microgels for the Encapsulation of Active

Ingredients;

ProzessNet- Chemical Nanotechnology Talks IX; Bio meets Nano;

Frankfurt am Main/D; November 03-04, 2008.


Cyclodextrin based Gels for the Complexation of Active Ingredients;

Bioannual Meeting of the GDCh-Division of “Macromolecular Chemistry”; Bio & Polymers;

Aachen/D; September 28-30, 2008.

Oral and Poster presentations:




Synthetic Fiber Talks, Advanced Processes for Advanced Fibres;

Aachen/D; April 29-30, 2012, (Poster).




5th

Aachen-Dresden International Textile Conference,

Aachen/D, November 25-26, 2011, (Poster).

(21) Karola Schäfer, Markus J. Kettel, Andrij Pich, Martin Möller;

Soft Nanotechnologie zur Funktionalisierung von Textilien;

8th


Münster/D, June 4, 2011, (Oral presentation).


Chlorhexidine loaded Cyclodextrin based Microgels as antimicrobial systems on textiles;

8th


Münster/D, June 4, 2011, (Poster).

X


Chlorhexidine loaded Cyclodextrin based Microgels as antimicrobial systems on textiles

Synthetic Fiber Talks, Fibers and Functions;



Chlorhexidine loaded Cyclodextrin based Microgels as antimicrobial systems on textiles;

4th

Aachen Dresden - International Textile Conference;

Dresden/D; November 25-26, 2010, (Poster).

(17) Karola Schaefer, Markus J. Kettel, Juergen Groll, Martin Moeller;

Cyclodextrin based Microgels as Carriers of Insecticides for Wool Textiles;

12th

Int. Wool Res. Conference (IWRC 2010),

Shanghai/PR of China, October 19-22, 2010, (Oral presentation).


Complexation of synthetic and natural insecticides in cyclodextrins for safely application

and controlled release;

12th

International Wool Research Conference (IWRC 2010)

Shanghai/China, October 19-22, 2010, (Poster).


Synthesis of Responsive Cyclodextrin Microgels via Batch Precipitation Polymerization;

ProzessNet-Jahrestagung 2010 und 28. DECHEMA-Jahrestagung der Biotechnologen

Aachen/D, September 21-23, 2010, (Poster).


Cyclodextrin based Nano and Microgels - Multifunctional colloidal Networks-;

3rd

EuCheMS Chemistry Congress, Chemistry-the Creative Force

Nürnberg/D; August 28- September 03, 2010, (Poster)


Cyclodextrin based Nano and Microgels;

Tagung des Wissenschaftlichen Beirats am DWI an der RWTH e.V.

Aachen/D; July 07-08, 2010, (Poster).


Complexation of Permethrin in Cyclodextrin based Nanogels and Application on Textiles;

Synthetic Fiber Talks, Membranes and Filters;


XI


Crosslinked cyclodextrin polymers and the creation of nano-and microgels;

Catalysis and Materials 2010: Developments in Industry and Academia

Aachen/D, February 05, 2010, (Poster).


Complexation of Permethrin in Cyclodextrin based Nanogels and Application on Textiles

(AiF 15123N);

3rd


Aachen, November 26-27, 2009, (Oral presentation / Poster).


Synthesis and Applications of Urethan Crosslinked Cyclodextrin Nanogels;

First European Cyclodextrin Conference;

Aalborg/DK; October 11-13, 2009, (Poster).


Synthesis of cross linked cyclodextrin polymers and the creation of nano- and microgels;

8th

International Converence on Advanced Polymers via Macromolecular Engineering (APME

2009);

Dresden/D, October 04-07, 2009, (Poster).


Cyclodextrin based Gels as carriers of sensitives additives for keratin fibers;

16th

International Hair Science Symposium (HairS 2009);

Weimar/D, September 09-11, 2009, (Poster).

(6) Markus J. Kettel, Jürgen Groll, Karola Schäfer, Martin Möller;

Cyclodextrin based nanogels as innovative encapsulation systems;

NanoMaterials 2009, New Technologies with Water

Bonn /D; June 16-18, 2009, (Poster).

(5) Markus J. Kettel, Karola Schäfer, Jürgen Groll, Martin Möller;

Cyclodextrin Gel Particles for the Encapsulation of Active Ingredients;

Synthetic Fibre Talks, New Technologies with water;

Vaalsbroek/NL; May 07-08, 2009, (Poster).

XII

(4) Karola Schäfer, Markus Kettel, Martin Möller;

New Concepts for the Insect Proofing of Textile Materials (AiF 15123 N);

2nd


Dresden, December 4-5, 2008, (Poster).


Synthesis of Cyclodextrin Based Nano and Microgels for the Encapsulation of Active

Ingredients;

ProzessNet- Chemical Nanotechnology Talks IX, Bio meets Nano;

DECHEMA- Haus Frankfurt am Main/D; November 03-04, 2008, (Poster).


Cyclodextrin Based Gels for the Complexation of active ingredients;

Bioannual Meeting of the GDCh-Division of “Macromolecular Chemistry”,

Bio & Polymers -New Polymer Technologies with Water;

Eurogress Aachen/D; September 28-30, 2008, (Poster).

(1) Markus J. Kettel, Karola Schäfer, Uwe Beginn, Martin Möller;

Encapsulation of active ingredients in cyclodextrin-based nanogels;

Synthetic Fibre Talks, Designing Properties - A Promise of Nanotechnology;

Vaalsbroek/NL; May 08-09, 2008, (Poster).

XIII

Summary

This thesis deals with the preparation and characterization of novel functional polymeric

nano- and microgels using cyclodextrin (CD) as multifunctional cross-linker and supramolecular

complexation domain for the uptake and release of potential chemical agents.

The incorporation of natural and vinyl functionalized α-, β- and γ-CDs were studied in

different polymerization reactions with different types of monomers for the preparation of stable

nano- and microgels with specific properties.

First, reactive α-, β- and γ-CD acrylates (CD-As) and CD methacrylates (CD-MAs) were

synthesized with an average amount of vinyl bonds per CD molecule of 2, 4, or 6. They were

used as cross-linkers to react with acrylate and methacrylate monomers by free radical

precipitation polymerization and in addition as complexation unit for the uptake of chemical

agents. Hydrophobic CD-MA nanogels based on methylmethacrylate (MMA), aqueous CD-A

nanogels based on 2-poly(hydroxyethyl methacrylate) (HEMA) and thermosensitive nanogels

based on CD-A and N-vinylcaprolactam (VCL) were prepared.

The preparation of CD containing nano- and microgels showed that the particle growth

and the formation of stable nanoparticle dispersions in organic as well as in aqueous solutions

are strongly influenced by the use of CD-A and CD-MA. Similar results in particle

characterization showed that the formation and the particle growth correlate with the increase of

the CD-A and CD-MA concentration in the reaction mixture. This led to a reduction of the final

hydrodynamic radii (Rh) of the nanoparticles from approximately 400 nm to 30 nm. The

increase of the vinyl group number per CD molecule induced the formation of smaller nanogels

(Rh = at 20 nm) and a considerable increase of the cross-linking degree.

Furthermore, nanogels with a high CD content were prepared by cross-linking of non-

functionalized natural CD with the reactive isocyanate (NCO)-terminated star-shaped

poly(ethylene oxide-stat-propylene oxide) (sP(EO-stat-PO)) prepolymer under smooth

surfactant-free condensation in water at RT. Optimization of the reaction parameters yielded in

ultrafine nanogels ( Rh = 25-150 nm) with a maximum content of 60 wt.-% active CD.

The complexation capacity and application possibilities of CD containing nano- and

microgels were investigated with regard to the uptake and release properties of potential organic

guest molecules like sensitive dyes (phenolphthalein, nile red), antimicrobial agents

(chlorhexidine), insecticides (permethrin) and pharmaceutical active ingredients (ibuprofen).

XIV

Results showed that CD units in nanogels have significant influence on the uptake of

organic guest molecules. The controlled inclusion complexation of the guests took place in

dependence on the CD content and on the ring size using α-, β- and γ-CDs. The monomer

composition and the degree of cross-linking of polymeric nanogel structure had an influence on

the uptake as well. Hydrophobic polymer backbone like poly(methyl metacrylate) (PMMA)

showed a high affinity to hydrophobic guest molecules and increased the uptake of

chlorhexidine. The hydrophilic backbone of poly(hydroxymethyl methacrylate) (PHEMA)

enabled the penetration of guest molecules in aqueous microgels whereas hydrophobic

ibuprofen is preferentially bounded by CD-A units. Phenolphthalein and nile red showed

changes in their characteristical color due to formation of specific CD complexes in aqueous

nanogels. In general, higher degree of cross-linking reduced the accessibility of CD domains for

guest molecules and thus, the uptake capacity of nanogels.

The release and stability of the guest molecule in nanogels depend on the external

environment. In general, the CD nanogel complexes using hydrophobic guest molecules like

permethrin, ibuprofen and nile red were stable in water. Slightly water soluble guest molecules

like chlorhexidine were slowly released by changes of the surrounding water concentration from

PMMA based nanogels. By extraction with organic solvents like methanol, release of

permethrin could be observed from aqueous CD/sP(EO-stat-PO) nanogels. Light sensitive

permethrin could be protected against UV irradiation due to complexation in CD nanogels.

Application on different surfaces such as polyester fibers or keratin fibers due to

physical self-bonding can be controlled by nanogel concentration in the dispersion and by

coating time. Bioassay tests of permethrin and chlorhexidine loaded nanogels treated surfaces

showed that the insecticidal and antimicrobial activity of the ingredients did not decrease after

complexation in cyclodextrin containing nanogels.

In additional work, a series of common and alternative active agents for insect-proofing

were studied with regard to the uptake and release properties by natural β-CDs. Computer-based

Monte Carlo force field simulations and experimental studies showed that greater molecules

form less stable complexes with β-CD than smaller molecules. Functional groups of the guest

molecule influenced the complexation. Furthermore, release studies of active agents from the

solid complexes show that the release process depends on the physical properties of guest

molecule e.g. vapor pressure and on the present water content.

XV

Zusammenfassung

Diese Arbeit befasst sich mit der Herstellung und Charakterisierung von neuartigen

funktionellen polymeren Nano- und Mikrogelen mit Cyclodextrin (CD)-Einheiten. CD wird

sowohl als multifunktionaler Vernetzer als auch als supramolekulare Komplexierungseinheit für

die Aufnahme und Abgabe von potentiellen Wirkstoffen eingesetzt.

Die Einbindung der natürlichen und mit unterschiedlicher Anzahl an Vinylgruppen

funktionalisierten α-, β-, und γ-CDen wurde in unterschiedlichen Polyreaktionen mit

verschiedenen Monomeren in der Herstellung von stabilen nanoskaligen Teilchen mit

spezifischen Eigenschaften untersucht.

Zunächst wurden reaktive α-, β- und γ-CD Acrylate (CD-A) und CD Methacrylate (CD-

MA) mit einem durchschnittlichen Substitutionsgrad von 2, 4 oder 6 Vinylgruppen pro CD

Molekül hergestellt. Diese wurden als Vernetzer eingesetzt, um Acrylat- und Methacrylat-haltige

Monomere durch die radikalische Fällungspolymerisation kontrolliert zu vernetzen und

zusätzlich als Komplexierungseinheit für die Aufnahme von chemischen Wirkstoffen zu

fungieren. Es wurden hydrophobe CD-MA Nanogele auf Basis von Methylmethacrylat (MMA),

wässrige CD-A Nanogele auf der Basis von 2-Poly(Hydroxyethylmethacrylat) (HEMA) und

temperatur-sensitive Nanogele basierend auf CD-A und N-Vinylcaprolactam (VCL) hergestellt.

Die Herstellung von CD-haltigen Nano- und Mikrogelpartikel zeigte, dass sowohl das

Partikelwachstum als auch die Bildung stabiler Nanopartikel-Dispersionen in organischer wie

auch in wässriger Phase stark durch die Verwendung von CD-A und CD-MA beeinflusst werden.

Ähnliche Ergebnisse der Partikelcharakterisierung zeigten, dass die Bildung und das

Partikelwachstum mit dem Anstieg der CD-A- und CD-MA-Konzentration in der

Reaktionsmischung korrelieren. Die Konzentrationserhöhung von CD-A und CD-MA führte zu

einer durchschnittlichen Reduktion des hydrodynamischen Radius (Rh) der Nanogele von 400 nm

auf 30 nm. Die Erhöhung der Anzahl an Vinylgruppen pro CD Einheit induzierte die Bildung

kleinerer Nanogele (Rh = 20 nm) und eine beträchtliche Erhöhung des Vernetzungsgrades.

Darüber hinaus wurden -CD-haltige Nanogele durch Vernetzung mit nicht-

funktionalisiertem, natürlichem -CD und einem reaktiven Isocyanat (NCO)-Endgruppen

funktionalisierten, sternförmigen Poly(ethylenoxid-stat-Propylenoxid) (sP(EO-stat-PO))

Prepolymer hergestellt. Die Reaktion wurde unter schonenden Bedingungen bei RT durch

tensidfreie Kondensation in Wasser durchgeführt. Die Optimierung der Reaktionsparameter

XVI

führte zu ultrafeinen Nanogelen (Rh = 25-150 nm) mit einem maximalen Gehalt von 60 Gew.-%

an aktivem CD in hohen Ausbeuten.

Die Anwendungsmöglichkeiten der CD-haltigen Nano- und Mikrogele wurden im

Hinblick auf die Aufnahme- und Abgabeeigenschaften potenzieller organischer Gastmoleküle

wie sensitive Farbstoffe (Phenolphthalein, Nilrot), antimikrobielle Wirkstoffe (Chlorhexidin),

Insektizide (Permethrin) und pharmazeutische Wirkstoffe (Ibuprofen) untersucht.

Die Ergebnisse zeigten, dass CD-Einheiten in Nanogelen einen signifikanten Einfluss auf

die Aufnahme von organischen Gastmolekülen haben. Die kontrollierte Einschlusskomplexierung

der Gäste erfolgte in Abhängigkeit vom CD-Gehalt und der Ringgröße mit α-, β-und γ-CD. Die

Zusammensetzung der Monomere und der Vernetzungsgrad der polymeren Nanogelstrukturen

zeigen auch einen Einfluss auf die Aufnahme. Eine hydrophobe polymere Grundstruktur wie von

Poly(Methylmethacrylat) (PMMA) zeigt eine hohe Affinität zu hydrophoben Gastmolekülen und

erhöhte die Aufnahme von Chlorhexidin. Das hydrophile Polymerrückgrat von

Poly(Hydroxymethylmethacrylat) (PHEMA) ermöglicht das Eindringen von Gast-Molekülen in

wässrige Mikrogele, während das hydrophobe Ibuprofen dort bevorzugt durch CD-A-Einheiten

angebunden wird. Phenolphthalein und Nilrot zeigten, aufgrund der Bildung von signifikanten

CD-Komplexen in wässrigen Nanogelen, deutliche Veränderungen ihrer charakteristischen

Farben. Im Allgemeinen verringert ein höherer Vernetzungsgrad die Zugänglichkeit der CD-

Einheiten für Gastmoleküle und damit die Aufnahmekapazität der Nanogele.

Die Freisetzung und Stabilität der Gastmoleküle in Nanogelen hängen von der äußeren

Umgebung ab. Im Allgemeinen waren die CD-Nanogel-Komplexe mit hydrophoben

Gastmolekülen wie Permethrin, Ibuprofen und Nilrot stabil in Wasser. Leicht wasserlösliche

Gastmoleküle wie Chlorhexidin wurden langsam durch Veränderungen der umgebenden

Wasserkonzentration aus CD-MA/PMMA-basierenden Nanogelen freigesetzt. Nach Extraktion

mit organischen Lösungsmitteln wie Methanol, konnte die Freisetzung und Auswaschung von

Permethrin aus wässrigen CD/sP(EO-stat-PO) Nanogelen beobachtet werden. Das

lichtempfindliche Permethrin konnte gegen UV-Bestrahlung durch Komplexierung in CD-

haltigen Nanogelen geschützt werden.

Nano- und Mikrogele konnten durch physikalische Selbstanbindung auf verschiedenen

Oberflächen wie Polyesterfasern oder Keratinfasern appliziert werden und durch die

Konzentration der Dispersion und durch die Beschichtungszeit gesteuert werden. Biologische

XVII

Tests von ausgerüsteten materialen Oberflächen, welche mit Permethrin und Chlorhexidin

beladenen Nanogelen behandelt wurden, zeigten, dass die insektizide und antimikrobielle

Aktivität der Inhaltsstoffe nach der Komplexierung durch CD-Nanogele nicht verringert wird.

In zusätzlichen Arbeiten wurde eine Reihe von synthetischen und alternativen

Wirkstoffen zur Insektenbekämpfung ausgewählt, um ihre Aufnahme- und Abgabeeigenschaften

mittels β-CD zu studieren. Computergestützte Monte Carlo Kraftfeldsimulationen und

experimentelle Untersuchungen zeigten, dass größere Moleküle weniger stabile Komplexe mit β-

CD als kleinere Moleküle bilden. Funktionelle Gruppen der Gastmoleküle beeinflussten die

Komplexierung. Weiterhin zeigten Freisetzungsstudien der Wirkstoffe aus festen Komplexen,

dass der Freisetzungsprozess von den physikalischen Eigenschaften wie vom Dampfdruck des

Gastmoleküls und von der Luftfeuchtigkeit beeinflusst wird.

1

Chapter 1

Introduction

1.1 Cyclodextrin as supramolecular host in nano- and microgels

Cyclodextrins (CDs) are cyclic oligomers based on glucopyranose subunits and provide

an important class of host guest systems in supramolecular chemistry. The special hydrophobic

cavities of CDs enable the sequestration of organic molecules by inclusion complexation due to

non-covalent host guest interactions [1,2].

Supramolecular host guest systems are characterized as interaction of two or more

organic molecules, ions or radicals in a unique structural relationship using forces other than

those of full covalent bonds. The commonly mentioned types of non-covalent host guest

interactions like hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic

interactions are observed in supramolecular structures [3,4,5,6].

The advantages of CDs over other potential supramolecular hosts like macrocyclic

cucurbiturils [7], calixarenes [8], pillararenes [9], cyclophanes [10], crown ethers [11],

porphyrins [12] and furthermore, cryptands [13], spherands [14], carcerands [15] is the rigid,

well-defined cyclic structure and the ability to bind a high number of many different low-

molecular-weight guest molecules [16].

Furthermore, CD is produced from renewable natural material in thousand of tones per

year in a relatively simple production process and it is offered in acceptable prices for most

industrial purposes. In addition, great experiences in use of CD in medical and pharmaceutical

field, in industrial products, technologies and research have been documented over many years

[17,18,19,20].

The negligible cytotoxic effects of CDs and the modification of the physical and

chemical properties of the guest molecule due to inclusion complex formation are important

aspects for potential applications. Thus, the use of CDs as carriers, solubilizer, stabilizer, drug

delivery and controlled release systems enables the target specificity and modulation of uptake

and release of chemical agents [21,22].

Organic molecules are usually complexed in aqueous solution. During the complexation,

water molecules that occupied the CD cavity can be substituted by appropriate guest molecules.

2

These guest molecules are less polar than water and the driving force of the complexation is the

substitution of high enthalpy water molecules [23,24].

The release takes place depending on the guest molecule by altering and by changing the

surrounding condition like solvent composition, surfactants, temperature and pH. Furthermore,

the decomplexation of the guest by other energetically preferred guest molecules and by

application on surfaces like tissue are observed [25,26].

The uptake and release properties of organic guest molecules may be modulated by

covalent embedding of CD in polymeric structure of aqueous nano- and microgels as additional

functional domains. Nano- and microgels are an important class of soft organic nanoparticles

with a swellable cross-linked polymeric structure and small size. The small size of several

nanometers enables working between molecular and cellular level and similar to CDs, nanogels

suggest high potential as carrier and delivery systems for many chemical agents [27,28,29,30].

Scheme 1. Incorporation of cyclodextrins as supramolecular host units in polymeric structure of

nano- and microgels.

The incorporation of CD with its perfect binding properties towards guest molecules in

small gel particles provides an interesting combination of supra- and macromolecular chemistry

with nanotechnology. The combination of CDs and nano- and microgels suggests some

advantages and may improve the properties and application possibilities for both. Hence, nano-

and microgels as carrier may promote self-bonding and fixation of CDs and the corresponding

inclusion complexes on different surfaces with the aim of target application, bioavailability in

solid phase and modulation of long time activation of the chemical agents. Furthermore, the

incorporation of CD hosts as multifunctional domains may moderate the controlled uptake,

permanent fixation or targeted release of chemical agents from nano- and microgels.

Cyclodextrin Nano/MicrogelsCyclodextrin

containing Nano/Microgels

3

1.2 Content of this thesis

This thesis deals with the preparation and characterization of novel functional polymeric

nano- and microgels using CDs as polymeric cross-linker and functional complexation domain

for the uptake and release of potential active agents. α-, β- and γ-CDs and CDs functionalized

with various numbers of vinyl groups are studied in combination with different monomer and

pre-polymers with the goal of stable nano-and microgels with specific properties in aqueous

solution. Furthermore, the application of CD containing nano- and microgel particles is

investigated with regard to the uptake and release of potential guest molecules like dyes,

antimicrobial agents, insecticides and pharmaceutical active ingredients as well as the coating on

different surfaces especially on textiles.

Chapter 2 gives a literature review about properties, applications and preparation

methods of nano-and microgels, natural CDs and CDs in polymeric- and nanostructures.

In Chapter 3, the incorporation of α-, β-, γ-CDs in nanogels based on cross-linked

poly(methyl methacrylate) (PMMA) combine the good complexation properties of CD, the

colloidal behavior of nanoparticles and the ability to work in organic as well as in aqueous

media. The nanogels are prepared by surfactant-free precipitation polymerization in organic

solvent. The particle formation and swelling in organic and aqueous solution are studied by

variation of the cyclodextrin methacrylate (CD-MA) concentration in the reaction mixture, the

number of vinyl groups at the CD ring and the differences by use of α-, β- or γ-CDs. The uptake

properties of the nanoparticles are studied by the dye adsorption method using phenolphthalein.

Chapter 4 deals with the uptake of the antimicrobial active agent chlorhexidine (CHX)

by CD-MA containing PMMA nanogels. CHX is an antiseptic and bacteriostatic agent that

shows activity against many bacteria and biofilms. The uptake of CHX by CD domains in

nanaoparticles is quantitatively described and the application on glass-surfaces is investigated.

Furthermore, release studies in water and biological tests with regard to antimicrobial efficiency

of the CHX nanoparticles are performed against Straphylococcus aureus.

In Chapter 5, the preparation of novel cyclodextrin acrylate (CD-A) containing

poly(hydroxyethyl methacrylate) (PHEMA) microgels for the application as drug carrier of the

anti-inflammatory drug ibuprofen is presented. The synthesis and the influence of -, - and -

CD-A with 2, 4 and 6 vinyl bonds is studied with regard to the particle size. Furthermore, the

uptake and complexation process of ibuprofen by -CD-A microgels in aqueous dispersion is

4

followed by UV-vis measurements and characterized by variation of the ibuprofen concentration

and by variation of the -CD-A content in microgels.

In Chapter 6, the incorporation of reactive CD-A in aqueous temperature-sensitive

nanogels based on poly(N-vinylcaprolactam) (PVCL) is studied by surfactant-free precipitation

polymerization process. α-, β- and γ-CDs were functionalized with acrylic groups and the

average amount of vinyl bonds per CD molecule was adjusted to 2, 4, or 6. The influence of the

CD-A on particle size, the volume transition point (VPTT) and the uptake of phenolphthalein is

demonstrated.

In Chapter 7, the incorporation of the fluorescent solvatochromic dye nile red (NR) in

thermo-responsible CD nanogels based on PVCL is described in detail. Series of nanogels with

-, - and -CD-A units are investigated with respect to the dye uptake capacity depending on

the ring size of the CD and the degree of cross-linking. The twisted intermolecular charge

transfer (TICT) of NR enables the investigation of the NR:CD complex structure in the CD

nanogels. Furthermore, temperature dependent studies at different degrees of swelling are

performed to observe changes in complexation structure and dye behavior in nanogels. The

adhesion of the CD nanogels to polyester fibers and their influence on the color of the fabric is

studied at different temperatures.

In Chapter 8, CD containing nanogels are prepared by surfactant-free condensation with

reactive prepolymers (NCO terminated sP(EO-stat-PO)) in water. The influence of the CD

content on the particle size and the optimization of reaction parameters yields ultrafine nanogels

are described. The maximum content of active CD is analyzed using dye sorption with

phenolphthalein to demonstrate the uptake capacity of these nanogels for hydrophobic molecules.

Chapter 9 deals with the modification of keratin fibers like wool fabrics and human hairs

with the common insecticide permethrin by -CD based nanogels from aqueous dispersion. The

uptake and release of permethrin in nanogels and on the fibers are studied quantitatively by

HPLC analysis. The permanence of sensitive insecticide is tested on the treated fibers with regard

to washing and UV fastness. Bioassay tests against the larvae of Tineola bisselliella and

Anthrenocerus australis are made to proof the activity of the treated keratin fabrics.

Chapter 10 is the Appendix to Chapter 9 and describes the theoretical and practical

formation of inclusion complexes based on a series of common synthetic and natural insecticides

with natural -CD.

5

1.3 References

1. Szejtli, J., Introduction and general overview of cyclodextrin chemistry, Chem. Rev. 1998, 98, 1743-1753.

2. Cram D. J.; Cram, J. M., Host-Guest Chemistry, Science 1974, 183, 803-809.

3. Rudkevich, D. M., Nanoscale molecular containers, Bull. Chem. Soc. Jpn 2002, 75, 393-413.

4. Lehn, J. M., Supramolecular Chemistry - Scope and Perspectives Molecules, Supermolecules, and

Molecular Devices (Nobel Lecture), Angew. Chem. Int. Ed. 1988, 27, 89-112.

5. Cram D. J., The Design of Molecular Hosts, Guests, and Their Complexes (Nobel Lecture), Angew. Chem.

Int. Ed. 1988, 27, 1009-1020.

6. Pedersen, C. J., The discovery of crown ethers (Noble Lecture), Angew. Chem. Int. Ed. 1988, 27, 1021-

1027.

7. Freeman, W. A.; Mock, W. L.; Shih, N.-Y., Cucurbituril, J. Am. Chem. Soc. 1981, 103, 7367-7368.

8. Shinkai, S., Calixarenes as new functionalized host molecules, Pure & Appl. Chem. 1986, 58, 1523-1528.

9. Ogoshi, T.; Kanai, S.; Fujinami, S.; Yamagishi, T. A.; Nakamoto, Y., para-Bridged symmetrical

pillar[5]arenes: Their Lewis acid catalyzed synthesis and host guest property, J. Am. Chem. Soc. 2008,

130, 5022–5023.

10. Collet, A., Cyclotriveratrylenes and Cryptophanes, Tetrahedron 1987, 43, 5725-5759.

11. Pedersen, C. J., Frensdorff, H. K., Macrocyclic Polyethers and Their Complexes, Angew. Chem. Int. Ed.

1972, 11, 16-25.

12. Sessler, J. L.; Seidel, D., Synthesechemie expandierter Porphyrine, Angew. Chem. 2003, 115, 5292-5333.

13. Lehn, J. M., Cryptates: The Chemistry of Macropolycyclic Inclusion Complexes, Acc. Chem. Res. 1978,

11, 49–57.

14. Cram, D. J.; Kaneda, T.; Helgeson, R. C.; Brown, S. B.; Knobler, C. B.; Maverick, E.; Trueblood, K. N.,

Host-Guest Complexation. 35. Spherands, the First Completely Preorganized Ligand Systems, J. Am.

Chem. Soc. 1985, 107, 3645-57.

15. Cram, D. J.; Karbach, S.; Kim, Y. H.; Baczynskyj, L.; Marti, K.; Sampson, R. M.; Kalleymeyn, G. W.,

Host-guest complexation. 47. Carcerands and carcaplexes, the first closed molecular container compounds,

J. Am. Chem. Soc. 1988, 110, 2554–2560.

16. Del Valle, E. M. M., Cyclodextrins and their uses: a review, Proc. Biochem. 2004, 39, 1033-1046.

17. Rajewski, R. A.; Stella, V. J., Pharmaceutical Applications of Cyclodextrins. 2. In Vivo Drug Delivery, J.

Pharm. Sci. 1996, 85, 1142-1169.

18. Loftsson, T.; Duchene, D., Cyclodextrins and their pharmaceutical applications, Int. J. Pharm. 2007, 329,

1–11.

19. Sanger, W., Cyclodextrin Inclusion Compounds in Research and Industry, Angew. Chem. Int. Ed. 1980, 19,

344-362.

20. Hedges, A. R., Industrial applications of cyclodextrins, Chem. Rev. 1998, 98, 2035-2044.

21. Uekama, K.; Otagiri, M., Cyclodextrins in drug carrier systems, Crit. Rev. Ther. Drug Carrier Syst. 1987, 3,

1-40.

6

22. Duchêne, D.; Wouessidjewe, D.; Ponchel, G., Cyclodextrins and carrier systems, J. Controlled Release

1999, 62, 263-268.

23. Liu, L.; Guo, Q.-X., The driving forces in the inclusion complexation of cyclodextrins, J. Incl. Phenom.

Macrocycl. Chem. 2002, 42, 1–14.

24. Rekharsky, M. V.; Inoue, Y., Complexation Thermodynamics of Cyclodextrins, Chem. Rev. 1998, 98, 1875-

1918.

25. Hirayama, F.; Uekama, K., Cyclodextrin-based controlled drug release system, Adv. Drug Delivery Rev.

1999, 36, 125-141.

26. Ma, M.; Li, D.-Q., New Organic Nanoporous Polymers and Their Inclusion Complexes, Chem. Mater.

1999, 11, 872-874.

27. Harada, A.; Takashima, Y.; Yamaguchi, H., Cyclodextrin-based supramolecular polymers, Chem. Soc. Rev.

2009, 38, 875-882.

28. Nayak, S.; Lyon, L. A., Soft nanotechnology with soft nanoparticles, Angew. Chem. Int. Ed. 2005, 44,

7686-7708.

29. Pich, A.; Adler, H. J., Composite aqueous microgels: an overview of recent advances in synthesis,

characterization and application, Polym. Int. 2007, 56, 291-307.

30. Oh, J. K.; Drumright, R.; Siegwart, D. J.; Matyjaszewski, K., The development of microgels/nanogels for

drug delivery applications, Prog. Polym. Sci. 2008, 33, 448-477.

7

Chapter 2

Literature Reviews

2.1 Nano-and Microgels

2.1.1 Multifunctional nanoparticles

In recent years continued development of the creation of complex materials has been

focused on the engineering over length-scales from molecular to the colloidal to the

macroscopic in order to improve their properties for specific applications. Nanoparticles are an

important class of nanomaterials and the extremely small size offers unique functions and

applications which are controlled by the high internal interface area [1]. Small particles are

defined by size. Coarse particles are sized between 10–2.5 µm and fine particles from 2500-100

nm [2]. Classical nanoparticles are ultrafine particles and range between 100-1 nm [3].

Most interest in nanoparticles is focused on the synthesis, modification, characterization

and applications of new advanced nanomaterials with specific functions and different responses

to changes in the near environment. Hence, multifunctional materials design focuses on methods

by which functional building blocks are assembled in complex architectures [4], with aim of

modifying the design and control of the compositions, morphology, nanostructure and

functionality of the nanomaterials [5]. The interdisciplinary conclusion of classical fields in

science promotes the development of inorganic, organic and hybrid nanosystems with novel

physical and chemical properties [6] all with the aim of creating new materials in different

dimensions with high complexity and some new functions e.g. self-organisation and self-

healing. Furthermore, nanobiological and nanomedical applications find enhanced interest in

public and research [7].

In the last years much of work in nanoparticles is concentrated on the design of “hard”

materials, such as inorganic nanoparticles, metals, carbon nanotubes, Buckyballs,

semiconductors, and organic or inorganic dielectrics. “Soft” materials with sizes on the

nanoscale consist of flexible and dynamic structures. They are designed by different polymer

compositions and show continued interest in the area of nanoscience. In particular, the

8

development of nanostructured hydrogels leads to a promising group of soft materials for

diverse applications [8].

2.1.2 Nano- and microgels

Swellable nanoparticles consisting of polymeric architectures with molecular building

blocks are an important class of soft colloidal functional materials in the range of several

nanometers. Swollen divinylbenzene microgel particles have already been discussed in the

literature by Hermann Staudinger in 1935 [9]. Later, polymeric nanoparticles have been

fundamentally investigated in the beginning of 1976 [10]. Ultrafine cross-linked polymeric

nanogels which are operating in the aqueous phase are primary developed and reported for drug

delivery in medical application [11].

In this context, interpenetrating polymer co-networks lead to the formation of small gel

particles with a three-dimensional (3D) spherical structure that can undergo particle swelling in

different mixable solvents. The most widely investigated so-called nano- and microgel systems

are small hydrophilic hydrogel particles. Multi-functionality of nano- and microgel particles can

be based on unique properties such as large surface area, high chemical functionality, controlled

porosity, ability to swell in different solvents and stimuli-sensitivity to changes in the

environmental conditions [12,13].

Figure 1. Morphology of thermo-sensitive VCL/AAEM nano- and microgels [16].

9

The ability to swell and de-swell in the aqueous phase and the consequent change of the

particle size by uptake and release of water is one of the most attractive properties of responsive

nano- and microgels. Therefore, interactions between the solvent and the polymer backbone and

interactions between the polymer chains such as degree of cross-linking of the gel architecture

influence the degree of swelling. The swelling properties of particles and furthermore, the

properties of the dispersion can be modified by the use of stimuli-sensitive polymers in the

macromolecular network which change their polymer-solvent interactions upon change of the

surrounding conditions. These effects lead to controlled changes in particle sizes and properties

[14]. Well known and well studied nano- and microgel systems combine the response to

temperature [15,16], pH [17], pressure [18], light [19], solvent components [20], magnetic field

[21], etc.

Figure 2. Hydrodynamic radii (a) and calculated swelling ratio (RhM

/RhSW

)3

(RhM

= measured

hydrodynamic radius at different temperatures; RhSW

= measured hydrodynamic radius for

maximally swollen nanogels at T = 20 °C) (b) as a function of temperature for temperature

sensitive PVCL/AAEM microgels [16].

The chemical functionality of polymeric nano- and microgels is extremely important for

their application in different areas of science and technology. Different polymeric architectures of

nano- and microgels with variation of monomer compositions [22], degree of cross-link [23],

20 25 30 35 40 45 50100

125

150

175

200

225

(Rh

M/ R

h

SW

)3

Particle size

Rh [n

m]

Temperature [°C]

0.0

0.2

0.4

0.6

0.8

1.0

Swelling ratio

10

incorporation of specific functional groups [24], surface modification [25], charging of particles

by ionic components [26], integration of biomacromolecules [27] or inorganic structures [28]

meet specific applications.

The progress in chemical design, modification and synthesis of nanoparticles as well as

the understanding of physico-chemical properties of nano- and microgels especially in aqueous

phase resulted in numerous chemical, bio-medical and technical applications such as for coatings

[29], personal care [30], food [31] and catalysis [32]. However, nano- and microgels can be used

in colloidal form as drug carrier for drug delivery [33] and for controlled release [34], as

antimicrobial agents [35], microreactors [36] and sensor [37], but also as building blocks for the

design of colloidosomes [38], hydrogels [39], films [40] and biomaterials [41].

2.1.3 Synthesis of Nano- and Microgels

Different synthesis routes have been developed to modify nano- and microgels or to

incorporate functional groups with different properties specifically in the core or at the surface of

the particles. Furthermore, the synthesis method enables the successful control of the particle

size, the particle size distribution (PSD) and the stability of the nanoparticle system [42].

One strategy is the reversible physical self assembly of pre-polymers to gel particles e.g. via

gelation method [43]. Another one is the chemically cross-linking of pre-polymers e.g. in

water/oil emulsion [44], by membrane emulsification [45] or by microfluidic techniques [46].

Monomers can be polymerized and cross-linked to nanoparticles by free or controlled

heterophase polymerization like precipitation polymerization [47] and by water/oil emulsion, an

inverse emulsion polymerization [48]. A new method to prepare nano- and microgels are the

photolithographic technique by particle replication in non-wetting templates (PRINT). The

creation of monodisperse gel particles takes place by prevention of a continuous hydrogel film

formation using elastomeric molds from a low surface energy network consisting of

perfluoropolyether [49].

The common synthesis techniques of nano- and microgels using covalently

polymerization of monomers and pre-polymers like precipitation polymerization and the

emulsion polymerizations (water in oil) and the inverse mini/microemulsion (oil in water) are

reviewed in more detail.

11

The surfactant free emulsion polymerization or the precipitation

polymerization/copolymerization are carrying out at higher temperatures. After initiation with

standard initiator for radical polymerization process the monomer is attacked by a radical. The

propagation and the chain growth starts. At the precipitation polymerization the polymer chain

collapses upon itself when the critical length is reached, because the polymerization temperature

is higher than the low critical solution temperature (LCST) of the polymer. The produced

precursor particles grow by further monomer addition, by aggregation of other precursor

particles, by capturing growing oligoradicals or by being captured by other existing particles [47].

Miniemulsion polymerization (oil-in-water) initiates the particle growth in nanodroplets

in the size range between 50 and 500 nm. The type and the amount of emulsifier mainly

influenced the size of the droplet and of the nanoparticles. Under high-pressure homogenization

or ultrasonication broadly distributed (macro)droplets are formed which are broken into narrowly

distributed, defined small stable nanodroplets. In each droplet the initiation of the polymerization

and the particle growth take place. An efficient surfactant and a co-stabilizer like long chain

alkanes and alcohols as (ultra)hydrophobes which are only soluble in the dispersed phase are

used to prevent the degradation of miniemulsion by Ostwald ripening and coalescence [48].

Inverse miniemulsion (water-in-oil) is the inverse case of the principal of miniemulsion.

Due to the change of the continuous phase from hydrophilic to hydrophobic, surfactants with low

hydrophilic–lipophilic balance (HLB) values are required. The Ostwald ripening is suppressed by

co-stabilizers such as lipophobe e.g. ionic compounds (salts or sugars) or non-ionic block

copolymer stabilizers that are insoluble in the continuous oily phase. Hydrophilic monomers and

hydrophilic polymers plus cross-linker (by preparation of gel particles) are miniemulsified in a

continuous hydrophobic phase followed by initiation of the polymerization either from the

hydrophilic droplet or from the hydrophobic phase [50,51].

The inverse microemulsion is based on the water-in-oil emulsion techniques and is used

to prepare ultrafine nanoparticles under 100 nm. In this case, a large excess of surfactant is used

e. g. for the preparation of microgels based on gelatin. The ratio of gelatin to surfactant can be as

high as 1:1600 [52].

12

2.2 Cyclodextrin

2.2.1 Natural Cyclodextrins

CDs are cyclic oligomers consisting of α-1,4-glycosidic bonded D-glycopyranose units

and were discovered by Villiers in 1891. He isolated crystalline CD from starch after bacterial

digestion for the first time and called it “cellulosine” due to its chemical behavior similar to

cellulose [53]. In 1903, Schardinger identified -CD as the “cellulosine” and published that the

Bacillus macerans was responsible for the formation of -CD and -CD [54]. In the following

decades, more CDs were discovered and chemical structures of the cyclodextrin subtypes such as

γ-CD were described [55].

Scheme 1. Natural α-, β- or γ-CD with cyclic α-1,4-glycosidic bonded 6, 7 or 8 D-glucopyranose

units [59].

CD can be obtained by degradation of starch. The technical synthesis is performed by use

of the cyclodextrin-glycosyl-transferase (CGTase). The CGTase cut the helical starch polymer in

small pieces consisting of a mixture with cyclic-linked 6, 7 or 8 D-glycopyranosese units (α-, β-

or γ-CDs) (Scheme 1) which can be isolated by precipitation and chromatographic methods [56].

The D-glucopyranose units create a truncated cone structure which provides a hydrophilic

exterior and a hydrophobic interior cavity (Scheme 2). The truncated cone is formed by the 4C1

chair conformation of the individual D-glucopyranose units. Hence, the primary hydroxy group is

O

OHHO

OH

O

O

OH

HO OH

O

O

OH

OH

OH

O

OO

OH

OH

HO

O

OH

OHHO

O

O

OH

HO

HO

O

C36H60O30Mol. Wt.: 972.84

O

OHHO

OH

O

O

OH

HO

OHO

OOH

OH

OH

O

O

OH

OH

OH

OO

OH

OH

HO

O

OOH

OH

HO

O

O

OH

HO

HO

O

C42H70O35

Mol. Wt.: 1134.98

O

OH

HO

OH

O

O

OH

HO

OHO

OOH

OH

OH

O

O

OH

OH

OH

O

O

OH

OH

HO

OOH

OH

HO

O

O

OH

HO

HO

O

O

OH

OH

HO

O

O

C48H80O40

Mol. Wt.: 1297.12

dprim= 4.7 Å

dsec = 5.2 Å

dprim= 6.0 Å

dsec = 6.2 Å

dprim= 7.5 Å

dsec = 8.3 Å

α-Cyclodextrin

6 glucose units

γ-Cyclodextrin

8 glucose units

β-Cyclodextrin

7 glucose units

C48H80O40

MW: 1297

C42H70O35

MW: 1135

C36H60O30

MW: 1297

13

located at the narrow opening and the secondary hydroxy groups in the second and third position

at the larger opening of the torus. All hydroxy groups of the glucose units are oriented to the

outer surface, which leads to exohydrophilic properties and thus, to the water solubility of the

CDs. The hydrophobic interior (endohydrophobic) caves have a diameter of 5-8 Å [57,58].

Scheme 2. Structure of natural cyclodextrin (n = number of glucose units, d = diameter of each

cavity, h = height) [58].

The hydrophilic outer surface and the hydrophobic interior surface of the cone structure

enable the complexation of various, amphiphilic and hydrophobic guest molecules in water which

have an appropriate molecular structure equivalent to the CD ring size [59]. The complexes exist

of non-covalent interactions such as hydrogen bonds, van der Waals forces and hydrophobic

interactions between the host and the guest molecule [60]. The driving force of the complexation

of guest molecule in the hydrophobic cavity of the CDs is controlled by the release of the

displaced water molecules from the torus. During the release and the increased mobility of water

molecules the entropy increases. Furthermore, the formation of new H-bonds between the water

molecules and the increase of the cohesion forces lead to decrease of enthalpy. Van der Waals

forces have only a very short range, so that inclusion compounds are more stable in general,

when the cavity of the CD is filled out perfectly by the guest molecule. Dipole-dipole interactions

stabilize only complexes of guests with strong dipole moments because of the axial dipole

moment of the CDs [61].

This unique property of the inclusion complex is commonly referred to as host:guest

complex formation. The association of the CD host and the guest molecules (D) as well as the

dissociation of the CD:D complex is characterized by thermodynamic equilibrium (eq.1). The

4,7 Ǻ - n = 6

dprim = 6,0 Ǻ - n = 7

7,5 Ǻ - n = 8

5,2 Ǻ - n = 6

dsec = 6,2 Ǻ - n = 7

8,3 Ǻ - n = 8

h = 8,0 Ǻ

dsec

dprim

14

stability of the host:guest interaction can be described by complex stability constant KS (eq.2). In

general, the complex stability constants for stable aqueous complexes are 102-10

4 M

-1 [62].

The complexation leads to the modification of the physical and chemical properties

(solubility, smell, reactivity etc.) of the guest molecules [63]. For example, the complexation of

pH-sensitive dye phenolphthalein by -CD results in fixation of the instable colorless

configuration whereas the non-complexed configuration presents a pink color at pH 10.5 in

aqueous solution (Scheme 3) [64].

Scheme 3. Structure of the phenolphthalein:-cyclodextrin complex and the complex properties

in respect to the color in aqueous solution [64].

Furthermore, the stability can be increased against oxidation, photodecomposition,

hydrolysis, dehydration as well as protection of guests from degradation in the gastrointestinal

tract [65]. Upon inclusion, poorly water-soluble molecules can be transferred in aqueous solution

and thus, the water solubility of the guest can increase as well as its bioavailability [66].

CDs find application as carrier-, delivery- and controlled release systems and improve the

efficacy of active ingredients such as drugs in the therapeutic medicine [67,68]. CDs are already

used in many fields of daily life and find enhanced applications in research (e.g. chromatographic

separation of enantiomers), industry and households (e.g. adsorber of fragrances) [69,70].

Phenolphthalein β-Cyclodextrin Phenolphthalein : β-Cyclodextrin-Complex

OO

O

O

OO

COO-

- - -

pH 10.5

-pink- -colorless-O

OHHO

OH

O

O

OH

HO OH

O

O

OH

OH

OH

O

OO

OH

OH

HO

O

OH

OHHO

O

O

OH

HO

HO

O

15

2.2.2 Cyclodextrin in polymeric systems

The use of CDs and CD derivates in polymeric systems leads to a variety of polymeric

networks and assemblies. CDs act as building blocks for the creation of new switchable materials

or as functional domains for uptake and release of active ingredients. Polymeric functional

materials based on CDs have been designed with different properties which differ in the type of

linking. In principal, CD can be integrated in polymers using physical and covalent linking [71].

The chemical covalent linkage of CD in polymer systems which can be seen as permanent linking

can be divided in condensation and polymerization reaction.

In condensation reaction CD can be exploited to prepare cross-linked polymer networks

(Scheme 4a). Thus, the covalent linking of the natural CD structure are made by reaction of the

large number of reactive hydroxyl groups or by hydroxyl, carboxylic acid or amine groups

containing CD derivatives with bi- or multifunctional agents such as aldehyde, ketone, isocyanate

or epoxide groups [71,72]. Furthermore, covalent polymer networks are prepared by cross-

linking of different polymer chains with functional groups [73,74].

The hydroxy groups of CDs can also be used to synthesize reactive CD monomers

suitable for radical polymerization reactions (Scheme 4b). One of the most widely used synthetic

method is the direct copolymerization using reactive CD monomers with vinyl groups and wide

variety of vinyl monomers such as MMA, HEMA, NIPAm, etc. [75,76]. Furthermore, CD with

reactive vinyl groups acts as low molecular weight cross-linking agent to a variety of pre-existing

polymers to prepare covalently linked networks by combining polymers and CDs [77,78].

Non-cross-linked linear polymers have been prepared by copolymerization of CD

derivates containing only one functional group with a variety of common monomers, mostly

acrylates. Some hydrogels are also functionalized with mono-substituted CDs with the aim to

increase the rotational freedom and the accessibility of the host to guest molecules [79,80].

A further method to create physical cross-linked polymers and hydrogels is the lock and

key-system due to physical self-assembly of polymers driven by inclusion complexation (Scheme

4c). Hence, self assembled polymer systems can be prepared due to mixing of two polymer

chains, one functionalized with covalently bonded CD and another polymer functionalized with

potential guest molecules for the CD complexation. Different guest molecules such as 4-tert-butyl

anilide, dodecyl, adamantly, cholesterol or aromatic rings were covalently attached on polymer

chains and studied for complexation with CD polymers [81-84]. The intramolecular cross-linking

16

of polymer chains by host:guest CD inclusion complexes leads to supramolecular hydrogels with

chemical (high stability upon dilution), desirable physical (reversibility) and switchable

properties [85,86].

Scheme 4. Preparation of CD containing polymers, hydrogels and polymeric nanostructures

using covalent linking via condensation (a) and polymerization reaction (b) or physical assembly

and cross-linking via key-lock interaction of CD units and guest minorities of polymers (c).

In some work, results showed that CDs control the formation of nano- and

microstructures of coalescent polymers [87]. In pharmaceutical formulations CDs are used to

improve the aqueous solubility, stability, dissolution rate, bioavailability and/or local tolerance

of drugs. Furthermore, CDs regulate the drug delivery in polymer systems [88,89].

The complexation properties of CD containing polymers differ in general from the free

low molecular weight CDs. Complexation studies showed that in some cases the complex

formation constant of CD embedded in polymers is decreased due to sterical effects and poor

Free-radical polymerization

Condensation

Cross-linking

Cross-linking

a

b

Key-lock-systemPhysical self-assembly

c

Cross-linking

17

accessibility of the CD cavity. In exceptional cases some cooperative effects of the polymer

backbone and the adjacent CDs can increase the complex stability constant (KS) [90,91].

Thus, cooperative or synergistic effects of the copolymer and the CD units can also be

expected. It is reported, that water-soluble polymers can be used to stabilize drug/CD complexes

through formation of ternary complexes. These ternary drug:cyclodextrin/polymer complexes

make it possible to enhance the complexation efficacy of CD and at the same time improve drug

bioavailability from CD containing drug formulations [92]. For example, these effects were

observed using poly(ethylene glycol) (PEG 400) during the complexation process of

progesterone and CD. Results showed that the solubility of the hydrophobic drug in CD/PEG

mixture could be increased and were up to 96 % higher than the theoretical solubility values

[93].

Furthermore, a comparison of the solubilization efficacy as well as the drug loading

capacity of two novel high molecular weight CD polymers, i.e. poly(ethylene glycol) (PEG/γ-

CD) and epichlorohydrin based γ-CD polymer (EPI/γCD) with natural γ-CD using

dexamethasone (Dex) as a model drug was higher than that of the parent natural γ-CD. The

results could be explained by the synergistic effects of the γ-CD and the polymer structure

which enable the upload of greater amounts Dex than the parent natural γ-CD [94].

Recently, a comparison between a CD-methacrylate (CD-MA) containing

poly(hydroxyethyl methacrylate) (PHEMA) hydrogel and a hydroxypropyl-CD (HPCD)

containing superhydrophilic hydroxyethyl methylcellulose (HPMC) network showed, that the

hydrophobic model drug thiosemicarbazones is increased incorporated by the CD-MA/PHEMA

hydrogel, because of additional hydrophobic interaction with the polymer network [95].

An alternative approach is to use CD-polymers, which can both enhance the aqueous

solubility of a drug and result in sustained drug release. The release properties can be adjusted

by polymeric structure and the CD minorities. With this aim, CD networks have been developed

to enhance the bioadhesion and to control the uptake and the sustained release of drugs. Thus,

hydroxyethyl methylcellulose (HPMC) hydrogels and semi-interpenetration networks (IPN)

based on polyacrylic acid sodium salt (PAcNa) have been developed by covalent incorporation

of γ-CD and cross-inking of the polymeric structures. It were reported that the drug loading

yield and the release properties of the co-networks depended on the capability to form inclusion

18

complexes with γ-CD. Here, the sustained release of the model drug Dex was controlled by CD

content and by resulting cross-linking degree of the network [96].

In conclusion, CD polymers may increase or reduce uptake and release of guest

molecules depending on the polymer composition, hydrophilicity/hydrophobicity of the polymer

structure, density/mesh size of the cross-linked network, accessibility of the CD host units and

effective formation of strong host:guest complexes.

2.3 Cyclodextrin based nano- and microgel particles

CDs have been structurally incorporated in polymers or hydrogels and nowadays the

preparation of CD containing nanoparticles open a new research field. CDs as supramolecular

complexation units in nanoscale particle systems possess advantages over low molecular weight

CD, hydrogels and nanoscalic particles. The incorporation of CD cavities into the polymer

network of small nanoparticles promotes improved uptake and release properties [97].

In the last years, three common methods have been established for the creation of CD

based nanoscalic particles combining the different methods classified by the already discussed

strategies for preparation of nano- and microgel particles as well as CD-polymers (Scheme 4).

CD containing nanogels have been prepared by physical key–lock interactions of different

polymer chains [98], by direct covalent cross-linking of natural CD using condensation with

suitable bi- or multifunctional agents [99], and by polymerization of CD monomers bearing

acrylic or vinyl moieties with other co-monomers [100]. The cross-linking of the polymeric

structure, the method of the particle formation, the monomer/polymer compositions and the

preparation with or without surfactants are important parameter for the particle synthesis. In

many cases surfactants are used for the creation of stable and uniform particles. Recently, results

have been reported in which CDs with their special intrinsic capability may act as surfactants

with emulsifying properties in the emulsions [101]. The literature of the recent years associated

with the preperation method with respect to the type of cross-linking are listed for CD containing

nano- and microparticles, microcapsules, nanosponges and nano scalic gels in a first review paper

by Moya et al. [102]. Now, most important results with respect to synthesis, cross-linker, particle

size, special properties and uptake/release mechanism of the used model guests are discussed.

First, nanogels have been obtained by physical self assembly using lock and key system.

Thus, by mixing of CD containing polymers with dextrans bearing alkyl side chains stable

19

nanogels of about 200 nm were formed. It was found that the formation and stability of these

self-assembled nanogels depended on the polymer concentration, the weight ratio of dextran to

CD polymer, the molar mass of the CD polymer, the number of the alkyl minorities of the

dextran and the number of carbons in the alkyl chains. Before mixing with dextran the CD units

of the polymer were loaded with drugs. The unloaded CD units of the polymer are then available

for the complexation of the alkyl units of dextrans. Nanogels have been studied with regard to the

uptake capacity of benzophenone and tamoxifen [98]. Results show that benzophenone as guest

molecule hinders the sequestration of the alkyl moieties of dextran by the CD in the polymer.

This effect does not lead to an impeding of the formation of associative nano-assemblies or

detraction of their stability [103]. The controlled disruption of the self-assembling microparticles

formed by CD polymer and hydrophobically modified dextran was studied with addition of

hydroxy-adamantane. Thus, the disruption occurred with the addition of the model guest in a

change of the spherical particle shape to a more random one [104].

The condensation reaction with epichlorohydrine (EPI) as cross-linker has been described

in detail by some work groups and is one of the most popular syntheses for CD gels. Recently,

CD containing microgels with a size of 50-200 µm based on poly(vinylalcohol) (PVA) and EPI

or with an interpenetrating network (IPN) based on poly(methyacrylic acid) (PMAA) have been

prepared by inverse emulsion. The functionality of CD minorities was tested for release of the

model drug methylorange (MO) by changing the pH value of the environmental condition.

Results showed that the drug release was driven by host guest interaction and is not related to the

volume phase transition of the pH sensitive microgels [105].

Nanoparticles with a hydrodynamic radii of 65-88 nm with controllable cationic

properties have been developed based on quaternary ammonium -CD (QA-CD), choline

chloride and cross-linking using EPI in a one-step condensation polymerization. With the aim to

treat brain disorders across the blood brain barrier using nanoparticles as drug delivery system,

the enhanced permeability across the bovine brain microvessel endothelial cell (BBMVEC)

monolayers being dependent upon the number of quaternary ammonium groups were found. The

permeability of the model drug doxorubicin (DOX) could be enhanced across BBMVEC

monolayers by 2.2 times. No toxic effect of CD nanoparticles (500 µg/mL) in BBMVEC was

detected and furthermore, nanoparticles could completely protect BBMVECs from cytotoxicity

of DOX. [106]. Furthermore, anionic -CD microparticles based on anionic polysaccharides

20

(carboxymethyl or sulfopropyl pullulan) were prepared using cross-linker 3-(glycidoxypropyl)

trimethoxysilane. The uptake and loading capacity of microparticles were studied to retain water

pollutants (phenol and benzoic acid derivatives, -naphthol), drugs (salicylic acid, indomethacin)

and proteins (lysozyme). Results showed that the retention of the guest molecules is influenced

by their inclusion in the cavity of cyclodextrin and in the pores of hydrogels, as well as through

the electrostatic forces toward anionic polyelectrolyte charges [107].

CD based microcapsules with a mean size in the 10–35 μm range were prepared by

interfacial cross-linking of -CDs with terephthaloyl chloride (TC) in 1 M NaOH using emulsion

technique. These microcapsules show high and rapid uptake of p-nitrophenol (pNP) reaching a

maximum within 1 h due to capsule structure. The maximal fixation was observed for small-sized

particles (≈11 μm) [108]. The controlled release of the guest molecule propranolol for several

hours was studied. Thus, drug release was more retarded using higher amount of added -CD

microcapsules [109].

Nanoporous CD containing nanosponges (CDNS) with a size ranged from 350 to 600 nm

with low polydispersity indices (PDI) have been prepared by use of active carbonyl compounds

(e.g. dimethylcarbonate or carbonyldiimidazole) as cross-linkers. Ultrasound-assisted

preparation method enables the creation of solid nanoparticles in crystalline form with a spherical

shape in DMF or DMSO [110]. CDNS with high swelling properties in aqueous phase are created

using pyromellitic dianhydride (PMA) as cross-linker. The pore sizes of CDNS could be

modulated by changing in the mole ratio of a CD cross-linker [111]. CDNS were investigated

mainly as drug carrier such as dexamethasone, flurbiprofen, doxorubicin, itraconazole, paclitaxel,

5-fluorouracil, tamoxifen, camptothecin, resveratrol [112] and furthermore, as adsorber for gases

[113]. Results showed that nanosponges can act as multifunctional carriers e.g. solubility

enhancement of drugs, protection of fragile molecules from light or degeneration, sustained

delivery and release system [112].

The oil in water miniemulsion method has been applied to prepare -CD nanospheres

with a size of 185–1600 nm cross-linked with isophorone diisocyanate (IPDI) in hexadecane and

sodium-n-dodecylsulfate (SDS) as surfactant. With the aim of water purification from organic

pollutants, toluene and phenol were used as model guest to study the adsorption and regeneration

of -CD nanospheres. Results showed that the maximum adsorption loading is achieved for

toluene within 8 h and for phenol within 14 h. The regeneration of the solid saturated

21

cyclodextrin nanospheres were studied by extraction in an ethanol solution for 24 h. The

cyclodextrin nanospheres exhibited a slightly lower adsorption loading of ca. 10% [114].

Recently, CD and hydroxypropyl -cyclodextrin (HPCD) and -CD based nanogels with a

size between 10 and 400 nm were prepared by reaction with hydroxypropyl methylcellulose

(HPMC) or agar and additional cross-linking with ethylene glycol diglycidyl ether (EGDE). The

synthesis took place in inverse miniemulsion (water in dichloromethane) with surfactant Span 80

using simultaneously an emulsification/solvent evaporation process. Results showed that

nanogels could also be formed without using a surfactant. This emulsion stabilizing effect of CDs

could be explained by formation of inclusion complexes with the organic solvent at the interface.

3-methylbenzoic acid (3-MBA) was selected for permeability tests which confirmed the

formation of inclusion complexes and the sustain release [99].

The radical copolymerization was also used as preparation method for the creation of

nano- and microgels. Hence, biodegradable and biocompatible nanogels with a particle size

between 60 and 260 nm were prepared by radical copolymerization of a polylactic acid (PLA)

macromonomer and CD monomer (from CD and 1-allyloxy-2,3-epoxy propane) with different

numbers of vinyl groups. The particle size depend on the PLA/CD ratio, whereas the

hydrophilicity of the microgels increased with increasing β-CD contents. Furthermore, an

increase of the cross-linking degree in microgels was controlled by increase of the vinyl groups

(1-7) on the CD monomer and led to a decrease in the degree of swelling and in the degradation

of microgels [115]. The two step precipitation polymerization has been applied to the synthesis of

CD-co-poly(N-isopropylacrylamide) core-shell nanogels (diameter = 133-142 nm). In the first

step the core microgel was prepared by precipitation polymerization of monovinyl-CD monomer

(GMA-EDA--CD), N-isopropylacrylamide (NIPAm), N,N methylenebis(acrylamide) (BIS,

cross-linker), sodium-n-docecylsulfate (SDS, surfactant) and ammonium persulfate (initiator) in

aqueous solution at 70 °C under stirring for 5 h. In the second precipitation polymerization step,

these nanogels (diameter = 106-115 nm) were subsequently used as core particles and seeds for a

further shell layer of PNIPAm. It has been shown that with increase of β-CD content the

temperature-induced deswelling of microgels decreases gradually (diameter = 90-65 nm). The

core–shell microgel was loaded with the drug paeonol, which has been used as an analgesic and

anti-inflammatory drug in aqueous solution. The sustained release was observed at 25 °C and

22

after collapsing of the microgels at 37 °C. Results showed that the inclusion complexation of

paeonol prevent the fast release at an early stage due to volume shrinking of gel particles [100].

However, nano- and microparticles, microcapsules, nanosponges and nano-structured

polymer material based on CD have been started to be discussed in weight range. Especially CD

containing nano- and microgels with characteristic swelling properties have a high potential for

further application in the area of soft nanomaterials. At the moment, nanoparticles have been

synthesized most often by lock key system and condensation reaction in organic solvent or with

surfactant using emulsions technique. The preparation of nanostructures with CD domains by

radical polymerization, especially the precipitation polymerization in water, have high potential

and should be investigated in more detail e.g. without surfactants and with variation of

comonomers. The roll of CD as cross-linker, stabilizer, and initiator for particle growth is not

investigated and published in detail and remains open.

The uptake of the discussed model drugs, guests and chemical agents by CD nanogels

actually based on the formation of inclusion complexes. The release mechanism of complexed

molecules from CD nanogels was controlled by the inclusion effect and simultaneously on the

prevention of drug release. Thus, similar to the CD containing bulk hydrogels and polymer

materials, the incorporated inclusion compounds with suitable guest molecules offer advantages

in effectiveness, long-term activity, and low effective concentrations of chemical agents.

The influence of the polymeric structure and comonomer compositions in nanogels on

the uptake and release behavior is not studied in detail. For modified applications, synergestic

effects with comonomers, variation in mash sizes of the cross-linked structure, improved

fixation on surfaces, bioavailability and improved possibility of specific application should be

investigated. Furthermore, the uptake and release irrespective of the volume of the nanogel and

by changing of the properties of the near environment have high potential for further

modifications.

23

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31

Chapter 3

Functional PMMA nanogels by cross-linking with cyclodextrin

methacrylate

Abstract: The incorporation of cyclodextrins (CDs) in nanogels based on cross-linked

poly(methyl methacrylate) (PMMA) combine the good complexation properties of hydrophilic

CDs, hydrophobic nature of PMMA, the colloidal behavior of nanogels and the ability to work

in organic as well in aqueous media. CD-MA nanogels are prepared by surfactant-free

precipitation polymerization in methanol. Solvent shift from the organic to the aqueous phase

using dialysis enables working in water. α-, β- and γ-CDs functionalized with methacrylic

groups (CD-MAs) are synthesized with an average amount of vinyl bonds per CD molecule of

2, 4, or 6 and they are used as cross-linker and as complexation unit. The formation and the

particle growth correlate with the increase of the CD-MA concentration in the reaction mixture.

This led to a reduction of the final hydrodynamic radius of nanoparticles from 363 nm to 36 nm

in aqueous phase. The number of vinyl groups per CD molecule influences the size of the

nanogels as well and induces the formation of smaller particles with a higher cross-linking

degree. The uptake properties of the CD-MA nanogels are studied by dye adsorption method

using phenolphthalein. Results show controlled complexation of the guest molecules in

dependence on the CD-MA content and the degree of cross-linking.

Keywords:nanogels, nanoparticles, methyl methacrylate, cyclodextrin, PMMA

AIBN

Cyclodextrin methacrylate (CD-MA)

O

R = O

O

RR

R

O

O

R

RRO

OR

R

R

O

O

RR

R

OO

R

R

RO

OR

RR

O

OR

R

R

O

MeOH, 65°C

Methyl methacrylate (MMA)

OO

Monomer

Cross-linker

R = OH

organic

solvent

H2O

Swollen

Collapsed

Variable parameter:

-CD-MA /MMA ratio,

-vinyl bonds (R)

-ring size

32

3.1 Introduction

The challenge in today`s research of small polymeric particles such as nano- and

microgels is the development of application-related particle structures, morphologies and

specific properties. Soft nanoparticles as carrier systems should have a high sensitivity to

changes of external environmental factors and enhanced uptake and controlled release properties

to chemical agents [1,2].

The creation of hydrophobic polymethyl methacrylate (PMMA) nanoparticles has been a

topic of research in the last two decades. The flexibility and the small size of several nanometers

have raised interest [3], and have got fundamental as well as applied perspectives especially in

the biomedical field [4]. In medical technologies PMMA nanoparticles are used because of the

very good toxicological safety record [5], furthermore, they are slowly biodegradable [6].

PMMA nanoparticles exhibit positive adjuvant effects with some antigens [7]. Applications as

vaccine adjuvants [8], oligonucleotide [9] and drug delivery systems [10], for protein separation

[11], analytical detection of soluble ions [12] and as carrier materials of pharmaceutical active

ingredients for controlled release have been studied [13].

Common preparation procedures for polymeric nano- and microparticles are performed

by mini- and micro-emulsion polymerization (EP) [14-16], inverse emulsion polymerization

[17,18], surfactant-free emulsion polymerization (SFEP) [19,20], surfactant-free precipitation

polymerization (SFPP) [21,22] or solvent shifting [23,24].

The creation of non-cross-linked PMMA nanoparticles based on the self assembly of

linear polymers has been particularly studied by some workgroups. Hence, surfactant free

precipitation polymerization has been shown to enable synthesis of polymer particles as small as

50 nm by microwave assisted polymerization at rather high solid content. Here, nanoparticles

based on PMMA homopolymers were synthesized by different procedures mentioned above

directly in water with different surfactants or in different solvent mixtures to overcome the

problem with the critical initial monomer concentration [25,26]. Only recently it has been

shown that similarly monodisperse sub 100 nm scale particles can also be obtained, when

hydrophilic co-units are introduced during polymerization and a less polar co-solvent is added to

the aqueous phase. Copolymeric PMMA particles were synthesized in acetone-water mixture

using a negatively charged persulfate initiator to improve the colloidal stability [20]. However,

33

the creation of size-controlled, stable colloidal dispersions of self-assembled linear polymers is

nevertheless a difficult procedure. Nanoparticles are obtained after some cleaning steps or

following precipitation in water and are formed via small hydrophobic aggregates of linear

polymer chains. Therefore, these linear PMMA based nanoparticles can only exist in dispersion

stabilized by hydrophobic and hydrophilic interactions in aqueous media.

Three-dimensional cross-linking of the polymer chains improves the stability of the

particle structure and the flexibility to swell in different solvents extends the general possibility

to work in organic as well as in aqueous media. Cross-linked PMMA particles have been

prepared in a size range of 50 nm up to several micrometers in common preparation procedure

discussed above using allyl methacrylate (AMA), 6-hexanediol diacrylate (HDDA), ethylene

glycol dimethacrylate (EGDM), tripropylene glycol diacrylate (TPGDA), ethylene glycol

diacrylate (EGDA) and N,N-methylenebis acrylamid (MBA, BIS) as cross-linker [27-30,18].

The incorporation of hydrophilic co-monomers in hydrophobic PMMA polymer network

could promote stability, the permeability in water and the increased loading of chemical agents in

aqueous phase. Thus, microgels based on PMMA have been prepared by combining hydrophobic

MMA and amphipilic HEMA. The PMMA core was covered with poly(2-hydroxyethyl

methacrylate) (HEMA) thin layer by soap-free emulsion polymerization. The polymerization

process and the particle size are influenced by temperature, initiator, monomer concentrations

and cross-linker content [31]. Furthermore, microgel particles have also been prepared by cross-

linking of poly(methyl methacrylate)-g-poly(ethylene oxid) (PMMA-g-PEO) macro-monomers.

The swelling properties the microgels were studied in different organic solvents [32].

The incorporation of cyclodextrins (CDs) as hydrophilic co-monomer, cross-linker and

complexation domain for many organic molecules in nanoparticle systems possesses some

advantages in combination with the hydrophobic PMMA. CDs (α-, β- or γ) are cyclic oligomers

with a hydrophilic outer surface which is formed by outstanding hydroxyl groups (18, 21 or 24)

from the -glycosidic linked glucose units (6, 7 or 8) [33,34]. The resulting hydrophobic interior

surface and the unique cavity act as macrocyclic complexation units for better binding and

uptake of hydrophobic molecules [35]. Complexation studies of many potential guest molecules

have been made [36], and thus CDs find application as carrier, delivery and controlled release

systems [37]. The chemical modification of CDs enables the covalent binding to polymers or

different kinds of materials e.g. textiles [38,39].

34

CDs with different numbers of vinyl bonds were used for the creation of cross-linked

polymers and hydrogels [40,41]. Mono-substituted CDs are used as co-monomers for the

synthesis of linear copolymers [42] e.g. based on PMMA [43]. The incorporation of

polymerizable CDs in small colloidal structures and particle systems has also been investigated

with different co-monomers [44-48]. Thus, Liu et al. synthesized linear MMA/β-CD copolymers

by radical copolymerization of MMA with mono-vinyl β-CD. Nanoparticles were obtained in a

mixture of DMF, water and acetone. The release of the model drug campthothecin (CPT) in

water could be described as controlled and mainly influenced by loaded amount of CPT in the

more hydrophobic particle core [49].

Studies of aqueous controlled release systems based on CD hydrogels show that the

release rate of hydrophobic drugs like dexamethocome [50] or of an antimicrobial

thioscimicarbenzone [51] can be retarded depending on the cross-linking density of the network.

The loading of the CD hydrogels can be increased if the carriers network is described in its

hydrophilicity. Whether the increased loading is based on a synergetic effect, e.g. enhancement of

the complexation by the more hydrophobic adjacent monomer units or simply by the hydrophobic

uptake of further drug molecules within the network remained open.

Here, a study on the preparation of CD containing nano- and microgels based on the

copolymerization of MMA and CD with methylacrylate substituents (CD-MA) is reported. The

concept for the nanogel preparation is based on the surfactant free precipitation polymerization in

methanol. It presents a new way of controlled synthesis of small, stable cross-linked

nanoparticles, which have the possibility to change their size upon change of the surrounding

medium. Because of the hydrophilic nature of the CDs it was anticipated that the resulting

particles can be disposed in water which enables further applications from aqueous solutions. The

results show the influence of the hydrophilic CD-MA cross-linker in PMMA based nanoparticles

on the particle size in aqueous and organic media, on the polymeric properties and on the uptake

characteristics of the model guest molecule phenolphthalein in aqueous systems.

35

3.2 Experimental

3.2.1 Materials

α-, β- and γ-CD produced by Wacker were dried at 80°C over 3 days before use.

Methacryloyl chloride was obtained from Merck and used as received. Solvents

dimethylformamide (DMF), methanol (MeOH) and acetone were purchased from VWR (DMF

and MeOH was degassed before use). Triethylamine (TEA) was purchased from Merck,

distilled before use and stored over 4 Å molecular sieve in N2-atmosphere. Methyl methacrylate

(MMA) was obtained from Aldrich and destabilized by column chromatography over aluminum

oxide; the further cross-linker N,N-methylene-bis-acryamide (BIS) from Aldrich was used as

received. The initiator, azobisisobutyronitrile (AIBN) from Aldrich was used without further

purifications. Phenolphthalein was used as received from Merck.

3.2.2 Modification of cyclodextrins

The modification of CD with reactive vinyl groups is published elsewhere [52]. For the

preparation of cyclodextrin methacrylates (CD-MA) with different degree of substitution,

Schlenk technique was used and the whole procedure took place under N2-atmosphere. CD was

diluted in 15 mL degassed DMF, and TEA was added to the solution. The mixture was stirred

and cooled down to 0°C, than methacryloyl chloride was given into the solution. After stirring

for 12 h, triethylamine hydrochloride was filtered and the clear solution was added into 200 mL

acetone to precipitate the modified CD. The filtrate was washed several times with acetone and

purified by Soxhlet extraction with acetone for three days. The obtained solid was dried, diluted

in a small amount of water and vacuum freeze-dried. In this work α-, β- and γ-CD-MA with an

average substitution degree of 2 and β-CD-MA with an average substitution degree of 4 and 6

were synthesized. Details of the cyclodextrin modification are summarized in the Supporting

Information (Scheme S1, Table S1, Figure S1).

3.2.3 Synthesis of cross-linked nanogels

The synthesis of cross-linked polymeric nanogels was performed by radical precipitation

polymerization technique in organic solvent. A three-necked flask equipped with a stirrer and a

reflux condenser was purged three times after evacuation with nitrogen. Solutions of the

36

monomers [19.98 mmol (2.00 g) MMA and variable amounts of modified CD-MA in 150 mL

methanol] were placed into the flask and stirred at 200 rpm for 15 minutes at 65 °C under

continuous purging with nitrogen. Afterwards, 0.12 mmol (0.02 g) initiator AIBN was added

under continuous stirring and the reaction was carried out for another 8 h under nitrogen

atmosphere. In some experiments 0.02 mmol (0.13 g) BIS was used as additional cross-linker to

compare properties of nanogels prepared only with addition of reactive CD-MA as cross-linker

or with combination of BIS and CD-MA.The ingredients used for the polymerization process

are summarized in Table 1.

Table 1. Reaction recipe for the preparation of CD-MA/MMA nanogels in 150 mL MeOH.

Nanoparticle samples are numbered as NMMAxCD(y), where N = run number; x = cyclodextrin

type, and y = substitution degree of cyclodextrin.

N

MMA,

[g] (mmol)

CD-MA,

[g] (mmol)

CD-MAtheor

,

[wt.-%]

BIS,

[g] (mmol)

AIBN,

[g] (mmol)

Yield,

[g]

1 MMA 2.00 (19.98) - - 0.02 (0.13) 0.02 (0.12) 0.56

2 MMAβCD(4) 2.00 (19.98) 0.05 (0.04) 2.39 0.02 (0.13) 0.02 (0.12) 0.48

3 MMAβCD(4) 2.00 (19.98) 0.1 (0.07) 4.67 0.02 (0.13) 0.02 (0.12) 0.44

4 MMAβCD(4) 2.00 (19.98) 0.3 (0.21) 12.82 0.02 (0.13) 0.02 (0.12) 0.74

5 MMAβCD(4) 2.00 (19.98) 0.6 (0.47) 22.73 0.02 (0.13) 0.02 (0.12) 0.68

6 MMAβCD(4) 2.00 (19.98) 0.05 (0.04) 2.42 - 0.02 (0.12) 0.60

7 MMAβCD(4) 2.00 (19.98) 0.1 (0.07) 4.72 - 0.02 (0.12) 0.44

8 MMAβCD(4) 2.00 (19.98) 0.3 (0.21) 12.93 0.02 (0.12) 0.78

9 MMAβCD(4) 2.00 (19.98) 0.6 (0.47) 22.90 - 0.02 (0.12) 1.08

10 MMAβCD(2) 2.00 (19.98) 0.3 (0.24) 12.50 0.02 (0.13) 0.02 (0.12) 0.64

11 MMAβCD(2) 2.00 (19.98) 0.3 (0.24) 12.61 - 0.02 (0.12) 0.62

12 MMAβCD(6) 2.00 (19.98) 0.3 (0.19) 12.50 0.02 (0.13) 0.02 (0.12) 1.01

13 MMAβCD(6) 2.00 (19.98) 0.3 (0.19) 12.61 - 0.02 (0.12) 0.90

14 MMAαCD(2) 2.00 (19.98) 0.3 (0.27) 12.61 - 0.02 (0.12) 0.81

15 MMAγCD(2) 2.00 (19.98) 0.3 (0.21) 12.61 - 0.02 (0.12) 0.94

After preparation, nanoparticle dispersions were filled into dialysis tubes with MWCO of

12 kDa (ZelluTrans - 12,0S 45 mm, MWCO: nominal 12,000-14,000; Carl Roth GmbH,

Karlsruhe) to remove unreacted monomers and smaller polymeric particles, and furthermore, to

37

replace methanol by water. Here, water (1 L) was changed after 12 h and 20 h, 32 h, dialyses

were stopped after 40 h. Hence, stable milky-transparent dispersions were formed (Figure S2).

3.2.4 Characterization of modified cyclodextrins and nanogels

1H-NMR and

13C-NMR spectra were recorded with a Bruker 400 NMR spectrometer

operating at 400 MHz. The samples were dissolved in deuterated dimethylsulfoxide (DMSO-d6)

(Figure S1).

RAMAN spectra were recorded with a Bruker RFS 100/s (spectral disintegration 4 cm-1

).

IR measurements were made with the help of a FTIR spectrometer: Nexus 470 (Thermo Nicolet)

(spectral disintegration 8 cm-1

).

Nanoparticle dispersions were dried by lyophilisation and pressed in KBr-pellets. The

quantitative determination of the cyclodextrin content in nanogels has been performed by

quantitative analysis of the IR band at 1032 cm-1

(C-O-C vibration of the glucose units) [53].

Differential scanning calorimetry (DSC) analyses were performed on Netzsch DSC 204

under nitrogen using 10 K/min scan rate. Samples were subjected to three cooling-heating cycles.

At first, the sample was heated up from 25 °C to 150 °C for removal of the thermal history, after

cooling, the sample was heated up to 400 °C to measure the glass transition point (Tg).

Thermogravimetric analyses (TGA) were performed under nitrogen atmosphere using

Netzsch TG 209c. Standard Netzsch alumina crucibles were used for 10 mg of dried samples and

empty crucibles for baseline correction. The samples were heated up to 100 °C, kept there for 5

min to remove residual water and heated further in 10 K/min steps to 500 °C.

The particle size of nanogels was analyzed by dynamic light scattering measurements

using Nano Zetasizer (Malvern) equipped with a He–Ne Laser (λ0 = 633 nm). Back scattering

light was detected using an angle of 173°. Hydrodynamic particle radius and the particle

distribution of the hydrodynamic radius were determined in water. Measurements were made in a

temperature range from 20 °C to 50 °C with a temperature equilibrium time of 120 s. Methanol

and chloroform were chosen as organic media. Here, hydrodynamic particle radii of freeze-dried

particles were measured 2 days after redispersion in the respective solvent. Water/methanol

solvent mixtures were chosen for swelling measurements. The concentration of methanol was

varied from 0 to 100 % (v/v) in water. The dielectricity constants and the viscosity values for the

solvent mixtures were taken as reported in the literature [54].

38

Measurements of the stability of nanoparticle dispersions were performed by using the

separation analyser LUMiFuge 114/116 (L.U.M. GmbH). Polystyrene cuvettes (10 mm) were

used for all measurements at an acceleration velocity of 2000 rpm. The sedimentation velocity in

µm/s and the calculated instability index were determined with the associated software.

The microscopical analysis was made with S-4800-Field-Emission-Scanning Electron

Microscope (Hitachi). One drop of the diluted nanoparticle dispersion was put on an aluminium

support and dried at room temperature in vacuum. After drying, samples were investigated at an

accelerating voltage of 1 kV and 8 mm disintegration.

For volumetric titration with phenolphthalein, a Brand Duran burette with a total volume

of 50 mL (± 0.05 mL, Ex +30 s) was used. A stock solution of 1 % phenolphthalein in ethanol

was prepared and an aliquot of 2.50 mL was dropped to 250 mL of 0.1 M sodium hydroxide to

obtain a 0.25 mmol/L alkaline phenolphthalein solution. All aqueous stock solutions were freshly

prepared and run within 12 h to ensure that absorbance changes due to any instability of

phenolphthalein did not contribute to experimental artifacts. β-CD stock solution in water was

prepared with a β-CD concentration of 8.8 mmol/L. Aliquots of the β-CD stock solution were

diluted with water to different concentrations with an end volume of 10 mL. To obtain a

calibration curve, titration of β-CD solutions with stock solution of phenolphthalein was

performed under stirring until the transition point from colourless to slightly pink was reached.

The nanoparticle samples (10 mL) with known particle concentration were titrated with 0.25

mmol/L alkaline phenolphthalein stock solutions under stirring until the transition point from

colourless to slightly pink was reached. The amount of β-CD in nanogels was calculated from the

calibration curve.

39

3.3 Results and Discussion

In this work, series of hydrophobic nanogels are prepared cross-linking of methyl

methacrylate (MMA) and cyclodextrin methacrylate (CD-MA) using radical precipitation

polymerization The polymerization and particle formation of hydrophobic (MMA) and

amphiphilic (CD-MA) monomers takes place in methanol without any further surfactants and

standard cross-linkers (Scheme S2 and Figure S2). For this, CDs were functionalized with

various numbers of vinyl bonds to ensure the strong embedding in polymeric networks by the

formation of covalent bonds with other acrylate monomers. Furthermore, CD-MA with more than

two vinyl groups is considered as functional cross-linking reagent and as potential nucleation

center for the particle formation in the early state of the polymerization process (Scheme 1). Due

to the bad solubility of natural CD in alcoholic solution the modification improves the solubility

of CDs in organic solvents as well as in water with the increase of the degree of substitution.

Thus, the modification of CD allows the free radical polymerization and the particle formation of

cross-linked poly(MMA-co-CD-MA) in organic solvents. After the reaction process, the organic

dispersions are purified with the help of dialysis and methanol is exchanged with water.

Shrinking of nanogels takes place slowly and stable milky dispersions of the nanogels in water

are obtained (Figure S3). The characterization of the CD-MA incorporation in the polymeric

structure, the investigation of the particle properties and the corresponding dispersions, the

swelling behavior of the nanogels in organic media and the uptake potential due to complexation

of the model dye phenolphthalein by the CD containing nanogels in water are described.

3.3.1 Incorporation of CD in PMMA nanogels

IR and RAMAN spectroscopy are suitable analytic methods to study the chemical

incorporation of CDs in the cross-linked polymeric structure of PMMA nanogels. Figure S4

shows the typical spectra of PMMA nanogels prepared with different CD content in the reaction

mixture. The IR spectra in Figure S4a present the synthesized nanogels which depict all

interesting peaks of the PMMA backbone and of the embedded CD-MA domains. The C=O

stretch vibration of methacrylates assigned to MMA and CD-MA is located at 1720 cm-1

. The

polymer backbone is represented by -CH2- vibration at 1250 cm-1

. The OH-groups of CD-MA are

shown at 3480 cm-1

and at 1630 cm-1

. The C-O-C vibration at 1032 cm-1

is unique band and

characterizes the presence of CD in the nanoparticle matrix [53]. This band is overlapped slightly

40

ba

1080 1040 1000 960 920

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.04

0

0.07

0.21

0.47

1032 cm-1

IR, A

bso

rba

nce

Wavenumbers [cm-1]

no CD-MA

0.04 mmol CD-MA

0.07 mmol -CD-MA

0.21 mmol CD-MA

0.47 mmol CD-MA

0 2 4 6 8 10 12 14 16 18 20 22 24

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Yie

ld [%

]

IR, Integral

Inte

gra

l of th

e s

ignal at 1032 c

m-1

Theoretical CD-MA content [%]

10

15

20

25

30

35

40

45 Yield

and is influenced by the C-O-CH3 band of PMMA at 1125 cm-1

. Similar results exhibit the

RAMAN spectra in Figure S4b. Here, the RAMAN spectra show the C-O-C vibration of CD with

a low intensity at 1032 cm-1

similar to the IR spectra. In comparison to the COO band of the

acrylate, the CD ratio in the polymer increases with increasing CD concentration in the reaction

mixture. Furthermore, RAMAN spectroscopy allows the investigation of the polymerization

progress and the analysis of the presence of unreacted monomers in the polymeric nanogels. The

vibrations of double bonds presented in the CD-MA and MMA monomers are located at 1637

cm-1

which disappear clearly after reaction and purification by dialysis. Thus, it can be concluded

that the obtained nanogels consist of a polymeric structure without further presence of reactive

vinyl bonds of monomers.

The incorporation of different amounts of CD-MA into PMMA nanogels can be studied in

more detail by quantitative investigation of the C-O-C band using IR spectroscopy. The exact

analysis and comparison of the band integral at 1032 cm-1

pro exact amount of 1 mg nanogels are

shown in Figure 1a. Due to the influence of the C-O-CH3 band of PMMA on the characteristic

CD band at 1032 cm-1

an exact quantitative determination of the CD content in the polymeric

structure by IR spectroscopy is not possible. But, the linear increase of the band integral with the

increase of the CD-MA content in the reaction mixture reflects the increase of CD-MA content

and a controlled incorporation of CD in the PMMA nanogels (Figure 1b).

Figure 1. The band at 1032 cm-1

in the FTIR spectra of 1 g -CD-MA containing PMMA

nanogels with different CD-MA (DS4) content (a). Integrals of the IR signals at 1032 cm-1

and

the yield of the PMMA nanogels after dialysis vs. the theoretically expected CD content (b).

41

Parallel to the increase of the CD content, the yield of stable CD-MA containing PMMA

particles increases after purification by dialysis. That means, a higher CD-MA content in the

reaction mixture leads to more incorporated CD in the polymer structure with higher cross-link

density and higher yield of stable particles.

Figure 2. The bands at 1032 cm-1

in the FTIR spectra of 1 mg -CD-MA containing PMMA

nanogels prepared with 0.3 mmol CD-MA content and different degrees of substitution (DS) with

vinyl groups (a). Integrals of the IR signals at 1032 cm-1

and the yield of the PMMA nanogels

after dialysis vs. the theoretically expected degree of substitution (b).

Figure 2a shows the FTIR spectra of nanogels prepared with the same amount of CD-MA

but with different numbers of vinyl groups. The increase of the IR band at 1032 cm-1

with

increase of the degree of substitution of CD-MA reflects the influence of the number of vinyl

groups and hence, the degree of cross-linking. Figure 2b shows that the increase of the CD-MA

integral is linear in dependence to the increase of the substitution degree. With the increase of the

amount of vinyl groups at CD-MA, the yield after purification increased, too. Thus, high numbers

of vinyl groups prefer the incorporation of CD-MA, increase the cross-link density and influence

the particle formation which results in high yield. Surprisingly, the use of the additional cross-

linker BIS does not have a strong influence on the yield and furthermore, BIS does not disturb the

incorporation of CD-MA in the polymer structure.

Taken together, a rising CD-MA content and an increase of vinyl groups per CD-MA in

the reaction mixture increases the degree of cross-linking and the incorporation of CD domains in

ba

1080 1040 1000 960 920

0.0

0.1

0.2

0.3

0.4

0.5

0.6

DS 6

DS 4

IR, A

bsorb

ance

Wavenumbers [cm-1]

DS 2

DS 4

DS 6

1032 cm-1

DS 2

2 4 6

0.5

1.0

1.5

2.0

2.5

3.0

3.5 IR, with BIS

IR, without BIS

IR, In

tegra

l of th

e s

ign

al at 1032

cm

-1

Substitution degree of CD-MA

10

15

20

25

30

35

40

45

50 Yield, with BIS

Yield, without BIS

Yie

ld [%

]

42

PMMA nanogels in a linear way. Hence, the embedding of CD-MA in nanogels is controllable by

stoichiometric ratios in the reaction mixture and by numbers of vinyl groups per CD-MA.

3.3.2 Thermal analysis

The influences of CD-MA on the thermoplastic properties of freeze-dried PMMA

nanogels are studied by investigating the shifts in melting point (Tg) and decomposition

temperature (Tdecomposition) using DSC and TGA measurements.

Figure 3. DSC measurements of nanoparticle samples containing different amounts of β-CD-

MA (4) (a); Tg of linear PMMA and of the cross-linked nanogels vs. the theoretically expected

CD content (b).

Figure 3a and 3b show the glass transition points of dry PMMA nanogels and the

corresponding thermographs analyzed by DSC. The theoretical Tg of commercial, linear atactic

PMMA is reported to be at 105 °C [54]. In agreement with the theory, the Tg increases slightly

with cross-linking by 0.02 mmol BIS to 106.1 °C [55]. Surprisingly, the incorporation of 0.04

mmol CD-MA reduces the Tg to 90.9 °C. In literature, the glass temperature of linear CD

containing polymers increases with increase of the mono-substituted CD content [47,49]. This

can be explained on the one hand by steric effects of the CD molecule as large voluminous

pendant group [56]. On the other hand by the stiffness of the polymer chain caused by

intermolecular hydrogen bonds of free CD rings connected to the carbonyl groups of MMA units

[43,57]. Here, the low incorporated CD content, the additional multi-substitution of

hydroxygroups on the CD molecule and hence, the handicapped possibility for free rotation may

a b

40 60 80 100 120 140 160 180 200 220

0.47 mmol-CD-MA

0.21 mmol-CD-MA

0.07 mmol-CD-MA

0.04 mmol-CD-MA

0.02 mmol BIS (no CD-MA)

linear PMMAexo

<=

DS

C [m

W/m

g] =

> e

nd

o

Temperature [°C]

PMMA 0 0.04 0.07 0.21 0.4780

90

100

110

120

130

140

Te

mpe

ratu

re [°C

]

Polymer and CD-MA content [mmol]

Tg

43

hinder the formation of hydrogen bonds. It seems that low content of the CD-MA cross-linker has

flexible influence on the network. The further increase of CD-MA molecules in the nanogels

results in sterical effects explained above and influences the cross-link density of the polymeric

network as well. Thus, the rigidity of the polymer network increases and as expected the Tg

increases from 90.9 °C (0.04 mmol) to 94.8 °C (0.07 mmol), then to 100.9 °C (0.21 mmol) and

finally to 125.0 °C (0.47 mmol). Furthermore, the clear transmission point disappears with higher

CD-MA content. This can be explained by the strong increase of cross-linking and hence, by a

loss of flexibility.

TGA results, which are shown in Figure 4a, describe the influence of the CD-MA content

on the temperature stability of PMMA nanogels. Here, cross-linked particles show a three-step

degradation beginning at 162 °C, at 265 °C and a significant degradation with a strong mass loss

at 406 °C. In comparison, linear PMMA shows a slight two-step degradation beginning at 288 °C

with a significant mass loss at 333 °C. The degradation steps may be generally explained by the

presence of head to head linkages, chain ends initiation from vinyl ends and the significant

degradation step by random scission [58].

Figure 4. TGA measurements of nanoparticle samples containing different amounts of β-CD-

MA (4) (a); Percentage mass loss vs. theoretically expected CD content (b).

In Figure 4b the procentual mass loss at the corresponding temperature is listed. Here, an

influence of cross-linking and of the CD-MA content is observed. After the first degradation step,

the polymeric network loses 15 % of its weight. PMMA polymers without and with different CD-

MA content reach the 15 % degradation point at different temperatures. Nanogels with CD-MA

a b

PMMA 0 0.04 0.07 0.21 0.47

150

180

210

240

270

300

330

360

390

420

450

480

Te

mp

era

ture

[°C

]

Polymer and CD-MA content [mmol]

15 %

25 %

50 %

75 %

85 %

120 160 200 240 280 320 360 400 440 480

0

10

20

30

40

50

60

70

80

90

100

Ma

ss [

%]

Temperature [°C]

linear PMMA

0.02 mmol BIS (no CD-MA)

0.04 mmol CD-MA

0.07 mmol CD-MA

0.21 mmol CD-MA

0.47 mmol CD-MA

44

content of 0.47 mmol lose 15 % of mass at a temperature of 258 °C. This temperature point

increases with the decrease of the CD-MA content to 268 °C (0.04 mmol CD-MA), further to 306

°C (0.02 mmol BIS) and then to 336 °C assigned to non cross-linked PMMA. This decrease of

the temperature with increasing CD-MA content is observed at mass losses of 15 % and 25 % in

a lower temperature range. However, the decrease is influenced by the first degradation step

shifts with the increase of the mass losses (50 %, 75 % and 85 %) to higher temperatures. Here,

the temperature points at mass losses of 50 %, 75 % and 85 % influenced generally by the third

degradation step increase with increase of the CD-MA content. Non cross-linked particles lose

their 85 % of their mass at 404 °C and the degradation point increases to 438 °C with cross-

linking using BIS. A slight drop to 426 °C using 0.05 mmol CD-MA is observed with an

subsequent increase to 431 °C (0.07 mmol CD-MA), then to 436 °C (0.1 mmol CD-MA) and

finally to 448 °C (0.47 mmol CD-MA) using CD-MA as cross-linker. Hence, cross-linked

nanogels, especially with higher CD-MA content show better temperature stability at higher

temperatures than lower cross-linked PMMA networks.

In conclusion, PMMA nanogels cross-linked with high amounts of CD-MA start to

degrade at lower temperatures caused by the first degradation step. Thus, it seems that cross-

linked polymers using BIS and especially CD-MA have more head to head linkages of the

monomer units than linear PMMA. On the other hand CD-MA and BIS containing polymers

show better stability over higher temperature range than linear PMMA influenced by the third

degradation step. It may be explained by deceleration of the random scission of the polymer

network caused by cross-linking.

3.3.3 Nanoparticle: Size, stability, morphology

Aqueous CD containing nanogels prepared in organic solvents exhibit good colloidal

stability in water after solvent shift. The aqueous milky dispersions of the nanogels are

colloidally stable. Solid nanogels obtained by lyophilisation are easily redispersable in water as

well as in organic solvents and the resulting dispersions are free of sediments, aggregates and

visual particles. In order to investigate the particle properties in aqueous and organic media DLS

measurements were performed. Figure 5a presents the particle size of the obtained nanogels in

water according to the CD-MA concentration in the reaction mixture. Here, PMMA particles with

embedded CD-MA show lower particle sizes than PMMA particles which are cross-linked only

45

with BIS, but which are prepared by the same synthesis method. By the use of CD-MA as cross-

linker and as comonomer the particle size decreases dramatically. Ratio of 1/500 CD-MA/MMA

(0.04 mmol CD-MA) in the reaction mixture reduces the hydrodynamic radii of the nanogels

from 363 nm to 138 nm. Higher contents of CD-MA reduce the hydrodynamic radii to 85.4 nm

(0.07 mmol), 66 nm (0.21 mmol) and to 36 nm (0.4 mmol).

Figure 5. Hydrodynamic radii (Rh in nm) of β-CD-modified nanogels prepared with and without

cross-linker BIS (β-CD with substitution degree 4; measurements performed at 20 °C) (a). Size

distribution curves of β-CD-modified nanogels prepared without cross-linker BIS (β-CD with

substitution degree 4; measurements performed at 20 °C) (b).

Furthermore, the additional use of BIS as further cross-linker was investigated. Here, BIS

does not influence the particle size and the hydrodynamic radius. The results of the particle sizes

do not indicate any difference of nanogels prepared with or without BIS. Taken together, the

growing process of PMMA nanogels and therefore, the particle formation is influenced only by

the CD-MA content. That means, CD-MA with different degree of vinyl groups acts as multiple

cross-linker and furthermore, as nucleation center for stable, round particles with a dense cross-

linked polymeric network. Raising CD-MA content in the reaction mixture assists the increase of

nucleation centers at the early stage of polymer growing and prefers the formation of many cross-

linked polymers and furthermore, of small particles in water.

The size distributions of nanogels with different CD-MA content show monomodal

functions (Figure 5b). Cross-linked PMMA particles without CD-MA have got a broad

distribution, but by increasing CD-MA concentration in the reaction mixture the degree of cross-

0 0.04 0.07 0.21 0.47

0

50

100

150

200

250

300

350

400

-CD-MA [mmol]

Rh [nm

]

with BIS

without BIS

10 100 10000

2

4

6

8

10

12

14

16

18

20

Inte

nsity [%

]

Rh [nm]

no CD-MA

0.04 mmol -CD-MA

0.07 mmol -CD-MA

0.21 mmol -CD-MA

0.47 mmol -CD-MA

a b

46

linking increases which results in smaller particle sizes and narrow size distributions. PMMA

nanogels prepared with a CD-MA content of 0.07 mmol show the narrowest size distribution.

Further, increasing CD-MA content results in broader distributions of the particle sizes.

Therefore, it can be concluded that a high CD content in the nanogels leads to minor aggregation

of particles due to glue effects in consequence of complexation of PMMA rests by CD domains

at the particle surface [59]. But the stability of the nanogels in aqueous environment is assured

and no optically visible aggregation and collapsing of the dispersion could be observed.

The dispersion stability of the nanogels with different CD-MA content was investigated in

detail by sedimentation kinetics using analytical centrifugation. Here, STEP-technology (Space +

Time resolved Extinction Profiles) developed by Lerche et al. allows simultaneous measurement

of the transmission profiles as function of time and position over the sample length within the

cuvette during rotation. The transmission profiles display the relative position of the phase

boundary between sediment and supernatant in the dispersion [60]. The transmission profiles of

the PMMA nanogels aqueous dispersions with different CD-MA content are shown in Figure 6.

Figure 6. The transmission profiles of aqueous dispersions of PMMA nanogels with different

CD-MA content (0.02 mmol BIS, 0.04 mmol CD-MA, 0.07 mmol CD-MA, 0.21 mmol CD-MA,

0.47 mmol CD-MA at 20 °C and a nanogel concentration of 1 g/L) as measured by using the

STEP-technology.

Figure 6a and 6b depict that the sedimentation process of PMMA nanogels is characterized by

polydisperse sedimentation without a sharp front in the transmission profiles. Furthermore,

Position [mm]

Tra

nsm

issio

n [

%]

No CD-MA 0.04 mmol CD-MA 0.07 mmol CD-MA 0.21 mmol CD-MA 0.47 mmol CD-MA

47

during the centrifugation the transmission of the dispersions gradually increases slightly with

time. Thus, particles with different size or different density sediment with different speed. Larger

particles with low transmission properties start to sediment faster than smaller particles with

higher transmission.

The transparency of the dispersions with the PMMA particles increases with the increase

of CD-MA content and hence, with decrease of the particle size (Figure S3 and 6a-e). Dispersions

of PMMA nanogels with low CD-MA content are polydisperse and the characteristic

sedimentation process presents dispersions which are stable to particle aggregation. The

increasing CD-MA content leads to small particles (<100 nm) with narrower size distribution as

analyzed by DLS and to a more hydrophilic polymer structure due to the high amount of CD

hydroxy groups. Hence, the transparency of the dispersions increases with a decreased

polydispersity and without any marked differences in transmission during the sedimentation

process. The transmission profiles of nanoparticle dispersions with high CD-MA content show

colloidally stable dispersions without substantial changes in transmission along the sample

length.

The time course of positional displacement of the sedimentation boundary exhibits the

sedimentation rate of each dispersion. The sedimentation rate gives a good impression of the

stability of dispersions and allow to study the influence of the CD-MA content on the stability of

the PMMA nanogels. The Stoke´s equation of very diluted dispersions describes the

determination of the sedimentation velocity of particles in the centrifugal field (eq.1) [22,61].

(1)

With r = particle radius; = dynamic viscosity of the fluid; (pp-pf) = difference of the particle

density (pp) and of the fluid density (pf). In general, g is the acceleration due to gravity and it is

shifted to centrifugal acceleration (a) in the centrifugal field (eq.2).

aZ = 2 R = 4

2 RN

2 (2)

48

In a rotating reference system with angular velocity acts on a body of mass, which is located at

a distance from the axis of rotation R. Centrifugal acceleration (a) relative to acceleration due to

gravity (g) is traditionally named "relative centrifugal force" (RCF) and is determined in

multiples of g (eq.3).

(3)

In these experiments, the centrifugal acceleration is set to be constant by the centrifuge,

furthermore the viscosity of the medium is constant for all aqueous dispersions with the same

particle concentration. Hence, the particle radius and the particle density, which are only

influenced by CD-MA in nanogels can change the sedimentation rate and the stability of the

dispersions.

The results are shown in Figure 7a. The sedimentation velocity of PMMA nanoparticle

dispersions with different CD-MA content and different radii decreases drastically with decrease

of particle size caused by increase of the CD-MA content in nanogels. Small amount of CD-MA

influences the sedimentation velocity nearly in a linear order, but with increased CD-MA content

the curve flattens slowly. Thereby, the stability of dispersion increases, while the instability index

decreases (Figure 7b).

Figure 7. Sedimentation rate (a) and instability index (b) vs. theoretically expected CD-MA

content.

a b

0.0 0.1 0.2 0.3 0.4 0.5

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Insta

bili

tyin

de

x

-CD-MA [mmol]

Instabilityindex

0

50

100

150

200

250

300

350

400

Particle Size Rh

Rh [n

m]

0.0 0.1 0.2 0.3 0.4 0.5 0.62

3

4

5

6

7

8

9

10

Sed

ime

nta

tio

n r

ate

[µ

m/s

]

-CD-MA [mmol]

Sedimentation Rate

0

50

100

150

200

250

300

350

400

Particle Size Rh

Rh [n

m]

49

The instability index characterizes the overall demixing of the dispersion and is quantified

by clarification as function of time divided by the maximum clarification possible. It is a

dimensionless number between 0 and 1. Here, completely or almost demixed samples represent

an instability index close to 1 (very instable); no or very slow demixing during the measurement

run is indicated by an instability index close to 0 (very stable). Easy ranking of samples is

obtained by comparing the values of the instability index for the same ROI (Range of the sample

analyzed), RCA and time of centrifugation. The instability index is calculated in four steps [62].

The first step is the clarification characterized in the profile difference (Tidiff

). Thus, the first

(initial) profile (T1) is subtracted from all profiles (Ti) (eq.4).

(4)

Change to the time Ti is the sum of values of the profile difference from the time Ti in the

position range rStart to rEnd set by the user (e.g. 105 to 110 mm). Index i corresponds to the

respective transmission profile number (index) (eq.5).

(5)

Maximum change possible calculated with the difference between 90 % and the mean

transmission from first profile multiplied with the difference between the end and start of the

position range (eq.6).

(6)

Instability index for the time Ti is the change to the time T1 and is divided by the maximum

change possible (eq.7).

(7)

50

Here, the instability index is greatly reduced by the use of CD-MA as cross-linker from

0.8 for PMMA particles just cross-linked with BIS to under 0.05 for particles prepared with 0.21

mmol and 0.47 mmol CD-MA. Therfore, the speed of demixing is reduced with decrease of

particle size and thus with CD-MA content in the PMMA nanogels.

These results lead to the conclusion that CD-MA does not only cross-link the PMMA

chains in the polymeric network. The CD-MA acts as nucleation center for small particles as

determined by DLS measurements. The experimental sedimentation data measured by

centrifugation confirm the theoretical explanations by Stoke´s equilibration. The small size

influences the formation of stable aqueous dispersions. Taken together, CD-MA reduces the

particle size and furthermore, it improves the stability of PMMA particles in water.

Different degree of substitution

In further studies, CD-MA monomers with different numbers of vinyl groups are prepared

and the influence of the substitution degree on the particle size is investigated. Figure 8a shows

the different hydrodynamic radii of nanogels synthesized with CD-MA monomers with an

average number of 2, 4 and 6 vinyl groups per monomer. Here, the size of the particles decreases

in an exponential order from 700 nm to 60 nm and then to 30 nm with an increasing substitution

degree. This may be explained by higher reactivity of CD-MA molecules with increasing number

of reactive vinyl groups. This results in faster nucleation of cross-linked polymers and higher

cross-linking density due to the increasing number of cross-linking points at the starting point of

the reaction. These effects may lead to many small particles with a compact volume after transfer

to water.

51

Figure 8. Hydrodynamic radii of CD-modified nanogels prepared with and without cross-linker

BIS: a) nanogels prepared with 0.3 g β-CD-MA with substitution degree 2 (0.24 mmol); 4 (0.22

mmol); and 6 (0.21 mmol); b) nanogels prepared with 0.3 g α-CD-MA (0.28 mmol); β-CD-MA

(0.24 mmol); and γ-CD-MA (0.21mmol) with substitution degree 2 (measurements performed at

20°C).

Different CD type

The influence of the ring size of CDs on the particle size is also studied by the use of α-,

β- and γ-CD with substitution degree of 2. Figure 8b shows the hydrodynamic radii of the

particles. Hence, the particle size decreases slightly with increase of the CD ring size. This may

be explained by the solubility of α-, β- or γ-CD and by the flexibility of their ring structure. Here,

natural and substituted α- and γ-CDs exhibit better solubility in water than β-CD. The same

solubility behavior is observed for substituted CDs in organic solvents. Here, the substitution

with methacrylate groups enables the polymerization and the cross-linking in alcoholic solution.

γ-CD exhibits the largest ring size and with its co-planar structure the highest flexibility in

methanol. The α-CD ring does not build a complete secondary H-belt formed by H-bonds of the

secondary OH-groups as one glucopyranoside unit is distorted. β-CD consists of such a rigid

secondary H-belt and this obstructs the interaction with the solvent. In this case, the most flexible

ring interacts much better with methanol and enables faster nucleation of the nanogels than ring

structures with inflexible orientation in solution.

a b

DS 2 DS 4 DS 6

0

100

200

300

400

500

with BIS

without BIS

Rh [nm

]


alpha beta gamma

150

200

250

300

350

400

450

500

Rh [nm

]

Cyclodextrin type

52

Morphology of applied particles

FESEM images of the particles are shown in Figure 9. Spherical particles with

homogeneous particle morphology are formed. Due to the hydrophobic polymer backbone of

PMMA the cross-linked polymer chains constrict at the particle centre and at the particle surface

constitute round particles with dense polymeric structure with restricted swelling properties.

These swelling properties depend on the CD-MA content in the nanogels. A higher CD-MA

content increases the OH content in the nanogels and promotes hence, a better diffusion of water

in and out of the particles.

As seen in Figure 9a-e, the particle size of the nanogels decreases with increase of CD-

MA content. PMMA particles (Figure 9a) only cross-linked with BIS are much larger than

particles (Figure 9b) just cross-linked with low molar ratio of CD-MA (1/500 CD-MA/MMA)

(0.04 mmol CD-MA (4)) in the reaction mixture. The particle size decreases further with

increasing CD-MA content to very small particles (Figure 9e) with a CD-MA ratio of 1/43 (0.47

mmol CD-MA (4)) to a size of several nm. Figure 9f shows CD-MA nanogels with a CD

substitution degree of 2. Here, round particles with a particle size of about 500 nm are

homogeneously distributed on a metal surface, here aluminum. The microscopic images confirm

the results measured by DLS although the slightly swollen particles in water lose size by release

of water during the drying process. Furthermore, nanogels show high affinity to surfaces, here

aluminum surfaces, due to physical self-bonding. Depending on the particle concentration,

PMMA nanogels distribute homogenously on the surface, either as single particles (Figure 9d)

from less concentrated dispersions or in form of particle films (Figure 9c) prepared from highly

concentrated dispersions.

53

Figure 9. FESEM images of β-CD-MA modified nanogels applied onto aluminum surfaces: a) no

CD-MA (4); b) 0.04 mmol β-CD-MA (4); c) 0.07 mmol β-CD-MA (4); d) 0.21 mmol β-CD-MA

(4); e) 0.47 mmol β-CD-MA (4) and f) 0.24 mmol β-CD-MA (2).

a b

dc

fe

54

3.3.4 Swelling behavior of CD-MA nanogels in organic solvents

PMMA nanogels with different CD-MA content have small hydrodynamic radii in water.

This can be explained by collapsing the polymeric cross-linked network during solvent shift from

methanol to water via dialysis. The hydrophilic PMMA polymer backbone is nearly water

insoluble, just the CD-MA with some hydroxyl groups is soluble in water. The cross-linked

network and the possibility to swell in organic solvents is important for further use of CD-MA

containing nanogels e.g. uptake and release of active agents (Scheme 1).

Scheme 1. Swelling and deswelling of the polymeric structure of PMMA nanogels cross-linked

with CD-MA in organic and aqueous media.

The influence of water mixable and water immiscible solvents on the dimension and on

the degree of swelling is part of further investigations. Figure 10 presents the results of DLS

measurements of redispersions of the freeze-dried nanogels in methanol and chloroform. In

Figure 10a, the hydrodynamic radii of PMMA nanogels with different amounts of CD-MA are

shown ½ hour and 2 days after redispersion in organic solvents. Hence, the particle sizes of the

nanogels increase after 1/2 hour slowly and after 2 days fully swollen nanogels are obtained. In

the same manner, the particle size decreases with the increase of the CD-MA content. Nanogels

with lower cross-linking degree and less CD-MA content show the largest difference in particle

size between the swollen and unswollen state. PMMA particles with the highest content of CD-

MA and with the highest degree of cross-linking do not distinguish in hydrodynamic radii and

thus, they do not swell. This indicates that the degree of cross-linking is very high and small

nanogels are not very flexible as well in water as in organic solvents.

organic solvent

H2O

swollen nanoparticles collapsed nanoparticles

55

Figure 10. Hydrodynamic radii (Rh in nm) of β-CD-MA modified nanogels in methanol or

chloroform after 1 h and 2 d of redispersion (β-CD with substitution degree 4; measurements

performed at 20 °C) (a). Degree of swelling of β-CD-modified nanogels in methanol or

chloroform in reference to the collapsed nanoparticle in water (β-CD with substitution degree 4;

measurements performed at 20 °C) (b).

The influence of the solvent type, here: methanol and chloroform, on the dimension and on the

degree of swelling is in both cases quite similar. After 1/2 hour, the size of methanol-swollen

nanogels is slightly larger than in case of chloroform-swollen particles. Thus, the smaller

methanol molecules are adsorbed faster and penetrate the particle network easier than the larger

chloroform molecules. The fully swollen particles show an inverted order after 2 days. Then,

nanogels fully adsorbed with chloroform molecules have a slightly larger particle size than fully

methanol swollen particles. Hence, the swelling degree of CD-MA containing PMMA nanogels

in chloroform is slightly higher than in methanol (Figure 10b).

The more detailed investigations of the influence of methanol on the particle size were

made in alcohol/water mixtures. Here, nanogels in water were transferred into different amount

of methanol and after 2 days the hydrodynamic radii were measured by DLS at 20 °C. Figure 11a

indicates that PMMA nanogels cross-linked with CD-MA are responsive to methanol in the

presence of water.

a b

0.0 0.1 0.2 0.3 0.4 0.5

0

5

10

15

20

25

30

35

40

CD-MA [mmol]

(Rh/R

h,0)3

CHCl3

CHCl3 2d

MeOH

MeOH 2d

0.0 0.1 0.2 0.3 0.4 0.50

50

100

150

200

250

300

350

400

450R

h [nm

]

CD-MA [mmol]

H2O

CHCl3

CHCl3 2d

MeOH

MeOH 2d

56

Figure 11. Hydrodynamic radii (Rh in nm) of β-CD-modified nanogels in methanol/water

mixtures after 2 d of redispersion (β-CD with substitution degree 4; measurements performed at

20 °C) (a). Degree of swelling of β-CD-modified nanogels in methanol/water mixtures in

reference to the collapsed nanoparticle in 100 % water (β-CD with substitution degree 4;

measurements performed at 20 °C) (b).

In all cases, the opposite effect was observed after addition of 10 % of methanol. The

particles shrink and start to collapse at the beginning (Figure 11b). The collapsing of all nanogels

upon addition of small amounts of alcohol may be explained by cononsolvency effects of the

solvent pair [32,63]. This effect was observed in previous investigations and reported for poly(N-

isopropylacrylamide), poly(vinylcaprolactam-co-acetoacetoxy methacrylate), poly(methyl

methacrylate-co-acrylic acid) and poly(methyl methacrylate)-g-poly(ethylene oxide) microgel

particles [64-65].

Particles with 0.41 mmol CD-MA content are not very sensitive and only after addition of

more than 70 % methanol the hydrodynamic radii increase slowly. Nanogels with a low content

of CD-MA (0.04 mmol) show a strong increase with further decrease of the radii after addition of

60 % alcohol. The particle size in a water/methanol mixture of 50/50 (v/v) is very high and

agglomerations cannot be ruled. The Rh of nanogels with 0.07 and 0.21 mmol CD-MA increases

after addition of 20 % methanol in a linear way.

a b

0 10 20 30 40 50 60 70 80 90 100

0

100

200

300

400

500

600

700

800 0.04 mmol -CD-MA

0.07 mmol -CD-MA

0.21 mmol -CD-MA

0.47 mmol -CD-MA

Rh [nm

]

MeOH [vol.-%]

0 10 20 30 40 50 60 70 80 90 100

02468

1012141618202224262830

0.04 mmol -CD-MA

0.07 mmol -CD-MA

0.21 mmol -CD-MA

0.47 mmol -CD-MA

MeOH [vol.-%]

(Rh/R

h,0)3

57

3.3.5 Uptake and complexation

CD is well known for the formation of host guest complexes with many hydrophobic

molecules. Here, CD does not act only as cross-linker for the synthesis of small PMMA nanogels.

The main function is the fitting of nanogels with additional pockets with a high binding activity

for the uptake and long term fixation of guest molecules for delivery and controlled release

processes. For the use of controlled uptake of different guests it is interesting to know the active,

complexable CD units in nanogels. The complexation of active CD containing matrices with

phenolphthalein is a common method for qualitative proof of the presence of CD. On this basis,

techniques were developed to study quantitatively the uptake of guests and the CD content of

different matrices. Dye sorption methods are already described in literature and the determination

of the active CD content in copolymers by titration with Phenolphthalein at pH 10 as well [66-

68]. The phenolphthalein uptake capacity of the nanogels is shown in Figure 12.

Figure 12. Phenolphthalein usage vs. theoretical CD-MA content in10 mL (1 g/L) nanogel

dispersion (a). Theoretical vs. active β-CD-MA content in nanogels determined by

phenolphthalein titration (b).

Figure 12a displays the consumption of phenolphthalein solution by the nanoparticle dispersion

(1 g/L) at pH 10. Hence, the consumption increases with increasing content of CD-MA

incorporated in nanogels. Nanoparticle dispersions with higher CD-MA content have a higher

potential for the uptake and complexation of phenolphthalein. The active, complexable CD-MA

content of the nanogels is determined in comparison to the calibration curve of the

phenolphthalein complexation by natural CDs. Figure 12b shows that the active CD-MA content

a b

0 2 4 6 8 10 12 14 16 18 20 22 24

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Usage o

f phenolp

hth

ale

in s

olu

tion [m

L]

theoretical CD-MA content [wt.-%]

0 2 4 6 8 10 12 14 16 18 20 22 24

0

2

4

6

8

10

12

14

16

18

20

22

24

theoretical CD-MA content

"active" CD-MA content

CD

-MA

conte

nt [w

t.-%

]

theoretical CD-MA content [wt.-%]

58

is lower than the theoretical expected values. This means, some incorporated CDs are not free for

complexation of guest molecules. One possible explanation is the occupation of the CD-MA due

to rotaxane formation. Furthermore, CD-MA container are strongly incorporated in highly cross-

linked polymeric structure and hence, these are not accessible for guest molecules.

The procentual active content of CD-MA in comparison with the theoretical CD-MA

content decreases with increase of the CD-MA content in the reaction mixture and with the

degree of cross-linking. Thus, complexation studies of nanogels prepared by the same

concentration (0.3 g) of CD-MA but with different numbers of vinyl groups are studied in more

detail (Figure 13a). Here, nanogels with CD-MA (DS2) complexed less phenolphthalein, than

CD-MA with substitution degree of 4 which contains the most active CD domains as determined

by titration. Nanoparticle dispersions made with CD-MA (DS6) have minimal complexation

properties.

Figure 13. Active CD-MA content vs. substitution degree of CD-MA in nanogels determined by

phenolphthalein titration (a); Active CD-MA/IR integral of the signal at 1032 cm-1

vs.

substitution degree of CD-MA in nanogels (b).

In comparison of the titration results in Figure 13a to the IR spectrometric results from

Figure 2, nanogels prepared with CD-MA (DS2) showed less incorporated CD-MA (active and

non-active) content than nanogels prepared with CD-MA (DS4). For this reason, a lower uptake

of phenolphthalein by CD domains than in nanogels with CD-MA (DS4) is expected. Nanogels

with CD-MA (DS6) have the highest content of CD-MA, but on the other hand the titration

experiments show the lowest uptake properties in comparison to nanogels prepared with CD-MA

(DS2 and 4).

a b

2 4 6

2

4

6

8

10

12

14

16

18


theoretical CD-MA content

with BIS

without BIS

Active C

D-M

A c

onte

nt [w

t.-%

]

2 4 6

2

4

6

8

10

substitution degree of CD-MA

with BIS

without BIS

Active C

D-M

A c

onte

nt

/ IR

-Inte

gra

l

59

Figure 13b shows the results of the complexation titration in comparison with the

integrals of the IR band at 1032 cm-1

. The resulting ratios of the active CD-MA content and the

IR-integral lead to the conclusion that nanogels prepared by CD-MA with a higher degree of

substitution exhibit lower complexation of guests. Hence, the covalently embedded CD-MA

domains are not accessible due to the highly cross-linked structure and furthermore, the inflexible

incorporation does not allow the orientation to the guest molecules. Nanogels prepared with CD-

MA with 2 vinyl groups exhibit a lower degree of cross-linking and thus, a more flexible mobility

of the CD domains in the polymeric structure. This results in better accessibility of CD by guest

molecules and is reflected in a better uptake of phenolphthalein. BIS shows a minimal influence

due to the slight increase of the cross-linking degree. But the influence decreases with increase of

the CD-MA content and thus, with increase of cross-linking. In case of CD-MA (DS 6), the

particles are already so highly cross-linked that BIS reveals no more a strong influence.

3.4 Conclusion

In conclusion, the controlled incorporation of CD-MA and cross-linking in PMMA

nanogels is presented. Series of nanogels with different CD-MA content and with different

numbers of vinyl groups at the CD-MA monomer were synthesized in organic solvent. Here, CD

functionalized with different numbers of vinyl groups acted as cross-linker and as nucleation

center with influence on the particle size. The particle size in water as well as in organic solvents

decreased with increasing CD-MA content and furthermore, with the number of vinyl groups per

CD molecule in the nanogels to less than 50 nm. The ring size of CD-MA influences the particle

size as well, nanogels with α- and β-CD-MA obtained larger particle size than those with γ-CD-

MA. Nanogels were swellable in water mixable and water immiscible solvents, their size

increases with increase of the portion of organic media. The uptake properties of CD-MA

nanogels were studied using the dye sorption method with phenolphthalein in aqueous alkali

solution. The uptake increased with increasing CD-MA content in the PMMA nanogels. Highly

cross-linked nanogels synthesized with CD-MA containing high number of vinyl groups showed

lower complexation behavior than slightly cross-linked nanogels prepared by CD-MA with low

amounts of vinyl groups.

60

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3.6 Supporting Information

1. Modification of cyclodextrin with vinyl groups

TEA, DMF, RT

Cyclodextrin methacrylate

OR =

OHO

Cyclodextrin

O Cl

Methacryloyl chloride

O

HO

OH

O

HO

6, 7, 8

O

R

R

O

R

6, 7, 8

Scheme S1. Modification of α-, β-, γ-cyclodextrin with methacryl groups.

Table S1. Chemicals used for the synthesis of CD-MA with different substitution degrees (DS).

Substance β-Cyclodextrin Methacryloyl chloride Triethylamine DS

β-CD-MA DS 2 2.0 g

(1.76 mmol)

0.51 mL, 0.55 g

(5.28 mmol)

0.62 mL, 0.45 g

(4.40 mmol) 2

β-CD-MA DS 4 2.0 g

(1.76 mmol)

0.68 mL, 0.74 g

(7.04 mmol)

0.81 mL, 0.59 g

(5.86 mmol) 4

β-CD-MA DS 6 2.0 g

(1.76 mmol)

1.70 mL, 1.84 g

(17.60 mmol)

2.03 mL, 1.48 g

(14.61 mmol) 6

α-CD-MA DS 2 2.0 g

(2.05 mmol)

0.59 mL, 0.64 g

(6.15 mmol)

0.71 mL, 0.52 g

(5.13 mmol) 2

γ-CD-MA DS 2 2.0 g

( 1.54 mmol)

0.44 mL, 0.48 g

(4.62 mmol)

0.53 mL, 0.39 g

(3.85 mmol) 2

65

Figure S1. 1H-NMR spectrum of α-CD-MA (DS2) in DMSO-d6

1H-NMR spectrum (DMSO-d6, 400 MHz):

(ppm): 1.87 (m, CH3); 3.29-3.40 (m, CH-2, 4); 3.60-3.64-3.77 (m, CH- 3, 5 CH2- 6); 4.50 (s, C-

OH-6); 4.80 (s, CH-1); 5.44-5.64 (s, C-OH-2, 3); 5.69 (m, CH-8), 6.05 (m, CH-9)

13C-NMR spectrum (DMSO-d6, 75 MHz):

ppm): 17.91 (C10) 59.93 (C6); 71.91 (C2); 72.06 (C5) 73.16 (C3); 82.05 (C4); 101.84 (C1);

126.82 (C=CH2); 135.64 (C-C=C); 166.30 (COO)

FTIR spectrum (KBr): ν (cm-1

): 3407, 2930, 1720 (C=O), 1635, 1406, 1364, 1297, 1201, 1157,

1079, 1032, 945, 859, 756, 580, 531

RAMAN spectrum: ν (cm-1

): 3253, 3107, 2931, 2913, 1717, 1637 (C=C), 1460, 1407, 1333

ppm (t1)

2.03.04.05.06.0

0

100000000

200000000

300000000

400000000

500000000

6.0

47

5.6

93

5.4

65

5.4

49

5.4

43

4.8

00

4.5

06

4.4

92

3.7

93

3.7

77

3.6

41

3.6

07

3.4

04

3.3

79

3.3

60

3.2

92

1.8

72

1.8

8

2.2

8

6.0

0

4.9

9

24

.49

24

.22

5.3

4

11

.31

Vinyl CH2-9, 8

OH-2, 3

CH-1

CH-5, 3CH2-6

CH-2, 4

CH3-7

OH-6

66

Scheme S2. Preparation of α-, β- or γ-CD-MA nanoparticles on the basis of PMMA and

Figure S2. FESEM image of β-CD-MA (2)-nanoparticles on aluminum.

Figure S3. Aqueous dispersions of PMMA nanogels with different CD-MA content.

Figure S4. FTIR spectra of nanoparticle samples containing different amounts of β-CD-MA (4)

(a); RAMAN spectra of nanoparticle samples containing different amounts of β-CD-MA (4) (b).

AIBN, MeOH, 65°C

Cyclodextrin methacrylate (CD-MA)

OR =

Methyl methacrylate (MMA)

OH

O

O

RR

R

O

O

R

RRO

OR

R

R

O

O

RR

R

OO

R

R

RO

OR

RR

O

OR

R

R

O

OO

0.02

mmolBIS

0.04

mmolCDMA

0.07

mmolCDMA

0.47

mmolCDMA

0.21

mmolCDMA

4000 3500 3000 2500 2000 1500 1000 500

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0.47 mmol

0.21 mmol

0.07 mmol

Absorb

ance

Wavenumbers [cm-1]

no CDMA

0.04 mmol CD-MA

0.07 mmol CD-MA

0.21 mmol CD-MA

0.47 mmol CD-MA

1032

1720

no CD-MA

0.04 mmol

1750 1500 1250 1000 750 500

0.0

0.1

0.2

0.3

0.4

0.5

0.6CD-MA (4)

0.47 mmol

0.21 mmol

0.07 mmol

0.04 mmol

Inte

nsity

Wavenumber [cm-1]

no CD-MA (PMMA) 0.04 mmol CD-MA 0.07 mmol CD-MA

0.21 mmol CD-MA 0.47 mmol CD-MA CD-MA DS 4

1637 1032

no CD-MA

a b

67

Chapter 4

Chlorhexidine loaded cyclodextrin containing PMMA nanogels as

antimicrobial coating and delivery system

Abstract: Antimicrobial nanogels, aggregates and films are prepared by complexation of the

antiseptic and bacteriostatic agent chlorhexidine (CHX) for clinical especially for ocular and

dental applications. A series of -, - and -cyclodextrin methacrylate (CD-MA) containing

hydrophobic poly(methyl methacrylate) (PMMA) based nanogels were loaded quantitatively with

CHX in aqueous dispersion. Results showed that CHX was enhanced complexed by use of CD-

MA domains in the particles structure. -CD-MA nanogels presented the highest uptake of CHX.

Furthermore, it was observed that the uptake of CHX in nanogels was influenced by the

hydrophobic PMMA structure. Large aggregates were formed during the complexation of CHX

in aqueous phase. CHX acted as external cross-linker of nanogels by formation of 1:2 (CHX:CD-

MA) inclusion complexes of two -CD-MA units on the surfaces from two different nanogels.

The aggregates adsorbed easily onto glass surfaces by physical self bonding and formation of a

dense cross-linked nanogel film. The release of CHX in water and PBS buffer solution was

quantitatively studied over 4 h whereas the release was strongly reduced in PBS buffer.

Biological tests of the applied CHX nanogels were made with regard to antimicrobial efficiency

and CHX nanogel films were successful performed against Staphylococcus. Aureus.

Keywords: nanogels, cyclodextrin, PMMA, chlorhexidine, antimicrobial, drug delivery

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NHH

N

NH

Cl

NH

NH

HN

HN

HN

H2O

Cl

NH

NH

HN

NH

HN

Cl

HN

HN

NH

HN

NH

Cl

HN

HN

NH H

N

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NHH

N

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NHH

N

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl N

H

NH

HN HN

HN

Cl H

N

HN

NH HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

n

Nanogels

in aqueous dispersion CHX loaded nanogels

68

4.1 Introduction

Many microorganisms initiate bacterial growth in even smallest parts of the micro- and

macro-cosm. Among them, many dangerous bacteria that pose a potential risk for human live can

be found. Thus, the use of antimicrobial materials for the reduction of bacterial formation is of

great importance in many fields of daily life. The application of antimicrobial agents and

materials are in the focus of interest and find already application in food, textile and plastic

products. In the cosmetic and pharmaceutical field as well as for bio and medical applications the

use of anti microbial agents is quite important to inhibit the growth or to kill aggressive bacteria

before they start to get dangerous [1-3]. Different strategies have been developed to control

bacterial formation. The combination of a microbial repellent agent and surface coating impairs

the development of biofilms on all kinds of material. The aim of the research is the inhibition of

bacterial adhesion and the killing of bacterial cells in the first state of biofilm formation [4].

Modification of surfaces with various antimicrobial substances such as antibiotics,

antiseptics or metals and enzymes which are grafted on various materials prevent the formation

of bacterial biofilms [5]. In clinical treatments, the common use of antibiotics helps reducing of

bacteria. But the potential risks in the development of bacterial resistance and hypersensitivity

requires effective alternatives for the future [6]. Beside these, sessile microorganisms often show

high resistance to biofilm inhibitors. Hence, the eradication of biofilms requires high

concentrations of disinfectants or antibiotics, causing severe environmental damages, emerging

multiresistance and further nosocomial infections. The concern of public health raises an urgent

need for developing new bacterial and biofilm resistant systems [7].

Therefore, alternatives, especially the further development of antiseptic products, find

more and more interest in research and clinical applications. New inclusion compounds of

potential antiseptics with a strong binding of the active agent have been developed to reduce the

toxicity and increase the effectiveness. Here, strong fixation of antiseptics by inclusion in

polymers or by complexation in cyclodextrins (CDs) are an interesting option for drug release

and delivery. One key component of the inclusion compounds is CD, a cyclic oligosaccharide.

The six (-CD), seven (-CD) and eight (-CD) glucopyranose units create a truncated cone

structure which provides a hydrophilic exterior and a hydrophobic interior cavity that allows the

formation of host-guest complexes. The complexes are stabilized by noncovalent interactions

such as hydrogen bonds, van der Waals forces and hydrophobic interactions [8,9]. The physical

69

and chemical properties of the guest can be changed by complexation and the stability can

increase against oxidation, photodecomposition, hydrolysis, dehydration as well as protection of

guests from degradation in the gastrointestinal tract [10]. By inclusion poorly water-soluble

molecules can be transferred in aqueous solution and thus, the water solubility of the guest can

increase as well as its bioavailability [11]. CD is already used in many fields of daily life and

improves the efficacy of active ingredients such as drugs in the therapeutically medicine [12,13].

These inclusion compounds offer advantages in effectiveness, long-term activity, and low

effective antimicrobial concentrations.

An interesting antiseptic molecule for the inclusion and encapsulation in polymer based

systems is chlorhexidine (CHX). It is a well known and studied active agent against many kinds

of bacterial strains. CHX is a chemical antiseptic which is effective against gram-positive and

gram-negative microbes [14,15]. The mechanisms of action of CHX are bactericidal as well as

bacteriostatic causing membrane disruption [16]. CHX is the “gold standard” and it is widely

used and receives great attention in periodontal therapy due to its potency against bacterial plaque

[17,18,19]. For the prophylaxis against wound infections, CHX is used as preoperative whole-

body disinfection [20,21]. Further studies have been made for skin, mucosal and vaginal

applications [22-24].

The inclusion of antiseptic CHX in CD has been studied with regard to its release and

effectiveness for antimicrobial applications. First, Loftson studied the interactions between

several water soluble CHX gluconate and 2-hydroxypropyl CD. Here, preservative molecules can

displace the drug molecules from the CD cavity, thus, reducing the solubilizing effects of the CD.

[25] Furthermore, the antimicrobial activity of the preservatives was reduced by formation of

preservative CD inclusion complexes [26]. The 1:2 (CHX:β-CD) complex was prepared and

characterized using X-ray crystallography, infrared spectroscopy, thermal analysis and nuclear

magnetic resonance. The minimum inhibitory concentration (MIC50) of the CHX:β-CD inclusion

compound against Streptococcus mutans, Eubacterium lentum, Fusobacterium nucleatum,

Bacteroides fragilis and Actinomices actinomycetemcomitans was determined. The CHX:β-CD

inclusion compound inhibited the bacterial growth at a low concentration [27-29].

Furthermore, CHX in CD inclusion compounds were embedded noncovalently in

polymers. The in vitro studies showed higher antimicrobial activity and long-term effectiveness

of this devices against oral microorganism pathogens [30].

70

Incorporation of CHX in urethane dimethacrylate and triethylene glycol dimethacrylate resin

were made. Here, the rates of release can be effectively controlled by the CHX diacetate content

and the pH [31]. In other studies, CD containing polymer (14 wt.-% CD) cross-linked with citric

acid (CTR) on polyvinylidene difluoride (PVDF) membranes was made to improve the

membrane properties to control the release and to increase the quantity of the antimicrobial agent

CHX diacetate deposited on the membrane. Raw membranes released all incorporated CHX in

few hours, whereas grafted membranes released more than tenfold of this quantity during 60–80

days [32].

Understanding the structure and properties of microbial surfaces at the nanometer level is

of great importance for the development of novel antimicrobial compounds. Hence, the

incorporation of CHX in nanogels and microspheres is in the focus of interest. Cross-linked

polymeric nano- and microparticles are highly functional materials, which can act as carrier and

deliverer of drugs and active agents. They have interesting properties such as small size, different

morphology, large surface area, high chemical functionality, ability to swell in different solvents

(e.g. water or alcoholic solution), and stimuli-sensitivity [33-36]. Furthermore, they show high

binding activity to surfaces by physical self-bonding. Nano- and microparticles can be used in

colloidal form as microreactors, drug carriers and antimicrobial agents but also as building blocks

for the design of colloidosomes, hydrogels, and films targeting such as applications in controlled

release, tissue engineering and optics [37-39]. The chemical functionality of polymer nanogels is

extremely important for their application in different areas of science and technology.

CHX containing cationic and anionic terminated polymeric beads based on poly-N-

isopropylacrylamides (PNIPAMs) with a size of several µm were also studied and the release

rates of CHX at temperatures below and above the volume phase transition temperature (VPTT)

of synthesized polymers were compared. The preparations, assessed in vitro below the VPTT,

release an initial burst of CHX at different periods of time between 120 and 240 min, followed by

a period of 24 h, when the rate of release remains approximately constant, approaching zero-

order kinetics [40].

In this way, mixtures of CHX acetate (CXA) and CHX gluconate (CXG) and the

corresponding -CD complexes in the ratio of 2:1 (-CD:CXA/G) were integrated in silica

nanogels with a size of about 745 nm to prepare and characterize a controlled release system and

to evaluate its antimicrobial activity. The kinetic release parameters of the drug showed that the

71

CHX system release profile followed zero order release up to 400 h after the burst effect in the

first 8 h. The therapeutic range of CHX was reached in nearly the first hour for all systems. The

CHX porous silica system was biologically active against Enterococcus faecalis and Candida

albicans in vitro [41].

Microparticles of poly(dl-lactic-co-glycolic acid) (PLGA) containing CHX free base,

CHX digluconate and their association or inclusion complex with methylated -CD and

hydroxypropyl -CD were prepared by single emulsion, solvent evaporation technique with a

size of 27-45 µm which increases to 30 % by incorporation of CHX. In this polymer-based

delivery system, the encapsulation efficiency, the release characteristics and the bioactivity of

antimicrobial agent were controlled by the complexation of the drug with CDs of different

lipophilicity. Preliminary studies have shown that the CHX released from PLGA chips is

biologically active against bacterial population that is relevant in periodontitis (P. gingivalis and

B. forsythus) and a healthy inhibition zone is maintained in agar plate assay over a period of at

least one week [42].

In this context, CDs are interesting functional units which can be integrated covalently in

a polymer network of nanogels. In previous publications, the high uptake of guest molecules by

CD host domains of nanogels has been studied [43-46].

In this work, the incorporation of CD into the polymer colloidal network of poly(methyl

methacrylate) (PMMA) based nanogels promotes improved uptake and release properties of

CHX. Furthermore, CHX loaded particles exhibit a simple and homogeneous adsorption onto

surfaces by physical self-bonding with high antimicrobial activity against Staphylococcus Aureus.

72

4.2 Experimental

4.2.1 Materials

α-, β- and γ-CD-MA with an average degree of substitution (DS) of 2 and β-CD-MA with

an average substitution degree of 4 are obtained by detailed preparation method in Chapter 2.

Methyl methacrylate (MMA) was obtained from Aldrich and destabilized by column

chromatography over aluminum oxide; a further cross-linker N,N-methylene-bis-acrylamide

(BIS) from Aldrich was used as received. The initiator, azobisisobutyronitrile (AIBN) from

Aldrich was used without further purifications. Chlorhexidine (N',N'''''-hexane-1,6-diylbis[N-(4-

chlorophenyl)(imidodicarbonimidic diamide) was obtained from Aldrich. The solvents (p.A.)

methanol and acetone were purchased from VWR.

4.2.2 Synthesis of CD-MA containing nanogels

The synthesis of cross-linked polymeric nanogels with CD-MA has been described in

detail in Chapter 2 and performed by the radical precipitation polymerization technique in

organic solvent. 19.98 mmol (2.00 g) MMA and modified α-, β-, γ-CD-MA were dissolved in

150 mL methanol and stirred at 200 rpm for 15 minutes at 65 °C under continuous purging with

nitrogen. Afterwards, 0.12 mmol (0.02 g) AIBN initiator was added under continuous stirring and

the reaction was carried out for another 8 h under nitrogen atmosphere. The ingredients used for

the polymerization process and the particle size are summarized in Table 1.

Table 1. Reaction recipe for the preparation of CD-MA/MMA nanogels in 150 mL MeOH using

for complexation studies with CHX. Nanogel samples are numbered as NMMAxCD-MA(y),

where N = run number, x = cyclodextrin type and y = substitution degree of cyclodextrin.

N

MMA,

[g] (mmol)

CD-MA,

[g] (mmol)

CDtheor

,

[wt.-%]

BIS,

[g](mmol)

AIBN,

[g](mmol)

Size

[nm]

1 MMA 2.00 (19.98) - - 0.02 (0.13) 0.02 (0.12) 363

2 MMAβCD(4) 2.00 (19.98) 0.3 (0.21) 12.93 - 0.02 (0.12) 66

3 MMAβCD(4) 2.00 (19.98) 0.6 (0.47) 22.90 - 0.02 (0.12) 36

4 MMAβCD(2) 2.00 (19.98) 0.3 (0.24) 12.61 - 0.02 (0.12) 353

5 MMAαCD(2) 2.00 (19.98) 0.3 (0.27) 12.61 - 0.02 (0.12) 345

6 MMAγCD(2) 2.00 (19.98) 0.3 (0.21) 12.61 - 0.02 (0.12) 219

73

After preparation, nanogel dispersions were purified by dialysis (dialysis tubes with MWCO of

12 kDa (ZelluTrans - 12,0S 45 mm, MWCO: nominal 12,000-14,000; Carl Roth GmbH,

Karlsruhe) to remove unreacted monomers and smaller polymeric particles. Furthermore,

methanol was replaced by water and stable milky dispersions were formed.

4.2.3 Complexation studies of CHX in CD

Different amounts of CHX diluted in acetone (2.08 g/L) were dropped under stirring to

aqueous α-, β- or γ-CD solutions (10 mg/10 mL) (Table S1). The mixture was stirred for 24 h;

after evaporation of the organic solvent the complexed CHX was kept in solution and the non-

diluted CHX amount precipitated. The filtered, clear solutions were analyzed by UV-vis

spectrophotometry [47]. The results are presented in Figure 1a and in the corresponding spectra

with the calibration curves in Figure S1.

4.2.4 Uptake of CHX by PMMA nanogels

The complexation of CHX in CD-MA nanogels was done in water. CHX was diluted in

acetone (2.08 g/L) and added in different volumes to the CD-MA containing nanogel dispersion

in the same way as used for its complexation by low molecular weight CD. After stirring and

evaporation of the solvent for about 24 h, non-complexed CHX precipitated and the filtered

loaded nanogel dispersions were measured by using UV-vis spectrophotmetry. The addition of

CHX and the the parameters of the nanogel dispersions are summarized in Table 2. The resulting

spectra are shown in Figure S2.

74

Table 2. Nanogel (NG), theoretical CD-MA content and added CHX concentrations of the

samples. α-, β- or γ-CD nanogels have a degree of substitution of 2 (DS2).

PMMA

NG

CD-

MA

CD-

MA

CD-

MA CHX CHX

det.

CHX

det.

CHX

CD-

MA

/CHX

CD-

MA/det.

CHX

[µg/ml] [wt.-%] [µg/ml] [µmol/ml] [µg/ml] [µmol/ml] [µg/ml] [µmol/ml]

1550.00 0.00 0.00 0.00 75.00 0.15 14.89 0.03 0.00 0.00

1550.00 0.00 0.00 0.00 100.00 0.20 30.37 0.06 0.00 0.00

1550.00 0.00 0.00 0.00 125.00 0.25 39.74 0.08 0.00 0.00

1550.00 0.00 0.00 0.00 150.00 0.30 45.96 0.09 0.00 0.00

1550.00 0.00 0.00 0.00 175.00 0.35 39.68 0.08 0.00 0.00

-CD-

MA NG

-CD-

MA %

-CD-

MA

-CD-

MA CHX CHX

det.

CHX

det.

CHX

-CD-

MA

/CHX

-CD-

MA

/det. CHX

[µg/ml] [wt.-%] [µg/ml] [µmol/ml] [µg/ml] [µmol/ml] [µg/ml] [µmol/ml]

1540.00 13.04 200.87 0.21 75.00 0.15 35.15 0.07 1.39 2.97

1540.00 13.04 200.87 0.21 100.00 0.20 46.81 0.09 1.04 2.23

1540.00 13.04 200.87 0.21 125.00 0.25 62.88 0.12 0.84 1.66

1540.00 13.04 200.87 0.21 150.00 0.30 63.83 0.13 0.70 1.64

1540.00 13.04 200.87 0.21 175.00 0.35 69.11 0.14 0.60 1.51

-CD-

MA NG

-CD-

MA %

-CD-

MA

-CD-

MA CHX CHX

det.

CHX

det.

CHX

-CD-

MA

/CHX

-CD-

MA

/det. CHX

[µg/ml] [wt.%] [µg/ml] [µmol/ml] [µg/ml] [µmol/ml] [µg/ml] [µmol/ml]

1550.00 13.04 202.17 0.18 75.00 0.15 38.12 0.08 1.20 2.36

1550.00 13.04 202.17 0.18 100.00 0.20 101.53 0.20 0.90 0.89

1550.00 13.04 202.17 0.18 125.00 0.25 119.77 0.24 0.72 0.75

1550.00 13.04 202.17 0.18 150.00 0.30 132.97 0.26 0.60 0.68

1550.00 13.04 202.17 0.18 175.00 0.35 151.15 0.30 0.51 0.60

-CD-

MA NG

-CD-

MA %

-CD-

MA -CD-MA CHX CHX

det.

CHX

det.

CHX

-CD-

MA

/CHX

-CD-

MA

/det. CHX

[µg/ml] [wt.%] [µg/ml] [µmol/ml] [µg/ml] [µmol/ml] [µg/ml] [µmol/ml]

1520.00 13.04 198.26 0.15 75.00 0.15 46.57 0.09 1.03 1.66

1520.00 13.04 198.26 0.15 100.00 0.20 61.63 0.12 0.77 1.25

1520.00 13.04 198.26 0.15 125.00 0.25 70.05 0.14 0.62 1.10

1520.00 13.04 198.26 0.15 150.00 0.30 79.97 0.16 0.52 0.97

1520.00 13.04 198.26 0.15 175.00 0.35 123.64 0.24 0.44 0.62

75

4.2.5 Release of CHX by PMMA nanogels

CHX loaded nanogels (2 mg/2 mL) were coated on glass surfaces (15 x 15 mm (225

mm2)) and dried at 0.1 mbar in the desiccator at 25 °C for about 12 h. Release studies were made

in water and in isotonic PBS-buffer solution (pH 7.4) for about 4 h (240 min). Here, 100 mL of

PBS-buffer solution were prepared by addition of 0.8 g sodium chloride (NaCl), 0.02 g potassium

chloride (KCl), 0.144 g sodium hydrogen phosphate (Na2HPO4) and 0.02 g kalium hydrogen

phosphate (KH2PO4) in 100 mL distilled water. The CHX coated glass surface was wetted with 4

mL dist. water or with 4 mL PBS-buffer solution which were changed every 30 min. The CHX

concentration of the solution was measured by using UV-vis spectrometry. The maxima at 260

nm represent the release of CHX from the surface in dependence on time. After 240 min the

release of CHX in 10 mL water was finished and the UV absorbance reached the zero point.

Absorption maxima of the resulting spectra are shown in Figure S3.

4.2.6 Characterization methods

The particle size of nanogels was analyzed by dynamic light scattering measurements,

using a Nano Zetasizer (Malvern) spectrometer equipped with a He–Ne Laser (λ0 = 633 nm).

Back scattering light was detected using an angle of 173°. Hydrodynamic particle radius and the

particle distribution of the hydrodynamic radius were determined in water. Measurements were

made in a temperature range from 20 °C to 50 °C with a temperature equilibrium time of 120 s.

Microscopical analyses were made with a S-4800-Field-Emission-Scanning Electron

Microscope (Hitachi). One drop of the diluted nanogel dispersion was fixed on an aluminum

support and dried at room temperature in vacuum. After drying, the samples were investigated at

an accelerating voltage of 1 kV and 8 mm disintegration.

IR measurements were made with the help of a FTIR spectrometer: Nexus 470 (Thermo

Nicolet) (spectral disintegration 8 cm-1

). CHX loaded CDs and nanogel dispersions were dried by

lyophilisation and pressed in KBr-pellets.

UV-absorption spectra of CHX containing dispersions were recorded by a JASCO V-630

spectrophotometer from 200-1000 nm using quartz cuvettes with a coating thickness of 1 cm. The

corresponding dispersion of the nanogel without loaded CHX was used as reference.

http://de.wikipedia.org/wiki/Kaliumchlorid

http://de.wikipedia.org/wiki/Kaliumchlorid

76

4.2.7 Biological tests

Chlorhexidine loaded CD-MA containing PMMA nanogels were studied for their

antimicrobial activity on surfaces by using biological tests. β-CD-MA nanogels with different β-

CD-MA content as well as α- and γ-CD-MA nanogels were loaded with 1.75 mg chlorhexidine in

10 mL (175 µg/mL) aqueous microgel dispersion. CHX loaded -, - and -CD-MA (DS2)

nanogels (140 µg CHX per sample) and -CD-MA (DS4)) nanogels (30-70 µg CHX per sample)

were coated on glass slides (15 x 15 mm (225 mm2)) from aqueous dispersion. The coated glass

substrates were placed into Petri dishes (Ø = 3 cm) and Staphylococcus aureus was inoculated

onto each surface under growth conditions in aqueous nutrient solution) [25 µL S. aureus (1 x 106

colony forming units / mL (cfu/mL) in 0.5 % pepton from casein, 0.3 % meat extract, pH 7.0)

(NL1]. A reference (uncoated glass substrate) was exposed with 25 μL S. aureus (1 x 106

cfu/mL). A sterility control (coated glass substrate) was exposed with 25 μL NL1 and the latter

was used in the leaching test (see below). A wetting agent was added to the exposure solution in a

concentration of 0.01 %. Exposure was performed at 25 °C for 2.5 h respectively. Thereafter, in

every Petri dish 1 mL NL1 was pipetted and the samples were shaken at RT and 150 rpm for 20

min. Then, from each Petri dish 200 μL were transferred to a well plate to study the residual

possibility for proliferation / growth. For the growth test, 200 µL of the shaking solution were

transferred to a microwell plate (2 parallel tests) and incubated for 20 h at 37 °C and 1000 s

agitation at 100 rpm per measuring cycle of 30 min with online measurement of the optical

density at 612 nm (OD612) = Measurement of the increase of OD 612, which is caused by the

growth of the micro-organisms.

77

4.3 Results and discussion

In this work, CD-MA containing nanogels were synthesized by radical polymerization of

methylmethacrylate (MMA) and cross-linking with CD-MA in methanol. Here, the substitution

of CDs with methacrylate groups enables solubility of the monomer, the polymerization and the

cross-linking in alcoholic solution. In previous work, the incorporation of -CD-MA into PMMA

nanogels was studied in more detail and described that the particle size of the nanogel (in the

range of 2 µm to 30 nm) can be controlled by the CD-MA content in the reaction mixture and by

the cross-linking degree using modified CDs with different (2, 4 and 6) vinyl groups.

Furthermore, results showed that also the ring size of the CD-MA monomer, i.e. the use of α-, β-

or γ-CD-MA, influence the size of the nanogels. Hence, the particle size decreases slightly with

increase of the CD-MA ring size. For targeted applications and complexation of hydrophobic

guest molecules in aqueous solution the organic solvents were replaced by water using dialysis.

The uptake behavior of β-CD-MA containing nanogels was characterized with the dye adsorption

method using the dye phenolphthalein. Here, the capacity of nanogels increase with increase of

the CD-MA content in nanogels.

4.3.1 Complexation of CHX in CD-MA nanogels vs natural CDs

The creation of antimicrobial nanogels is made by loading of antiseptic and bacteriostatic

CHX. The CD-MA domains with a substitution degree of 2 embedded in the polymeric PMMA

structure of the nanogels act as complexation unit for the fixation of the antimicrobial agent. It is

well known that physical, chemical and size related properties of the guest molecules influence

the host-guest interactions of the complexation. Furthermore, size related properties of α-, β- or γ-

CD have also enormous influence on the uptake and release of guest molecules. Thus, simple

complexation tests of CHX in PMMA nanogels contain α-, β- or γ-CD-MA have to be done as

well as in the corresponding low molecular weight CD. The investigation of the complexation

behavior and of the stability of the complexes depend on the ring size of CD (α-, β- or γ-CD) and

on guest molecule. Here, solutions of CHX in acetone were dropped under stirring to aqueous

solutions of natural α-, β- or γ-CD and to dispersions of PMMA nanogels with or without the

corresponding CD-MA domains. The mixture was stirred for 24 h; after evaporation of the

organic solvent the complexed CHX was kept in solution and the non-dissolved CHX amount

78

precipitated. Thus, the complexation resulted in a solubility increase of CHX in water which

contained α-, β- or γ-CD host molecules.

IR spectroscopic analysis

The IR spectra of the freez-dried CHX loaded α-, β- and γ-CD-MA nanogels in

comparison with the CHX: α-, β- and γ CD inclusion complex are shown in Figure 1. In Figure

1a-b the IR spectra of the CHX complexed by natural α-, β- and γ-CD complexe are shown. Here,

CHX:CD complexes present the characteristic bands of CHX and CD. The -NH bands at 3400

cm-1

and the C=C stretching bands at 1650, 1600,1550 and 1500 cm-1

can be assigned to the

aromatic rings of CHX. The OH groups of the CD ring are presented at 3500-3300 cm-1

and

overlap with the -NH band of CHX. The band at 1032 cm-1

is the C-O-C stretch vibration of the

glucose unit of CD. The sharpness of the CD bands at 3500-3300 cm-1

and 1032 cm-1

characterize

the complex formation. In literature, physical mixtures of free CHX and free CD showed broad

bands, which are sharpened by complexation due to the breakdown of hydrogen bonding [27].

Due to complexation, the C=C bands of CHX at 1612 cm-1

shift to higher and the band at1550

cm-1

to lower wavenumbers. In contrast to the sharpening of the CD bands, the characteristic

CHX bands get broader.

The IR spectra of the CHX containing CD-MA nanogels show all characteristic bands of

CHX, CD-MA and the PMMA backbone of the polymeric structure(Figure 1 c-d). Here, the

sharp CD-MA band of the O-C-O group at 1032 cm-1

is observed in CHX containing α-, β- and γ-

CD-MA nanogels and characterizes that CHX is complexed by CD-MA domains. The typical

CHXs band are presented at 1612 cm-1

and 1550 cm-1

with a high and low shift, similar to the

natural CHX:CD complex shown above. A further band appears at 1650 cm-1

, which can be

assigned to the CD-MA and to the PMMA backbone. The band at 1720 cm-1

is the characteristic

acrylate band of MMA and CD-MA. The stretch vibration of the ester appears in the spectra with

high intensity.

A rough comparison of the CHX band at 1512 cm-1

and the characteristic CD band at

1032 cm-1

enables a comparison of the complexed CHX content between α-, β- and γ-CD-MA

nanogels (Figure 1c). Here, α-, β- and γ-CD-MA nanogels shows a higher maxima of the CHX

band at 1512 cm-1

than the CD band at 1032 cm-1

. These results are reflected by the natural

CHX:β- and γ-CD complexes in Figure 1a. In case of α-CD-MA nanogels, the ratio of the band at

79

1512 cm-1

to the α-CD band at 1032 cm-1

shows that the CHX content is much higher by uptake

in nanogels than in the natural α-CD (Figure 1a.). Here, the band at 1512 cm-1

is much lower than

the CD band at 1032 cm-1

. That means, the polymeric structure of hydrophobiv PMMA has also

an influence on the uptake of CHX and helps α-CD with the smaller cone to fix the guest

molecule in the nanogel.

Figure 1. FTIR spectra of CHX complexed by -, β- and -CD (a) and details with CHX bands

between 1700 and 1500 cm-1

(b). FTIR spectra of CHX containing -, β- and -CD-MA nanogels

(NG) (c) and detail with CHX bands between 1700 and 1500 cm-1

(d).

a b

c d

4000 3500 3000 2500 2000 1500 1000 500

0

1

2

3

4

5

6

CHX:-CDMA NG

CHX:-CDMA NG

CHX:-CDMA NG

1650-1500

1032

1720 -CDMA NG

CHX:-CDMA NG

CHX:-CDMA NG

CHX:-CDMA NG

IR, A

bsorb

ance

Wavenumbers [cm-1]

3400

-CDMA NG

1700 1650 1600 1550 1500 1450

0

1

2

3

4

5

6

1578 1537 1637

CHX:-CDMA NG

CHX:-CDMA NG

-CDMA NG

CHX:-CDMA NG

1490

IR, A

bsorb

ance

Wavenumbers [cm-1]

1612

1700 1650 1600 1550 1500 1450

0

1

2

3

4

5

6

7

IR, A

bsorb

ance

Wavenumbers [cm-1]

1637 1537 149015781480

CHX

CHX:-CD

CHX:-CD

CHX:-CD

15551612

4000 3500 3000 2500 2000 1500 1000 500

0

1

2

3

4

5

6

IR, A

bsorb

ance

Wavenumbers [cm-1]

CHX:-CD

CHX

CHX:-CD

CHX:-CD

1032

1650-1500 CHX

CHX:-CD

CHX:-CD

CHX:-CD

80

4.3.2 Quantitative complexation of CHX by CD-MA nanogels vs. natural CDs

Here, the complexation properties of natural α-, β- and γ-CD and the corresponding

nanogels with regard to CHX were studied by solubility tests and UV-vis spectrometric

measurements in aqueous dispersion. As the solubility of CHX in water is very low (0.08 g/L),

the solubility should increase during inclusion in α-, β- or γ-CD or in hydrophobic polymeric

nanogels. The CHX containing solutions and stable dispersions were measured by UV-vis

spectroscopy.

The complexation studies of CHX in natural α-, β- or γ-CD are shown in Figure 3a and b.

The poorly water-soluble CHX can be dissolved in higher and in different concentrations in water

due to controlled host-guest complexation by CD. Figure 2a shows the maxima of the absorbance

at 255 nm of solutions with different amounts of complexed CHX. The incorporation of CHX in

-CD and -CD is stericaly favored caused by the ring size of the cone. It is well known that -

CD forms energetically stable host-guest systems particularly with aromatics [8,9]. The

complexation of CHX has already been studied in previous work and 1:1, 1:2, and 1:3

complexation has been proven by different analytical methods [27,25].

By addition of up to 150 µg/mL, the ratio of CHX/CD should form a 3:1 complex and 200

µg/mL or 250 µg/mL addition of CHX should create a 2.5:1 and 2:1 complex. In this case, full

complexation of CHX by CD takes place and high solubility is expected. The strong increase of

the absorbance and of the solubility degenerates after addition of 300 µg/mL and 350 µg/mL

CHX, when a 1.5:1 and 1:1 CHX:CD complex is preferred. Further addition leads to a decrease

of solubility, the absorbance stagnates and the maximum of diluted CHX in water is reached. The

non-complexed rest precipitates and sediments to the bottom. Increase of the CHX absorbance

due to complexation by -CD domains documents that an incomplete complexation of the guest

molecule caused by the small cavity also increases the water solubility.

81

Figure 2. UV-vis spectra of CHX (250 µg/mL) complexed by natural -, - or -CDs in water

(absorbance of CHX in water is measured of a saturated aqueous CHX solution by addition of 3.5

mg to 10 mL water) (a) and UV absorption maxima of different CHX concentrations complexed

by -, - or -CDs (b). UV-vis spectra of CHX (100 µg/mL) complexed by -, - or -CD-MA

nanogels (c), absorption maxima of different CHX concentrations complexed by nanogels with

-, - or -CD-MA and PMMA nanogels without CD-MA content in water (d).

225 240 255 270 285 3000.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Absorb

ance

Wavelength [nm]

without CD-MA NP:CHX

-CD-MA NP:CHX

-CD-MA NP:CHX

-CD-MA NP:CHX

225 240 255 270 285 3000.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Absorb

ance

Wavelength [nm]

CHX in H2O

-CD:CHX

-CD:CHX

-CD:CHX

a b

c d

0 50 100 150 200 250 300

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Absorb

ance m

axim

a

CHX addition [µg/mL]

-CD:CHX

-CD:CHX

-CD:CHX

50 75 100 125 150 175

0

25

50

75

100

125

150

175

200 -CD-MA NP:CHX

-CD-MA NP:CHX

-CD-MA NP:CHX

without CD-MA NP:CHXC

HX

up

take [

µg/m

L]


82

The complexation of CHX in -, - and -CD-MA nanogels was done in aqueous

dispersion. CHX was diluted in acetone and added in different concentrations to 10 mL CD-MA

containing nanogels dispersion (1.5 mg/mL) in the same way as used for its complexation in low

molecular weight CD. After stirring and evaporation of the solvent for about 24 h, non-

complexed CHX precipitated and the filtered loaded microgels were measured by UV-vis

spectrometry. Figure 2c shows the UV-vis spectra of -, - and -CD-MA containing PMMA

nanogels loaded with the same concentration of CHX. Here, it occurs that the addition of CHX to

nanogels increases the absorbance maxima at 260 nm. Thus, the enhanced solution in water and

the uptake of CHX takes place for all nanogels with and without CD-MA content whereas, the

absorbances and thus, the complexion yields are higher for - and -CD-MA nanogels.

The absorbance maxima of different concentrations of CHX in PMMA nanogels with and

without containing -, - and -CD-MA is shown in Figure S3. It is found that PMMA nanogels

without embedded CD-MA show low absorbance and therefore, low uptake of CHX without

significant increase by addition of different amounts of the guest molecule. -CD-MA containing

PMMA nanogels show low absorbance but slight increase with increase of CHX content. - and

-CD-MA containing PMMA nanogels show higher absorbance after addition of CHX. However,

the uptake leads to strong increase of the absorbance maxima with rising CHX content.

The UV absorbance of different concentrations of completely dissolved CHX in water by

complexation in natural -, - and -CD allows the calculation of a straight calibration curve.

The comparison of the absorbance measured for CHX containing nanogels with the calculated

calibration curve of -, - or -CD allows the quantitative determination of the exact uptake of

CHX in the PMMA nanogels. In Figure 2d the calculated amounts of CHX in the nanogels are

listed. Here, nanogels with -CD-MA domains show the best uptake of CHX and have the

strongest influence on the complexation of CHX. The uptake increases from 38.12 µg/mL with

rising addition of CHX to 151.15 µg/mL. -CD-MA domains influence also the adsorption of the

guest molecule caused by almost linear increase of the CHX content from 46.56 µg/mL to 123.64

µg/mL. It seems that the sterically preferred -CD ring stabilizes energetically and fixes the guest

in the polymeric structure more than the -CD ring. -CD-MA domains influence the uptake of

CHX in the nanogels as well. The CHX uptake increases with rising addition of CHX from

46.81µg/mL to 69.11 µg/mL, but not as strong as in case of - and -CD-MA. Nanogels without

83

CD-MA content but with the hydrophobic PMMA backbone show also slight uptake (14.89

µg/mL to 39.68 µg/mL) of hydrophobic CHX. Here, the course of the uptake with increasing

addition of CHX can be described as degressive. It seems that PMMA nanogels favor the

adsorption over the sedimentation process of water insoluble molecules. Thus, the CD-MA

domains in the nanogels have a significant influence on the CHX adsorption and increase the

uptake possibilities for hydrophobic nanogels. Furthermore, CD-MA complexation keeps the

molecule and stabilizes the CHX in the nanogels.

The investigation of the influence of the CD-MA content and the influence of the

hydrophobic polymer backbone of PMMA in the nanogel on the complexation is shown in Figure

3 in detail. The theoretical molar possible CD-MA content which is given in percentage ratio of

the reaction mixture is presented. The molar addition of CHX to the theoretically expected molar

amount of the CD-MA domains in the nanogels is calculated as 1:1 complex at the beginning.

Then the CHX addition is increased to higher concentration in the dispersion. Here, the extreme

CHX excess in relation to the CD-MA domains in nanogels enables the investigation of the

influence of the hydrophobic structure of the PMMA backbone. It is shown in Figure 3a that

PMMA with its hydrophobic polymer backbone can also adsorb CHX in small amounts.The

influence of -CD-MA in nanogels is not very high (Figure 3b). Here, the adsorbed CHX is less

than the theoretical -CD-MA content. In Figure 3c, -CD-MA shows a linear increase of the

CHX uptake, the adsorbed CHX content is less than the theoretical value of the added CHX but

increases and exceeds the molar -CD-MA content. -CD-MA shows high adsorbance affinity to

CHX (Figure 3d). The CHX concentration increases linearly and exceeds the molar ratio of the

determined as well as the theoretical CD-MA content to a great extent. In this case, the influence

of -CD-MA is obvious and furthermore, the combination of the hydrophobic PMMA backbone

and the CD-MA domains which work together and favor a good complexation and adsorbance of

CHX in these nanogels.

84

Figure 3. Molar uptake of different CHX amounts in PMMA nanogels in comparison with the

molar CHX addition and the theoretical molar CD-MA content in the nanogels in mL. Uptake of

CHX in PMMA nanogels without CD-MA (a), in α-CD-MA/PMMA nanogels (b), β-CD-

MA/MMA nanogels (c), γ-CD-MA/PMMA nanogels (d).

a b

c d

75 100 125 150 1750.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35


CH

X c

onc. [µ

mol/m

l] without CD-MA

CHX addition

CHX uptake

75 100 125 150 1750.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35


CD

-MA

, C

HX

con

c. [µ

mol/m

l]

-CD-MA

CHX addition

CHX uptake

75 100 125 150 1750.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35 -CD-MA

CHX addition

CHX uptake


CD

-MA

, C

HX

con

c. [µ

mol/m

l]

75 100 125 150 1750.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

-CD-MA

CHX addition

CHX uptake


CD

-MA

, C

HX

con

c. [µ

mol/m

l]

85

4.3.3 Influence of the internal cross-linking degree of nanogels on the CHX uptake

Studies on the uptake properties of nanogels with a higher cross-link density prepared

with cross-linker -CD-MA (DS4) with 4 vinyl groups are also part of the investigation. The

increase of the degree of cross-linking caused by increased substitution degree of -CD-MA

influences the colloidal properties. Thus, the particle size, the degree of swelling and the uptake

properties depend on the degree of cross-linking.

Here, transparent dispersions which contain ultrafine PMMA nanogels prepared by cross-

linking of -CD-MA (DS4) became cloudier upon addition of CHX than dispersions with

nanogels synthesized with substitution degree of 2. Interestingly, the optical stability of the

dispersions kept constant after addition. In Figure 4, the absorbance spectra of CHX added to

nanogels prepared by -CD-MA with 4 vinyl groups are shown. Figure 4a shows nanogels

prepared with 0.21 mmol -CD-MA (DS4) and Figure 4b displays nanogels prepared with 0.47

mmol -CD-MA (DS4) which are loaded with CHX. The baseline of the UV-vis spectra

increases from 0.0 (pure particle dispersion) to higher absorbance. It is observed that the

wavelength of the absorbance at 260 nm is shifted with a low increase of the maximum to higher

wavelength with increase of CHX content in the dispersion due to the CHX complexation in

nanogels. Due to the strong shift in the baseline and the red shift of the absorbance with the

increase of the CHX concentration in the nanogel dispersions, the CHX content is quantitatively

not comparable with the calibration curves made by complexation of CHX by natural -CDs.

The increase of the turbidity is explainable by the cross-linking of particles to a particle

network by CHX as linker. Thus, CHX forms a 1:2 (CHX: -CD-MA) complex with 2 -CD-MA

containers from 2 different nanogels. This external cross-linking of different nanogels takes place

when CHX is complexed directly by -CD-MA container at the particle surface.

For -CD-MA nanogels (DS4) with a higher cross-link density and with a dense polymeric

network can be expected that the penetration of the guest molecule in the internal particle

structure is more hindered than -CD-MA nanogel (DS2) prepared with lower degree of cross-

linking. Hence, it can be assumed that CHX is almost complexed at the CD-MA containing

nanogel surface. The difference in the hydrophobicity of the environmental particle surface, the

interaction with the solvent and other -CD-MA containing nanogels leads to strong shifts of the

typical CHX band at 260 nm to higher wavelength.

86

Figure 4a (0.21 mmol (13 wt.-%)-CD-MA) clarifies the absorbance shift from 255 nm to

higher wavelength which stagnates at 280 nm upon addition of 150 µg CHX. Figure 4b (0.47

mmol (23 wt.-%) -CD-MA) shows that the absorbance shift does not stagnate, the wavelength

increases from 255 nm to over 300 nm upon addition of CHX. These results displays a controlled

uptake and cross-linking of the nanogels by CHX guest molecules by variation of CD-MA units

with a the saturation point using nanogels with a lower -CD-MA content.

Figure 4. UV-absorption spectra of CHX complexed by β-CD-MA/PMMA nanogels. Nanogels

are prepared by CD-MA with degree of substitution of 4. The nanogels contain different β-CD-

MA content of (0.21 mmol (13 wt.-%) (a) and 0.47 mmol (23 wt.-% ) (b).

Scheme 1. Aggregation and cross-linking of nanogels due to external complexation of CHX.

200 250 300 3500.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0 mmol CD (DS4) 75 µg/mL CHX

100 µg/mL CHX

25 µg/mL CHX

150 µg/mL CHX

175 µg/mL CHX

Absorb

ance

Wavelength [nm]

a b

200 250 300 3500.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0mmol CD (DS4)

Absorb

ance

Wavelength [nm]

5 µg/mL CHX

100 µg/mL CHX

125 µg/mL CHX

150 µg/mL CHX

175 µg/mL CHX

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NHH

N

NH

Cl

NH

NH

HN

HN

HN

H2O

Cl

NH

NH

HN

NH

HN

Cl

HN

HN

NH

HN

NH

Cl

HN

HN

NH H

N

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NHH

N

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NHH

N

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl N

H

NH

HN HN

HN

Cl H

N

HN

NH HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

Cl

HN

HN

NH

HN

NH

Cl

NH

NH

HN

HN

HN

n

Nanogels

in aqueous dispersion CHX loaded nanogels

87

4.3.4 Morphology of CHX loaded nanogels in dispersion and on glass surfaces

The influence of the CHX uptake and complexation by CD-MA nanogels in aqueous

dispersion is studied by FESEM spectroscopy. The addition of CHX to aqueous -CD-MA

nanogels with DS2 shows a slight decrease in transparency of the nanogel dispersions. This is

explainable by external cross-linking of the nanogel by CHX complexed in two different -CD-

MA cones of two different nanogels.

Figure 5a presents the 0.24 mmol -CD-MA (DS2) nanogel dispersion without and Figure

5b after addition of the 70 µg/mL CHX guest molecules. Multiple interaction of nanogels are

detectable which lead to dense formation and agglomeration of particles in concentrated

dispersion. The interactions are visible clearly between the single 350 nm sized -CD-MA

particles. A strong cross-linking effect is observed by the use of ultrafine -CD-MA nanogels

with Rh under 50 nm (Figure 5c-d). Here, after addition of 70 µg/mL CHX to a transparent

dispersion of 0.47 mmol -CD-MA nanogels (DS4) agglomeration takes place and large particle

structures and aggregates are formed. The electron microscopic results are in accordance to the

UV-vis spectra wherein the strong shift in the turbidity of the dispersion prevents an accurate

determination of the the CHX content (Figure 5a-b).

A further advantage of polymeric CD-MA nanogels compared to low molecular weight

CDs is the improved adsorption and fixation on different surfaces. The polymeric character of the

small particles enables the fixation by physical self-bonding and allows the application of the

complexed ingredients on the surface with good permanence under dry conditions. CHX loaded

PMMA nanogels can be applied by dip-coating and padding from aqueous dispersions onto

surfaces. The photography in Figure 5e shows the applied 0.47 mmol -CD (DS4) nanogels with

different CHX content on glass plates. Similar to the corresponding nanogel dispersion (Figure

5c-d) the transparencies of the nanogel film decrease with the increase of the CHX content. That

means the film density of the connected nanogel increases. The particle film of the highest CHX

content (70 µg) are shown in Figure 5f. Here, the single nanogels are three-dimensional cross-

linked to other particles which forms a dense CHX gel film on the glass surface.

88

Figure 5. FESEM images of 0.24 mmol CD-MA (DS2) nanogels for (a) and after complexation

with 70 µg/mL CHX on aluminum surface. (b). Cryo-FESEM image of 0.47 mmol CD-MA

(DS4) nanogels for (c) and after complexation with 70 µg/mL CHX (d). Photography of 0.47

mmol CD-MA (DS4) nanogels with different CHX content coated on glass plates (e) and FESEM

images of the nanogel film consisting of the 0.47 mmol CD-MA (DS4) nanogels with 70 µg/mL

CHX (f).

0 µg 30 µg 40 µg

50 µg 60 µg 70 µg

e f

dc

ba

89

4.3.5 Release of CHX from coated surfaces

The release of CHX from coated surfaces was investigated and is presented in Figure 6a-

b. Studies were made in water as well as in PBS-buffer solution at RT. Glass plates coated with

6.7 mg CD-MA containing nanogels with CHX uptake of 74-153 µg/mL were given into 4 mL

aqueous solutions which were changed every 30 min in a period of time of 4 h. The CHX

concentration of the solution was measured by using UV-vis spectrophotometry in which the

maxima at 253 nm represents the release of pure non-complexed CHX from the surface in

dependence on time.

The release curves of the free base CHX show a typical trend in a 4 h period. The release

in water is quite strong in the first 30 min (burst effect) and the CHX concentration on the surface

decreases rapidly. It is observed that the maximum release in the first 30 min is similar or below

the maximum solubility (80 µg/mL) of the free CHX base in water. The following released CHX

concentration decreases more slowly with time and keeps an average of 2.5 µg/mL (-CD-MA) 5

µg/mL (-CD-MA) and 7.5 µg/mL (-CD-MA) over a period from 90 min to 210 min. After 210

min, the release of CHX in 10 mL water is almost finished and the released CHX in water

reaches the zero point.

In the PBS-buffer solution, CHX is released more slowly with a smaller amount of CHX

than in water. The trend of release in PBS is similar to that in water but in the first 30 min the

agent is delivered in lower concentrations. After 120 min, the delivery remained static and

decreased constantly but slowly until the end of the measurement series.

The comparison of the totally released amount with the theoretical CHX amount (which

was added) and with the determined real uptake are shown in Figure 6c-d. Here, -CD-MA

nanogels took up 42 % (74 µg/mL) of the added CHX and release even the same amount 94 %

(69 µg/mL) of the uptake in water. In PBS the total amount of CHX release is 44 % (32 µg/mL)

of the uptake and thus, much less than in water. -CD-MA nanogels take up 73 % (127 µg/mL) of

the added CHX and release about the same amount of the uptake in water i.e. 97 % (123 µg/mL).

But only 30 % (38 µg/mL) of the uptake are released in PBS buffer solution. -CD-MA nanogels

have an uptake of 88 % (153 µg/mL) of CHX and release of 95 % (145 µg/mL) which is similar

as found for -, and -CD-MA nanogels In PBS-buffer the release is prevented and is 60 % (91

µg/mL) of the incorporated CHX.

90

These results led to the explanation that the free phosphate ions in PBS buffer solution form with

the free CHX base an insoluble salt upon exposure [42]. This salt precipitates or fixes the CHX

molecule in the nanogel.

Figure 6. UV absorption maxima at 253 nm of free CHX released from CHX loaded CD-MA

nanogels (140 µg CHX / 13 wt.-% β-CD-MA content) coated on glass surfaces in every 4 mL

water [a] and in every 4 mL PBS buffer solution [b] every 30 minutes over 4 h.Total CHX release

in water and in PBS after 4 h in µg/mL in comparison with the theoretical total CHX uptake [c]

and the determined CHX uptake in -, - and -CD-MA nanogels [d].

a b

c d

alpha CD beta CD gamma CD0

25

50

75

100

125

150

175

200CHX Addition vs. Release

CHX Addition Release in H2O Release in PBS

CH

X c

onte

nt [µ

g/m

L/2

40m

in]


25

50

75

100

125

150

175

200

CH

X c

onte

nt [µ

g/m

L/2

40m

in]

CHX Uptake Release in H2O Release in PBS

CHX Uptake vs. Release

0 30 60 90 120 150 180 210 240

0

10

20

30

40

50

60

70

80

90

100CHX Release in PBS

-CD-MA NG

-CD-MA NG

-CD-MA NG

Re

lea

se

[µ

g/m

L]

Time [min]

0 30 60 90 120 150 180 210 240

0

10

20

30

40

50

60

70

80

90

100

-CD-MA NG

-CD-MA NG

-CD-MA NG

Re

lea

se

[µ

g/m

L]

Time [min]

CHX Release in H2O

91

4.3.6 Antimicrobial properties

CHX loaded CD-MA containing PMMA nanogels have been studied for their

antimicrobial activity on surfaces by using biological tests. α-, β- and γ-CD-MA containing

nanogels as well as nanogels with different β-CD-MA content were coated on glass slides (140

µg CHX per sample) from aqueous dispersion. Staphylococcus aureus was inoculated onto each

particle surface under growth conditions in aqueous nutrient solution. residual possibility of

proliferation / growth.

-CD-MA (DS2) containing nanogels without CHX do not show any influence on the

bacterial growth (Figure 7a). Thus, after exposure of S. aureus to those nanogels residual growth

is comparable to the growth after exposure to the uncoated reference. On the other hand, all CHX

loaded -, - and -CD-MA (DS2) nanogel samples inhibited the typical growth of S. aureus

completely after exposure under the test conditions and in the investigation period. Hence, all

CD-MA nanogel have the capacity for the uptake of the right amount of CHX which is active

against bacterial growth of S. areus.

In Figure 7b nanogels films prepared by ultrafine CHX containing CD-MA nanogels with

-CD-MA (DS4) are studied with respect to different CHX contents. Here, all samples with an

CHX amount of 30 µg to 70 µg pro 225 cm2 show an inhibition of the growth of S. aureus. That

means CHX can not only control the external cross-linking of the nanogels and the film

formation, but also CHX loaded nanogels show high effectiveness against the bacterial growth

especially in low CHX concentrations of 30 µg/225 cm2.

Release tests, discussed above, show that CHX is not only active on the material surface,

it can be released in low amounts in the aqueous environment by decomplexation and

redispersation of particles. Quantitative release studies are compared with antimicrobial leaching

tests. Therfore, the coated surface are exposured to the nutrient solution without S. aureus and

aliquots of the solution are inoculated with S. aureus and a growth test are performed. The

amount of CHX released to the exposure solution showed antimicrobial activity and inhibits the

bacterial growth after inoculation with S. aureus (Figure 7c-d). Thus, the antimicrobial activity is

not only restricted to the surface, CHX acts in high efficiency in the environment by release.

92

Figure 7. Growth curves of S. aureus after exposure on surfaces coated with CHX loaded

nanogels; samples exposed to S. aureus and transferred to growth tests of nanogels with different

CD types [a] and different CHX content [b]; samples exposed without bacteria and afterwards

inoculated to exposure/ shaking solution with S. aureus before growth tests of nanogel with

different CD type [c] and different CHX content [d].

a b

c d

0 5 10 15 20 25 30 35 40 45

0.1

0.2

0.3

0.4

0.5

0.6

OD

61

2

Time in cycles a 30 min

Ref. without CHX

unloaded -CD-MA (4.8 wt%) NG

CHX -CD- MA (4.8 wt%) NG

CHX CD-MA (4.8 wt%) NG

CHX -CD-MA (4.8 wt%) NG

0 5 10 15 20 25 30 35 40 45

0.1

0.2

0.3

0.4

0.5

0.6O

D6

12


Ref. without CHX

unloaded -CD-MA (4.8wt%) NG

CHX -CD-MA (4.8wt%) NG

CHX CD-MA (4.8wt%) NG

CHX -CD-Ma (4.8wt%) NG

0 5 10 15 20 25 30 35 40 450.0

0.2

0.4

0.6

0.8

1.0

1.2

OD

612


Ref. without CHX

30 µg CHX -CD-MA NG





0 5 10 15 20 25 30 35 40 45

0.0

0.2

0.4

0.6

0.8

1.0

1.2

OD

612


Ref. without CHX






93

4.4 Summary

In this paper, CD-MA nanogels loaded with CHX for the application on surfaces with the

aim of achieving antimicrobial functionality are presented. Aqueous PMMA nanogels with

different content of CD-MA were prepared in organic solvent. In addition to β-CD-MA, α- and γ-

CD-MA were used as cross-linker and complexation units. After transfer of the nanogels to the

aqueous phase, the PMMA nanogels with CD-MA complexation minorities were complexed with

the antimicrobial agent CHX in water. The uptake of CHX can be controlled by the CD-MA

content in the nanogels. PMMA nanogels with more CD-MA units show better uptake of the

active CHX guest molecule than nanogels with lower CD-MA amounts. Large aggregates were

formed during the complexation of CHX in aqueous phase. The loaded nanogels and aggregates

can be applied on glass surfaces and fixed by physical self-bonding results in dense film

formation. It was shown, that CHX was released from the surface into aqueous solutions.

Biological studies with Staphylococcus aureus proved the antimicrobial activity of the CHX

nanogel coatings. However, the antimicrobial activity is not only located on the coated surface,

but also in the aqueous environment.

94

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hydroxypropyl-β-cyclodextrin, Drug Dev. Ind. Pharm. 1992, 18, 1477-1484.

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Pharm. Res. 1994, 11, 1207-1210.

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controlled release system of chlorhexidine and chlorhexidine:bcd inclusion compounds based on porous silica,

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Inclusion Effects, Macromol. Rapid. Commun. 2004, 25, 1912-1916.

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1399.

97

4.6 Supporting information

Table S1. Natural CD and CHX concentrations plus the CD/CHX ratios as well as the

absorbance of the used samples.

CD α-CD

/CHX

β-CD

/CHX

γ-CD

/CHX CHX CHX

CHX:

α-CD

CHX:

β-CD

CHX:

γ-CD

[µg

/mL]

[mol-

ratio]

[mol-

ratio]

[mol-

ratio]l

[µg

/mL]

[µmol

/mL] [Abs.max] [Abs. max] [Abs. max]

1000 10.40 8.91 7.79 50 0.10 0.3299 0.2754 0.3132

1000 5.20 4.45 3.90 100 0.20 0.5225 0.5508 0.5502

1000 3.47 2.97 2.60 150 0.30 0.6966 0.7344 0.7336

1000 2.60 2.23 1.95 200 0.40 0.8797 0.8546 0.8352

1000 2.08 1.78 1.56 250 0.49 0.9542 1.0200 1.0422

1000 1.73 1.48 1.30 300 0.59 0.9853 1.0267 1.0622

98

Figure S1. UV-absorption spectra of different CHX concentrations complexed by -CD (a), -

CD (b) and -CD (c) in water (right) with the corresponding calibration curves (left).

a

b

c

200 225 250 275 300 325 3500.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2 50 µg/mL CHX

100 µg/mL CHX

150 µg /mLCHX

200 µg /mLCHX

250 µg /mLCHX

300 µg /mLCHX

Ab

so

rba

nce

Wavelength [nm]

-CD

200 225 250 275 300 325 3500.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2 50 µg/mL CHX

100 µg/mL CHX

150 µg /mLCHX

200 µg /mLCHX

250 µg /mLCHX

300 µg /mLCHX

Ab

so

rba

nce

Wavelength [nm]

-CD

200 225 250 275 300 325 3500.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2 50 µg/mL CHX

100 µg/mL CHX

150 µg /mLCHX

200 µg /mLCHX

250 µg /mLCHX

300 µg /mLCHX

Ab

so

rba

nce

Wavelength [nm]

-CD

y = 270.96x1.5416

R² = 0.987

0

50

100

150

200

250

300

350

0 0.2 0.4 0.6 0.8 1 1.2

CD

co

nc.

[µg

/mL

]

Absorbance

CHX:-CD

y = 247.22x1.3056

R² = 0.9735

0

50

100

150

200

250

300

350

0 0.2 0.4 0.6 0.8 1 1.2

CD

co

nc.

[µg

/mL

]

Absorbance

CHX:-CD

y = 249.14x1.424

R² = 0.9887

0

50

100

150

200

250

300

350

0 0.2 0.4 0.6 0.8 1 1.2

CD

co

nc.

[µg

/mL

]

Absorbance

CHX:-CD

99

Figure S3. UV-absorption spectra of different concentrations of CHX incorporated in PMMA

nanogels (a), -CD-MA (b), β-CD-MA (c) and -CD-MA (d) containing nanogels in water.

Figure S4. UV absorption maxima at 253.5 nm of different CHX concentrations released by CD-

MA containing nanogels with different type of CD-MA in water (a) and in PBS solution (b).

a b

c d

200 220 240 260 280 300 320 340

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Absorb

ance

Wavelength [nm]

75 µg/mL CHX

100 µg/mL CHX

125 µg/mL CHX

150 µg/mL CHX

175 µg/mL CHX

-CD-MA NG

200 220 240 260 280 300 320 340

0.0

0.2

0.4

0.6

0.8

1.0

Ab

so

rba

nce

Wavelength [nm]

75 µg/mL CHX

100 µg/mL CHX

125 µg/mL CHX

150 µg/mL CHX

175 µg/mL CHX

-CD-MA NG

200 220 240 260 280 300 320 3400.0

0.2

0.4

0.6

0.8

1.0

1.2

Ab

so

rba

nce

Wavelength [nm]

75 µg/mL CHX

100 µg/mL CHX

125 µg/mL CHX

150 µg/mL CHX

175 µg/mL CHX

-CD-MA NG

200 220 240 260 280 300 320 340

0.0

0.1

0.2

0.3

0.4

0.5A

bsorb

ance

Wavelength [nm]

75 µg/mL CHX

100 µg/mL CHX

125 µg/mL CHX

150 µg/mL CHX

175 µg/mL CHX

without CD-MA NG

0 30 60 90 120 150 180 210 240

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35 CHX Release in PBS

-CD-MA NG

-CD-MA NG

-CD-MA NG

Ab

so

rba

nce

ma

x.

Time [min]

0 30 60 90 120 150 180 210 240

0.0

0.1

0.2

0.3

0.4

0.5

CHX Release in H2O

-CD-MA NG

-CD-MA NG

-CD-MA NG

Ab

so

rba

nce

ma

x.

Time [min]

a b

101

Chapter 5

Ibuprofen loaded cyclodextrin containing poly(2-hydroxyethyl

methacrylate) nano/microgels

Abstract: In this work, the creation of novel nano-drug-carrier for ibuprofen on the basis of

cyclodextrin acrylate (CD-A) and 2-poly(hydroxyethyl methacrylate) (PHEMA) is presented.

The incorporation of different amounts of -, - and -CD-A with different number of vinyl

bonds (2, 4 and 6) in aqueous PHEMA based microgels is investigated. The synthesis is

performed by surfactant-free precipitation polymerization in aqueous solution. The increase of

the CD-A concentration in reaction mixture led to the reduction of the final hydrodynamic radius

of microgels from 347 nm to 114 nm. Furthermore, rising number of vinyl groups per CD-A

molecule decrease the size of the particles. The improved uptake properties of microgels are

shown by complexation with a common anti-inflammatory drug ibuprofen. The drug loading in

microgels is controllable by CD-A content and degree of cross-linking and allows the quantitative

uptake and fixation of ibuprofen in microgels.

Keywords: microgels, 2-poly(hydroxyethyl methacrylate), cyclodextrin, ibuprofen, drug

adsorption

AMPA

Cyclodextrin acrylate (CD-A)

O

R = O

O

RR

R

O

O

R

RRO

OR

R

R

O

O

RR

R

OO

R

R

RO

OR

RR

O

OR

R

R

O

H2O, 70°C

Hydroxyethylmethacrylate (HEMA)

OO

Monomer

Cross-linker

R = OH

OH

Variable parameter:

-CD-A /HEMA ratio,

-vinyl bonds (R)

-ring size

Ibuprofen

Ibuprofen

CD-Complex

CH3

HO OH

H3C

OH

OH⇌

102

5.1 Introduction

The developments in the area of drug delivery systems drive forward the creation of new

drug carriers such as nanparticles, hydrogels and insert films. These drug carriers have the aim of

improving drug application in vivo such as the prolongation of drug actions, decrease of drug

metabolism, reduction of drug toxicity and the controlled-release by targeted delivery [1,2].

Among these drug carriers aqueous nanoparticles such as internal cross-linked nano- and

microgels are particularly attractive because of their nanoscalic size with its large interfacial

surface area which enables working between the molecular and cellular level. The ability for

uptake and release of drugs and other active ingredients by nano- and microgels is given due to

their high porosity and swell ability in different solvents especially in aqueous media. The

interesting properties as stimuli-sensitivity to the environment, the chemical and biomedical

functionality results in controlled handling of drugs with regard to the prolongation of the

residence time, improve bioavailability and the convenient use of targeted application. The

chemical functionality and the internal structure of polymeric nano- and microgels is extremely

important for uptake of drugs and the further application in the aqueous phase [3,4].

An interesting matrix for the development of nanocalic hydrogel particles is poly(2-

hydroxyethyl methacrylate) (PHEMA). The monomer (HEMA) is characterized by its functional

hydroxy group and the vinyl bond. These molecular properties of HEMA enable water solubility

of the monomer and the polymerization in aqueous media [5].

Cross-linked PHEMA was one of the first components of hydrogels. Since 1960 PHEMA

is already used as medicated soft hydrogel as basis of contact lenses and as carrier for the

delivery of ophthalmic drugs [6,7]. Furthermore, PHEMA is used as ocular surgery, prostheses,

sature coatings, artificial internal organs and drug delivery systems [8-10].

The hydrophilicity and porous nature qualifies PHEMA as extremely good polymeric

material for controlled drug delivery. Additionally, PHEMA hydrogels have a number of further

properties that improve material application with respect to long-term device performances. The

enhanced biocompatibility [11], bioadhesive activity [12], low toxicity [13], and high degree of

swelling [14] have already been studied and reported in many publication.

However, PHEMA hydrogels have some limitations in the application of long time

therapy. Although PHEMA hydrogels are characterized by high water uptake which can be

compared with the water content of living cells [15], the uptake of drugs is strongly limited and

103

the release rate of drugs is very fast [16,17]. Hence, the copolymerisation of HEMA with other

acrylate, methacrylate and acrylamide monomers and different cross-linking agents results in

copolymeric materials such as hydrogels that vary in the physical, chemical and mechanical

properties with the aim of controlling the uptake and release of potential drugs. Therefore,

HEMA were copolymerized with monomers as styrene [18] and methyl methacrylate (MMA)

[19] to yield more hydrophobic structures or with stimuli-sensitive monomers as N-

isopropylacrylamid (NIPAM) [20] and N-[3-(dimethylamino)propyl]methacrylamid (DMAPMA)

[21] for the creation of temperature- or pH-responsive polymeric hydrogels.

The development of special polymeric architectures or the incorporation of functional

monomers with hydrophobic and strong binding affinities to potential active ingredients in

PHEMA hydrogel structure were developed [22]. Charged polymer systems are made to increase

the interaction between hydrogel and drugs so that the drugs had more difficulties diffusing from

the hydrogel e.g. by incorporation of 4-vinylpyridine (VP) or ionic monomer such as N-(3-

aminopropyl)methacrylamide (APMA) [23].

In this context, cyclodextrins (CD) are interesting functional units which can be integrated

in polymer network of PHEMA hydrogels. Due to its torus-shaped cone structure formed by

cyclic -1,4 glycosidic bonded glucose units, the internal hydrophobic cavity enables the

complexation and fixation of active ingredients [24-26]. CD-methacrylate with different numbers

of vinyl bonds (CD-MA) and mono functionalized CDs with only one vinyl group are

copolymerized with PHEMA or cross-linked to hydrogels [27-29]. CD containing hydrogels

based on PHEMA find already applications as matrix for contact lenses with the aim of drug

incorporation and release [30]. Furthermore they are used as platforms for antibacterial agents

[31].

Successfully applications and developments of PHEMA as hydrogel offer good

requirements as strong candidate for the creation of flexible aqueous nano- and microgel systems.

Spherical particles of PHEMA were already developed by Horak in 1986, studied about their

medico-biological properties and later PHEMA particle were tested as non-toxic to living cells

[32,13]. Until now, some PHEMA based microgels are prepared, e.g. as double hydrophilic

particles by block-copolymerization with polyethylenoxide (PEO) and as termo-responsive

microgel by copolymerization with NIPAM [33-36]. Recently, dual stimuli-responsive PHEMA

104

microgels are prepared which are characterized by pH-depended swelling and light-induced

degradation [37].

The functionality may be enhanced by combining the complexation properties of CDs, the

hydrogel properties of PHEMA and the small size of nanoscalic particles. The covalently

incorporation of CD into the polymer network of PHEMA based nano- and microgels is not

studied in detail and provides improved uptake and prolongation of the drug activity by use as

drug carrier.

In this work, the preparation of novel nano/microgels containing CD-A and the polymeric

structure of PHEMA is of particular interest. The nano/microgels are prepared by surfactant free

precipitation polymerization in the aqueous phase. CD-A functionalized with 2, 4 and 6 groups

acts as cross-linking agent and as binding minority for active ingredients [38].

For uptake and complexation studies, the model drug ibuprofen ((RS)-2-(4-(2-

methylpropyl)phenyl)propanoic acid) is selected. Ibuprofen is a non-steroidal anti inflammatory

drug (NSAID) and is used for relief of symptoms e.g. arthritis, dysmenorrheal, fever, and

furthermore as an analgesic. It inhibits non-selectively the cyclooxygenases I and II (COX-1 and

COX-2) which are responsible for the formation of inflammation-mediating prostaglandins.

Ibuprofen is widely used as painkiller (analgesic), anti-inflammatory, swelling and antipyretic

(antipyretic) in the body [39,40]. An controlled uptake of ibuprofen by nano/microgels in water

due to complexation in CD-A minorities can be expected.

105

5.2 Experimental

5.2.1 Materials

Hydroxyethyl methacrylate (HEMA) was received from Aldrich and purified and

destabilized by column chromatography over aluminum oxide, a further cross-linker N,N-

methylenbisacryamid (BIS) from Aldrich was used as received. The initiator, 2,2-azobis(2-

methylpropioamidine) dihydrochlorid (AMPA) from Aldrich was used without further

purifications. α-, β-, γ-cyclodextrin (CD) produced by Wacker were dried by 80 °C over 3 days

in oven for usage.

5.2.2 Modification of Cyclodextrin

The modification of CD (Wacker) to cyclodextrin acrylate (CD-A) is published elsewhere

[38]. For the preparation of CD-A with different degree of substitution Schlenk technique were

used at all reaction procedures. The synthesis of β-CD-A with a substitution degree of 2, 4 and 6

were made and controlled by NMR (more details in Chapter 6, Supporting Information).

5.2.3 Microgel Synthesis

The batch precipitation polymerization technique is reported and already used for

different microgel systems [4]. Solution of the monomers (2 g,HEMA) and variable amounts of

modified CD-A (0.05 g, 0.1 g and 0.3 g) in 150 ml water were placed into a double-wall glass

reactor equipped with a stirrer (Scheme 1). The reactor was floated with nitrogen and the reaction

mixture was heated up to 70 °C. After 30 min, the reaction was started by adding of 0.05 g

initiator (AMPA). Polymerization was carried out at constant stirring rate of 200 rpm and

temperature of 70 °C for 6 hours. Obtained stable microgel dispersions were purified by dialysis.

The dispersions were filled in dialysis tubes (ZelluTrans - 12,0S 45 mm, MWCO: Nominal

12.000-14.000; Carl Roth GmbH, Karlsruhe) and placed in a water tank of 2 L volume. Water

was changed after 24 h and 48 h, dialysis were stopped after 96 h. The ingredients used for

polymerization process are summarized in Table 1. The nanogel samples are numbered as

NVCLxCD-A(y), with N = run number, x = CD type, and y = substitution degree of CD.

106

Table 1. Reaction recipe for the preparation of CD-A/HEMA nanogels in 150 g water.

N HEMA,

[g] (mmol)

CD,

[g] (mmol)

CDtheor

,

[wt.-%]

BIS,

[g](mmol)

AMPA,

[g](mmol)

Yield

[g]

1HEMA 2.00 (15.37) - - 0.02 (0.13) 0.02 (0.07) 1.95

2HEMAβCD(4) 2.00 (15.37) 0.05 (0.04) 2.39 0.02 (0.13) 0.02 (0.07) 1.04

3HEMAβCD(4) 2.00 (15.37) 0.1 (0.07) 4.67 0.02 (0.13) 0.02 (0.07) 2.07

4HEMAβCD(4) 2.00 (15.37) 0.3 (0.22) 12.82 0.02 (0.13) 0.02 (0.07) 1.82

5HEMAβCD(4) 2.00 (15.37) 0.05 (0.04) 2.42 - 0.02 (0.07) 1.56

6HEMAβCD(4) 2.00 (15.37) 0.1 (0.07) 4.72 - 0.02 (0.07) 1.81

7HEMAβCD(4) 2.00 (15.37) 0.3 (0.22) 12.93 - 0.02 (0.07) 1.68

8HEMAβCD(2) 2.00 (15.37) 0.3 (0.24) 12.82 0.02 (0.13) 0.02 (0.07) 1.40

9HEMAβCD(2) 2.00 (15.37) 0.3 (0.24) 12.93 - 0.02 (0.07) 1.00

10HEMAβCD(6) 2.00 (15.37) 0.3 (0.21) 12.82 0.02 (0.13) 0.02 (0.07) 1.23

11HEMAβCD(6) 2.00 (15.37) 0.3 (0.21) 12.93 - 0.02 (0.07) 1.18

12HEMAαCD(2) 2.00 (15.37) 0.3 (0.28) 12.82 0.02 (0.13) 0.02 (0.07) 1.28

13HEMAαCD(2) 2.00 (15.37) 0.3 (0.28) 12.93 - 0.02 (0.07) 1.33

14HEMAγCD(2) 2.00 (15.37) 0.3 (0.21) 12.82 0.02 (0.13) 0.02 (0.07) 1.38

15HEMAγCD(2) 2.06 (14.72) 0.3 (0.21) 12.93 - 0.02 (0.07) 1.18

5.2.4 Uptake of Ibuprofen by Microgels

Different amounts of a stock solution of ibuprofen (16.5 g/l in methanol) were added to 10 ml of

microgel solutions of a given concentration of (13.1 mg/mLl) to evaluate the drug uptake

capacity of the modified microgels (Table 2). A control experiment with microgels without

cyclodextrin was also carried out. The solutions were equilibrated for 48 h, during this time

methanol was slowly evaporated.

Table 2. Calculated parameter of the ibuprofen complexion by -CD-A (0.22 mmol, DS4) based

microgels in aqueous dispersions.

N MG

mg/mL

CD-A

mg/mL

CD-A

mmol/mL

Îbuprofen

µL/10mL

Îbuprofen

µg/mL

Îbuprofen

mmol/mL

1 13.10 1.69 1.36 25 41.25 0.20

2 13.10 1.69 1.36 50 82.50 0.40

3 13.10 1.69 1.36 75 123.75 0.60

4 13.10 1.69 1.36 100 165.00 0.80

5 13.10 1.69 1.36 125 206.25 1.00

6 13.10 1.69 1.36 150 247.5.0 1.20

7 13.10 1.69 1.36 175 288.75 1.40

8 13.10 1.69 1.36 200 330.00 1.60

9 13.10 1.69 1.36 225 371.25 1.80

10 13.10 1.69 1.36 250 412.50 2.00

11 13.10 1.69 1.36 275 453.75 2.20

107

5.2.5 Characterization Methods

IR measurements were made by FTIR-Spectrometer: Nexus 470 (Thermo Nicolet)

(spectral disintegration of 8 cm-1

). Nanogel dispersions were dried by liyophilisation and

privileged studied in KBr-pellets. Studies about the different band integrals and the ratios of

HEMA/CD-A were made by enclosed software.

Particle size of microgels was analyzed by dynamic light scattering measurements, using

Nano Zetasizer (Malvern) spectrometer. The spectrometer consist of a He–Ne Laser (λ0 = 633

nm). Back scattering light was detected using an angle of 173°. Hydrodynamic particle radius and

the particle distribution of the hydrodynamic radius were determined. Measurements were made

at 20 °C.

The microscopy analysis was made with S-4800-Field-Emission-Scanning Electron

Microscope (Hitachi). One drop of the microgel solution was put on an aluminium support and

dried at room temperature in vacuum. After drying, samples were investigated at accelerating

voltage of 1 kV and 8 mm disintegration.

UV-absorptions spectra of ibuprofen containing dispersions were recorded by JASCO V-

630 Spectrophotometer from 200-1000 nm using silica cuvettes with coating thickness of 1 cm.

The corresponding microgel dispersion without loaded ibuprofen attended as reference.

108


The surfactant free precipitation polymerization of CD-A with HEMA, initiated by

AMPA is shown in Scheme1. The approaches of the investigation and the amounts of reagents

used for preparations are summarized in Table 1.

All synthesis were made by constant HEMA, AMPA and water content. Stirring rate at 200 r/min

and temperature at 70 °C are kept constant as well. The reactions were initiated in water by

AMPA, a water soluble azoinitiator and start immediately after addition. A stable milky

dispersion is build. BIS is used as additional cross-linker to study cross linking properties of CD-

A as functional unity and the influence of the degree of cross-linking on the uptake behavior of

microgels. Furthermore, the influence as cross-linker on the particle size, size distribution and

microgel morphology is studied.

Scheme 1. Synthesis of Cyclodextrin microgels based on PHEMA via batch precipitation

polymerization (the additional cross-linker BIS was used to study further cross-linking properties

of the colloids ).

Figure 1. FESEM image of PHEMA microgels functionalized by β-CD-A.

The addition of CD-A in different stoichiometric ratios to HEMA 0.04 mmol (0.05 g)

CD-A to 2 g HEMA (1/40) over 0.07 mmol (0.1 g) CD-A (1/20), till an amount of 0.22 mmol

(0.3 g) CD-A to 2 g HEMA (1/7) was performed.

The investigation of the impact of different vinyl groups at CD-A (approximately 2, 4 and

6 vinyl groups per CD ring) and the effect of different ring sizes by use of α-, β-, γ-CD-A on the

copolymerization, cross linking process and furthermore on the microgel properties is in focus of

interest.

*

O

NH

*

O

NH

AMPA, H2O, 70°C

N,N-Methylenbis(acrylamide) (BIS)

O O

Hydroxyethyl-methacrylate

(HEMA)

OH

Cyclodextrinacrylate(CD-A)

O

R =

O

O

O

RR

R

O

O

R

RRO

OR

R

R

O

O

RR

R

OO

R

R

RO

OR

RR

O

OR

R

R

O

H

109

The cross-linked polymers were characterized by IR and Raman spectroscopy. The

particle size of the swollen microgels in aqueous dispersion was studied by dynamic light

scattering and cryo FESEM. The morphology and the particle size such as the application

properties of dried microgels were investigated after coating on different surfaces. Furthermore,

the complexation of a common model drug ibuprofen by CD containing microgels were

investigated und studied with regards to the uptake capacity of the particles in aqueous

dispersion.

5.3.1 Investigation of different cyclodextrin acrylat content in microgels

The IR spectra of the dried HEMA microgels containing CD-A in different contents are

shown in Figure 2. The wavenumber of 1720 cm-1

is equivalent to the C=O vibration of CD-A

and furthermore, the vibrations of ketone and ester carbonyls from PHEMA backbone are

arranged. The characteristic band of the hydroxy groups of CD is located at 1623 cm-1

. The band

at 1032 cm-1

characterizes the C-O-C vibration of the glucose units of CD-A and proofs that CD-

A can be incorporated in microgels [38]. So far, an increasing of the band at 1032 cm-1

can be

observed. It results in the elevation of the CD-A content in microgels, which can be controlled by

addition of different CD-A amounts to the reaction mixture.

Figure 2. FTIR-spectra of microgel samples containing different amounts of β-CD-A in

comparison with natural β-CD and PHEMA.

4000 3500 3000 2500 2000 1500 1000 500

0

1

2

3

4

5

6

7

8

9

-CD HEMA (1:20)

-CD HEMA (1:40)

Poly(HEMA)

-CD

Ab

so

rban

ce

Wavelenght [cm-1

]

Poly(HEMA)

-CD

-CD-A-HEMA (1:40)

-CD-A-HEMA (1:20)

1032 cm-1

1654 cm-1

1720 cm-1

110

5.3.2 Influence of the CD-A content on the particle size

The influence of the reactive CD-A content on the size of PHEMA microgel particles was

studied by dynamic light scattering (DLS) method. The results are summarized and plotted in a

diagram shown in Figure 3a.

Figure 3. Hydrodynamic radii of β-CD (DS4) modified microgels prepared with and without

additional cross-linker BIS (a). Size distribution index (b) and size distribution curves of β-CD-

modified microgels prepared without (c) and with cross-linker BIS (d), measurements performed

at 20°C.

The data and the resulting graph show a decrease of the microgel size with increase of the β-CD-

A content. The synthesis of microgels based on PHEMA prepared with N,N-

methylenbisacryamide (BIS) as cross-linker leads to particles with a hydrodynamic radii of 347

nm. First, a low concentration of CD-A does not influence the size of microgels dramatically.

0 0.04 0.07 0.22

100

200

300

400

CD-A content [mmol]

Rh [n

m]

BIS

without BIS

a b

10 100 10000

4

8

12

16

20

24

28

Inte

nsity [%

]

Rh [nm]

no CD

0.04 mmol CD-A (DS4) BIS



10 100 10000

4

8

12

16

20

24

28

Inte

nsity [%

]

Rh [nm]

no CD

0.04 mmol CD-A (DS4)



c d

0 0.04 0.07 0.220.00

0.05

0.10

0.15

with BIS

without BIS

PD

I

CD-A content [mmol]

111

The hydrodynamic radii of microgels decrease slowly by the use of 2.4 % (0.04 mmol ) β-CD-A

to 337 nm. By use of 4.7 % (0.07 mmol ) β-CD-A in the reaction mixture the experimental data

show a reduction of the hydrodynamic radii to 282 nm. Further increase of the CD concentration

up to 12.9 % (0.22 mmmol) reduces the size strongly to microgels with radii of 114 nm.

BIS as additional cross-linker besides CD-A was used to study the influence of more

cross-linking points in microgels on the particle size. The particle sizes are not influenced

strongly by BIS. Here also, the trend to smaller microgels is mainly influenced by rising CD-A

content in microgels. Differences are detectible at a low (0.04 mmol) and a high CD-A content

(0.22 mmol). At lower CD-A (0.04 mmol) content microgels with BIS are smaller. It can be

assumed that CD-A polymerizes slower than methacrylate containing BIS with HEMA. Thus,

BIS cross-links faster at the beginning of the particle nucleation process which lead to dense

cross-linked core and a low cross-linked outer surface mainly cross-linked by CD-A. The smaller

particle size by use of a high CD-A (0.22 mmol) content without BIS indicate that the outer

surface of PHEMA is highly cross-linked by CD-A. CD-A containing microgels without BIS

suggest it because in opposite to CD-A/VCL (Chapter 6) and CD-MA/MMA (Chapter 5) particle

systems the trend curve of the decrease in particle size with increase of the CD content can be

described as progressive decreasing.

The particle size distribution index (PDI) in Figure 3b and the corresponding size

distribution curves are presented in Figure 3c-d. These results show that the CD-A content does

not influence the size distribution of the microgels strongly. The size distribution of microgels

increases slowly with the decrease of the particle size and thus by increase of CD-A

concentration.

The results indicate that CD-A modified with reactive vinyl groups participate actively in

polymerization process (as cross-linker and nucleation center) and promotes formation of

colloidal polymeric microgels. The enhanced water-solubility and reactivity of CD-A leads to the

higher number of growing polymer particles in aqueous phase and finally results in formation of

very small microgels. Therefore, it can be concluded that the presence of reactive CDs in

precipitation polymerization allows regulating microgel size in a broad range without using

surfactants. Obtained colloidal particles are well-stabilized in aqueous phase and no aggregation

phenomena were detected.

112

5.3.3 Morphology of dry microgel on surfaces and size related swelling properties in water

The investigation of the microgel morphology, the particle size under dried condition

and the particle properties after coating on surface is made by scanning electron microscopy

(FESEM). Microgels with different -CD-A content were applied on aluminum surface and

dried at RT and vacuum. The FESEM images are presented in Figure 4. All microgel particles

exhibit a spherical, small particle form and show a homogeneous distribution on the surface by

physically self-bonding.

Particle sizes of PHEMA microgels with different CD-A content show the same trend

under dry conditions applied on surfaces as seen in aqueous dispersion studied by DLS. Figure

4a,c,e (with BIS) and Figure 4b,d,f (without BIS) show that the particle size decreases with

increase of CD-A in the reaction mixture. In the totally collapsed form after release of water the

particles shrink to ultrafine particles with sizes of under 100 nm. Microgels without CD-A have

got a Rh of 350 nm (images are not shown) under dry conditions. The incorporation of a low

amount of CD-A (0.04 mmol (1/40)) in microgels prepared with and without BIS led to similar

particle sizes with Rh between 500 and 100 nm. The narrow size distribution in the aqueous

phase measured by DLS cannot be observed in the collapsed state. On the other hand microgels

with a high amount of CD-A characterized with a small size and a higher PDI in water show

very homogeneous particle size distributions in the collapse state. The ultrafine gel particles

have a very small size with Rh of approximately 25 nm under dry conditions (Figure 4e-f). BIS

don’t show any influence after release of water , the morphology and the sizes of collapsed

microgels are similar to microgels without additional cross-linker.

113

Figure 4. FESEM images of modified PHEMA microgels with different β-CD-A content. 0.04

mmol CD-A (a) plus additional cross-linker BIS (b), 0.07 mmol CD-A (c) plus additional cross-

linker BIS (d) and 0.22 mmol CD-A (e) plus additional cross-linker BIS (f).

a b

c d

e f

114

5.3.4 Incorporation of CD-A with different vinyl groups

The influence of CD-A with different numbers of vinyl groups is presented in Figure 5a.

Microgels which prepared by CD-A (DS4) with four vinyl groups get smaller hydro dynamic

particle diameter (114 nm) than microgels prepared by CD-A (DS2) with a substitution degree

of 2 (446 nm). Synthesis used CD-A (DS6) with 6 vinyl groups present ultrafine microgels with

the smallest particle size of 33 nm. It is conclude that with increasing vinyl groups per CD-A the

degree of cross-linking in microgels increases which influences the density of the internal

polymeric structure and thus, the swelling properties of the microgels. The degree of cross-

linking increases with DS2<DS4<DS6 while the particle size decreases. Here, BIS does not play

important factor during the synthesis. Hence, the higher the degree of cross-linking is, the less

influence BIS the internal particle structure and thus, the particle size.

Figure 5. Hydrodynamic radii (at 20°C) of microgels prepared with β-CD-A using different

substitution degree (a) and different CD type (b) with and without cross-linker BIS.

5.3.5 Properties of microgels prepared by CD-A with different ring sizes

The influence of the ring size on the particle properties and the complexation behavior is

also quite interesting and shown in Figure 5b. First, it is possible to produce microgels with α-

CD-A (DS2) and γ-CD-A (DS2) as well. They can also be used as cross-linkers for microgels.

Polymeric colloid particles, made by α-CD-A (DS2) and γ-CD-A (DS2) are smaller than

microgels made by β-CD-A (DS2). The hydrodynamic radius of microgels which contains α-

CD-A (DS2) is 440 nm, microgels synthesized with γ-CD-A (DS2) get a size of 374 nm and

microgels with β-CD-A (DS2) content have a size of 446 nm.

a b

alpha beta gamma300

350

400

450

500 with BIS

without BIS

Rh [nm

]

Cyclodextrin type

DS 2 DS 4 DS 60

100

200

300

400

500 with BIS

without BIS

Rh [n

m]

Substitution degree of CD-A

115

5.3.6 Particle growth and stability

The precipitation polymerization and the initial growth of microgels was studied of the -

CD-A (DS2) microgel. The time-dependent polymerization process and the particle size were

followed by DLS measurements beginning after reaction start for the next 2 h in detail (Figure 6).

In the first 15 minutes the hydrodynamic radius of the particles increases fast to 150 nm. In steps

of approximately 50 nm per 15 min the growth curve is linear up to 400 nm in 60 min. The

growth curve flattens during the next two hours and the particles reach their final hydrodynamic

radius of 480 nm after 2 h.

Figure 6. Growth curve of the hydrodynamic radii of microgels using DLS measurements.

Microgels do not disintrigate into small particles or polymer chains. Hence, particles are

not aggregates and do not consist of many individual particles. Single colloidal microgel particles

are internally stabilized by cross-linking using CD-A as cross-linker. Otherwise, microgels are

stabilized against agglomeration which does not take place by increasing temperature. Thus, the

external stabilization can be explained using ionic initiator AMPA and OH groups of HEMA and

CD-A units in the polymeric microgel which prevent the coagulation of particles in aqueous

dispersion.

0 3 0 60 90 1 20 150 18 0

0

1 00

2 00

3 00

4 00

5 00

6 00

7 00

8 00

0 .2 2 m m ol - CD - A (D S2)

Rh

[n

m]

T im e [m i n]

116

5.3.7 Uptake of Ibuprofen

A further advantage of CD modified microgels compared to microgels without

hydrophobic cavities should be the improved adsorption of hydrophobic guest molecules and

their stabilization in aqueous phase. The uptake of potential drugs in CD based microgels show

the application potential as drug carrier for drug delivery and controlled release processes.

Ibuprofen is a representative example of such a hydrophobic drug. The complexation of

ibuprofen in natural CDs is investigated and reported as an 1:1 inclusion complex and find

already applications and drug delivery [41,42].

Scheme 2. Complexation of ibuprofen by CD-A containing PHEMA based microgels.

In this work, the uptake of ibuprofen in PHEMA microgels prepared with a -CD-A

amount of 0.07 mmol and 0.22 mmol in aqueous solution is studied. The water insoluble

ibuprofen was added to the microgel dispersion in the diluted form of a minimum of methanol

solution and after agitation over 48 h and sedimentation of non-complexed molecules the filtered

dispersion was investigated by UV-vis spectrometry (Figure 7). Non-diluted ibuprofen does not

show any absorbance in water, but the diluted natural CD:Ibuprofen complexes show an

absorbance band of the aromatic at 272 nm (Figure 7a).

This typical absorbance can also be observed by ibuprofen complexed in -CD containing

microgels. The band at 272 nm is presented slightly broader as the absorbance band of the natural

CD complexes (Figure 7b). Furthermore, Figure 7b shows an increase of the absorbance at 272

nm with an increase of the -CD-A content in microgels by addition of 0.08 mmol/mL ibuprofen

O

RR

R

O

O

R

RR

O

OR

R

R

O

O

RR

R

OO

R

R

R

O

OR

RR

O

O

R

R

R

O

O

RR

R

O

O

R

RR

O

OR

R

R

O

O

RR

R

OO

R

R

R

O

OR

RR

O

O

R

R

R

O

Ibuprofen

β-CyclodextrinIbuprofen-

β-Cyclodextrin-Complex

CH3

HO OH

Ibuprofen-β-Cyclodextrin-Complex

CH3

HO OH

H3C

OH

OH

117

to an calculated -CD-A concentration of 0.015 mmol/mL (0.22mmol -CD-A) in dispersion.

The spectroscopic data indicate that hydrophobic cavities of CD-A are accessible for

complexation and stabilization of hydrophobic molecules in aqueous phase. Preliminary results

indicate that the complexation is controllable and allows the quantitative uptake and fixation of

hydrophobic drugs in microgels.

Figure 7. UV-absorption spectra of ibuprofen (1.65mmol/mL) complexed by natural CDs (a) and

β-CD-A-modified-microgels (b) in water.

In Figure 8a, the uptake of ibuprofen by CD-A microgels can be tracked over time.

Microgels prepared with 0.07 mmol CD-A with and without additional cross-linker BIS are

studied with respect to the uptake in dependence of the time. In the first hour the absorbance of

the ibuprofen containing dispersion increases strongly and thus, most ibuprofen can be

complexed in the first state of the addition. The absorbance curve continues to rise in the next 3

hours and flattens slightly. These results show that the complexation maximum and the uptake

capacity of the microgel is reached. By use of additional cross-linker BIS the absorbance curve

flattens after 2 h and a further uptake of ibuprofen cannot be observed. Hence, the increase of the

cross-linking degree using the additional cross-linker BIS influenced the uptake capacity of CD-

A microgels. It may be explained by the reduced accessibility of CD-A domains in the microgel

core and by the reduced ibuprofen diffusion to CD-A container caused by stronger cross-linking

of the polymer backbone.

250 260 270 280 2900.0

0.1

0.2

0.3

0.4

Absorb

ance

Wavelength [nm]

CD

CD

CD272 nm

250 260 270 280 2900.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

272 nm

Absorb

ance

Wavelength [nm]

no CD-A

0.07 mmol -CD-A BIS

0.22 mmol -CD-A BIS

0.07 mmol -CD-A

0.22 mmol -CD-A

a b

118

The influence of the CD-A content in microgels is investigated by variation of the

calculated CD-A content (Figure 8b). It is observed that the absorbance of ibuprofen and thus the

uptake increases with the increase of the CD-A content by addition of higher amounts of

ibuprofen. The uptake of microgels prepared with BIS shows a reduced uptake than microgels

prepared without BIS, while the uptake trend which is influenced by the CD-A content is not

disturbed. Hence, the adsorption of guest molecules can be controlled by the CD-A content and

thus, by binding possibilities for the guest molecules in microgels.

The modified microgels functionalized with 12.9 % (0.22 mmol) CD-A are studied with

respect to the complexation capacity by variation of the ibuprofen concentration in dispersion

(Figure 8d). The molar amount of CD-A container, theoretically calculated on the ratio in the

reaction mixture, is compared with the molar increase of added ibuprofen concentration in

aqueous dispersion expecting an 1:1 inclusion complex.

The UV-vis results are shown in Figure 8a. The rising addition of ibuprofen results in a

linear increase of the absorbance and thus in linear increase of the uptake. At the point when an

1:1 complex is expected, the absorbance flattens slightly and stagnates. Further addition does not

increase the absorbance. The results show that the uptake is mainly controlled by CD-A domains

in microgels and by the diffusion possibilities of ibuprofen influenced by the degree of cross-

linking into the hydrophobic CD-A cavities. Contrary, non-modified microgels do not show any

absorbance and thus, no uptake of ibuprofen.

119

Figure 8. UV-absorption maxima of ibuprofen (1.65mmol/mL) complexed by β-CD-A (0.07

mmol CD-A) modified microgels (with and without additional cross-linker BIS) in water at

different stirring times (a) and at variation of the CD-A amount using 0.07 mmol and 0.22 mmol

CD-A content (b). The rising molar addition of ibuprofen with respect to the molar ratio of the

theoretically calculated CD-A content in microgels is presented (c) and the experimental

absorbance maxima in aqueous dispersion (d).

0 2 4 6 8 10 12 14 16 18 20 22 24 26

0.00

0.02

0.04

0.06

0.08

0.10

0.12A

bso

rban

ce

Time [h]

with BIS

without BIS

0.00 0.05 0.10 0.15 0.20 0.25

0.0

0.1

0.2

0.3

0.4

0.5

Ab

so

rban

ce

CD-A content [mmol]

with BIS

without BIS

0 100 200 300 400 5000.0

0.4

0.8

1.2

1.6

2.0

2.4

Ho

st:G

ue

st con

cen

tratio

n [

mm

ol/m

L]

Ibuprofen [µg/mL]

Ibuprofen

CD-A

a b

c d

0 100 200 300 400 500

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Ab

so

rban

ce

Ibuprofen [µg/mL]

Ibuprofen: 0.22 mmol -CD-A (DS4)

120

5.4 Conclusion

Poly(2-hydroxyethyl methacrylate) microgels functionalized with reactive cyclodextrin

units (CD-A) have been prepared by surfactant-free precipitation polymerisation in water. The

incorporation of different amounts of -, - and -CD-A with different number of vinyl bonds (2,

4 and 6) in aqueous PHEMA based microgels was investigated. The results indicate that the

increase of the CD-A concentration in reaction mixture led to the reduction of the final

hydrodynamic radius of microgels from 347 nm to 114 nm. Increase of the number of vinyl

groups per CD-A molecule induced formation of smaller nanogels (Rh = 22.5 nm) and

considerable increase of the cross-linking degree. The use of additional cross-linker showed less

significant influences on the particle properties. The complexation properties of CDs

incorporated in microgel networks were demonstrated by the uptake of hydrophobic drug

ibuprofen. The experimental data indicated that hydrophobic cavities of cyclodextrin are active

for complexation of hydrophobic molecules in aqueous phase. The drug loading in microgels was

controllable by CD-A content and degree of cross-linking and allows the quantitative uptake and

fixation of ibuprofen in microgels.

121

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13. Horák D.; Červinka M.; Bedard, V. P., Hydrogels in endovascular embolization: VI. Toxicity tests of poly(2-

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15. Smyth, G.; Quinn, F. X.; McBrierty V. J., Water in hydrogels. 2. A study of water in poly (hydroxyethyl

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16. Xinming L.; Yingde C.; Lloyd A. W.; Mikhalovsky S. V.; Sandeman S. R.; Howel C. A.; Liewen L.,

Polymeric hydrogels for novel contact lens-based ophthalmic drug delivery systems: a review, Cont. Lens.

Anterior. Eye. 2008, 31, 57-64.

17. Li, C. C.; Chauhan, A., Modeling Ophthalmic Drug Delivery by Soaked Contact Lenses, Ind. Eng. Chem. Res.

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18. Okano, T.; Katayama, M.; Shinohara, I., The influence of hydrophilic and hydrophobic domains on water

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19. Vargün, E.; Sankir, M.; Aran, B.; Sankir, N. D.; Usanmaz, A., Synthesis and Characterization of 2-

Hydroxyethyl Methacrylate (HEMA) and Methyl Methacrylate (MMA) Copolymer Used as Biomaterial, J.

Macromol. Sci., A: Pure and Appl. Chem. 2010, 47, 235-240.

20. Quynh, T. M.; Yoneyamab, M.; Maki, Y.; Dobashi, T., Poly(N-isopropylacrylamide-co-hydroxyethyl

methacrylate) Graft Copolymers and their application as carriers for drug delivery system, J. Appl. Polym.

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21. Mishra, R. K.; Ramasamy, K.; Majeed, A. B. A., pH-responsive poly(N[-3(dimethylamino)propyl]

methacrylamide-co-2-hydroxyethyl methacrylate)-based hydrogels for prolonged release of 5-fluorouracil, J.

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22. Kakwere, H.; Perrier, S., Design of complex polymeric architectures and nanostructured materials/hybrids by

living radical polymerization of hydroxylated monomers, Polym. Chem. 2011, 2, 270-288.

23. Andrade-Vivero, P.; Fernandez-Gabriel, E.; Alvarez-Lorenzo, C.; Concheiro, A., Improving the loading and

release of NSAIDs from PHEMA hydrogels by copolymerization with functionalized monomers, J. Pharm. Sci.

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28. dos Santos J. F.; Couceiro R.; Concheiro A.; Torres-Labandeira, J. J.; Alvarez-Lorenzo, C., Poly(hydroxyethyl

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thermosensitive P(NIPAAm-co-HEMA)/β-CD based hydrogels via click chemistry, Macromol. Rapid

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30. Xu, J.; Li, X.; Sun, F.,Cyclodextrin-containing hydrogels for contact lenses as a platform for drug

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Concheiroc, A.; Sosnik, A., β-Cyclodextrin hydrogels for the ocular release of antibacterial

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34. Quan, C.-Y.; Sun, Y.-X.; Cheng, H.; Cheng, S.-X.; Zhang, X.-Z.; Zhuo, R.-X., Thermosensitive P(NIPAAm-

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35. Gan, T.; Zhang, Y.; Guan, Y., In Situ Gelation of P(NIPAM-HEMA) Microgel Dispersion and Its Applications

as Injectable 3D Cell Scaffold, Biomacromolecules 2009, 10, 1410-1415.

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hydroxyethyl(meth)acrylates, J. Pharm. Pharmacol. 2010, 62, 1356-1357.

37. Klinger, D.; Landfester, K., Dual Stimuli-Responsive Poly(2-hydroxyethyl methacrylate-co-methacrylic acid)

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inflammatory drugs in cancer prevention: A critical review of non-selective COX-2 blockade, Oncology

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41. Chow, D.-N. D.; Karara, A. H., Characterization, dissolution and bioavailability in rats of ibuprofen-β-

cyclodextrin complex system, Int. J. Pharm. 1986, 28, 95-101.

42. Mura, P.; Bettinetti G. P.; Manderioli, A.; Faucci, M. T.; Bramanti, G.; Sorrenti, M., Interactions of ketoprofen

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125

Chapter 6

Thermosensitive aqueous nanogels modified with cyclodextrinA

Abstract: In this work, the incorporation of reactive cyclodextrins acrylates (CD-As) in

aqueous nanogels based on poly(N-vinylcaprolactam) (VCL) is studied by surfactant-free

precipitation polymerization process. α-, β-, γ-CDs were functionalized with acrylic groups and

the average amount of vinyl bonds per CD molecule was adjusted to 2, 4, or 6. The increase of

the CD-A concentration in the reaction mixture led to the reduction of the final hydrodynamic

radius of nanogels from 227 nm to 62 nm. The increase of the vinyl groups number per CD

molecule induced formation of smaller nanogels (Rh = 22.5 nm) and considerable increase of the

cross-linking degree. Obtained CD-A-modified nanogels show temperature sensitivity in

aqueous medium with a volume transition point around 30 °C. The complexation properties of

CD-As incorporated in nanogel networks were proved by titration with phenolphthalein.

Keywords: nanogel, microgel, cyclodextrin, N-vinylcaprolactam

A. Reproduced with permission from Kettel, M. J.; Dierkes, F.; Schäfer, K.; Moeller, M., Pich, A., Aqueous

nanogels modified with Cyclodextr, Polymer 2011, 52, 1917-1924. Copyright Elsevier.

O

RR

R

O

O

R

RR

O

OR

R

R

O

O

RR

R

OO

R

R

R

O

OR

RR

O

O

R

R

R

O

R:

Variable parameter: -ring size (α-; β-; γ-cyclodextrin); -substitution degree (R) (2; 4; 6)

O

OH

Oor

Nanogel with grafted CDReactive CD

0 0.04 0.07 0.2240

60

80

100

120

140

160

180

200

220

240

-CD, [mmol]

Rh,

[nm

]

TEM image of VCL/CDex microgel.

500 nm

1 µm

126

6.1 Introduction

In recent years continued development of the creation of complex materials has been

focused on the engineering over length-scales from molecular to the colloidal to the

macroscopic in order to improve their properties for specific applications. Today’s efforts in

nano- and micro-structured and multifunctional materials design focus on methods by which

functional building blocks are assembled in complex architectures. Aqueous nano- and

microgels are outstanding polymer materials because of their interesting properties such as small

size, high porosity, large surface area, high chemical functionality, ability to swell in different

solvents (e.g. water), and stimuli-sensitivity [1-2]. Nano- and microgels can be used in colloidal

form as microreactors [3-4], drug carriers [5-6], and antimicrobial agents but also as building

blocks for design of colloidosomes [7-8], hydrogels [9-10] and films [11] targeting such

applications as controlled release, tissue engineering and optics.

The chemical functionality of polymer nano- and microgels is extremely important for their

application in different areas of science and technology. So far colloidal polymer networks have

been modified by incorporation of ionisable or reactive groups [12-15], integration of

biomacromolecules [16-17] or nanoparticles [18-19].

In this context, cyclodextrins (CDs) are interesting functional units which can be integrated

in polymer network of nano- and microgel colloids [20-21]. Cyclodextrins are composed of α-

1,4-coupled six, seven or eight D-glucose units which form cyclic oligomers. The caves of these

host molecules have a diameter of 5-8 Ǻ and exhibit a hydrophobic internal cavity and a

hydrophilic outer layer [22]. This structure and the hydrophobic cavity of CDs can act as host

for various, suitable guest molecules. This unique property is commonly referred to as inclusion

complex formation. The complexation leads to modification of the physical and chemical

properties (solubility, smell, reactivity etc.) of the guest molecules [23-24].

So far, CDs were structurally incorporated in nanoparticles [25], polymers [26] or hydrogels

[27-33] to modulate complexation and release of small molecules such as dyes or drugs.

Alternatively, CDs were used for design of physically assembled polymer systems by formation

of inclusion complexes between polymer-modified CDs and various guest molecule

functionalized polymers. Widely used method for preparation of polymer networks containing

CDs is the copolymerization of vinyl- or (meth)acryloyl-modified CDs with other vinyl

monomers or macromonomers such as 2-hydroxyethyl methacrylate (HEMA) [27-29], N-

127

isopropylacrylamide (NIPAM) [30-31], acrylic acid (AA) [32], (meth)acrylated hyaluronic acid

or poly(lactide) [33] or N-vinylpyrrolidone (NVP) [34]. Self-assembled polymer systems such

as supramolecular hydrogels [35-37] and nano- and microparticles [38-40] have been prepared

based on host-guest CD inclusion complexes. Recently, the incorporation of CDs in aqueous

colloidal nano- and microgels was reported. Microgels based on β-CD and poly(vinyl alcohol)

(PVA) were prepared in inverse emulsion system by cross-linking of β-CD and PVA with

epichlorhydrine. Alternatively, interpenetrating polymer network (IPN) microgels were

synthesized by polymerizing methacrylic acid in β-CD/PVA microgels. It has been reported that

the size of obtained microgel particles can be controlled by variation of stirring rate and

emulsifier concentration between 50 µm and 200 µm [41].

The biodegradable polylactic acid-β-cyclodextrin (PLA-β-CD) cross-linked copolymer

microgels were prepared by radical copolymerization of PLA macromonomer and polymerizable

β-CD derivatives [42]. The β-CD derivatives with variable numbers of polymerizable vinyl

groups were used in polymerization process. It has been shown that the hydrophilicity of the

microgels increased with increasing β-CD contents, while the swelling ratios and degradation rate

decreased.

The synthesis of aqueous microgels by precipitation polymerization of N-

isopropylacrylamide (NIPAM) in presence of mono-vinyl substituted β-CD has been reported

by Liu et al. [43]. β-CD/PNIPAM core–shell microgels were obtained by a two-stage

precipitation polymerization in aqueous solution. At the first stage, core microgels with CD

moieties were synthesized by precipitation copolymerization of NIPAM and mono-vinyl β-CD

monomer with use of sodium-n-docecylsulfate (SDS) as surfactant. At the second stage, using

the core particles as seeds, PNIPAM shell was further prepared by NIPAM polymerization. The

diameter of β-CD/PNIPAM core particles varied between 106 nm and 115 nm. It has been

shown that with increase of β-CD content the temperature-induced deswelling of microgels

decreases gradually.

Copolymer microgels were synthesized by surfactant-free precipitation copolymerization of

N-vinylcaprolactam (VCL) with a mono-vinyl β-CD monomer in aqueous solutions [44]. It has

been reported that addition of reactive CD could reduce the particle size and size distribution of

the copolymer microgels. Obtained copolymer microgels exhibit thermally responsive changes of

128

particle size in aqueous solutions, but similarly to PNIPAm system [43] the incorporation of the

β-CD monomer into a PVCL microgel reduced its thermal sensitivity.

So far, the behavior of reactive cyclodextrins in precipitation polymerization was not

investigated in details. Therefore, the primary aim of this study is the investigation of α-, β-, and

γ-CDs with different numbers of vinyl groups in precipitation polymerization process and their

incorporation in nano- or microgel particles. For this study, vinylcaprolactam (VCL) was selected

as main monomer which has been already effectively used for synthesis of different microgel

systems [45-47].

In this work, the characterization of CD-acrylate (CD-A) modified nanogels with respect to

their size, size distribution, swelling degree and morphology. The accessibility of CD-A units in

nanogel particles was evaluated by titration with model dyes. The first results indicate that it is

possible to reduce the size of CD-A-modified colloidal networks down to 45 nm by keeping their

most attractive properties such as temperature-sensitivity, colloidal stability and narrow size

distribution unaltered. It is expected that cyclodextrin based nanogels provide useful

functionalities such as effective bioconjugation, good adhesion to surfaces, controlled

complexation and release of drugs, cosmetic ingredients, dyes or antimicrobial agents.

6.2 Experimental

6.2.1 Materials

N-Vinylcaprolactam (VCL) was obtained from Aldrich and distilled by vacuum

distillation. Acetoacetoxyethyl methacrylate (AAEM) was received from Aldrich and purified

and destabilized by column chromatography over aluminum oxide; a further cross-linker N,N-

methylene-bis-acrylamide (BIS) from Aldrich was used as received. The initiator, 2,2-azobis(2-

methylpropionamidine) dihydrochloride (AMPA) from Aldrich was used without further

purifications. α-, β-, γ -Cyclodextrins (CD) produced by Wacker were dried at 80°C over 3 days

before use. Solvents dimethylformamide (DMF), acetone were purchased from VWR (DMF

was degassed before use). Triethylamine (TEA) was purchased from Merck, distilled before use

and stored over 4 Å molecular sieve in N2-atmosphere. Acrylic acid chloride and

phenolphthalein were obtained from Merck and used as received.

129

6.2.2 Modification of cyclodextrins

The modification of cyclodextrin with reactive vinyl groups is published elsewhere [48-

49]. For the preparation of cyclodextrin acrylates with different degree of substitution Schlenk

technique was used and the whole procedure took place under N2-atmosphere. CD was diluted in

15 mL degassed DMF and TEA was added to the solution. The mixture was stirred and cooled

down to 0°C, then acrylic acid chloride was given into the solution. After stirring for 12 h the

mixture triethylamine hydrochloride was filtered and the clear solution was added in 200 mL

acetone to precipitate the modified cyclodextrin. The filtrate was washed several times with

acetone and purified by Soxhlet-extraction with acetone for three days. The obtained solid was

dried, diluted in a small amount of water and vacuum freeze-dried. In this work α-, β-, γ-CD-A

with an average substitution degree of 2 and β-CD-A with an average substitution degree of 4 and

6 were synthesized. Details of the cyclodextrin modification are summarized in Supporting

Information (Scheme S1, Table S1).

6.2.3 Nanogel Synthesis

The synthesis of nanogels in aqueous solution was performed by batch precipitation

polymerization technique (Scheme S2) [45]. A double-wall glass reactor equipped with a stirrer

and a reflux condenser was purged with nitrogen. Solution of the monomers 14.72 mmol (2.06 g)

VCL, 0.23 mmol (0.05 g) AAEM, and variable amounts of modified CD-A in 147 mL water was

placed into the reactor and stirred at 200 rpm for 1 h at 70 °C under continuous purging with

nitrogen. Afterwards, 3 mL aqueous solution of initiator (0.07 mmol (0.02 g) AMPA) was added

under continuous stirring. Reaction was carried out for another 12 h. In some experiments 0.02

mmol (0.13 g) BIS was used as additional cross-linker to compare properties of nanogels

prepared only with reactive CD-A as cross-linker or combination of BIS and CD-A. After

preparation the stable, milky nanogel dispersions were purified by ultrafiltration to remove

unreacted monomers and smaller polymeric particles. The nanogel dispersion in water was

pumped through a membrane with molecular weight cut off of 30 kDa (Millipore, Pelicon XL

Device, Ultracel 30 PLCHK Membrane, regenerated cellulose with NMWCO 30 kDa). The

ultrafiltration time was adjusted to 48 h. The ingredients used for polymerization process are

summarized in Table 1.

130

Table 1. Reaction recipe for the preparation of CD-A/VCL/AAEM nanogels in 150 g water. The

nanogel samples are numbered as NVCLxCD(y), where N = run number; x = cyclodextrin type;

and y = substitution degree of cyclodextrin.

N VCL,

[g](mmol)

AAEM,

[g](mmol)

CD-A,

[g](mmol)

CD-A,

[wt %]

BIS,

[g](mmol)

AMPA,

[g](mmol)

Yield

[g]

1VCL 2.06 (14.72) 0.05 (0.23) - - 0.02 (0.13) 0.02 (0.07) 1.02

2VCLβCD(4) 2.06 (14.72) 0.05 (0.23) 0.05 (0.04) 2.27 0.02 (0.13) 0.02 (0.07) 1.34

3VCLβCD(4) 2.06 (14.72) 0.05 (0.23) 0.1 (0.07) 4.44 0.02 (0.13) 0.02 (0.07) 1.16

4VCLβCD(4) 2.06 (14.72) 0.05 (0.23) 0.3 (0.22) 12.24 0.02 (0.13) 0.02 (0.07) 0.84

5VCLβCD(4) 2.06 (14.72) 0.05 (0.23) 0.05 (0.04) 2.29 - 0.02 (0.07) 0.33

6VCLβCD(4) 2.06 (14.72) 0.05 (0.23) 0.1 (0.07) 4.48 - 0.02 (0.07) 0.31

7VCLβCD(4) 2.06 (14.72) 0.05 (0.23) 0.3 (0.22) 12.35 - 0.02 (0.07) 0.43

8VCLβCD(2) 2.06 (14.72) 0.05 (0.23) 0.3 (0.24) 12.24 0.02 (0.13) 0.02 (0.07) 1.38

9VCLβCD(2) 2.06 (14.72) 0.05 (0.23) 0.3 (0.24) 12.24 - 0.02 (0.07) 1.27

10VCLβCD(6) 2.06 (14.72) 0.05 (0.23) 0.3 (0.21) 12.35 0.02 (0.13) 0.02 (0.07) 0.53

11VCLβCD(6) 2.06 (14.72) 0.05 (0.23) 0.3 (0.21) 12.35 - 0.02 (0.07) 0.43

12VCLαCD(2) 2.06 (14.72) 0.05 (0.23) 0.3 (0.28) 12.24 0.02 (0.13) 0.02 (0.07) 1.22

13VCLαCD(2) 2.06 (14.72) 0.05 (0.23) 0.3 (0.28) 12.24 - 0.02 (0.07) 1.27

14VCLγCD(2) 2.06 (14.72) 0.05 (0.23) 0.3 (0.21) 12.35 0.02 (0.13) 0.02 (0.07) 1.39

15VCLγCD(2) 2.06 (14.72) 0.05 (0.23) 0.3 (0.21) 12.35 - 0.02 (0.07) 1.39

6.2.4 Characterization of modified Cyclodextrins and Nanogels

1H-NMR and

13C-NMR spectra were recorded with Varian Mercury 300 NMR

spectrometer operating at 300 MHz. The samples were dissolved in deuterated dimethylsulfoxide

(DMSO-d6) (Figure S1).

RAMAN spectra were recorded with a Bruker RFS 100/s (spectral disintegration 4 cm-1

).

IR measurements were made by FTIR spectrometer: Nexus 470 (Thermo Nicolet) (spectral

disintegration 8 cm-1

). Nanogel dispersions were dried by lyophilisation and pressed in KBr-

pellets. The quantitative determination of the cyclodextrin content in nanogels has been

performed by quantitative analysis of the band at 1032 cm-1

(C-O-C vibration of the glucose units

[50]). The integrals of this band measured for the nanogel samples were compared with a

calibration curve obtained by mixing different amounts of β-CD (0.07 mmol, 0.11 mmol, 0.33

mmol, 0.44 mmol, 0.66 mmol) with KBr.

131

Particle size of nanogels was analyzed by dynamic light scattering measurements using

Nano Zetasizer (Malvern) spectrometer equipped with a He–Ne Laser (λ0 = 633 nm). Back

scattering light was detected using an angle of 173°. Hydrodynamic particle radius and the

particle distribution of the hydrodynamic radius were determined. Measurements were made in a

temperature range from 20 °C to 50 °C with a temperature equilibrium time of 120 s.

The microscopical analysis was made with S-4800-Field-Emission-Scanning Electron

Microscope (Hitachi). One drop of the diluted nanogel dispersion was put on an aluminium

support and dried at room temperature in vacuum. After drying samples were investigated at

accelerating voltage of 1 kV and 8 mm disintegration.

For volumetric titration with Phenolphthalein a Brand Duran burette with a total volume

of 50 mL (± 0.05 mL, Ex +30s) was used. A stock solution of 1 % phenolphthalein in ethanol

was prepared and an aliquot of 2.50 mL was dropped to 250 mL of 0.1 M sodium hydroxide to

obtain a 0.25 mmol/L alkali phenolphthalein solution. All aqueous stock solutions were freshly

prepared and run within 12 h to ensure that absorbance changes due to any instability of

phenolphthalein did not contribute to experimental artifacts. β-CD stock solution in water was

prepared with a β-cyclodextrin concentration of 8.8 mmol/L. Aliquots of β-CD stock solution

were diluted with water to different concentrations with an end volume of 10 mL. To obtain a

calibration curve titration of β-CD solutions with stock solution of phenolphthalein under stirring

until the transition point from colorless to slight pink was performed. The nanogel samples (10

mL) with known particle concentration were titrated by 0.25 mmol/L alkali phenolphthalein stock

solutions under stirring until the transition point from colorless to slight pink. The amount of β-

CD in nanogels was extracted from the calibration curve.

132


A series of nanogels containing cyclodextrin acrylates (CD-A) were prepared by precipitation

polymerization in aqueous phase. The modification of cyclodextrins with reactive vinyl groups

was aimed to ensure fixation of CD in the nanogel network by formation of covalent bonds and

effective incorporation in nanogels during radical polymerization process. Due to the fact that

modified CDs used in the present study are functionalized with numerous acrylate groups, they

can be considered as functional cross-linking reagents that can substitute conventional cross-

linkers such as BIS. Several parameters such as CD type and substitution degree, CD-A content,

presence of additional cross-linking agent BIS have been varied to study the influence of the

reactive CD-A on properties of colloidal nanogels. The synthesis details are given in Table 1.

6.3.1 Incorporation of CD in nanogels

To evaluate the incorporation of reactive CD-A in nanogels during the polymerization

process FTIR spectroscopy was used. The normalized IR spectra of nanogel samples prepared by

varying the concentration of β-CD-A (substitution degree 4) as well as a reference microgel

prepared without CD are presented in Figure 1a.

Figure 1. FTIR spectra of nanogel samples containing different amounts of β-CD-A (4) (a);

integral of the signal at 1032 cm-1

vs. theoretically expected CD-A content (b).

The characteristic signal for the amide band of VCL is located at 1623 cm-1

. The signal at 1720

cm-1

is equivalent to the C=O vibration of CD-A but overlaps strongly with the vibrations of

ba

0 2 4 6 8 10 12 14

0

2

4

6

Inte

gra

l o

f th

e s

ign

al a

t 1

032

cm

-1

theoretical CD-A content [%]

4000 3500 3000 2500 2000 1500 1000 500

0.0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

Abso

rba

nce

Wavenumbers [cm-1]

no CD-A

0.04 mmol CD-A

0.07 mmol CD-A

0.22 mmol CD-A 1720

16231032

133

ketone and ester carbonyl groups from AAEM. The band at 1032 cm-1

originates from C-O-C

vibrations of the glucose units of CD-A and proofs qualitatively that CD-A is incorporated in

nanogels [49]. Figure 1b indicates that the intensity of the band at 1032 cm-1

increases if the

concentration of reactive CD-A in reaction mixture increases. This shows that the CD-A content

in nanogels can be controlled by concentration of reactive CD-A in the reaction mixture. The

integrals of the bands at 1032 cm-1

from FTIR spectra were used to determine the CD-A content

in nanogels. In this case a calibration curve of low molecular β-cyclodextrin (see Experimental

Section) was used to determine quantitatively the CD-A content in the copolymer structure.

Obtained data are plotted vs. theoretical CD-A content in Figure 2. Figure 2 shows that the

experimental CD-A content is smaller compared to theoretically expected values. According to

the FTIR data the incorporation efficiency of CD-A in nanogel structure is 46.8 % (using 0.04

mmol β-CD-A), 59.7 % (using 0.07 mmol β-CD-A), and 80.4 % (using 0.22 mmol β-CD-A). It

occurs that with increasing CD-A in the reaction mixture the efficiency of CD-A incorporation

in the terpolymer nanogel increases. It can be predicted that the increase of the CD

concentration in the reaction mixture will reduce considerably the particle nucleation period and

increase the number of precursor particles. Thus the overall polymerization rate is expected to

be higher in the presence of reactive CDs. This acceleration of the polymerization rate probably

increases the incorporation efficiency of CDs into nanogels.

An alternative possibility to evaluate the amount of cyclodextrin units in the nanogel

structure is based on the ability of β-CD to form colorless complexes with phenolphthalein in

aqueous solutions. Phenolphthalein is a well known and studied dye, which shows a pink color in

alkaline solution due to the delocalization of the π-electrons of the chinoid system.

Phenolphthalein is complexed by cyclodextrin very well and fixed in the cavity of ring structure.

Because of the complexation, the delocalization of the π-electrons is disturbed and the pink color

disappears in alkaline solution. Titration and quantitative studies of materials containing

cyclodextrin especially polymeric matrices have been made by complexation with

phenolphthalein [34,51-53]. Therefore, titration of nanogels with known amount of

phenolphthalein can provide information about accessibility of CD-A units in nanogel structure.

By comparing the titration results with the data obtained by IR spectroscopy the amount of

“active” and sterically hindered CD-A can be evaluated. In this case the “activity” is related to

the ability of CD-A forming host-guest complexes with different organic molecules. Sterically

134

hindered CD-A units in nanogels lose their complexation ability and cannot be utilized later for

uptake, transportation and release of organic molecules.

To make sure that complexation of phenolphthalein does not influence the colloidal

behavior of nanogels the nanogel size of the nanogels complexed with phenolphthalein was

measured (Supporting Information Figure S2). Phenolphthalein loaded CD-A-modified nanogels

do not change their size compared to original nanogel samples. No particle aggregation effects

were detected by light scattering experiments after titration with phenolphthalein.

Figure 2. Theoretical vs. measured β-CD-A content in nanogels.

As shown in Figure 2 the content of active CD-A (CD-A molecules able to build host-guest

complexes) in nanogels determined by titration with phenolphthalein is lower compared to the

values obtained from FTIR measurements. The fraction of sterically hindered CD-A increases

with the CD-A concentration in the reaction mixture. Since CD-A can act as cross-linking agent

it can be assumed that nanogels prepared with higher CD-A content exhibit higher cross-linking

density. This effect probably leads to reduction of the CD-A accessibility in colloidal polymer

networks. Certain fraction of CD-A groups can be sterically hindered in nanogel network due to

location in the nanogel core having higher cross-linking density compared to the corona. On the

other hand CD-A groups may interact with polymer chains of nanogel network. In both

scenarios described above the complexation ability of CD-A units is decreased and will

ultimately lead to lower “active” CD-A content. Experimental data presented in Figure 2

indicate that the amount of the reactive CD-A in the polymerization process influences both

incorporation efficiency and accessibility of CD-A units.

0 2 4 6 8 10 12 14

0

2

4

6

8

10

12

14 theoretical CD-A content

"active" CD-A content

CD-A content

CD

co

nte

nt [w

t.-%

]

theoretical CD content [wt.-%]

135

6.3.2 Nanogel size and morphology

Obtained nanogels exhibited excellent colloidal stability in water. Independently from

the content of CD or CD type prepared nanogel dispersions were stable over months and do not

show agglomeration or precipitation. Interestingly, freeze dried nanogels can be easily

redispersed in water and form aggregate-free dispersions.

To evaluate the influence of the reactive cyclodextrin on the size of nanogel particles

light scattering experiments of dialyzed dispersions were performed. The experimental data

shown in Figure 3 indicate strong reduction of the nanogel size with increase of the β-CD-A

concentration in reaction mixture. In this case, the hydrodynamic radii of nanogels decrease

from 227 nm to 102 nm by increase of the β-CD-A concentration up to 2.27 wt.-% (0.04 mmol).

Further increase of the CD-A concentration up to 12.24 wt.-% (0.22 mmol) resulted in decrease

of the size of nanogels to 62 nm. Similar behavior was observed for nanogels containing α- and

γ-CD-A. Interestingly, presence of cross-linker BIS does not influencing the hydrodynamic radii

of nanogels. The size distribution of nanogels increases with increase of CD-A concentration.

The size distribution curves are presented in Supporting Information Figure S3.

Figure 3. Hydrodynamic radii (Rh in nm) of β-CD-A-modified nanogels prepared with and

without cross-linker BIS (β-CD-A with substitution degree 4; measurements performed at 20 °C).

The experimental data shown in Figure 3 indicate that reactive cyclodextrin molecules

participate actively in the particle formation process. The availability of reactive vinyl groups and

the hydrophilicity of modified CDs induce formation of larger amount of nucleation centers at the

0 0.04 0.07 0.2240

60

80

100

120

140

160

180

200

220

240

-CD [mmol]

Rh [n

m]

with BIS

without BIS

136

early stages of precipitation polymerization process. This leads to higher amount of growing

polymer particles in aqueous phase and finally results in the formation of very small nanogel

particles. Therefore, it can be concluded that the presence of reactive CD-As in precipitation

polymerization allows regulating nanogel size in a broad range without use of surfactant.

Additionally, the results presented in Figure 3 lead to the conclusion that in the present system

nanogels functionalized with CD-As can be prepared without use of further cross-linker. The

integration of BIS in the reaction recipe does not influence strongly the nanogel dimensions. It

can be assumed that the reason for this phenomenon is multifunctionality and higher reactivity of

CD-A compared to BIS. In this case the cross-linking density in nanogels will be strongly

influenced by CD-A concentration in reaction mixture.

Figure 4. FESEM images of β-CD-A-modified nanogels prepared with cross-linker BIS: a) no

CD-A; b) 0.04 mmol CD-A; c) 0.07 mmol CD-A; d) 0.22 mmol CD-A.

a

d

b

c

137

Figure 4 shows FESEM images of β-CD-A-modified nanogels. In good agreement with

light scattering results discussed above, microscopy data indicate that with increase of the β-CD-

A concentration nanogel size decreases. Nanogel particles are spherical and flatter on the solid

substrate after removal of water by evaporation.

Another important parameter that may influence nanogel properties is substitution degree

of CD-A or average number of reactive vinyl bonds. Figure 5a shows hydrodynamic radii of

nanogels prepared with similar amount of β-CD-A having different substitution degrees. The

nanogel size decreases in linear order with increase of the substitution degree. This effect can be

explained by increased reactivity of CD-A having higher substitution degree. This may lead to

faster particle nucleation and increase of the number of growing particles in reaction system.

Additional parameter that may enhance reduction of nanogel size is decrease of the water-

solubility of CD upon substitution of OH groups by acrylic groups. In this case it is expected that

positioning of the CD-A units in nanogels will be shifted toward particle interior.

Figure 5. Hydrodynamic radii of CD-A-modified nanogels prepared with and without cross-

linker BIS: a) nanogels prepared with 0.3 g β-CD-A with substitution degree 2 (0.24 mmol); 4

(0.22 mmol); and 6 (0.21 mmol); b) nanogels prepared with 0.3 g α- CD-A (0.28 mmol); β-CD-A

(0.24 mmol); and γ-CD-A (0.21mmol) with substitution degree 2 (measurements performed at 20

°C).

a b

DS 2 DS 4 DS 6

20

40

60

80

100

120

140

with BIS

without BIS

Rh [nm

]

Substitution degree of CD-A

alpha beta gamma

120

140

160

180

200

220

with BIS

without BIS

Rh [n

m]

CD-A type

138

Figure 5b shows hydrodynamic radii of nanogels prepared with α-, β- and γ-CD-A having

substitution degree 2. Decrease of the nanogel size with increase of the CD-A ring size can be

observed. Nanogel particles prepared with β-CD-A exhibit the smallest hydrodynamic radius at

room temperature. This effect can be explained by different solubility of α-, β- and γ-CD-A in

water. The C-2-OH group of one glucopyranoside unit in CD-A can form a hydrogen bond with

the C-3-OH group of the adjacent glucopyranose unit. In the β-CD-A molecule, a complete

secondary belt is formed by these H-bonds; therefore, the β-CD has a rather rigid structure [53].

This intra-molecular H-bond formation is probably the explanation for the observation that β-CD

has the lowest water solubility of all CDs. (appr. 0.2 g/L). The H-bond belt is incomplete in the α-

CD molecule, because one glucopyranose unit is in a distorted position. Consequently, instead of

the six possible H-bonds, only four can be established simultaneously (water solubility of α-CD

is 1.5 g/L). γ-CD is a non-coplanar, consists of more flexible structure; therefore, it is the most

soluble of the three CDs (water solubility of γ-CD is 2.4 g/L). The solubility of different

cyclodextrins in water is shown in Figure S4 [54].

6.3.3 Temperature-Sensitivity

As reported in previous work VCL-based microgels exhibit temperature-responsive properties

[44]. VCL forms hydrogen bonds with water molecules through the carbonyl groups. At the same

time the methylene groups of the VCL ring induce hydrophobic structuring of the water and this

leads to entropy-controlled polymer-polymer interactions. If the solvent-polymer interactions are

stronger than the polymer-polymer interactions PVCL chains exhibit a random-coil structure. If

the hydrogen bonds to water break (due to temperature increase) the release of the structured

water takes place and polymer-polymer interactions become dominant leading to the coil-globule

transition. The temperature at which the phase separation of linear polymers takes place is called

the lower critical solution temperature (LCST). The nanogels consisting of cross-linked PVCL

chains exhibit thermo-responsive and volume phase transition temperature (VPTT) close to

LCST of non-cross-linked VCL chains (app. 32 °C). PVCL-based nanogels swell if the

temperature is below VPTT and shrink if temperature increases above VPTT. Light scattering

experiments performed at different temperatures allow tracking the change of the nanogel size

and calculate their swelling ratio [55].

139

Figure 6a shows hydrodynamic radii of β-CD-A-modified nanogels prepared with variable β-

CD-A amounts with use of cross-linker BIS. β-CD-A-modified nanogels show similar behavior

as the reference microgel and shrink if temperature increases. Figure 6b shows the calculated

swelling ratio defined as (RhM

/RhSW

)3 where Rh

M = measured hydrodynamic radius at different

temperatures; RhSW

= measured hydrodynamic radius for maximally swollen nanogels at

T = 20 °C. These results indicate that increase of the CD-A content does not influence the

swelling-deswelling properties of nanogels. The volume phase transition for all samples occurs in

a broad temperature range.

Figure 6. Hydrodynamic radii (a) and calculated swelling ratio (b) as a function of temperature

for β-CD-A-modified nanogels prepared with addition of cross-linker BIS (β-CD-A with

substitution degree 4).

The temperature-dependent behavior of nanogel samples prepared with CD-As having

different substitution degree was analyzed. Figure 7 indicates that the response of nanogels to the

temperature change becomes smaller if substitution degree of CD-A increases. This is reflected in

reduction of nanogel size (Figure 7a) and increase of the swelling ratio at temperatures above

VPTT (Figure 7b) for nanogels prepared with higher substituted CD-As. This indicates that the

cross-linking degree in nanogels increases with increase of the substitution degree in CD-As.

This results in an extreme case (substitution degree 6) in linear reduction of nanogel size and

swelling ratio with temperature. Presence of cross-linker BIS does not influence nanogel behavior

at different temperatures.

20 25 30 35 40 45 50

25

50

75

100

125

150

175

200

225

250no CD-A

0.04 mmol -CD-A (4)

0.07 mmol -CD-A (4)

0.22 mmol -CD-A (4)

Rh [n

m]

Temperature [°C]

ba

20 25 30 35 40 45 500.0

0.2

0.4

0.6

0.8

1.0no CD-A

0.04 mmol -CD-A (4)

0.07 mmol -CD-A (4)

0.22 mmol -CD-A (4)

Sw

elli

ng

ra

tio

Temperature [°C]

140


for nanogels prepared with β-CD-A having substitution degree 2 (0.24 mmol); 4 (0.22 mmol);

and 6 (0.21 mmol) (0.3 g CD-A was used for nanogel synthesis).

Nanogel samples prepared with α-, β- and γ-CD-A having substitution degree 2 exhibit similar

temperature response (Figure 8). The swelling ratio for nanogel samples is not influenced by the

cyclodextrin type. The swelling ratio for nanogel samples is not influenced by the cyclodextrin

type and shows similar behavior within investigated temperature region.


for nanogels prepared with 0.3 g α-CD-A (0.28 mmol); β-CD-A (0.24 mmol); and γ-CD-A

(0.21mmol) with substitution degree 2 (0.3 g CD-A was used for nanogel synthesis).

a b

20 24 28 32 36 40 44 48 5215

30

45

60

75

90

105

120

CD-A (2) with BIS

CD-A (2) without BIS

CD-A (4) with BIS


CD-A (6) with BIS


Rh [n

m]

Temperature [°C]

20 25 30 35 40 45 500.0

0.2

0.4

0.6

0.8

1.0 CD-A (2) with BIS

CD-A (6) with BIS

Sw

elli

ng

ra

tio

Temperature [°C]

a b

20 25 30 35 40 45 50

60

80

100

120

140

160

180

200

220 CD-A (2) with BIS


CD-A (2) with BIS


CD-A (2) with BIS


Rh [n

m]

Temperature [°C]

20 25 30 35 40 45 500.0

0.2

0.4

0.6

0.8

1.0

1.2 CD-A (2) with BIS


CD-A (2) with BIS


CD-A (2) with BIS


Sw

elli

ng

ra

tio

Temperature [°C]

141

The results shown in Figueres 7 and 8 show that the cyclodextrin type and swelling degree for

obtained nanogels can be varied independently. This property of nanogels is very important for

their use as a container for uptake/release of hydrophobic guest molecules.

Size related properties of cyclodextrin based nanogels enable working between molecular

and cellular dimensions, and the large surface area of the small particles leads to a higher

cyclodextrin content at the interface than in case of three-dimensional bulk gels. It is expected

that cyclodextrin based aqueous nanogels provide useful functionalities such as effective

bioconjugation, good adhesion to different surfaces, controlled complexation and release of

drugs, cosmetics, dyes or antimicrobial agents. They can also be used for limited drug delivery, in

this case is excepted oral delivery of drugs is excepted, because these nanogels are not

biodegradable. Preliminary tests indicate that CD-A-modified nanogels can be transferred to

organic solvents. This opens a possibility to complex water-insoluble compounds by CD [56] and

utilisation of nanogels in catalysis or separation processes.

6.4 Conclusion

Poly(N-vinylcaprolactam) nanogels functionalized with cyclodextrin units have been

prepared by surfactant-free precipitation polymerization in water. Series of α-, β-,γ-CDs

functionalized with acrylic groups having the average amount of vinyl groups per CD-A

molecule adjusted to 2, 4, or 6 were prepared.The experimental data indicate that the increase of

the CD-A concentration in reaction mixture lead to reduction of the final hydrodynamic radius

of nanogels from 227 nm to 62 nm. Increase of the number of vinyl groups per CD-A molecule

induce formation of smaller nanogels (Rh = 22.5 nm) and considerable increase of the cross-

linking degree. Obtained cyclodextrin-modified nanogels show temperature sensitivity in

aqueous medium with a volume transition point around 30 °C and superior colloidal stability.

The complexation properties of CDs incorporated in nanogel networks were proved by titration

with phenolphthalein. This procedure allows the determination of “active” CD-A units, e.g. able

to build inclusion complexes with hydrophobic guest molecules.

142

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46. Häntzschel, N.; Zhang, F.; Eckert, F.; Pich, A.; Winnik, M. A., Poly(N-vinylcaprolactam-co-glycidyl

methacrylate) Aqueous Microgels Labeled with Fluorescent LaF3:Eu Nanoparticles, Langmuir 2007, 23,

10793-10800.

47. Schachschal, S.; Balaceanu, A.; Melian, C.; Demco, D. E.; Eckert, T.; Richtering, W.; Pich, A., Polyampholyte

Microgels with Anionic Core and Cationic Shell, Macromolecules 2010, 43, 4331–4339.

48. Hamcerencu, M.; Popa, M.; Riess, G.; Ritter, H.; Alupei, V., Unsaturated esters of cyclodextrin – synthesis

and characterization, Cellulose. Chem. Technol. 2005, 39, 389-413.

49. Harada, A.; Furue, M.; Nozakura, S., Cyclodextrin-Containing Polymers. 1. Preparation of Polymers,




51. Taguchi, K. Transient binding of phenolphthalein-.beta.-cyclodextrin complex: an example of induced


52. Topchieva, I. N.; Spiridonov, V. V.; Kalashnikov, Ph. A.; Kurganov, B. I., The detection of cyclodextrin self-

association by titration with dyes, Colloid Journal 2006, 68, 98-105.

145



54. Szejtli, J., Past, present and future of cyclodextrin research, Pure Appl. Chem. 2004, 76, 1825–1845.

55. Crowther, H. M.; Vincent, B., Swelling behavior of poly- N-isopropylacrylamide microgel particles in

alcoholic solutions, Colloid Polymer Sci. 1998, 276, 46-51.

56. Kida, T.; Fujino, Y.; Miyawaki, K.; Kato, E.; Akashi M., 6-O-Modified β-Cyclodextrin Enabling Inclusion

Complex Formation in Nonpolar Media, Org. Lett. 2009, 11, 5282–5285.

146

6.6 Supporting information

1. Modification of cyclodextrin with vinyl groups (CD-A)

Scheme S1. Modification of α-, β-, γ-cyclodextrin with vinyl groups.

Table S1. Chemicals used for the synthesis of CD-A with different substitution degrees (DS).

Substance β-Cyclodextrin Acryloyl chloride Triethylamine DS

β-CD-A DS 2 2.0 g

(1.76 mmol)

0.43 mL, 0.48 g

( 5.28 mmol)

0.62 mL, 0.45 g

(4.40 mmol)

2

β-CD-A DS 4 2.0 g

(1.76 mmol)

0.57 mL, 0.64 g

(7.04 mmol)

0.81 mL, 0.59 g

( 5.86 mmol)

4

β-CD-A DS 6 2.0 g

(1.76 mmol)

1.42 mL, 1.59 g

( 17,60 mmol)

2.03 mL, 1.48 g

(14.61 mmol)

6

α-CD-A DS 2 2.0 g

(2.05 mmol)

0.50 mL, 0.56 g

( 6.15 mmol)

0.71 mL, 0.52 g

(5.13 mmol)

2

γ-CD-A DS 2 2.0 g

( 1.54 mmol)

0.37 mL, 0.42 g

( 4.62 mmol)

0.53 mL, 0.39 g

(3.85 mmol)

2

TEA, DMF, RT

Cyclodextrin acrylate

O

R =

OH O

Cyclodextrin

O Cl

Acryloyl chloride

O

HO

OH

O

HO

6, 7, 8

O

R

R

O

R

6, 7, 8

147

Figure S1. 1H-NMR spectrum of β-CD-A (2) in DMSO-d6

1H-NMR spectrum (DMSO-d6, 300 MHz):

(ppm): 3.21-3.35 (m, CH-2, 4); 3.51-3.62 (m, CH-5, 3, CH2- 6); 4.53 (s, C-OH-6); 4.83 (s, CH-

1); 5.62-5.66 (s, C-OH-2, 3); 5.87 (m, CH-8), 6.15 (m, CH-9); 6.25 (m, CH-7)

13C-NMR spectrum (DMSO-d6, 75 MHz):

(ppm): 59.88 (C6); 71.99 (C2); 72.38 (C5) 73.02 (C3); 81.48 (C4); 101.88 (C1); 128.07 (C-

CH2-8); 131.68 (C-CH-7); 165.34 (COO)

FTIR-spectrum (KBr): ν (cm-1

): 3380, 2928, 1721 (C=O), 1633, 14106, 1364, 1297, 1198, 1154,

1079, 1032, 945, 859, 756, 580, 531

RAMAN-spectrum: ν (cm-1

): 3253, 3103, 2946, 2913, 1721, 1634 (C=C), 1461, 1461, 1407,

1334, 1112, 1083, 947, 858, 788, 669, 577, 482, 446

ppm (t1)

3.504.004.505.005.506.00

0

50

100

150

200

250

300

6.2

51

6.1

46

5.8

96

5.8

63

5.6

63

5.6

40

5.6

18

4.7

70

4.3

89

3.5

73

3.2

81

6.1

5

7.0

0

13.3

9

4.8

4

28.0

3

43.7

7

Vinyl H-7, 9, 8

OH-2, 3

CH-1

OH-1

CH-5, 3, 6

CH-2, 4

148

2. Synthesis of nanogels

Scheme S2. Preparation of nanogels by α-, β- or γ-CD-A with active vinyl groups

(inset shows FESEM image of α-CD-A nanogels).

3. Hydrodynamic radii of nanogels before and after complexation with

phenolphthalein

Figure S2. Hydrodynamic radii of β-CD-A-modified nanogels with and without added

phenolphthalein (β-CD-A with substitution degree 4; measurements performed at 20 °C).

O

NH

O

NH

NO

Vinylcaprolactame (VCL)

AMPA, H2O, 70°C

O

R

R

O

R

6, 7, 8

Cyclodextrin acrylate

O

R =O

O

O

O

O

Acetoacetoxyethyl-methacrylateOH

N,N-Methylenbis(acrylamide) (BIS)

O

0.04 0.07 0.2240

60

80

100

120

-CD-A [mmol]

Rh [n

m]

CD-A Nanogel

CD-A Nanogel + Phenolphthalein

149

4. Size distribution of nanogels with variable cyclodextrin content

Figure S3. Size distribution curves of β-CD-A-modified nanogels prepared with and without

cross-linker BIS (β-CD-A with substitution degree 4; measurements performed at 20 °C).

5. Solubility of α-, β-, γ- cyclodextrin in water

Figure S4. Solubility of α-, β-, γ-cyclodextrin in water (from Szejtli J, Pure Appl Chem

2004;76(10):1825–1845) [54].

10 100 10000

2

4

6

8

10

12

14

16

18

20

22

Inte

nsity [%

]

Rh [nm]

no CD-A

0.04 CD-A (4)

0.07 CD-A (4)

0.22 CD-A (4)

b

10 100 10000

2

4

6

8

10

12

14

16

18

20

22

Inte

nsity [%

]

Rh [nm]

no CD-A

0.04 CD-A (4) BIS

0.07 CD-A (4) BIS

0.22 CD-A (4) BIS

a


4

8

12

16

20

24

so

lub

ility

[g

/ 1

00m

L]

natural cyclodextrins

151

Chapter 7

Thermochromic cyclodextrin containing nanogels by complexation

of solvatochromic dye Nile Red

Abstract: In this work, permanent incorporation of the solvatochromic dye nile red (NR) is

presented into temperature responsive CD-A containing nanogels based on poly(N-

vinylcaprolactam) (PVCL). The uptake studies of NR in series of -, - and -CD acrylate

containing nanogels showed that the complexation is controlled by degree of cross-linking, CD-

A content and CD type in nanogels. The structure of the NR:CD complexes in nanogels could

be characterized as inclusion complexes and as capped complexes found for -CD and -CD

domains with high uptake capacity. The fluorescence of NR was influenced by different

structures of the NR:CD complexes and thus, by twisted intermolecular charge transfer (TICT).

The fluorescence emission was much higher for NR complexed by CD in nanogels than by

natural CDs. A large and reversible bathochromic color shift while shrinking and swelling of the

NR loaded -CD nanogels at the volume transition temperature (VPTT at 32-37°C) was

observed and characterized as reversible restructuring of the NR:-CD complex in nanogels.

Furthermore, NR containing CD nanogels show good adhesion to polyester fibers. After wet

coating onto textiles, an irreversible change in color was obtained upon increase of temperature.

Keywords: nanogel, cyclodextrin, N-vinylcaprolactam, nile red, solvatochrom, thermochrom

N

O

N

CH3

O

CH3

H

H

O

O

H

O

N

O

N

CH3

O

CH3

H

H

O

O

H

O

N

O

N

H3C

O

H3C

H

H

O

O

H

O

€

NO

N

CH3

O

H3C

H

O

O

RR

R

O

O

R

RR

O

OR

R

R

O

O

RR

R

OO

R

R

R

O

OR

RR

O

O

R

R

R

O

O

RR

R

O

O

R

RR

O

OR

R

R

O

O

RR

R

OO

R

R

R

O

OR

RR

O

O

R

R

R

O

β-CyclodextrinNile RED-

Cyclodextrin-Complex

N

O

N

CH3O

H3C

Nile Red

Nile RED-β-Cyclodextrin-

1:1 Inclusion-ComplexNile RED-γ -Cyclodextrin-

1:1,1:1 and 2:1 Capped-Complexes

N

O

N

H3C

O

H3C

H

H

O

O

H

O

20 24 28 32 36 40 44 4850

60

70

80

90

100

110

120

130

140

Wa

ve

len

gth

[n

m]

Temperature [°C]

550

560

570

580

NR : CD-A (DS2) NG

Rh [n

m]

152

7.1 Introduction

The incorporation of photochromic, solvatochromic and thermochromic dyes in various

polymeric structures finds more and more interest in research and application [1]. Controlling of

photophysical and photochemical properties of dyes by changing the near environment by pH,

temperature, polarity, or structure is part of interest. Nile red (NR) is such a sensitive

solvatochromic and fluorescent dye which acts mainly as material for white light emitting diodes

(LEDs) [2], as marker and as staining agent for biological systems [3,4]. NR is well known for its

solvatochromic properties and remarkable sensitivity to the microenvironment by changing the

absorption and fluorescence [5]. The structure of NR consists of a diethylamino group which acts

as one electron donor. The electron donor is attached to the electron with drawing rigid aromatic

system by a single bond. Photoexitation leads to internal rotation of the diethylamino group along

the single bond, which tends to result in a ‘twisted intramolecular charge transfer (TICT)’ state

[6]. The TICT state can be observed in N,N-dimethylaniline derivatives in polar solvents and

characterized by dual emission in fluorescence spectra. Two typical emission bands can be found,

one characterizes the locally excited state (LE) and another band is attributed to the TICT state.

Here, large Stoke’s shift characterizes the difference to normal emission bands [7]. In polar

solvents, the NR molecule does not show a dual fluorescence unlike most other TICT molecules.

In the excited electronic state of NR the TICT is the main nonradiative decay process [8,9]. The

restricted environment of NR, especially the geometry and structure of the surroundings have

also an important influence on the photophysical properties [10-12].

Hence, NR is a good marker and sensor to study the polarity of different systems and can

be used for many investigations of interesting carrier and release systems. Up to now, it has

already been used to study the local polarity in micelles [13,14] and reverse micelles [9] as well

as Langmuir–Blodgett films [15], zeolites [8,10,16], membranes [17], microgels [18,19] and

supramolecular assemblies [11]. Complexation studies of NR by α-, β- and γ-CD have been made

by different work groups which show that the photochromic properties depend on the complex

structure which is influenced by the different ring size of α-, β- and γ-CD [20-22].

The role of CD domains in responsive CD containing nanogels and the development of

potential thermochromic applications e.g. as nanosensor can be studied by the uptake of the

fluorescent dye NR. In this work, studies about the uptake of the NR by CD nanogels and,

especially the complexation in CD domains are studied in detail by UV-vis and fluorescence

153

spectroscopy. Furthermore, the application of NR loaded nanogels on polyester fabrics shows the

good adhesion on surfaces. Release and restructuring of NR from CD domains in nanogels by

changing the temperature are also part the of investigation in aqueous solution and on polyester

fibers. The properties of dye loaded nanogels are studied at the volume phase transition

temperature (VPTT) of VCL nanogels.

7.2 Experimental

7.2.1 Materials

α-, β- and γ-cyclodextrins acrylate (CD-A) with an average substitution degree (DS) of 2

and furthermore, β-CD-A with a DS of 4 were obtained from α-, β- and γ-CDs (Wacker) by

modification using a standard synthesis recipe from Chapter 6 [23]. N-Vinylcaprolactam (VCL)

was obtained from Aldrich and distilled by vacuum distillation. Acetoacetoxyethyl methacrylate

(AAEM) was received from Aldrich and was purified and destabilized by column

chromatography over aluminum oxide; a further cross-linker N,N-methylene-bis-acrylamide

(BIS) from Aldrich was used as received. The initiator, 2,2-azobis(2-methylpropionamidine)

dihydrochloride (AMPA) from Aldrich was used without further purifications. The solvents

(p.A.) tetrahydrofuran (THF) and acetone were purchased from VWR. Nile red (NR) was used

as received from Aldrich. Standard polyester fabric (Dacron 54, approx. 126 g/m2 fabric weight)

of testex Prüftextilien GmbH, Bad Muenstereifel/D, was used.

7.2.2 Nanogel synthesis

The synthesis of nanogels in aqueous solution was performed by batch precipitation

polymerization technique and is already reported in detail Chapter 6 [23]. For the synthesis of

CD nanogels, a double-wall glass reactor equipped with a stirrer and a reflux condenser was

purged with nitrogen. A solution of the monomers 14.72 mmol (2.06 g) VCL, 0.23 mmol (0.05

g) AAEM, and variable amounts of CD-A in 147 mL water were placed into the reactor and

stirred at 200 rpm for 1 h at 70 °C under continuous purging with nitrogen. Afterwards, 3 mL

aqueous solution of the initiator (0.07 mmol (0.02 g) AMPA) were added under continuous

stirring. The reaction was carried out for another 12 h. In some experiments 0.02 mmol (0.13 g)

BIS was used as additional cross-linker to compare the uptake properties of the nanogels

154

prepared only with reactive CD-A as cross-linker or combination of BIS and CD-A. After

preparation, the stable, milky nanogel dispersions were purified by ultrafiltration to remove

unreacted monomers and smaller polymeric particles. The nanogel dispersion in water was

pumped through a membrane with molecular weight cut off of 30 kDa (Millipore, Pelicon XL

Device, Ultracel 30 PLCHK membrane, regenerated cellulose with NMWCO of 30 kDa). The

ultrafiltration time was adjusted to 48 h. The ingredients used for polymerization process are

summarized in Table 1. The nanogel samples are numbered as NVCLxCD-A(y), with N = run

number, x = CD type, and y = substitution degree of CD.

Table 1. Reaction recipe for the preparation of CD-A/VCL/AAEM nanogels in 150 g water.

N VCL,

[g](mmol)

AAEM,

[g](mmol)

CD-A,

[g](mmol)

CD-A,

[wt.-%]

BIS,

[g](mmol)

AMPA,

[g](mmol)

Yield

[g](%)

1VCL 2.06 (14.72) 0.05 (0.23) - - 0.02 (0.13) 0.02 (0.07) 1.02 (47)

2VCLαCD(2) 2.06 (14.72) 0.05 (0.23) 0.3 (0.28) 12.24 0.02 (0.13) 0.02 (0.07) 1.22 (50)

3VCLαCD(2) 2.06 (14.72) 0.05 (0.23) 0.3 (0.28) 12.35 - 0.02 (0.07) 1.27 (52)

6VCLβCD(2) 2.06 (14.72) 0.05 (0.23) 0.3 (0.24) 12.24 0.02 (0.13) 0.02 (0.07) 1.38 (56)

7VCLβCD(2) 2.06 (14.72) 0.05 (0.23) 0.3 (0.24) 12.35 - 0.02 (0.07) 1.27 (52)

4VCLβCD(4) 2.06 (14.72) 0.05 (0.23) 0.3 (0.22) 12.35 - 0.02 (0.07) 0.43 (18)

5VCLβCD(4) 2.06 (14.72) 0.05 (0.23) 0.6 (0.44) 21.98 - 0.02 (0.07)

8VCLγCD(2) 2.06 (14.72) 0.05 (0.23) 0.1 (0.07) 4.44 0.02 (0.13) 0.02 (0.07) 0.97 (43)

9VCLγCD(2) 2.06 (14.72) 0.05 (0.23) 0.1 (0.07) 4.48 - 0.02 (0.07) 0.96 (43)

10VCLγCD(2) 2.06 (14.72) 0.05 (0.23) 0.3 (0.21) 12.24 0.02 (0.13) 0.02 (0.07) 1.39 (57)

11VCLγCD(2) 2.06 (14.72) 0.05 (0.23) 0.3 (0.21) 12.35 - 0.02 (0.07) 1.39 (57)

12VCLγCD(2) 2.06 (14.72) 0.05 (0.23) 0.6 (0.43) 21.82 0.02 (0.13) 0.02 (0.07) 1.51 (55)

13VCLγCD(2) 2.06 (14.72) 0.05 (0.23) 0.6 (0.43) 21.98 - 0.02 (0.07) 1.23 (45)

155

7.2.3 Complexation studies of NR by natural CDs

Different amounts of NR diluted in a mixture of acetone/THF (1/2) (1 g/L) were dropped

under stirring to aqueous α-, β- or γ-CD solutions (10 mg/10 mL). The mixture was stirred for 24

h for full complexation of NR and evaporation of the organic solvent. The complexed NR was

kept in solution and the non-diluted NR amount precipitated. The filtered, clear solutions were

analyzed by using UV-vis and fluorescence measurements.

7.2.4 Uptake of NR by CD-A containing nanogels

The complexation of NR in α-, β- or γ-CD-A nanogels was done in aqueous dispersion.

NR diluted in a mixture of acetone/THF (1/2) (1 g/L) was added in different concentrations to the

CD-A nanogel dispersion in the same way as used for its complexation by low molecular weight

natural CD. After stirring and evaporation of the solvent for about 24 h, non-complexed NR

precipitated and the filtered loaded nanoparticle dispersions were measured by using UV-vis and

fluorescence spectrometry. The particle size of the NR loaded nanogels was characterized by

dynamic light scattering.

7.2.5 Application of NR loaded nanogels on polyester fabrics

Dispersions with γ-CD-A nanogels (10 mg/mL) and a NR concentration of 20 µg/mL

were used for further coating experiments on polyester fibers. The coating process took place in a

special beaker in which the dispersion was placed. The polyester fabrics (126 mg/cm2) were

dipped in 5 mL NR loaded nanogel dispersion by variation of the time (15, 30, 45, 60 or 120

min). After that, the coated fabric was dried for 48 hours at RT under vacuum and were packed

waterproof and stored protected from light. The treated fabrics were analyzed by FESEM and

color measurements in dependence of the coating time.

7.2.6 Characterization of modified cyclodextrins and nanogels

The particle size of nanogels was analyzed by dynamic light scattering measurements,

using a Nano Zetasizer (Malvern) spectrometer equipped with a He–Ne Laser (λ0 = 633 nm).

Back scattering light was detected using an angle of 173°. The Hydrodynamic particle radius and

the particle distribution of the hydrodynamic radius were determined. The measurements were

made in a temperature range from 20 °C to 70 °C with a temperature equilibrium time of 120 s.

156

The microscopical analysis was made with a S-4800-Field-Emission-Scanning Electron

Microscope (Hitachi). One drop of the diluted nanogel dispersion was put onto an aluminum

support and dried at room temperature in vacuum. After drying, the samples were investigated at

an accelerating voltage of 1 kV and 8 mm disintegration.

UV-vis spectra were performed with the help of a JASCO V-630 spectrophotometer from

200-1000 nm using quartz cuvettes with coating thickness of 1 cm. The corresponding nanogel

dispersion without loaded NR was used as reference.

Fluorescent spectra were measured by a Fluoromax-4P, (HORIBA Jobin Yvon GmbH,

Unterhaching/D) spectrometer.

The color measurements were performed using a Datacolor Spectraflash SF600 plus CT

calorimeter from Datacolor, Marl. The measurements were made without luster and with a blend

of 20 mm. They were performed using the illuminant D65 and 10 ° observer angle. Fabrics were

measured in two layers. For each sample, three measurements were made and the mean values

were calculated. The color values are calculated according to the Datacolor formula based on the

CIELAB system for illuminant.Temperature dependence color measurements of the treated fabric

were preformed from 25-80 °C by Thermosystem FP 900 (Mettler Toledo GmbH,

Schwarzenbach/CH).

157


Nanogels functionalized with CD-A domains were prepared by surfactant-free

precipitation polymerization in water which is already reported and investigated in detail in

Chapter 6 [23]. CD-A acts as cross-linker in the synthesis of polymeric nanogels and functional

domain for the dye uptake. NR as solvatochromic guest agent presents good complexation

properties by natural β-CD and especially by γ-CD. Hence, in this work γ-CD containing

nanogels are in the focus of interest. Here, α-, β- and γ-CD-A (DS2) were prepared with average

2 numbers of reactive vinyl groups (and also 4 vinyl groups in case of β-CD-A (DS4) nanogels)

for covalent incorporation in the nanogel network during radical polymerization. The number of

vinyl groups per CD-A molecule and the incorporated CD-A content influence the cross-link

density of the polymeric network and thus, the polymerization process. Several parameters such

as the CD-A type (α-, β- or γ-CD-A), the CD-A content and the presence of the additional cross-

linking agent BIS were varied to study the influence of CD-As on the properties of the colloidal

nanogels with regard to the uptake of NR. All nanogels which are used for further studies in this

work and the corresponding details of the synthesis are given in Table 1.

7.3.1 Nanogel size, temperature-sensitivity and morphology

The synthesized nanogels with different γ-CD-A content are studied with regards to their

colloidal behavior in water (Figure 1a). In recent publication, the influence of the β-CD-A (DS4)

content in PVCL nanogels on the particle size and furthermore, the comparison of the

hydrodynamic radii of -, - and -CD-A nanogels (prepared from 12.35 mmol CD-A (DS2))

was studied in Capter 6 [23]. Here, nanogels prepared with -CD-A contain a smaller particle size

(Rh=134 nm) than nanogels prepared with -CD-A (Rh=183 nm) and a larger particles size than

-CD-A (Rh=119 nm). So far, nanogels with different content of γ-CD-A (DS2) show a similar

trend as β-CD-A (DS4) nanogels which were already studied. The hydrodynamic radius

decreases with increase of the γ-CD-A (DS2) amount in the reaction mixture. In all cases, the

nanogels prepared with γ-CD-A (DS2) are slightly larger than nanogels prepared with -CD-A

(DS4), but they show the same colloidal stability in water. The hydrodynamic radii of nanogels

prepared with -CD-A and -CD-A plus an additional cross-linker BIS show that the use of a

further standard cross-linker does not have influence on the particle formation and size.

158

Figure 1b shows the hydrodynamic radii of γ-CD-A modified nanogels prepared with

variable γ-CD-A (DS2) amounts in dependence of the temperature. γ-CD-A modified nanogels

present a similar behavior as the reference nanogel without CD-A and shrink during the VPTT as

the temperature increases. Figure 1c shows the calculated swelling ratio defined as (RhM

/RhSW

)3,

where RhM

= measured hydrodynamic radius at different temperatures, RhSW

= measured

hydrodynamic radius for maximally swollen nanogels at T = 20 °C. These results indicate that an

increase of the -CD-A content does not influence the swelling-deswelling properties of the

nanogels. The volume phase transition for all samples occurs in a broad temperature range.

Figure 1. Hydrodynamic radii of nanogels with different γ-CD-A (DS2) content in comparison

with nanogels with different -CD-A (DS4) content (a) and -, - and -CD-A (DS2) nanogels

studied in Chapter 6 (b) [23]. Hydrodynamic radii (c) and calculated swelling ratio (d) as function

of temperature for nanogels prepared with different γ-CD-A (DS2) content (0.07 mmol; 0.21

mmol and 0.43 mmol CD-A).

c d

a b

20 25 30 35 40 45 500.0

0.2

0.4

0.6

0.8

1.0

without CD-A

0.07 mmol -CD-A (DS2)



Sw

elli

ng

ra

tio

Temperature [°C]

20 25 30 35 40 45 500

25

50

75

100

125

150

175

200

225

250 without CD-A




Rh [n

m]

Temperature [°C]

alpha beta gamma

120

140

160

180

200

220

BIS

without BIS

Rh [n

m]

CD-A type

0.0 0.1 0.2 0.3 0.4 0.5 0.60

25

50

75

100

125

150

175

200

225

250

Rh, [n

m]

-CD-A (DS4)

-CD-A (DS2)

without CDA

CD-A [mmol]

159

Nanogels with different -CD-A (DS2) content were coated onto aluminum plates by dip-

coating from dispersion to study the morphology of totally collapsed γ-CD-A nanogels by

FESEM. Here, nanogels have good physical self-bonding properties due to the gel character of

the cross-linked polymeric backbone. The homogeneous distribution of the surface can be

controlled by the nanogel concentration in the aqueous dispersion. The particles have a round,

spherical structure and the particle size decreases by the use of rising -CD-A content in the

nanogels (Figure 2a-d).

Figure 2. -CD-A containing PVCL nanogels coated on aluminum plates. PVCL nanogels are

prepared without CD-A (a), with 0.07 mmol -CD-A (b), with 0.21 mmol -CD-A (c) and with

0.43 mmol -CD-A (d).

The results are readily comparable and in agreement with the size of swollen nanogels measured

by DLS in water. Here, dried nanogels are presented in the totally collapsed state, whereas -CD-

A nanogels measured in dispersion consist of different amounts of incorporated water. The totally

a

c

b

d

160

collapsed state of the -CD-A nanogels after release of water by drying in vacuum shows similar

size related properties as deswollen nanogels after reaching the volume phase transition

temperature between 30-40 °C in water. The results of the morphology and the particle size of -

CD-A nanogels are in good agreement with studies of PVCL nano- and microgels presented

described in the literature [23-27].

7.3.2 Uptake and complexation of nile red in cyclodextrin and CD nanogels

The solvatochromic NR guest molecule is a water insoluble dye which shows high

sensitivity for changes in the microenvironment, which enables investigation of the complexation

and uptake properties of CD-A containing PVCL nanogels. The complexation of NR by natural

CD has already been studied and reported in detail [20-22]. In these publications, the

stoichiometries and geometries of the inclusion and the capped host:guest complexes were

determined using of UV-vis absorption spectroscopy, steady state/time resolved fluorescence

spectroscopy, mass ionization spectroscopy and theoretical calculations of the complex by

molecular modeling. The solvation dynamics of NR in aqueous solution in presence of CD and

the effect of twisted intermolecular charge transfer (TICT) in the NR complexation were studied

in detail.

In these work, the focus of interest is the role of embedded CD-A in nanogels on the

uptake properties of NR. A handicapped rotation of the covalently bound CD-A rings, the

influence of the PVCL backbone on the complexation and the influence of the cross-linked

structure of the polymer network on the accessibility of CD domains by guest molecules are

further factors which may lead to interesting results. Scheme 1a presents the theoretical uptake of

NR in nanogels by complexation in CD domains. Here, the complexation and the uptake of NR

were characterized by UV-vis and steady state fluorescence spectroscopy.

161

Scheme 1. Theoretical uptake of nile red in CD-A containing nanogels (a) and possibilities of the

complex formation of NR in - and -CD (b).

First, pre-studies on the complexation of NR by natural -, - and -CD in water using

UV-vis spectroscopy were made to get references for the uptake of NR by CD-A containing

nanogels and further, to understand the function of the CD domains in the nanogels structure.

An exact amount of NR was given to aqueous solutions with the exact amount of natural -, -

and -CD. Due to the low solubility in water the red solid precipitated in aqueous solution. Thus,

in UV-vis spectrum (Figure 3a) a low absorbance with a maximum at ~ 0.02 at 600 nm is

observed [22]. In agreement with the literature, α-CD complexes NR in a very low amount. The

corresponding UV-vis spectrum does not show high absorbance in water at 585 nm. Furthermore,

the water insoluble dye precipitate as red solid. Thus, -CD attributed no strong binding effects

to NR and it seems that the cavity is not suitable for the complexation of the NR molecule [21].

-CDs complex NR molecules in higher amount, which results in water-soluble complexes with

slightly red color and an absorbance at 585 nm. NR containing -CD solutions show a strong blue

color at 552 nm without sedimentation. Hence, the observation indicates that the complex

formation of all guest molecules takes place. -CD increases the solubility of NR in water and

N

O

N

CH3

O

CH3

H

H

O

O

H

O

N

O

N

CH3

O

CH3

H

H

O

O

H

O

N

O

N

H3C

O

H3C

H

H

O

O

H

O

€

NO

N

CH3

O

H3C

H

O

O

RR

R

O

O

R

RR

O

OR

R

R

O

O

RR

R

OO

R

R

R

O

OR

RR

O

O

R

R

R

O

O

RR

R

O

O

R

RR

O

OR

R

R

O

O

RR

R

OO

R

R

R

O

OR

RR

O

O

R

R

R

O

β-CyclodextrinNile RED-

Cyclodextrin-Complex

N

O

N

CH3O

H3C

Nile Red

Nile RED-β-Cyclodextrin-

1:1 Inclusion-ComplexNile RED-γ -Cyclodextrin-

1:1,1:1 and 2:1 Capped-Complexes

N

O

N

H3C

O

H3C

H

H

O

O

H

O

b

a

162

presents the best complexation behavior to NR. The complexation of NR by - and -CD is

explained in the literature by different complexation possibilities. Hence, the NR molecule can be

incorporated with the whole structure in the - and -CD cone; otherwise the guest molecule is

placed on the cone and is fixed by hydrogen bonds. The so-called capped complex with a

stoichiometry of 2:1 and 1:1 is favored in case of -CD, while for -CD a 1:1 inclusion complex

is preferred [21-22]. Scheme 1b shows the formation of the host:guest complex and the

possibilities of complex formation.

Figure 3. UV-vis spectra of 20 µg/mL NR in 10 mL solutions of α-, β- and γ-CD (a) and in 10

mL dispersions with α-, β- and γ-CD-A containing nanogels (NG) (b), measured at room

temperature.

The uptake studies of NR in CD-A containing nanogels were carried out similarly to

investigations with natural CD molecules. The nanogels differ in the CD type (-, - and -CD),

in the content of CD-A domains or in the degree of cross-linking prepared with and without

additional cross-linker BIS. The uptake of exact amounts of NR by -, - and -CD nanogels was

studied. In Figure 3b, comparable UV-vis spectra of NR containing α-, β- and γ CD nanogel

dispersions with spectra of solutions with NR complexed by natural CDs are detectable.

Nanogels without CD (only cross-linked PVCL backbone) do not show any high absorbance in

the UV-vis spectrum. Thus, the uptake of NR by the polymeric structure takes place, but strong

fixation by complexation is missing. This lead to the conclusion that the cross-linked structure of

the polymer VCL backbone do not have significant binding properties for NR.

a b

400 450 500 550 600 650 700 750 8000.00

0.05

0.10

0.15

0.20

0.25

Ab

so

rban

ce

Wavelength [nm]

-CD

-CD

-CD

H2O

450 500 550 600 650 7000.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

Ab

so

rban

ce

Wavelength [nm]

CD-A (DS2) NG

CD-A (DS2) NG

CD-A (DS2) NG

NG without CD-A

163

Interesting results are found for α-CD nanogels. The absorbance of NR in α-CD nanogels

at 585 nm is much higher than the absorption of NR in natural α-CD solution at 585 nm. The

combination of the cross-linked PVCL structure with embedded α-CD domains leads to fixation

of the NR molecules in the gel particles, although the direct complexation is handicapped by the

ring size of α-CD. NR loaded β-CD nanogels and especially γ-CD nanogels present both higher

absorbance maxima at 552 nm which indicates a better uptake of guest molecules. Interestingly,

differences in the complexation structure can be seen for β- and γ-CD nanogels and the

corresponding natural CDs. The absorbance spectrum of NR in β-CD nanogels shows beside the

band at 585 nm a strong second band at 558 nm which indicates the present of an further capped

complex. On the other hand, -CD nanogels show beside the band at 552 nm a smaller second

band at 595 nm which conclude the presence of an inclusion complex. These absorbance are

additionally influenced by interaction of NR with the polymer PVCL backbone. The absorbance

maxima of natural β- and γ-CD are slightly higher than that of the corresponding nanogels with

the same NR concentration. Thus, the calculated CD content in the nanogels (in case of 1:1

incorporation of CD-A and VCL during the polymerization) complexes less NR than natural

CDs. This can be explain that many CD units in the nanogels are not free or not accessible for

full complexation of NR.

Controlled uptake of rising NR concentrations by α-, β- and γ-CD containing nanogels is

studied in detail by determination of the absorption maxima and the corresponding wavelengths

of characteristic NR bands in aqueous dispersion. Results are shown in Figure 4.

Nanogels without any CD domains show a very low increase of the absorbance at 585 nm

(Results are not shown.). Similar results are observed for α-CD containing nanogels, which

present slight increase of the absorbance with increased NR concentration but with a low optical

density at 585 nm. β- and γ-CD show higher absorbance maxima with increase of the NR content

in the dispersion. Rising NR addition increases the absorbance maximum of all nanogel samples

and thus, slightly the uptake of NR in nanogels. The uptake curve flattens at the addition point of

10 µg/mL NR indicating that the uptake is saturated. The absorbance maxima of NR in α-, β- and

γ-CD nanogels are observed at different wavelengths (585 nm (α-CD), 558 nm (β-CD) and 552

nm (γ-CD) which do not shift by increase of the NR concentration. Hence, the increase of guest

molecules do not influence the structure of the complexes.

164

Taken into account that the same degree of substitution of the CD units (DS2), the same

cross-linking points in the network and the same content of CD rings (0.21 mmol) present

different absorption maxima at different wavelength. Thus, different kinds of uptake and

complexation take place which are results of different complexation structure between NR and α-

, β- or γ-CD nanogels.

Figure 4. Absorbance maxima and the corresponding wavelengths of different NR

concentrations taken by nanogels with (0.3 g) -, -, -CD-A or without CD-A content (a-b) and

nanogels with (0.3 g) -, - and -CD-A plus additional cross-linker BIS (c-d).

Another focus of interest in NR uptake studies is to recognize the influence of the degree

of cross-linking on the uptake and on the formation of complexes with CD domains using

additional cross-linker BIS. Results in Chapter 6 showed less influence of BIS on the particle size

and on the degree of swelling [23]. The absorption maxima and the corresponding wavelengths of

different concentrations of NR in dispersions of CD (DS2) containing nanogels are shown in

c d

a b

0 2 4 6 8 10 12 14 16 18 200.00

0.01

0.02

0.03

0.04

0.05 -CD-MA (DS2) NG BIS

-CD-MA (DS2) NG BIS

-CD-MA (DS2) NG BIS

Ab

so

rba

nce

Nile Red Addition [µg/mL]

0 2 4 6 8 10 12 14 16 18 20540

550

560

570

580

590

600

610

620 -CD-MA (DS2) NG BIS

-CD-MA (DS2) NG BIS

-CD-MA (DS2) NG BIS


Wa

ve

len

gh

t [n

m]

0 2 4 6 8 10 12 14 16 18 20540

550

560

570

580

590

600

610

620 -CD-MA (DS2) NG

-CD-MA (DS2) NG

-CD-MA (DS2) NG


Wa

ve

len

gh

t [n

m]

0 2 4 6 8 10 12 14 16 18 200.00

0.02

0.04

0.06

0.08

0.10

0.12 -CD-MA (DS2) NG

-CD-MA (DS2) NG

-CD-MA (DS2) NG

Ab

so

rba

nce


165

Figure 4c-d. The use of BIS results in a decrease of the absorbance maxima of all NR containing

nanogel dispersions in comparison to nanogels without BIS. A large shift in the wavelength is

only significantly detectable in case of NR loaded -CD-A nanogels compared to the absorbance

wavelength of NR loaded -CD nanogels without BIS. Here, the presence of only one absorbance

band leads to the conclusion that only the inclusion complexation take place in whereas the

capped complex does not occur. The lower optical density leads to the assumption that only CD

domains on the particle surface are accessible for complexation. This can also be seen in case of

- and -CD nanogels with BIS. BIS does not change the structure of the NR:CD complex, but a

higher degree of cross-linking hinders the uptake and the accessibility of the CD domains by

guest molecules in the polymer network. In conclusion, a rising degree of cross-linking disturbs

the uptake and complexation by the CD domains.

In Figure 5, the detailed uptake of different NR concentrations in CD nanogel dispersions

is described and the different wavelengths of the NR absorbance provide information about

differences in the polar environment of NR and thus, of the uptake and complexation by CD

domains in polymeric nanogels.

Figure 5a-b shows the comparison of NR in -CD nanogels prepared with and without

BIS in more detail. The wavelength at 585 nm of the natural CD:NR complex which shifts

slightly to 590 nm upon uptake of NR in nanogels characterizes some interactions of NR with the

polymeric structure of PVCL and -CD domains. The slight blue shift compared to water (~600

nm) indicates an increase of the hydrophobic environment of NR. This can be explained by the

uptake in the polymeric network and low complexation behavior of NR by α-CD domains. The

absorbance of NR increases slightly with rising NR content and is kept constant at an uptake of 6

µg/mL. The absorbance maxima of all NR concentrations in presence of -CD nanogels prepared

with BIS show that all maxima are among the maxima of α-CD nanogels prepared without BIS.

Thus, high degree of cross-linking decreases the NR uptake capacity of -CD nanogels.

In Figure 5c-d, the complexation of NR by β-CD domains in nanogels leads to a blue shift

to 558 nm by the use of 0.24 mmol β-CD (DS2) in comparison to the NR absorbance in water

(~600 nm). This observation explains the enhanced dissolution of a capped complex by hydrogen

bondings with an increased hydrophobic environment. The second band at 585 nm leads to the

conclusion that dditionally a 1:1 inclusion complex by surrounding of NR with the hydrophobic

166

cavity of β-CD exists. Nanogels prepared with 0.24 mmol -CD consisting of 2 vinyl groups

(DS2) show higher absorbance maxima and thus, a better uptake than nanogels prepared with -

CD (DS2) and the additional cross-linker BIS. The increase of cross-linking points by the use of

BIS as additional cross-linker shifts the absorbance maxima from 558 nm to 592 nm. Thus, the

increase of cross-linking complicates the penetration of NR into the nanogel structure and

disables the formation of the capped complex (which is characterizes at absorbance maxima at

552 nm). Here, NR is stronger complexed by β-CD units at the surface of the nanogels and the

more hydrophobic PVCL backbone of the polymeric network has no strong influence on the NR

molecule. The hydrophilic properties of the β-CD molecules increase with the increase of the

substitution degree per β-CD unit and usage of BIS. Therefore, the absorbance shifts to 595 nm

by the same β-CD units and the same high cross-linking degree.

Studies with nanogels synthesized with 0.22 mmol β-CD with 4 vinyl groups (DS4)

reflect the results. In Figure 5c-d the absorbance maxima show the lowest absorption and thus, a

low uptake and complexation of NR. The 100 % increase of cross-linking points in the polymeric

structure by use of the same CD concentration decreases the uptake possibilities for NR

drastically. Furthermore, the fixation of the CD in the nanogels increases with rising amounts of

vinyl groups of the CD monomer. In comparison to natural free β-CDs the free orientation of the

β-CD host to the guest molecule is also handicapped by increased fixation of β-CD in the

polymeric structure by covalent bonds. Hence, β-CD (DS4) with 4 vinyl groups is stronger fixed

in the polymeric structure and less flexible than β-CDs (DS2) with 2 vinyl groups. An increase of

β-CD (DS4) units in the nanogels with the use of 0.44 mmol β-CD (DS4) in the reaction mixture

shows higher absorbance with increase of NR concentration compared to nanogels with lower β-

CD content (0.22 mmol β-CD DS4 or 0.24 mmol β-CD (DS2)). Here, a large number of

complexation units compensates the rising degree of cross-linking. But nanogels with 0.24 mmol

β-CD (DS2) with the lowest degree of cross-linking show the best NR uptake and complexation

properties. These results lead to the conclusion that the degree of cross-linking is more important

for the uptake of NR than the number of β-CD units.

167

Figure 5. Absorbance maxima and the corresponding wavelengths of different NR

concentrations in dependence of the CD containing nanogel (NG) dispersions. -CD-A nanogels

are prepared with or without the additional cross-linker BIS (a, b). -CD-A nanogels are prepared

by cross-linking with 2 or 4 vinyl groups per CD-ring (DS2 or DS4), with different β-CD-A

content (0.24 mmol) or (0.47 mmol) and with or without additional cross-linker BIS (c, d). -CD-

A nanogels with different -CD-A content are prepared with or without additional cross-linker

BIS (e, f).

e f

c d

a b

0 2 4 6 8 10 12 14 16 18 200.00

0.01

0.02

0.03

0.04

0.05


0.28 mmol -CD-A (DS2) NG

0.28 mmol -CD-A (DS2) NG BIS

Absorb

ance

0 2 4 6 8 10 12 14 16 18 20575

580

585

590

595 0.28 mmol -CD-A (DS2) NG



Wavele

ngh

t [n

m]

0 2 4 6 8 10 12 14 16 18 200.00

0.01

0.02

0.03

0.04

0.05






Ab

so

rba

nce

0 2 4 6 8 10 12 14 16 18 20550

560

570

580

590


Wa

ve

len

gh

t [n

m]





0 2 4 6 8 10 12 14 16 18 20

0.0

0.2

0.4

0.6

0.8

1.0 0.07 mmol -CD-A (DS2) NG







Ab

so

rba

nce

0 2 4 6 8 10 12 14 16 18 20540

545

550

555

560

565

570 0.07 mmol -CD-A (DS2) NG







Wa

ve

len

gh

t [n

m]

168

-CD is well known for the formation of capped complexes with NR [21-22]. Here, the

investigation of NR complexation by -CD nanogels (DS2) in dependence of the -CD content

are in the focus of interest (Figure 5e-f). The complexation of NR by natural free -CD is

characterized by an absorbance wavelength at 552 nm. In nanogels, a complexation of NR by -

CD domains is detected at 555 nm after use of 0.07 mmol -CD (DS2), 550 nm after use of 0.21

mmol -CD (DS2) and 546 nm after use of 0.43 mmol -CD (DS2) content. The blue shift due to

the increase of the -CD content is explainable by the increase of complexation possibilities

which change the NR environment. Capped complexes of NR and -CD domains are mainly

fixed by hydroxy bonds (Scheme 1b) in which the molecule lies flat on the open side of the CD

cavity [22]. Here, the NR:-CD complex mainly occurs in 1:1 and also in 2:1 stoichiometric

ratios. It seems that the complexation changes the environment around NR which gets more

hydrophobic than NR complexed by -CD and -CD as well as in case of free NR in water.

The maxima of the absorbance increases with the increase of the NR concentration. The

complexation is saturated at approximately 10 µg/mL NR and the curve flattens which means that

the uptake contingent and the dye fixation by complexation is exhausted.

The kind of complexation is not changed by BIS as the wavelength does not shift significantly in

comparison to the corresponding nanogels without BIS. Impressively, the amount of complexed

NR decreases with the increase of cross-linking. The absorbance maxima of NR complexed by

0.07 and 0.21 mmol -CD nanogels plus BIS are much lower than the corresponding nanogels

prepared without BIS. This leads to the conclusion that BIS and the increase of cross-linking

prevent the complexation, but do not change the formation of the favored capped structure of the

NR:-CD complex.

In summary, the uptake and the complexation properties of NR by CD containing

nanogels can be studied by observing of the near environment of NR after addition to nanogel

dispersions in water. The environment can be studied by absorbance spectroscopy which is

influenced by the polarity of the NR surrounding and the structure of complex. These depend on

the complexation unit, here the ring size of the CD type and further the degree of cross-linking

which is important for the assessment of NR molecules and for the water uptake. Pure PVCL

nanogels without CD domains and nanogels with α-CD bind NR only via the porous structure of

the nanogels in water. γ-CD containing nanogels prefer capped complexation of NR via hydroxyl

169

bonds in the nanogel structure. β-CD nanogels fix NR via capped complexation and by inclusion

complexation which is shown in the characteristic wavelength in comparison with natural CD:NR

complexes. An increasing CD content and decreasing cross-linking degree increase the

complexation capacity and the access ability of NR in CD domains of nanogels.

7.3.3 Fluorescence of NR loaded α-, β- and γ CD nanogels

In Figure 6 the fluorescence spectra of 2 µg/mL NR in natural -, - and -CD solution

and in the corresponding nanogels dispersion are shown. In Figure 6a, NR : -CD complex

fluoresces at a wavelength of 652 nm after excitation at 585 nm. NR loaded α- or -CD nanogels

fluoresce at 661 nm with a lower intensity than NR complexed in -CD nanogels with excitation

at 552 nm and 585 nm. In Figure 6b, NR complexed in -CD nanogels fluoresces at a wavelength

of 648 nm, α-CD at 646 nm and VCL nanogels without CD content at 652 nm after excitation at

585 nm. NR loaded -CD nanogels fluoresce at 561 nm with the lowest intensity after excitation

at 552 nm. Interestingly, because -CD nanogels exhibit the best uptake possibility for NR in

nanogels as measured by absorbance spectroscopy. NR loaded nanogels without CD domains

show the highest fluorescence although these nanogels exhibit the lowest NR uptake. The results

are in good agreement with the fluorescence of NR complexed by natural CD and with results

and explanations about the TICT state of NR in the literature [21-22].

Figure 6. Fluorescence emission spectra of 2 µg/mL NR in 10 mL solutions of α-, β- and γ-CD

(a) and in 10 mL dispersions with α-, β- and γ-CD containing nanogels (b), measured at room

temperature.

a b

600 625 650 675 7000

1x107

2x107

3x107

4x107

5x107

6x107

Flu

ore

sce

nce

Inte

nsity (

a.u

.)

Wavelength [nm]

-CD-A (DS2) NG

-CD-A (DS2) NG

-CD-A (DS2) NG

NG without CD-A

600 625 650 675 7000.0

2.0x106

4.0x106

6.0x106

8.0x106

1.0x107

1.2x107

-CD

-CD

-CD

H2O

Flu

ore

sce

nce

Inte

nsity (

a.u

.)

Wavelength [nm]

170

Similar to the studies about the absorbance of NR loaded nanogels, the detail

investigation on the fluorescence emission of the controlled successive addition of NR to similar

concentrated aqueous natural CD and CD nanogel dispersions are shown in Figure 7.

Figure 7. Fluorescence intensities of different NR concentrations complexed in α-, β- and γ CD

(a,b) and of α-, β- and γ CD containing nanogels (c,d).

Figure 7a shows the intensity of different NR concentrations complexed by natural -, -

and -CD in water. Here, the successive addition of NR to aqueous - and -CD solutions leads

to an increase of the emission intensity. The fluorescence intensity of NR complexed by -CD is

much less than that of NR complexed by -CD. The increase of intensity upon the increase of NR

concentration is characterized by linear order which suggests a 1:1 complex formation. The

emission of NR complexed by -CD increases at first, followed by a decrease of fluorescence

intensity with the increase of dye concentration. These results lead to the conclusion that

guest:host complexes of 1:1 followed by 1:2 are formed, which are reflected by a decrease in

0 2 4 6 8 10 12 14 16 18 20

2.0x106

4.0x106

6.0x106

8.0x106

1.0x107

1.2x107

1.4x107

1.6x107

1.8x107

Flu

ore

sce

nce

Inte

nsity [a.u

.]


-CD

-CD

-CD

0 2 4 6 8 10 12 14 16 18 20650

652

654

656

658

660

662

664

666

668

670 -CD

-CD

-CD

Wa

ve

len

gth

[nm

]Nile Red Addition [µg/mL]

c d

a b

0 2 4 6 8 10 12 14 16 18 20640

642

644

646

648

650

652

654

656

658

660NG without CD-A

-CD-A (DS2) NG

-CD-A (DS2) NG

-CD-A (DS2) NG

Wavele

ngth

[nm

]


0 2 4 6 8 10 12 14 16 18 201x10

7

2x107

3x107

4x107

5x107

6x107

7x107

8x107

9x107

Flu

ore

sce

nce

Inte

nsity [a.u

.]


NG without CD-A

-CD-A (DS2) NG

-CD-A (DS2) NG

-CD-A (DS2) NG

171

fluorescence intensity due to self-quenching inside the CD cavity. Here, the emission of NR:-

CD complex shows the highest intensity followed by NR complexed of -CD and -CD. The

results of the absorbance and emission spectra of NR:-CD and NR:-CD complexes are

contrary. This phenomenon is explainable by the TICT mechanism which is a non-radiative

deactivation pathway in case of NR. Here, the TICT state is changed caused by different structure

types of the CD complexes. According to the literature, the fluorescence of NR is influenced by

hydrogen bonds, in which an increase of the hydrogen bonds donating capacity of the medium

reduces significantly the quantum yield and life time [28]. The capped complex of NR on the

cavity of -CD is mainly fixed by hydrogen bonds. Outside of the cavity, the rotational freedom

of the diethylamino group is weakly hindered and the emission is less than in the inclusion

complex. Here, the formation of a 1:1 or 1:2 guest:host complex of NR with -CD which fixes

the dye by full inclusion in the cone, with less hydrogen bonds, prevents the rotational freedom of

the diethylamino group of NR in which the polarity is decreased inside the cavity. The

fluorescence emission increases and the non-radiative rate is retarded. NR in β-CD shows

fluorescence at 655 nm, whereas the emission intensities of -CD and -CD are measured at 660

nm and 662 nm. A dramatical shift in the emission wavelength by increase of the NR

concentration is not observed (Figure 7b).

In Figure 7c, the fluorescence intensities of NR loaded - and -CD nanogels at

wavelengths at 642-650 nm upon excitation of 585 nm are depicted. Fluorescence intensities of

NR loaded -CD nanogels are measured at the same wavelength as for NR complexed in -CD

nanogels but with excitation at 552 nm. The fluorescence intensity of NR in PVCL nanogels

without CD domains shows at first a strong increase followed by a decrease with increase of NR

concentration. In case of -CD nanogels, the emission intensity increases at the beginning and

decreases after further addition of NR continuously. γ-CD nanogels show increased fluorescence

intensity with rising NR concentration, at 12 µg/mL saturation of the dye uptake is observed. -

CD nanogels show similar to PVCL nanogels a strong increase at the beginning of the NR

addition followed by a slight decrease at 8 µg/mL NR. The fluorescence intensity of NR

containing nanogels is higher than NR complexed by natural CD. The corresponding wavelength

increases and decreases with the fluorescence intensity (Figure 7d). The highest wavelength shift

is shown after the uptake of NR by VCL nanogels from 642 nm to 654 nm back to 650 nm

172

followed by γ-CD nanogels with a shift from 646 nm to 656 nm. NR complexes with - and -

CD depict a slight wavelength shift between 646 nm and 650 nm. In comparison with NR

complexed by natural CD the wavelength of the fluorescence of NR in nanogels is lower. The

fluorescence intensity curves of -CD nanogels show similar saturation properties with an

increase of NR content compared to natural -CD. -CD nanogels present similar increasing and

decreasing fluorescence intensities compared to natural CD (Figure 7a,c).

Figure 8. Fluorescence intensities of NR complexed in nanogels with different γ-CD content in

dependence on the NR concentration.

The -CD content of the nanogels plays a big role on the uptake and fixation of NR

molecules. The same amount of NR in aqueous nanogel dispersions with 0.07 mmol (0.1 g), 0.21

mmol (0.3g) or 0.43 mmol (0.6 g) -CD content fluoresces in different intensities at different

wavelengths (Figure 8a). Here, the highest intensity which increases with the NR concentration is

observed for nanogels with a 0.07 mmol -CD content at 653 nm, the lowest for nanogels with a

0.43 mmol -CD content at 649 nm. For all NR concentrations, the curve of fluorescence

intensities flattens with increased γ-CD content. Figure 8b documents the wavelength shift of the

fluorescence emission in parallel to the fluorescence intensity increase which shifts to higher

wavelength with an increase of NR concentration and decreases with increased CD content.

a b

0 2 4 6 8 10 12 14 16 18 20 221.5x10

7

2.0x107

2.5x107

3.0x107

3.5x107

4.0x107

4.5x107

5.0x107

Flu

ore

scence Inte

nsity [a.u

.]





0 2 4 6 8 10 12 14 16 18 20 22640

642

644

646

648

650

652

654

656

658

660 0.07 mmol -CD-A (DS2) NG



Wa

ve

len

gth

[nm

]


173

7.3.4 Particle size of NR loaded α-, β- and γ-CD containing nanogels

The change of particle size was also investigated after the uptake of NR. Here, the

influence of the NR concentration on the hydrodynamic radius of the nanogels with different CD-

types was studied with 0.24 mmol (0.3 g) β-CD (DS2) and 0.21 mmol (0.3 g) γ-CD (DS2)

content. (Figure 9a-b).

Figure 9. Hydrodynamic radii (Rh) of β- (a) and γ-CD (b) nanogels with complexed NR.

-CD nanogels do not change the hydrodynamic radii with the uptake of NR. The radii remain

constant at 77 nm. By complexation of -CD nanogels, the hydrodynamic radius increased

slightly with the increase of NR concentration in nanogels from 133 to 149 nm. Here, it is

assumed that the increased amount of NR molecules in different complexation structures expands

the cross-linked polymeric structure of the whole particle. 1:1 Inclusion complexation of NR by

-CD domains does not depict such an expansion. The capped NR complex, which is favored by

-CD shows such swelling of the particle. Here, the NR guest molecule lies onto the CD cone and

complexes with 2 NR guest molecules and 1 CD container are possible as well. Lowly cross-

linked polymeric structures enable these expansions but a higher degree can handicap the dye

uptake and furthermore, the expansion of the polymeric particle structure.

a b

0 2 4 6 8 10 12 14 16 18 200

20

40

60

80

100

120

Rh

[n

m]



0 2 4 6 8 10 12 14 16 18 200

20

40

60

80

100

120

140

160

180


Rh

[n

m]


174

7.3.5 Solvatochromic interaction of NR by temperature response of nanogels

Temperature-sensitive nano- and microgels release their ingredients by shrinking at the

volume phase transition point (VPTT) [29]. By increase of temperature and the resulting release

of water, the loaded ingredients push out of the collapsing gel particle in the near environment.

Incorporated CD domains have the possibility to fix molecules by complexation [30]. The release

of NR should be reduced due to complexation by CD domains of nanogels. The ingredients

should be kept in the nanogels. Changes in temperature and water concentration in the

nanoparticles lead to changes in the interior environment of the nanogels and hence, of the guest

molecule e.g. due to restructuring of the complex or beginning of the release process. The CD

containing nanogels consist of a PVCL backbone which shows temperature sensitivity between

30-40 °C. The hydrodynamic radii of the nanogels shrink to approximately 50 % of their volume

(Figure 10). Investigations of NR in γ-CD nanogels are made at different temperatures to study

the behavior of the guest molecule during the collapse of the nanogels at the VPTT.

Figure 10a shows that nanogels without CD domains, which are just cross-linked with

BIS, do not change the characteristic dye absorption wavelength significantly during the

shrinking of the nanogel. Thus, the change in temperature and in water concentration has no

influence on the near environment of NR. On the other side, NR complexed by high γ-CD

content in nanogels also does not change the environment markedly as well which results in

slight increase of the absorbance at 545 nm to 549 nm at the VPTT (Figure 10b). These suggests

that the NR:γ-CD capped complexes are stable and fix NR by hydrogen bonds in the nanogels.

Interestingly, Figures 10 c-d show nanogels with 0.21 mmol covalently embedded γ-CD

domains with and without additional cross-linker. Here, changes in the absorption wavelength of

NR over more than 20 nm are detectable. Hence, the structure of the NR:γ-CD complex is

changed by shrinking of the nanogels caused by change of the inner environment.

175

Figure 10. Hydrodynamic radii and wavelengths of absorbance of NR loaded nanogels without

CD (a), with 0.43 mmol γ-CD (b), with 0.21 mmol γ-CD (c), with 0.21 mmol γ-CD + BIS (d)

measured at different temperatures.

Explanations for this phenomenon of color change caused by the shrinking process of the

nanogels with medium content of γ-CD are presented in detail by temperature-dependent studies

of the solvatochromic dye in Figure 11. Here, the UV-vis absorption maxima and the

corresponding wavelengths of NR in nanogels are depicted depending on the temperature and

compared with NR complexed in natural CD, incorporated by VCL nanogels without CD

domains or dissolved in mixtures of acetone and THF. The change of polarity in the environment

by change in temperature leads to a color change of NR due to the polarity dependence of NR.

c d

a b

20 24 28 32 36 40 44 48

100

120

140

160

180

200

220

240

Wavele

ngth

[nm

]

Temperature [°C]

540

550

560

570

580

590NR in NG without CD-A

Rh [nm

]

22 26 30 34 38 42 46 50

70

80

90

100

110

120

130

140

150

160

170

Wa

ve

len

gth

[n

m]

Temperature [°C]

540

550

560

570

580

590NR : 0.21 mmol CD-A (DS2) NG BIS

Rh [n

m]

20 24 28 32 36 40 44 4850

60

70

80

90

100

110

120

130

140

Wa

ve

len

gth

[n

m]

Temperature [°C]

550

560

570

580

NR : 0.21 mmol CD-A (DS2) NG

Rh [n

m]

20 24 28 32 36 40 44 4840

45

50

55

60

65

70

75

80

85

90

Wavele

ngth

[nm

]

Temperature [°C]

540

550

560

570

580

590NR : 0.47 mmol CD-A (DS2) NG

Rh [nm

]

176

Figure 11. Maxima of absorbance in the UV-vis spectra of NR in PVL nanogels (a), THF/Aceton

(b), natural - (c) and γ-CD (d) measured from 25-55 °C. Results of γ- CD containing nanogels

prepared with 0.21 mmol (e), 0.47 mmol CD-A (f) and with 0.21 mmol CD plus additional cross-

linker BIS measured from 25-55 °C (g) and back from 55-25 °C (h) are shown.

e f

c d

a b

g h

25 30 35 40 45 50 550.015

0.016

0.017

0.018

0.019

Wa

ve

len

gth

[nm

]

Temperature [°C]

540

550

560

570

580

590NR in NG without CD-A

Absorb

ance m

ax [a

.u.]

25 30 35 40 45 50 55

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

Wavele

ngth

[nm

]

Temperature [°C]

540

550

560

570

580

590NR0.21mmol -CD-A (DS2) NG BIS

Absorb

ance m

ax [a.u

.]

60 50 40 30 250.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

0.13

Wavele

ngth

[nm

]

Temperature [°C]

540

550

560

570

580

590NR0.21 mmol -CD-A (DS2) NG BIS

Absorb

ance m

ax [a.u

.]

25 30 35 40 45 50 550.30

0.31

0.32

0.33

0.34

0.35

0.36

Wavele

ngth

[nm

]

Temperature [°C]

540

550

560

570

580

590NR0.21 mmol -CD-A (DS2) NG

Absorb

ance m

ax [a.u

.]

25 30 35 40 45 50 55

0.52

0.54

0.56

0.58

0.60

Wavele

ngth

[nm

]

Temperature [°C]

540

550

560

570

580

590NR0.47 mmol -CD-A (DS2) NG

Absorb

ance m

ax [a.u

.]

25 30 35 40 45 50 550.29

0.30

0.31

0.32

0.33

0.34

0.35

Wa

ve

len

gth

[n

m]

Temperature [°C]

540

550

560

570

580

590NR in Aceton/THF (2:1)

Absorb

ance

max [a

.u.]

25 30 35 40 45 50 55

0.06

0.07

0.08

0.09

0.10

0.11

Wavele

ngth

[nm

]

Temperature [°C]

540

550

560

570

580

590NR:-CD

Absorb

ance m

ax [a.u

.]

25 30 35 40 45 50 55

0.184

0.188

0.192

0.196

0.200

Wavele

ngth

[nm

]

Temperature [°C]

540

550

560

570

580

590NR-CD

Absorb

ance m

ax [a.u

.]

177

In organic solvent using a mixture of acetone and THF (2:1), the NR absorbance and the

corresponding wavelength (540 nm) decrease slightly with an increase of temperature (Figure

11b). The hypsochromic shift indicates a slight decrease of the polarity of the solvent with rising

temperature.

The complexation of NR by -CD in water, which is characterized by an absorbance at

552 nm in the UV-vis spectrum, shows also a decrease of absorbance and a minimal

hypsochromic shift. With the increase of temperature, the polarity decreases of the environment.

Thus, the hydrogen bonds, which stabilize the capped complex, remain intact. Further interaction

with a related second CD ring leads to the formation of a 2:1 (CD:NR) complex. Hence, an

increase of the hydrophobic interaction of the fully covered NR molecule can be assumed (Figure

11d).

On the opposite, the NR:-CD complex, which is characterized by UV-vis absorbance at

576 nm also shows a minimal hypsochromic shift but an increase of the absorbance beginning at

45 °C. It occurs that the solubility of the complex increases, by breaking the interior ring of H-

bonds formed by secondary OH-groups of the CD-ring, which are typical for -CD and

responsible for the low solubility in water in contrast to - and -CD [30]. The breaking of H-

bonds enables the restructuring of the complex with an increase of the environmental

hydrophobicity of NR (Figure 11c).

NR dissolved in the cross-linked PVCL structure of nanogels without CD domains shows

a decrease of absorbance with an increase of temperature. The wavelength depicts a slight

bathochromic shift which suggests that the NR molecules are pushed out from the hydrophobic

nanogel structure by simultaneous release of water. During the release process and precipitation

of NR the solubility in water is reduced (Figure 11a).

In Figure 11f, NR which is complexed by 0.47 mmol -CD nanogels shows similar

behavior by increase of temperature like the natural -CD. The change in temperature is

characterized by a decrease in UV absorbance, but with a minimal bathochromic shift of the

wavelength from 545 nm to 549 nm. Thus, the increase of temperature decreases slightly the

hydrophilicity of the NR environment. This means, the hydrogen bonds of CD domains in the

nanogel, which stabilize the capped complex, are destroyed slowly by increase of the temperature

and shrinking of the nanogel. The slight red shift can be explained by additional inclusion

complex formation or by interactions with the PVCL backbone. But, the high number of hydroxy

178

groups from the high -CD content in nanogel may lead to further interaction of NR with

adjactent CD domains. Thus, the break of interactions between NR and hydroxy groups of one

CD host lead to a further capped complexation and interaction with other potential CDs. High CD

content in nanogels prevent the release and the destructuring of NR in nanogels.

However, NR complexed by 0.21 mmol -CD (DS2) domains in the nanogels shows a

strong change in absorbance intensity and wavelength upon increase of temperature and decrease

of particle size (Figure 11e). 20 µg/mL NR incorporated in 0.3 -CD nanogels show a strong red

shift reaching the VPTT of PVCL nanogels. Between 34-40 °C, the wavelength of the absorbance

maximum shifts from 552 nm to 576 nm. The absorbance intensity increases simultaneously.

Hence, the nanogel shrinks by release of water and the hydrogen bonds of the NR:-CD complex

are destroyed due to interior changes in the polymeric structure. In contrast to NR complexed by

nanogels with a high -CD content, the reduced complexation possibilities with other adjacent

CD domains lead to new orientation in the cross-linked PVCL structure. Due to the fact that the

absorbance increases and the wavelength shifts to 576 nm, which is comparable with

characteristic absorbance of the 1:1 -CD complex, it can be expected that NR guest molecules

are pushed into the cone of -CD during shrinking process. Thus, the formation of a 1:1 complex

is preferred.

The observed color effect is reversible upon decrease of temperature. From 60 °C to 30

°C a strong hypsochromic effect (from 575 nm to 550 nm) is detectable with a simultaneous

decrease in absorbance (Figure 11g-h). At the VPTT, the structure of the totally collapsed

nanogel is opened and enables the uptake of water by the formation of hydrogen bondings

between H2O molecules and the PVCL backbone. During the uptake of water from the

environment, water molecules push out the guest molecule from the -CD ring and enable the

new formation of a more stable capped complex inside the nanogel stabilized by hydrogen bonds.

179

7.3.6 Nile Red loaded nanogels coated on polyester fabrics

NR loaded nanogels were applied onto polyester fibers to investigate the coating process

on synthetic fiber materials which can find many application possibilities in different fields e.g.

as color sensor for biological systems. Here, the application was made by dip-coating and self-

fixation due to physical self-bonding due to the typical gel character of the nanogels. The coating

was controlled in dependence on time and studied in periods of 15, 30, 45, 60 min and finished

after 120 min. Figure 12 (left) shows the fabrics after dip-coating of -CD nanogels (10 mg/mL)

loaded with 20 µg/mL NR solution at RT. Here, NR:-CD nanogel modified fibers have got a

red-purple color. The color of the samples after coating for 15 min is less strong and increases

with the coating time. After 45 min the color difference is not visually detectable any more. On

the nanoscale, the microscopical images in Figure 12 (right) show similar results. Here,

nanoparticles with a small size of approximately 50-100 nm are distributed homogenously on the

polyester fibers. The round nanogels glue by physical self-bonding on the fiber surface.

Comparisons of the FESEM images reflect the colored images of optical photography in Figure

12 (left). Here, fibers coated for 15 min show lower particle concentrations on the fibers than

fibers coated by dip-coating in 30 min. The particle concentration increases with the coating time.

180

Figure 12. Photography (right) and FESEM images (left) of polyester fabrics coated with 20

µg/mL NR loaded -CD nanogels (10 mg/mL) in dependence on time and studied in periods of 0

(a), 15 (b), 30 (c), 45 (d), 60 (e) min and finished after 120 (f) min at RT.

a

c

b 15 min

30 min

0 min

d 45 min

e 60 min

f 120 min

181

The application of NR loaded nanogels to polyester fabrics during the dip coating process

was also studied by color measurements. The adsorption of NR nanogels on polyester was

followed by diffuse reflexion measurements in dependence on time. Here, the color of polyester

fabrics modified with -CD nanogels with 20 µg/mL loaded NR was measured at different

coating times. The results are shown in the diagram of Figure 13a. Furthermore, the principal of

the color measurement is depicted in Figure 13b. It was found that the polyester turns redder (“a”

value is more positive) and bluer (“b” value becomes more negative) after coating. Furthermore,

the sample becomes darker in comparison to the white pure polyester fabric (“L” value

decreases). Intensities of the red and blue colors, and furthermore, the lightness of the fibers

decrease with the coating time. The saturation curves show that after 30 min of coating a

saturation process takes place and the increase of the color intensity flattens. Thus, the physical

self-bonding of the nanogels to polyester during dip-coating starts directly at the beginning of the

dipping process and gets slower after 30 min. The difference in color intensity of fabrics coated

for 60 min and those coated for 120 min is minimal. Thus, further coating does not lead to higher

efficiency in the color intensity. The color measurements of the coated polyester fabrics are in

good agreement with the optical and the FESEM images given in Figure 12.

Figure 13. Color measurements of polyester fabrics coated with NR loaded -CD nanogels at RT

in different application times (a). Principal of color determination by quantitative characterization

of the lightness (L values), the red/green “a”-values and the yellow/blue “b”-values of the sample

(according to CIELAB).

ba

0 20 40 60 80 100 120-8

-6

-4

-2

0

2

4

6

8

10

12

14

16

a,

b

Dipcoating pro time [min]

a

b

78

79

80

81

82

83

84

85

86

87

88 L

L

182

7.3.6 Temperature sensitivity of applied Nile Red nanogels

The temperature sensitivity of the polyester fabrics modified with NR loaded -CD

nanogels was tested by color measurements at different temperatures. Here, the polyester fabrics

coated with maximally NR loaded nanogels (20 µg/mL) were heated up to 80 °C. The color

change was measured in steps of 5 °C as shown in Figure 14. In the range of 35 to 45 °C the “a”

values (Figure 14c) increase and the “b” values (Figure 14b) decrease. That means the modified

polyester fabrics become redder and bluer. Thus, incorporated water starts to release from the

nanogel and restructuring of the complexed NR in the gel particles change takes place. The

environment of NR becomes more hydrophobic and hydrogen bondings consist only between NR

and the -CD domains. This means interior -CD domains complex NR. The hydrophobic coating

caused by shrinking of the nanogel during water release keeps NR in the nanogels. Then, from 50

to 65 °C fabrics become brighter and the “a” and “b” values increase markedly. Hence, the color

becomes less red and less blue. NR and the whole polymeric nanogel structure dried on the

fabrics and the surface of the particle becomes whiter. The non-transparent surface of nanogels

covers the NR in the core and results in the increased brightness of the fibers. This can be seen in

an increase of the “L” values (Figure 14a). At 65 °C, the fibers become redder as demonstrated

by a strong increase of the “a” values and a slight increase of “b” values. It seems that NR

precipitates as red solid inside the polymeric structure.

By cooling from 80 °C back to 25 °C, the red color intensity decreases again (Figure 14c

left) together with a further decrease of the blue color (Figure 14b left). Hence, dry nanogels with

strong complexed NR applied on the fabric does not take up water directly from the environment.

Thus, the process is not reversible unlike the NR behavior in aqueous -CD nanogels studied in

water.

183

Figure 14. Color measurements of polyester fabric coated with NR loaded nanogels at different

temperatures are described in 5 °C steps by the “L” (a), “b” (b) and “a” (c) values.

30 40 50 60 70 80

12.5

13.0

13.5

14.0

14.5

15.0

15.5

16.0

Temperature [°C]

a

a

blue

yellow

black

white

red

green

c

b

a

75 65 55 45 35 2512.5

13.0

13.5

14.0

14.5

15.0

15.5

16.0

Temperature [°C]

a

a

75 65 55 45 35 2581.0

81.5

82.0

82.5

83.0

83.5

84.0

Temperature [°C]

L

L

75 65 55 45 35 25-7.0

-6.5

-6.0

-5.5

-5.0

-4.5

-4.0

-3.5

-3.0

b

Temperature [°C]

b

30 40 50 60 70 8081.0

81.5

82.0

82.5

83.0

83.5

Temperature [°C]

L

L

30 40 50 60 70 80

-7.0

-6.5

-6.0

-5.5

-5.0

-4.5

-4.0

-3.5

-3.0

b

Temperature [°C]

b

184

7.4 Conclusion

Here, a series of nanogels prepared by cross-linking of different content of -CD ( DS2)

acrylate with vinylcaprolactam were synthesized and compared with the corresponding - and

-CD nanogels. The properties of the CD nanogels were studied with regard to their uptake

properties and complexation behavior by using the solvatochromic dye nile red (NR). Nanogels

without CD domains or with -CD units showed a decreased dye uptake, while - and -CD

nanogels presented an increased dye uptake. Furthermore, the results showed that the uptake of

NR increases with rising CD content in nanogels. A rising degree of cross-linking by the use of

an additional cross-linker N,N-methylene-bis-acryamide (BIS) reduced the uptake capacities of

the CD nanogels. The fluorescence emission rate of NR could be controlled and was influenced

by different structures of the CD complexes. The NR inclusion complex, which was preferred

by -CD shows high fluorescence because of the more hydrophobic environment of the guest

molecules. -CD domains fixed NR by formation of hydrogen bonds, which supports the twisted

intermolecular charge transfer (TICT) and non-radiative rate. Furthermore, reversible

restructuring of NR in -CD nanogels was observed by shrinking of the -CD nanogels in

aqueous dispersion at the volume phase transition temperature (32-37 °C), which was

characterized by a strong red shift in absorbance spectrum. The application onto polyester

fabrics showed good adhesion and fixation of the dye loaded nanogels by physical self-bonding.

The wet coated NR nanogels on the fabrics showed also irreversible change in color upon

increase of temperature and release of water.

185

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187

Chapter 8

Surfactant-free preparation of nanogels with high functional β-

cyclodextrin contentA

Abstract: The preparation of ultrafine (Rh=50-150 nm) nanogels through surfactant-free

condensation of reactive prepolymers with β-cyclodextrin (β-CD) in water was presented. These

nanogels possess a maximum content of 60 wt.-% functional β-CD that can form inclusion

complexes as demonstrated by dye sorption with phenolphthalein. Aside of this extremely high

uptake capacity to hydrophobic molecules, the nanogels also show good adhesion to surfaces in

homogeneous distribution with size of Rh of 25 nm under dry conditions.

Keywords: nanogels, cyclodextrin, reactive prepolymers, host-guest-systems

A. Reproduced with permission from Kettel, M. J.; Hildebrandt, H.; Schaefer, K.; Moeller, M.; Groll, J., Tenside

free preparation of nanogels with high functional β-cyclodextrin content, ACS Nano, 2012, 6(9), 8087-8093.

Copyright American Chemical Society.

O

RR

R

O

O

R

RR

O

OR

R

R

O

O

RR

R

OO

R

R

R

O

OR

RR

O

O

R

R

R

O

O

RR

R

O

O

R

RR

O

OR

R

R

O

O

RR

R

OO

R

R

R

O

OR

RR

O

O

R

R

R

O

Phenolphthalein -pink-

Phenolphthalein in β-Cyclodextrin

-colorless-

β-CyclodextrinPhenolphthalein-

β-Cyclodextrin-Complex

pH= 10.5

β-Cyclodextrin-sP(EO-stat-PO)-Nanogel

Phenolphthalein loaded β-Cyclodextrin-

sP(EO-stat-PO)-Nanogel

OO

O

O

OO

COO-

- - -

pH 10.5

188

8.1 Introduction

Cross-linked polymeric hydrogel particles, called nano- or microgels, are functional

materials that may be prepared in a wide range of chemical compositions [1,2]. These colloidal

polymer networks exhibit pronounced swelling in water, which allows transport processes into

the particles and makes them especially interesting for applications such as sequestration or

release [3,4]. For that, specific binding activities have to be embedded into the nanogels.

Cyclodextrins (CDs) are interesting and versatile binding motives and are well-known for

the formation of supramolecular inclusion complexes with many organic molecules [5]. The

complexation of sensitive or volatile ingredients, for example, drugs, pharmaceutical products,

flavoring agents, perfumes, and insecticides are of particular interest [6,7]. Natural CDs consist of

six, seven, or eight D-glucose units which are α-glycosidic linked to a cyclic oligomer and named

R, β, or γ-CD, respectively. Since the interior hydrophilic part of the torus-shaped CD facilitates

complexation with the guest molecules, the reactive hydroxy groups on the outer part of the

molecules can be used for modification, functionalization, and copolymerization. This way, CDs

have been used as cross-linker for the creation of three-dimensional bulk hydrogels [8].

Nanogels offer an increased surface area in combination with shorter diffusion lengths

which leads to a higher CD accessibility and faster complexation as compared to three-

dimensional bulk gels [9]. Moreover, they can be applied from aqueous solution to form films

and coatings. Radical polymerization techniques are usually applied for the preparation of nano-

and microgels [10-13].

Accordingly, CDs containing polymeric particles and microgels have been prepared by

radical copolymerization with methyl methacrylate (MMA) in organic solution, with N-

isopropylacrylamide (NIPAm) by mini-emulsion via addition of surfactant, and by precipitation

polymerization with N-vinylcaprolactam (VCL) in aqueous solution [14-17]. Another method to

prepare nanogels with γ-CD or hydroxypropyl-β-CD is by an emulsification/solvent evaporation

process. Here, the aqueous phase consists of a fix CD concentration of 20% (w/w) with or

without hydroxypropyl methylcellulose (HPMC) or agar at various concentrations.

Ethyleneglycol diglycidyl ether (EGDE) acts as cross-linking agent and is essential for the

formation of nanogels [18]. Polydisperse CD-nanoparticles in water and ethanol have also been

obtained by self-assembly of a water insoluble polymer, which was made by the reaction of CD

and toluene-2,4-diisocanate (TDI) in organic solvent dimethyl sulfoxide (DMSO) [19,20]. The

189

preparation of CD containing nanogels in water without direct polymerization from monomers is

often performed by exploiting the complexation ability of CDs, so that the CD molecules act as

cross-linkers themselves. This way, the majority of CD molecules is blocked for uptake of guest

molecules into the nanogels, and the overall complexation capacity is moderate. It has been

recently demonstrated a preparation technique for nanogels based on the addition reaction among

functional hydrophilic prepolymers [21].

Here it is presented a new method to design nanogels with high β-CD content via

polyaddition directly in aqueous surfactant-free solution at room temperature (RT) without the

use of organic solvents. The method was optimized to yield nanogels with a hydrodynamic radius

between 50 and 150 nm. Owing to the choice of prepolymers and nanogel preparation technique,

the nanogels contain up to 60 wt.-% β-CD that is functional and can take up guest molecules as

demonstrated with the sorption of phenolphthalein. Finally, also film formation of these nanogels

is demonstrated, enabling the preparation of ultrathin films with strong loading and release

capacity for hydrophobic guest molecules.

8.2 Experimental

8.2.1 Preparation of β-CD containing nanogels

Preparation of β-CD Containing Nanogels. β-CD (Wacker) (0.75 g, 0.66 mmol) was dried

at 80 °C for about 48 h and dissolved in deionized water (50 mL). Six-arm NCO-terminated star

P(EO-stat-PO) prepolymers were synthesized using published procedures [22]. An aqueous β-CD

mixture was added to (0.75 g, 0.06 mmol) NCO-terminated prepolymer under stirring. The

aqueous phase was stirred continually at 300 rpm over 24 h at RT using a glass paddle connected

to an overhead stirrer (all reaction conditions in Table 1).

The aqueous reactant mixture got a clear nanogel dispersion, which was purified by dialysis in

water using a dialysis tube with MWCO of 12 kDa (ZelluTrans-12,0S 45 mm. MWCO: Nominal

12000-14000; Carl Roth GmbH, Karlsruhe). The water (2 L) was changed after 24 and 48 h;

dialyses were stopped after 96 h.

Yield: 40-70%.

190

Table 1. Reaction recipe and conditions for the preparation of nanogels with β-CD by constant

stirring rate of 300 rpm and RT.

8.2.2 Analysis of the reaction

IR spectra were obtained using an FTIR spectrometer: Nexus 470 (Thermo Nicolet)

(spectral disintegration of 8 cm-1

). Nanogel dispersions were dried by liophilization and

privileged studied in KBr pellets.

IR (KBr): ν/cm-1

= 3356 (m, OH), 2870 (m), 1720 (m, vs C=O), 1677 (m, N-H), 1638 (m, OH),

1458 (m), 1350 (m), 1301 (m), 1248 (m), 1147 (s), 1101 (s), 1081 (s), 1032 (s, C-O-C), 1004 (s),

945 (m), 863 (m).

8.2.3 Particle characterization

Analysis of particle size was made by dynamic light scattering measurements (DLS),

using a Nano Zetasizer (Malvern) spectrometer. The spectrometer consists of a HeNe Laser (λ0 =

633 nm). Back scattering light was detected using an angle of 173°. Hydrodynamic particle

radius and the particle distribution of the hydrodynamic radius were determined. All DLS

measurements were made at 25 °C.

β-CD

1135 g/mol

[g],(mmol)

sP(EO-stat-PO)

12000 g/mol

[g],(mmol)

wt.-ratio

CD/

sP(EO- stat-PO)

mol-ratio

CD/

sP(EO-stat-PO)

water

[mL]

educts/

water

[wt.-%], (g/mL)

- 0.600 (0.05) 0/1 0/1 20 3 (0.03)

0.375 (0.33) 1.125 (0.09) 1/3 4/1 50 3 (0.03)

0.500 (0.44) 1.000 (0.08) 1/2 5/1 50 3 (0.03)

0.750 (0.66) 0.750 (0.06) 1/1 11/1 50 3 (0.03)

1.000 (0.88) 0.500 (0.04) 2/1 21/1 50 3 (0.03)

1.125 (0.99) 0.375 (0.03) 3/1 32/1 50 3 (0.03)

0.250 (0.22) 0.250 (0.02) 1/1 11/1 10 5 (0.05)

0.1125 (0.1) 0.375 (0.03) 1/3 4/1 5 10 (0.1)

191

FESEM and Cryo-FESEM electron microscopic analyses were made using a model S-

4800 field emission scanning electron microscope (Hitachi) with a high disintegration and cryo-

function. Swollen particles in aqueous solution were studied by cryo-scanning electron

microscopy. A droplet was taken from an aqueous particle dispersion and after shock freezing in

liquid nitrogen the freeze droplet was cut and the surface of the cut was sublimated in vacuum.

The morphology of the swollen nanogels was studied at a temperature of -167 °C, accelerating

voltage of 1 kV, and 8mm disintegration. Dry nanogels are studied after coating onto aluminum

surface. The Al carrier was dipped 15 min into the aqueous particle dispersion. After being dried

at RT and by vacuum, samples coated with nanogels are studied by FESEM at RT and an

accelerating voltage of 1 KV and 8 mm disintegration. Particle diameter and radius are

determined by enclosed software.

8.2.4 Preparation and analysis of Phenolphthalein Complexes

A stock solution of 1% phenolphthalein (Wacker) in ethanol was made, and aliquots of

2.50 mL were dropped into 250 mL of 0.1 M sodium hydroxide to obtain a 0.25 mmol/L alkali

phenolphthalein solution. All aqueous stock solutions were freshly prepared and run within 12 h

to ensure that absorbance changes due to any instability of phenolphthalein did not contribute to

experimental artifacts. The β-CD stock solution was prepared in water with a β-CD concentration

of 8.8mmol/L. Aliquots of β-CD stock solution were diluted with water to different

concentrations with an end volume of 15 mL. Different concentrated β-CD solutions were titrated

by 0.25 mmol/L alkali phenolphthalein stock solution under stirring until the transition point

from colorless to slight pink. Dispersions of nanogels containing β-CD were used as received

after dialysis and diluted in water. A 10mL aliquot of each prepared nanogel dispersion with

known particle concentration was titrated by 0.25 mmol/L alkali phenolphthalein stock solution

under stirring until the transition point from colorless to slight pink. For all volume titration a

buret was used from Brand Duran; DIN, with a total volume of 50 mL, ± 0.05 mL, Ex +30s.

UV-vis-absorptions spectra were recorded by Varian Cary 100 UV-vis spectrophotometer of Fa.

Varian, Darmstadt. A silica cuvette with a coating thickness of 1 cm was used. Water attended as

reference.

192


Amphiphilic 12 kDa six-arm isocyanate NCO-terminated star-shaped poly(ethylene

oxide-stat-propylene oxide) with 80% EO content (NCO-sP(EO-stat-PO)) [22] was used as

reactive polymeric cross-linker for β-CD (Figure 1a). In water, isocyanates hydrolyze to amine

groups that further react with other NCO groups to urea bridges between the prepolymers. This

process leads to formation of a three-dimensional polymer network and may be used for the

preparation of hydrogels, [23] in which also biopolymers that possess nucleophilic groups such as

hyaluronic acid may be embedded resulting in biopolymerpolymer hybrid hydrogels [24]. Here,

β-CD was added to aqueous solutions of NCO-sP(EO-stat-PO). Hence, aside the hydrolysis-

aminolysis cross-linking reaction described above, reaction between the NCO groups and the

hydroxy groups of β-CD leads to cross-linked polyurethane networks. Since alcoholysis is

kinetically favored compared to hydrolysis, the cross-linking via β-CD is the main network-

forming mechanism.

With this method, β-CD-nanogels may be prepared by a simple one-pot preparation

method in water without the use of surfactants (Figure 1b). This process strongly depends on

reaction parameters. The influence of the stoichiometric ratio of the reactant and the

concentration has been examined at a constant stirring rate of 300 rpm at room temperature

(Table 1). After 24 h reaction and subsequent dialysis against water, between 40 and 70 wt.-% of

educts remained as cross-linked nanogels in solution. Generally, the adoption of sP(EO-stat-PO)

in a higher molar ratio to β-CD in the reaction mixture leads to a higher yield of nanogels after

dialysis.

Infrared-spectroscopy of freeze-dried purified nanogels shows a higher β-CD content with

an increase in the stoichiometric ratio of β-CD to sP(EO-stat-PO) in the reaction mixture. This is

evidenced by increasing intensity of the typical β-CD hydroxy bands (3350 cm-1

and 1680 cm-1

)

and the characteristic stretching vibration peak of C-O-C from β-CD (1032 cm-1

) [25]. The

absence of a NCO band at 2265 cm-1

and the presence of the typical band for urea (1690 cm-1

)

and urethane (1720 cm-1

) confirm chemical cross-linking (Figure 1c).

193

Figure 1. Reaction Scheme of β-CD with NCO-sP(EO-stat-PO) to urethane and urea cross-linked

nanogels (a). Schematic structure of aqueous nanogels with active -CD domains based on

sP(EO-stat-PO) (b). IR-spectra of nanogels with increasing β-CD content (c).

4000 3500 3000 2500 2000 1500 1000

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4/1

5/1

11/1

21/1

tra

nsm

issio

n

wavenumber [cm-1]

4/1 CD/sP(EO-stat-PO)





32/1

H2O

1.

H2O R2 NH2

R2 NH2 R2

HN

HN

R2O

R2 N C O

R2 N C OR1 OH R2

HN O R1

O2.

R1 = - CD

= sP(EO-stat-PO)

-Cyclodextrin (CD)

R2

R2 N C O

OR O

R

OR

OR

OR

OR

OO

O NH

O

NCO

[Propylenoxid] [Ethylenoxid]

NCO terminated sP(EO-stat-PO)

Isophorone Isocyanate

R =

n n

O

OHHO

HO

O

O

HO

HO

OH

O

OHO

HO OHO

O

HO

OH

OH

O

O

OH OH

HO

O

O

OH

OH

HOO

OOH

OH

HO

O3.

Urethane and Urea Cross-linked Nanogels

a

b c 3350

1690

1720 1032

Structure of aqueous nanogel particle

with active -CD domains

194

The stability of the prepared nanogels was characterized at 3 wt.-% concentration in

aqueous dispersion by dynamic light scattering (DLS; Figure 2a). Low β-CD content in the

nanogels (β-CD/sP(EO-stat-PO) molar ratio of 4/1) leads to particle dispersions with a

multimodal, large size distribution over length scales. This corresponds to the multimodal size

distribution curve of nanogels prepared from pure sP(EO-stat-PO). By increasing the β-CD

content to β-CD/sP(EO-stat-PO) molar ratios of 11/1, 22/1, and 32/1, uniform nanogels with a

narrow size distribution and an increasing hydrodynamic radius of Rh = 118.6 ± 12.8 nm (11/1) to

Rh = 175.9 ± 5.3 nm (21/1) and further to Rh = 218.8 ± 12.5 nm (32/1) are obtained. Obviously,

β-CD supports and stabilizes the formation of more uniform structures and subsequently

chemically cross-links the nanogels.

Optical stability of nanogel dispersion with higher β-CD content is given over months in

low concentrated aqueous solution. DLS of aqueous nanogels (11/1 molar ratio β-CD/sP(EO-stat-

PO) in water at RT reflects a monomodal size distribution with Rh = 118.1 nm. After 5 d dialysis

Rh remains constant at 118.6 nm, indicating stability of the nanogels (Figure 2b). Cryo FESEM

images of nanogels in swollen state prepared in 3 wt.-% aqueous dispersion (Figure 2c) reveal

uniform particle morphology and confirm the nanogel size of Rh = 50-150 nm as determined by

DLS (Rh = 118.6 (11/1)). For studies of collapsed particles, aluminum slides are dip-coated in

nanogel dispersions for 15 min, dried, and investigated by FESEM (Figure 2d). In the dry state,

the nanogels shrink to an average diameter of 50nm (Rh = 25 nm). The calculated volumetric

(de)-swelling ratio defined as (RhD/Rh

S)3 (Rh

D = hydrodynamic radius under dry conditions; Rh

S =

hydrodynamic radius for maximally swollen nanogels in water) averages 0.009 [26]. Thus, the

nanogels exhibit excellent swelling properties in water up to more than four times their own

particle size from the collapsed state. Furthermore, they display a good adhesion to the surface

and a homogeneous distribution. At higher concentrations (5 wt.-%), nanogels show an affinity

for agglomeration (Supporting Information, Figure S1a). This may be explained by formation of

inclusion complexes between β-CD and hydrophobic moieties of the prepolymer that are

available at the nanogels surface, as multiple interactions between single nanogels are more

probable at higher concentrations. Such β-CD mediated interfacial phenomena between hydrogels

have recently been demonstrated to physically “glue” macroscopic hydrogels [27]. At very high

concentration (10 wt.-%) 3-dimensionally cross-linked bulk hydrogels are formed (Supporting

Information, Figure S1b).

195

Figure 2. DLS-measurements of nanogels with β-CD units in aqueous dispersion at 25 °C; size

distributions of nanogels with different β-CD content (a) and size distribution of nanogels (11 β-

CD/ 1 sP(EO-stat-PO)) in aqueous dispersion 1 h after reaction and after 5 d dialysis (b). Cryo-

FESEM images of swollen nanogels containing β-CD in ratio of (11 β-CD/1 sP(EO-stat-PO))

with a diameter of 100-300 nm (Rh=50-150 nm) made in 3 wt.-% aqueous dispersion (c). FESEM

images of the nanogels (11 β-CD/ 1 sP(EO-stat-PO)) made in 3 wt.-% aqueous dispersion with an

average diameter of 50 nm (Rh=25) in the dry collapsed state coated on aluminum (d).

10 100 1000

0

4

8

12

16

20

24

28

32

1h after reaction

5d after dialysis

Inte

nsity

Rh [nm]1 10 100 1000

0

4

8

12

16

20

24

28

32

Inte

nsity

Rh [nm]

sP(EO-stat-PO)





c d

a b

196

The number and availability of inclusion sites of β-CD in nanogels are crucial for their

sorption properties in water. β-CDs which are occupied and bound in rotaxane-like structures are

not accessible for host molecules. An easy way to characterize the amount of functional β-CD in

nanogels is the dye sorption method, for example by using the complexation of phenolphthalein

by β-CD. Phenolphthalein shows an absorbance at 553 nm in alkaline aqueous solution (pH 10.5)

due to delocalization of the π-electrons. Upon complexation of phenolphthalein in β-CD, this

delocalization is disturbed by lactonization of the ionized form. As a consequence, the

absorbance changes and the pink color disappears [28,29]. The creation of colorless 1:1

complexes in alkaline aqueous solution can thus be used for the quantitative determination of β-

CD-content in polymeric materials [30-32].

Table 2. Determination of CD content in nanogel dispersions after dialysis by titration of

Phenolphthalein in aqueous solution at pH 10.5.

Supporting Information, Figure S2 shows a qualitative proof for complexation of

phenolphthalein by β-CD nanogels in alkaline aqueous solution. For quantitative determination of

active β-CD in nanogels in comparison to free β-CD, the volumetric titration of phenolphthalein

in aqueous dispersion at pH 10.5 has been performed.

mol-ratio

CD/sP(EO-stat-PO)

nanogel conc.

[mg/mL]

Phenolphthalein solution

[mL/10mL]

CD content

[mg/mL]

CD content

[µmol/mL]

4/1 8.18 0.45±0.10 0.14±0.05 0.13±0.04

5/1 9.71 1.13±0.15 0.72±0.17 0.63±0.15

11/1 8.37 1.53±0.60 1.22±0.08 1.07±0.07

21/1 7.00 1.98±0.10 1.90±0.16 1.68±0.14

32/1 5.40 2.70±0.23 3.30±0.25 2.91±0.22

197

Figure 3. Amount of Phenolphthalein-titrated β-CD in nanogel dispersions in comparison with

the amount of β-CD added during preparation of nanogels in different molar ratios to sP(EO-stat-

PO) (a). Active β-CD content [wt.-%] in prepared nanogels based on complexion studies

compared with the theoretical content in the reaction mixture (b). Yield of prepared nanogels

after dialysis based on liophilisation and yield of active β-CD based on complexation studies with

respect to the educts (c).

2 3 4 5 6 7 8 9 10 11

0

1

2

3

4

32/1

21/1

11/1

5/1

titra

ted

C

D in

dis

pe

rsio

n [µ

mo

l/mL

]

CD added to the reaction mixture [µmol/mL]

4/1

4/1 5/1 11/1 21/1 32/10

10

20

30

40

50

60

70

Yie

ld w

ith r

esp

ect

to the e

duct

s [%

]

-CD/sP(EO-stat-PO) in the reaction mixture

nanogel

functional CD

4/1 5/1 11/1 21/1 32/1

0

10

20

30

40

50

60

70

80

functional CD

therotical CD

C

D c

on

ten

t [w

t.-%

]

CD/sP(EO-stat-PO) in reaction mixture

b

c

a

198

Aqueous solutions of free β-CD in different concentrations have been titrated in water at

pH 10.5 until the transition point to obtain a calibration curve (Supporting Information, Figure

S3). Afterwards, nanogels containing β-CD have been titrated until the transition point, and the

results were compared to the calibration curve obtained from free β-CD. A higher amount of β-

CD added during preparation of the nanogels also results in a higher amount of functional β-CD

in the nanogels dispersion, with the highest concentration of active β-CD in the case of a molar

ratio of 32/1 β-CD/sP(EO-stat-PO) corresponding to a 3.3 mg/mL solution of free β-CD (Table 2

and Figure3a).

Correlating this value to the expected amount of β-CDs per nanogel reveals, in agreement

with the IR-data, an increased β-CD content of nanogels from molar ratio 4/1 to 32/1 β-

CD/sP(EO-stat-PO) (Figure 3b). Nanogels prepared with a higher β-CD ratio (32/1 CD/sP(EO-

stat-PO)) also show a higher content of incorporated, active β-CD than nanogels prepared with

lower ratio (11/1 and 4/1 β-CD/sP(EO-stat-PO)). The maximal content of functional β-CD is

reached with the molar ratio 32/1 β-CD/sP(EO-stat-PO) with about 60 wt.-% functional β-CD in

the nanogels. Hence, 60 wt.-% of each nanogel in this sample are β-CD that are able to take up

guest molecules. However, the yield of the nanogel preparation process, meaning the amount of

obtained nanogels with respect to the educts, increases with decreasing amount of β-CD in the

reaction mixture. This trend is accompanied with a decreasing amount of functional β-CD in the

nanogels (Figure 3c). For the nanogels prepared with the molar ratio 32/1 β-CD/sP(EO-stat-PO),

30 wt.-% of the β-CD added to the reaction mixture for nanogel preparation is incorporated into

the gels in a functional way. With decreasing content of β-CD in the reaction mixture, the yield of

nanogels with respect to the educts increases, while the amount of functional β-CD in the

nanogels as well as the yield of functional β-CD in the nanogels with respect to the amount added

to the reaction mixture decreases. Taking into account the IR results that show a continuous

decrease of β-CD content with decreasing amount of β-CD in the reaction mixture, it is

concluded that, although some β-CD may be bound in the nanogels in a nonfunctional manner,

most of the nondetectible β-CD is not integrated into the nanogels during preparation and thus

removed during dialysis.

199

8.4 Conclusion

In conclusion, it was presented the preparation of β-CD containing nanogels through an

aqueous surfactant-free process by reaction between NCO-functional hydrophilic prepolymers

and β-CD. This process allows production of uniform nanogels with narrow size distribution and

a hydrodynamic radius of 50-150 nm. Coatings on aluminum showed nanogels with a good

adhesion to surfaces in homogeneous distribution with size of Rh = 25 nm under dry conditions.

The efficiency and the advantages of the nanogels in respect to the uptake of guest molecules

were investigated by dye sorption method with phenolphthalein. This method enabled the

quantitative determination of complexable β-CD units in colloidal polymeric material. Taking the

results together, low β-CD content in the reaction mixture leads to a high yield of nanogels, but

with a large, multimodal particle size distribution and low complexation properties. With the use

of a high β-CD amount in the reaction mixture, nanogels with small uniform particle size and

high uptake properties to hydrophobic molecules can be produced, with a maximum content of 60

wt.-% active β-CD per nanogel. Owing to this extremely high content of active β-CD in aqueous

dispersion, these nanogels are very promising materials for a variety of applications. Ongoing

research focuses on the application of these materials for technical surface modification such as

textiles as well as sustained release in agriculture and drug delivery.

200

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22. Goetz, H.; Beginn, U.; Bartelink, C. F.; Gruenbauer, H. J. M.; Moeller, M., Preparation of

isophoronediisocyanate terminated starpolyethers, Macromol. Mater. En. 2002, 287, 223-230.

23. Dalton, P. D.; Hostert, C.; Albrecht, K.; Moeller, M.; Groll, J., Structure and Properties of Urea-Crosslinked

Star Poly[(ethylene oxide)-ran-(propylene oxide)] Hydrogels, Macromol. Biosci. 2008, 8, 923-931.

24. Dhanasingh, A.; Salber, J.; Moeller, M.; Groll, J., Tailored hyaluronic acid hydrogels through hydrophilic

prepolymer cross-linkers, Soft Matters 2010, 6, 618-629.





27. Harada, A.; Kobayashi, R.; Takashima, Y.; Hashidzume, A.; Yamaguchi, H., Macroscopic self-assembly

through molecular recognition, Nat. Chem. 2011, 3, 34-37.



29. Buvári, A.; Barcza, L., Complex formation of phenolphthalein and some related compounds with β-cyclodextrin,

J. Chem. Soc. Perkin Trans. 1988, 2, 1687-1690.

30. Topchieva, I. N.; Spiridonov, V. V.; Kalashnikov, Ph. A.; Kurganov, B. I., The detection of cyclodextrin self-

association by titration with dyes, Colloid Journal 2006, 68, 98-105.



32. Trellenkamp, T.; Ritter, H., Poly(N-vinylpyrrolidone) Bearing Covalently Attached Cyclodextrin via Click-


2010, 43, 5538-5543.

202

8.6 Supporting Informations

Figure S1. Cryo-FESEM Image of agglomerated nanogels in concentrate dispersion prepared in

5 wt.-% reaction mixture with average diameter of 500 nm (a). Surface of 3 dimensional bulk

sP(EO-stat-PO) hydrogel containing β-CD made in 10 wt.-% aqueous reaction mixture (b).

Figure S2. UV/Vis spectra of 1 mL phenolphthalein in alkaline (pH 10.5) aqueous solution, in

presents of free β-CD and nanogels with β-CD in different molar ratios to sP(EO-stat-PO). (a).

Photography of Phenolphthalein containing aqueous samples (pH 10.5) with β-CD nanogels with

a molar ratio of 11/1 CD/sP(EO-stat-PO) (right) and without β-CD nanogels (left) (b).

a b

500 520 540 560 580 6000.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

Absorb

ance

Wavelength [nm]

aqueous solution

free CD



Phenolphthalein/

waterPhenolphthalein/

β-CD-nanogel

ba553 nm

203

Figure S3. Calibration curve of natural β-CD was obtained by Phenolphthalein titration of

different concentrated aqueous β-CD solutions at pH 10.5.

y = 5.7333x1.7624

R2 = 0.9972

0

5

10

15

20

25

30

35

40

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25

Usage of phenolphthalein solution [ml]

β-C

D [

mg

/10

ml]

β-CD straight calibration curve

205

Chapter 9

Application of β-cyclodextrin containing nanogels as carriers of

permethrin on keratin fibers

Abstract: In this work, the application of nanogels with high functional -cyclodextrin (CD)-

content as new and versatile method for the modification and protection of textiles is presented.

The complexation potential of covalently embedded -CD in nanogels is demonstrated for the

common insecticide permethrin in aqueous environment. It is shown that permethrin containing

-CD nanogels can be applied easily, homogeneously and safely on keratin fibers like wool

fabrics and human hairs. The permethrin concentration on fibers is directly controlled by the

permethrin content in nanogels. The permanence of permethrin on treated fibers with regard to

washing and UV fastness were tested. Our results show that permethrin complexed in nanogels is

removed from the textile during washing, but that the complexation of permethrin by -CD

domains in the nanogels protects the active ingredient from UV-degradation. Bioassay tests

against the larvae of Tineola bisselliella and Anthrenocerus australis show that the activity of the

ingredients does not decrease after complexation in -CD gels and it results in protection of the

wool fibers against degradation by the insect larvae.

Keywords: nanogels, cyclodextrin, host-guest-systems, phermetrin, keratin fibers, textiles

Perm. CD nanogel Perm. (Ref.)0

10

20

30

40

50

60 trans-Perm.

cis-Perm.

Perm

. lo

ss o

n f

ibers

[w

t.-%

]

Permethrin (Perm.) loss

on fibers after UV-irradiation

Application on

keratin fibers

- permethrin -CD-nanogels

1 µm

16 % 20 %

43 % 38 %

206

9.1 Introduction

Nano- and microgels are small cross-linked polymeric particles, which can be considered

as colloidal hydrogels if they are composed of water soluble/swellable polymer chains. Specific

properties like the degree of swelling, particle size or responsibility to changes in the next

environment of the nanogels can be controlled by variation of the functional monomers, the

degree of cross-linking and the preparation method [1,2]. Furthermore, the embedding of

functional molecules with specific binding properties for guest molecules finds more and more

interest and application possibilities [3,4].

For example, incorporation of cyclodextrins (CDs) as specific binding domain into the

polymer network promotes improved uptake and release properties of micro- and nanogels and

has proven superior to low molecular weight CDs [5]. CDs are well known for typical cone

structure, which consist of a hydrophilic outer surface and hydrophobic interior cave with

diameter of 5-8 Å. The typical structure properties of CDs enable the formation of host-guest

complexes with suitable hydrophobic molecules [6]. Complexation leads to modification of the

physical and chemical properties (solubility, smell, reactivity etc.) of the guest molecules [7]. In

many fields, CDs find application as adsorber and/or switchable release domains [8,9]. Hence,

non-bonded natural CDs or covalently bonded modified CDs are already used for encapsulation

of bad odors or the fixation and controlled release of perfumes e. g. in textile industry [10,11].

CD containing nanogels have improved properties and advantages compared to low molecular

weight CDs. A modified release behavior controlled by copolymerization, an improved fixation

on surfaces by physical binding and an improved possibility for specific application might be

expected for polymeric CD carrier and delivery systems [12]. Nanogels are known for their

ability to adsorb on surfaces and form thin films [13,14], in which they should conditionally raise

fast with a homogeneous distribution onto textiles. On surfaces, nanogels are physically

connected due to their gel character. Thus, in contrary to covalently bound and non-modified

CDs [15,16]. CD containing nanogels should be applicable to all types of fabrics and surfaces due

to the good adhesion to surfaces caused by the internal cross-linked gel character. The coating of

textile surfaces with nanogels has advantages over the application of polymers as it results in very

thin and homogeneous gel layers or particles. Polymer application results often in thick and

inhomogeneous layers which has negative impact on the haptic properties and on the

performance of the textiles. The application of conventional polymers, e. g. acrylates, results

207

often in textiles with stiff handle [17,18]. The ability to form stable nanogel dispersions in water

is a further major advantage over polymers which are often applied from organic solvents. Thus,

loaded and unloaded -CD nanogels can be applied on different surfaces from aqueous

dispersion. This process enables an easy handling and a save and nontoxic application process.

Furthermore, physically bonded nanogels loaded with specific ingredients should also be

removable and exchangeable.

One major interest for textiles is the protection of human beings, animals and the textile

material itself against insect attack. Permethrin (3-phenoxybenzyl (1RS)-cis,trans-3-(2,2-

dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate) which belongs to the synthetic pyrethroids,

is one commonly used agent and finds applications as common insecticide, acaricide and

repellent agent with a broad range in its activity spectra [19,21]. It acts as neurotoxin and is

responsible for prolongation of the sodium channel activation. The unimpeded flow of Na+ ions

through the cell membranes leads to uncontrolled nerve impulses, incoordination and finally

paralysis of the organism [22,23]. Permethrin is used in agriculture to protect plants and to kill

livestock parasites [24,25]. Further, it is effective for the industrial domestic insect control. As

insect repellent, it finds application against ticks, mosquitoes and in pet flea preventative collars

[26,27]. As personal protective, permethrin is used as repellent in cloth impregnation and finds

application primarily for the treatment of military uniforms and mosquito nets [28]. In medicine,

permethrin is a first-line treatment for scabies and is used on humans to eradicate parasites such

as head lice and mites [29-31].

The complexation of insecticides and especially of permethrin by -CD is well studied by

different workgroups [32-34]. Experimental studies in cooperation with theoretical calculations

show that in low -CD concentrations a 1:1 complex and in higher concentrations a 1:2 complex

is formed. The complexes are ordered in nanorods when the surplus of -CD and thus, the

complex density of 1:2 increases [35]. Also the application of permethrin:-CD complexes onto

textiles, especially on cotton and polyester fabrics [36-38], or their incorporation in mosquito nets

have been investigated [39]. The results show that the activity of the agent is not reduced by

complexation but it is observed that -CD which possesses high biocompability enhances the

activity.

However, the use of β-CD containing nanogels for complexation of permethrin and as

protective coating for textiles has not yet been explored. Coating possibilities with permethrin

208

complexed by β-CD containing nanogels for different fiber types offer a new application method

for insecticides, aiming further at the protection of textiles against the attack of the larvae of

moths or beetles or to protect humans against mosquitoes and head lice. The active agent is

released during contact or attack of insects on the fabrics, thus, it acts as stomach or contact

poison. Rapid, uncontrolled release and early decomposition of the active agents on the fabric

should be prevented by the complexation in -CD domains [40].

It have been shown before that by the use of reactive multifunctional pre-polymers, -CD

can be cross-linked in a water-based organic solvent-free approach to nanogels with up to 60 wt.-

% active -CD content [41]. Aim of the present work is the complexation of permethrin as model

insecticide into these nanogels and their use for coating textiles as protection against insects.

Studies regarding the permethrin uptake, coating on keratin fibers and the permanence of the

treated textiles are presented and discussed. Furthermore, the activity of the permethrin -CD

nanogel treated keratin fabric is tested in bioassays against the larvae of Tineola bisselliella

(clothes moth) and Anthrenocerus australis (carpet beetle).

209

9.2. Experimental

9.2.1 Materials

β-CD was obtained from Wacker GmbH, Burghausen, Germany, and dried before use at

80 °C for about 48 h in a drying cabinet. Isocyanate-terminated star-shaped poly(ethylene oxide-

stat-propylene oxide) (NCO-sP(EO-stat-PO)) was prepared as described before [42]. Here, star-

shaped polyethers with a backbone of statistically copolymerized 80 % ethylene oxide and 20 %

propylene oxide have a molecular weight of 12000 g/mol (PDI = 1.15) and are terminated with

isocyanate (NCO) groups at the distal ends of the arms. Trans- and cis-permethrin was obtained

from Sigma Aldrich, Taufkirchen, Germany, and used as received. A mixture of trans- and cis-

permethrin with the common ratio of 75 % (trans) to 25 % (cis) which are used for standard

treatment of wool textiles was diluted in minimum of methanol. The used wool fabrics (179.35

g/m2, tissue thickness: 0.845 mm; raw white) was provided by Becker & Fuehren Tuche GmbH,

Aachen, Germany. The used human hair (European hair type) was received from the author.

9.2.2 Preparation of β-CD containing nanogels

β-CD containing nanogels were synthesized according to the published preparation

procedure [41,42]. 502 mg (0.44 mmol) β-CD were dried at 80 °C for about 48 h and dissolved in

deionized water (50 mL). The aqueous β-CD solution was added to 504 mg (0.028 mmol) of the

NCO-terminated sP(EO-stat-PO) pre-polymer under stirring. The aqueous phase was stirred

continually at 300 rpm over 24 h using a glass paddle connected to an overhead stirrer. The clear

dispersion was filled in a dialysis tube (ZelluTrans - 12,0S 45 mm, MWCO: Nominal 12000-

14000; Carl Roth GmbH, Karlsruhe, Germany) which was put into water (2 L). The water was

changed after 24 h and 48 h, the dialysis was stopped after 96 h. The purified dispersion was

filled up to a 250 mL end volume. Yield: 748.7 mg (74 %). Particle size: 50-200 nm.

Further analytical data are given in the Supporting Information.

210

9.2.3 Complexation of permethrin by nanogels

Trans- (75 %) and cis- (25 %) permethrin containing methanolic solutions were added in

different concentrations to 50 mL aqueous β-CD containing nanogel dispersion (3 mg/mL). The

reaction mixtures were stirred for 12 h at RT for full complexation and evaporation of the

methanolic solvent. After few minutes, white, cloudy, stable particle dispersion was formed. The

stable dispersions are separated from non-complexed permethrin sediments and freeze-dried or

applied directly onto keratin fibers for further analysis. Table 1 shows the total permethrin

concentrations in the β-CD nanogel dispersion for the complexation process. The added molar

permethrin concentration was calculated for the expected molar β-CD content in nanogels by

theoretical conversion of a 1:1 ratio of β-CD and NCO-sP(EO-stat-PO).

Further analytical data are given in the Supporting Information.

Table 1. Permethrin (Perm.) concentrations in β-CD/sP(EO-stat-PO) nanogel (NG) dispersions

for the complexation process. For quantitative analysis of the permethrin content on treated fibers

the percentage weight ratio of permethrin and wool textile (Tex.) (3.79 g ≈ 200 cm2) is shown.

NG calc. CD* Perm. calc. CD Perm. Perm./NG NG/Tex. Perm./Tex.

mg/mL mg/mL mg/mL µmol/mL µmol/mL wt.-% wt.-% wt.-%

2.99 1.50 0.00 1.32 0.00 0.00 0.38 0.00

2.99 1.50 0.06 1.32 0.15 2.00 0.38 0.08

2.99 1.50 0.12 1.32 0.31 4.01 0.38 0.16

2.99 1.50 0.30 1.32 0.77 10.02 0.38 0.39

2.99 1.50 0.75 1.32 1.92 25.04 0.38 0.98

2.99 1.50 1.50 1.32 3.83 50.09 0.38 1.94

* calc. CD: calculated -CD content in -CD nanogels based on an expected conversion of 1:1

ratio β-CD: NCO-sP(EO-stat-PO)

211

9.2.4 Application of permethrin loaded nanogels onto textiles

A series of nanogel dispersions with different particle and permethrin concentrations was

used for further coating experiments. The quantitative permethrin application with the associated

nanogel concentration on textiles is shown in Table 1. The nanogel concentrations and the

associated permethrin content used for different coatings are listed in Table 2. The coating

process took place in a special beaker in which the dispersion was put. The wool textiles (3.79 g

≈ 200 cm2) were padded with 50 mL of a dispersion containing permethrin-loaded nanogel

particles for half an hour using a mini-foulard (in-house development) (Scheme 1c). After that,

the coated fabric was dried at RT for half an hour and further for half hour at 80 °C. The tissue

samples were packed waterproof and stored under ambient conditions protected from light.

Table 2. -CD/sP(EO-stat-PO) nanogel (NG) and permethrin (Perm.) concentrations of aqueous

dispersion for the coating by padding with the help of a mini-foulard plus the percentage weight

ratio of permethrin and wool textile (Tex.) (3.79 g ≈ 200 cm2) is listed.

NG calc. CD*

Perm. calc. CD Perm. Perm./NG NG/Tex. Perm./Tex.

mg/mL mg/mL mg/mL µmol/mL µmol/mL wt.-% wt.-% wt.-%

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.60 0.30 0.06 0.26 0.15 10.02 0.78 0.08

1.50 0.75 0.15 0.66 0.38 10.02 1.94 0.20

2.99 1.50 0.30 1.32 0.77 10.02 3.80 0.39

12.00 6.00 1.20 5.29 3.07 10.02 13.67 1.56

* calc. CD: calculated -CD content in –CD nanogel based on an expected conversion of 1:1

ratio β-CD: NCO-sP(EO-stat-PO)

212

9.2.5 Application of Permethrin-Based Insecticide onto Wool Textiles (Reference Treatment)

Permethrin-based insecticides (here: Eulan™ SPA-01, Tanatex GmbH, Leverkusen, Germany,

and Mystox™ CMP, Catomance Technologies Ltd., Stevenage, United Kingdom, were applied

onto wool fibers or fabrics in dosages of 0.3 – 1.0 % (wt.-% on weight of wool) from an aqueous

liquor (liquor ratio = 1:30) which contained 4 % (wt.-% on weight of wool) (NH4)2SO4, the pH-

value was adjusted to 6 using acetic acid, by bath exhaustion procedure. The treatment was

performed at 98 °C for 60 min using an Ahiba Turbomat (Type TM6) laboratory dyeing machine

(Ahiba, Birsfelden, Switzerland). The wool samples were rinsed after the treatment with warm

water (50 °C) for 10 min and with cold tap water for 20 min. The samples were squeezed off and

dried at ambient conditions protected from light exposure. In parallel, blank treatments without

addition of insecticides were performed.

9.2.6 Characterization of -CD containing nanogels, permethrin loaded nanogels and keratin

fibers and fabrics treated with permethrin

IR spectra were obtained using the FT-IR-spectrometer: Nexus 470 (Thermo Nicolet,

Neu-Isenburg, Germany) (spectral disintegration: 8 cm-1

). Nanogel dispersions were dried by

lyophilisation and studied preferentially in KBr-pellets.

Raman spectra were measured with a spectral resolution of 4 cm-1

with the help of a

Bruker RFS 100/s Raman spectrometer (Bruker Optics, Ettlingen, Germany) using a Nd:YAG

laser (wavelength: 1064 nm). The solid samples were measured in an aluminum pan.

1H- and

13C-NMR measurements were carried out using a AC400 spectrometer (Bruker,

Ettlingen, Germany); 1H-NMR spectra were recorded at 400 MHz and

13C-NMR spectra at 100

MHz. DMSO-d6 was used as solvent; the signal of the non-deuterated solvent was used as

standard.

UV-vis spectra were measured with the help of a Varian Cary 100 UV-vis-

spectrophotometer (Varian, Darmstadt, Germany) using quartz cuvettes (optical path length: 1

cm). The respective solvent (here: water or ethanol) was used as reference.

Analysis of the particle size was made by dynamic light scattering measurements (DLS),

using the Nano Zetasizer (Malvern Instruments GmbH, Herrenberg, Germany) spectrometer. The

spectrometer contains a He–Ne laser (λ0 = 633 nm). Back scattering light was detected using an

213

angle of 173°. The hydrodynamic particle radius and the particle distribution of the

hydrodynamic radius were determined. All DLS-measurements were made at 25 °C.

The electron microscopic analyses were made using the S-4800-Field-Emission-Scanning

Electron Microscope (Hitachi Ltd., Tokyo, Japan) with high disintegration and cryo-

function. Dry nanogels were studied after coating onto the surface of keratin fibers. After

application of nanogels by padding or dip-coating, the treated fibers were dried further at RT and

in vacuum. The samples coated with nanogels were studied by SEM at RT using an accelerating

voltage of 1 KV and 8 mm disintegration. The particle diameter was determined with the help of

the SEM enclosed software.

The permethrin concentration of -CD based nanogels was determined by RP-HPLC-

analysis with the help of Nucleosil 100-5 C18 columns (Macherey & Nagel GmbH, Dueren,

Germany) using the eluent: methanol/water - 87/13 (v/v), and a flow rate of 0.5 mL/min at RT.

The exact concentration was determined by the use of UV-detection at 206 nm and a straight

calibration line of pure permethrin. The permethrin content of treated textiles and of freeze-dried

nanogels was determined by HPLC analysis after extraction with methanol (1 g textile/20 mL

methanol; 30 min at 80 °C).

The fastness to washing of treated textiles was determined according to DIN EN ISO 105-

C10 at 40 °C (30 min). 5 g needle soap was used for the washing of 1 g wool in 50 mL soap

solution. The textile was then further washed with water for 10 min and 2 times with distilled

water. The evaluation of the insecticide resistance to washing was performed by determination of

the residual content of the parasite protection products after washing fastness tests by HPLC

analysis after extraction of the wool.

The light fastness testing of treated fabrics was performed according to DIN EN ISO 105-

B06. Here, permethrin treated fabrics were irradiated for 48 h in a Atlas Weather-Ometer ES25

equipment (ATLAS Material Testing Technology, Chicago, USA) at a light intensity of 0.27

W/m2/nm (recorded at 340 nm). The evaluation of the insecticide resistance to light was

performed by determination of the residual content of the parasite protection products on treated

wool after the light fastness test by HPLC analysis after extraction of the textile.

The color measurement was performed using the colorimeter Datacolor Spectraflash

SF600 plus CT from Datacolor, Marl, Germany. The measurements were made without luster

and with a 20 mm blend. They were performed using illuminant D65 and the 10° observer.

214

Fabrics were measured in two layers. For each sample, five measurements were made and the

mean values were calculated. The whiteness was calculated as W-CIE, the yellowness values as

G-DIN 6167 and the yellowness differences as ΔG-DIN 6167. The color values were calculated

according to the Datacolor formula based on the CIELAB system and for illuminant D65.

The biological activity of wool fabrics coated with permethrin containing nanogels was

determined according to ISO 3998-1977 (E) (in accordance with the Wools of New Zealand Test

Method 25) against the larvae of moths (Tineola bisselliella) and against the larvae of the

Australian carpet beetle (Anthrenocerus australis) at AgResearch, Inc., Hamilton, New Zealand.

215


Aim of this work was to evaluate and exploit the application potential of nanogels as

surface-active carriers and anti-insect coatings for textiles. For this, nanogels were chosen to act

as carrier materials for permethrin as model compound with regard to fixation and as coating

systems due to self-bonding on fiber materials. The whole application procedure is subdivided in

3 main steps (Scheme 1a-c).

Scheme 1. Scheme of the synthesis of β-CD containing nanogels based on NCO-star P(EO-stat-

PO) in aqueous solution (a), incorporation of permethrin into β-CD containing nanogels in

aqueous solution (b), and nanogel application on textile surfaces (c). 7

Preparation and purification of nanogels with high active -CD content is followed by

complexation of permethrin into the nanogels. Finally, the treatment of fabrics by coating with

the loaded nanogels from washing liquors and thermofixation of the treated materials was

performed, and their bioactivity was tested.

O

O

Cl

Cl

O

aqueous cyclodextrin

mixture stirrer

24 h

gel-particles in water

RTNCO-

sP(EO-stat-PO)

stirrerpermethrin

solutionactive ingredients

in nanogelspermethrin

textile

aqueousnanogel

dispersion

a

b

c Rh = 25 -100 nm

Rh = 50 -200 nm

216

9.3.1 Cyclodextrin based nanogels

Nanogels are prepared in water by cross-linking of β-CD with a NCO-terminated star-

shaped pre-polymer on the basis of polyethylene oxide (80 %) and polypropylene oxide (20 %)

(sP(EO-stat-PO)) as described before [41,42]. The colloidal polymeric particles are created and

stabilized by incorporation of β-CD domains in the polymer backbone of cross-linked sP(EO-

stat-PO) in aqueous solution via covalent urethane bonding between the NCO groups of the

prepolymer and the hydroxy groups of the -CD units [41]. Furthermore, their size of several

nanometers can be controlled by the β-CD content, as an increasing β-CD concentration in the

reaction mixture leads to an increase of the size from 50 nm to several hundred nanometers. The

uptake capacity of these nanogels and quantitatively determined the complexation properties of

the -CD units in the nanoparticles by the dye-sorption method using the pH sensitive dye

phenolphthalein were investigated before.

For the present study,whether these nanogels do attach onto keratin fibers when applied

from aqueous dispersion it was initially checked. (Figure 1: nanogel adsorption on wool fibers).

Figure 1. β-CD nanogels coated on wool fibers (a) and uncoated wool fibers as reference (b).

The -CD containing nanogels can be coated onto fabrics by padding resulting in highly

homogeneous distribution as analyzed by field emission electron microscopy. Only FESEM with

a high resolution enables the optical detection of the applied nanogels. After application,

homogeneous coating of the wool fabrics with unloaded gel particles of the size of approx. 100

nm (hydrodynamic radii (Rh ≈ 50 nm)) is determined.

a b

1 µm 1 µm

217

9.3.2 Complexation and release of permethrin by/from -CD nanogels

Complexation of permethrin by natural low molecular weight CDs has already been

studied in detail by some workgroups. Stoichiometric formation of a 1:1 permethrin:-CD

complex is described using higher permethrin concentration and lower -CD content in aqueous

solution. By increase of the -CD ratio in comparison to permethrin the formation of a 2:1

complex and the formation of nanorods was described [32,33].

In this work, the complexation of permethrin by -CD nanogels is made in aqueous

dispersion (Scheme 2). Different concentrations of permethrin diluted in alcoholic solution were

dropwise added to -CD containing nanogels in a clear aqueous dispersion. The complexation of

the hydrophobic molecule is observed directly from the decrease of transparency. Hence,

hydrophobic molecules bind to the hydrophobic interior surfaces of covalently embedded -CD,

which makes nanogels more hydrophobic. The water insoluble ingredients increase their water

solubility by complexation and are distributed homogeneously in the whole dispersion. (Figure

S1).

Scheme 2. Uptake and complexation process of cis/trans permethrin in -CD containing star

P(EO-stat-PO) nanogels in aqueous dispersion.

The qualitative uptake of permethrin by β-CD nanogels was studied by IR, Raman, UV

and NMR spectroscopy of the freeze-dried permethrin-loaded β-CD nanogels (Figure 2).

Infrared spectroscopy shows beside the characteristic bands of the stretching vibration of

C-O-C bonds associated with β-CD (1030 cm-1

) the typical amide bands (1677 and 1535 cm-1

)

O

RR

R

O

O

R

R

RO

OR

R

R

O

O

R

R

R

O

O

R

R

R

O

O

R

RR

O

OR

R

R

O

O

RR

R

O

O

R

R

RO

OR

R

R

O

O

R

R

R

O

O

R

R

R

O

O

R

RR

O

OR

R

R

O

β-Cyclodextrin

Permethrin-β-Cyclodextrin-

Complexβ-Cyclodextrin-sP(EO-stat-PO)-Nanogel

Permethrin loaded β-Cyclodextrin-sP(EO-stat-PO)-Nanogel

O

O

Cl

Cl

O

O

O

Cl

Cl

O

cis/trans-Permethrin

218

and the urethane amide I band (1720 cm-1

) which confirm the chemical cross-linking and the

covalent bonding of β-CD into the sP(EO-stat-PO) based nanogels. The complexation of

permethrin is documented by the IR bands at 1585, 1487 and 1457 cm-1

which can be assigned to

aromatic residues. The C=O band of the permethrin ester group at 1720 cm-1

is overlapped by the

amide I band of the urethane groups in the nanogel (Figure 2a).

The Raman signal at 1003 cm-1

originates from breath vibrations of the benzene ring; it is

associated with the complexed permethrin. Furthermore, the Raman bands at 1595, 1617, 1732

and 3055 cm-1

can be referred to the complexed permethrin as these signals are due to the C-C-,

C=C- (olefinic or aromatic), ester and C-H (aromatic) residues in the permethrin molecule. The

successful incorporation of cyclodextrins into sP(EO-stat-PO) gels is proven by the presence of

the out-of-plane deformation of the glucose ring at 480 cm-1

in the Raman spectrum (Figure 2b).

The UV spectra of -CD nanogels, which are loaded with permethrin, exhibit a

characteristic UV maximum at 278 nm which originates from the aromatic residues of the

permethrin molecule (Figure 2c). The complexation of permethrin by natural -CD and -CD

nanogel resulted in a slight red shift of the UV absorption band compared to that of permethrin

(273 nm). This effect was be explained by the high electron density of the permethrin guest

molecule located inside the -CD host cavity [25,33].

The 1H-NMR spectra of -CD nanogels with complexed permethrin depict characteristic

peaks of the sP(EO-stat-PO) backbone at 1.04 ppm and 3.35 ppm which is overlapped by the CH

and CH2 protons of the CD ring in the area of 3.2-3.6 ppm. Furthermore, β-CD shows the

characteristic peak at 4.83 (s, CH-1) and the peaks of the OH groups at 4.53 (s, C-OH-6) and

5.62-5.66 (s, C-OH-2, 3). The complexed permethrin in the nanogel is depicted at 1H-NMR

signals at 1.164 ppm (CH3, cis/trans-permethrin), 2.04 (C-H, cyclopropane, permethrin), 5.11

(COO-CH2-Ar), 5.703 (HC=CCl2), 7.01 (CHar,), 7.40 (CHar) (Figure 2d).

From all the data of the spectrometric analysis and in comparison with pure cis/trans-

permethrin, the permethrin cyclodextrin complex and the unloaded β-CD nanogel, it can be

concluded that permethrin is complexed mainly by the β-CD nanogels. Furthermore, it is difficult

to analyze how much permethrin is complexed by the β-CD units with this analytical methods

and otherwise, how much is encapsulated by the cross-linked structure of the sP(EO-stat-PO)

polymer backbone. In this case, synergistic effects between the β-CD domains and the polymer

enhance the uptake process.

219

Figure 2. IR spectra of -CD and the permethrin:-CD inclusion-complex (Perm. CD) in

comparison with -CD nanogels (CD-NG) and permethrin:loaded nanogels (Perm. CD-NG) in

DMSO-d6 (a). Raman spectra of the permethrin:loaded (Perm. CD-NG) and unloaded -CD

nanogels (CD-NG) (b). UV absorption spectra of permethrin in ethanol and the permethrin:-CD

inclusion complex (Perm. CD) in water in comparison with -CD nanogels (CD-NG) and

permethrin-loaded nanogels (Perm. CD-NG) in aqueous dispersion (c). 1H-NMR spectra of

permethrin and the permethrin:-CD inclusion complex (Perm. CD) in comparison with -CD

nanogels (CD-NG) and permethrin-loaded nanogels (Perm. CD-NG) in DMSO-d6 (d).

In water, the β-CD nanogel complex is quite stable because the hydrophobic guest

molecule prefers the hydrophobic interior cavity of -CD molecules. By use of organic/water

mixtures permethrin release is expected due to the re-dispersion of the β-CD nanogels in water.

Hence, -CD nanogels (2.99 mg/mL) with included permethrin are tested for their release

behavior in methanolic solutions. Here, the freeze-dried permethrin loaded nanogels are extracted

a

c

b

d

2000 1500 1000 500

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

(4)

(3)

(2)

IR-T

ransm

issio

n

Wavenumber [cm-1]

CD-NG (4)

Perm. CD-NG (3)

CD (2)

Perm. CD (1)

(1)

240 260 280 300 3200.00

0.05

0.10

0.15

0.20

(4)

(3)

(2)

UV

-Absorb

ance

Wavelenght [nm]

CD-NG (4)

Perm. CD-NG (3)

Perm. CD (2)

Permethrin (1)

(1)

273

278

8 7 6 5 4 3 2 1 0

(1)

(2)

(3)

NM

R-I

nte

nsity

ppm

CD-NG (4)

Perm. CD-NG (3)

Perm. CD (2)

Permethrin (1)

(4)

2000 1500 1000 5000.00

0.03

0.06

0.09

0.12

0.15

4801732

16171595

Ram

an-T

ransm

issio

n

Wavenumber [cm-1]

CD-NG (2)

Perm. CD-NG (1)

1003

(2)

(1)

220

in methanolic solvent (150 mg/20 mL) and are washed by variation of time at 80 °C under

permanent shaking of the mixture. Then, the methanolic extraction solution is filtered and

analyzed by HPLC analysis, to determine the permethrin content quantitatively similar to the

literature [43,44].

In Figure 3a, the HPLC diagrams of permethrin released from nanogels in comparison to

unloaded nanogels in methanol are shown. The -CD nanogels without permethrin do not show

any significant peaks. This means that only released permethrin is detected at 206 nm. The

permethrin-loaded nanogels release the guest molecule, and in the HPLC diagrams, cis- and

trans-permethrin can be detected by the strong peaks at 17 min (trans) and at 21 min (cis) in a

ratio of 75 % (trans) to 25 % (cis). This ratio of the cis/trans isomers is similar to the ratio (3/1

trans/cis) of the original permethrin solution which is added to the -CD nanogel dispersion.

Thus, the -CD nanogel complex releases trans- and cis-permethrin without any preference of

one of the isomers.

Figure 3. Release of permethrin (Perm.) from -CD nanogels in methanolic solutions determined

by HPLC analysis. HPLC-diagram of trans- and cis-permethrin released from loaded -CD

nanogels in comparison with unloaded -CD nanogels in methanol (a). Trans- and cis-permethrin

release from -CD nanogels by variation of the methanol/water content of the extraction solvent

and variation of extraction time (b).

In Figure 3b, the variation of the methanol/water content in the extraction solvent and the

extraction time are studied. Here, a mixture of water and methanol (1:1) shows a slight release of

permethrin from -CD nanogels. Change of extraction time does not lead to an increase of the

0 5 10 15 20 25 30 35 40 45

0

200

400

600

800

1000

1200

Inte

nsity [m

V]

(at

20

6n

m)

Time [min]

Permethrin in CD nanogels

CD nanogels

cis-permethrin

trans-permethrin

ba

0 5 10 15 20 25 30

0

5

10

15

20

25

30

Pe

rm.

rele

ase f

rom

Na

no

ge

l [µ

g/m

g]

Extraction time [min]

trans-Perm. theo. cis-Perm. theo.

trans-Perm. MeOH cis-Perm. MeOH

trans-Perm. H2O/MeOH cis-Perm. H

2O/MeOH

221

released permethrin content. On the opposite, the pure methanolic solution increases the release

of trans- and cis-permethin significantly. Here, the extraction time can promote the released

amount of trans- and cis-permethrin. The total content of the complexed permethrin is rinsed out

by pure methanol. The hydrophobic properties of the guest molecule in a less hydrophilic

environment plus the high hydrodynamic pressure due to the surplus of the solvent lead to a

rising release of permethrin in organic solvents. The concentration of the released permethrin in

pure methanol increases to 100 % of the expected values. These values are 90 % higher than the

permethrin release in a water/methanol mixture (1:1).

9.3.3 Application onto textiles

Stable aqueous nanogel dispersions loaded with permethrin suggest a good possibility for

the application on surfaces. The treatment of textiles, especially of wool fabrics, with permethrin

loaded nanogels presents a controlled, but effective insect-proofing with safe application. The

coating process is made by dip-coating and padding. Keratin fibers like wool or human hairs can

be treated easily with permethrin from aqueous liquors. The coatings are studied using aqueous

dispersions with different β-CD nanogel concentrations (Table 1). The surface of wool fabrics

which were treated with permethrin loaded -CD containing nanogels is analyzed by field

emission electron microscopy (Figure 4). Slightly coated fabrics are obtained by padding using

0.6 mg/mL (0.8 wt.-% permethrin/textile) permethrin loaded nanogel dispersion (Figure 3b). In

comparison to the untreated fabric (Figure 4a) the surface shows the presence of small round

particles. The particle concentration and thus, the permethrin concentration on fibers increase

with the increase of the permethrin loaded particle concentration in the aqueous dispersion

(Figure 4b-d). An increase of the nanogel concentration to 1.5 mg/mL (2 wt.-%) results in a

higher particle concentration on the surface. Heavily coated fibers which can be seen in Figure 3d

are obtained with nanogel concentration of 3 mg/mL (4 wt.-%). Here, agglomerations and dense

distribution characterize the surface of the fabric. Very heavily coated fabrics obtained by using

nanogel concentrations of 12 mg/mL (16 wt.-%), exhibit the presence of a gel film which adheres

to the fabric and cross-links the individual wool fibers (Figure 4e). In case of coatings with low

nanogel concentrations, individual gel particles can be identified with relatively homogeneous

distribution on the wool fibers. In high resolution the particles show a homogenous distribution

222

with dimensions of 100 to 200 nm. The nanogels are difficult to detect in low resolution. The

morphology of the particles on the surface can be described as spherical.

Figure 4. FESEM images: Uncoated (a), slightly coated with clearly identifiable permethrin

loaded β-CD nanogels on wool fibers (b-f) and heavily coated wool fibers (e). Figure (f) shows

permethrin loaded nanogels applied onto human hairs. (FESEM images of untreated human hairs

are given in the Supplementary Information as Figure S2).

a b

dc

fe

40 µm 40 µm

40 µm

10 µm 10 µm

10 µm 10 µm

10 µm 5 µm

40 µm 40 µm

40 µm

223

In Figure 4f, human hairs which are treated with permethrin containing nanogels by dip-

coating are shown. Here, a low concentrated nanogel dispersion was used which results in slight

coating with particles. The treatment of human hairs with permethrin containing β-CD nanogels

might be a possible application against head lice. Permethrin loaded nanogels can be used in

shampoos or water based conditioners. The fixation of permethrin by nanogels on hairs exhibits

long activity and minimizes skin irritations caused by a reduction of application repetitions and

uncontrolled adsorption on the skin.

The coating of wool fabrics with permethrin containing nanogels is performed with

different permethrin concentrations (Table 2) in stable β-CD nanogel concentration. The

permethrin content of the wool fabrics is determined quantitatively by HPLC analysis after

extraction of the textile with pure methanol solution. Wool fabrics (3.79 g ≈ 200 cm2) are treated

with -CD nanogel dispersions (3 mg/mL 4 wt.-% on the textile) containing permethrin with

0.00 mg/mL (0 wt.-% on the textile), 0.06 mg/mL (0.08 wt.-% on the textile), 0.12 mg/mL (0.16

wt.-% on the textile), 0.29 mg/mL (0.39 wt.-% on the textile), 0.74 mg/mL (0.98 wt.-% on the

textile) or 1.45 mg/mL permethrin (1.94 wt.-% on the textile). The determined permethrin

concentrations in methanol extracts of treated wool fabrics are listed in Figure 5a.

Figure 5. Quantitative determination of the permethrin content of β-CD nanogel particles

obtained by extraction of treated wool fabrics with methanol and detection at 206 nm using

HPLC analysis in dependence on the applied permethrin concentration (= theo.) of the β-CD

nanogel dispersion. Measurements after treatment (a) and determination of the permethrin

amount after 3 years storage (b).

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

total permethrin concentration in dispersion [wt.-%]

perm

eth

rin c

onte

nt on fib

ers

[w

t.-%

] trans-Perm. (theo.)

cis-Perm. (theo.)

trans-Perm.

cis-Perm.

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

total permethrin concentration in dispersion [wt.-%]

perm

eth

rin

con

tent

on

fib

ers

[w

t.-%

] trans-Perm. (theo.)

cis-Perm. (theo.)

trans-Perm.

cis-Perm.

ba

224

In comparison to the linear order of the maximally expected values of the permethrin

content in the aqueous dispersion, the concentration is similar to the calculated maximally

possible concentration. Only the highest selected concentration of permethrin in the nanogels

(1.94 wt.-%) results in lower permethrin content in the wool extracts than added. Thus, the higher

calculated molar permethrin ratio in -CD domains in the nanogels shows, that the nanogel and

especially the -CD capacity for the complexation is reduced to take up a surplus of guest

molecules. Trans- and cis-permethrin are applied in the expected ratios on the fabrics due to the

good adsorption properties of the -CD nanogels on the fiber surface. Hence, the concentration of

permethrin can be controlled on the keratin fibers by the application of β-CD nanogels from

aqueous dispersion.

Figure 5b presents the results of the same samples measured after three years storage at

RT under ambient laboratory conditions. In the literature, it has already been reported that the

stability of permethrin is low due to decomposition primarily by microorganisms, but also by

photolysis [45]. In water, permethrin is broken down by photolysis into 3-phenoxybenzyl alcohol

(PBA) and dichlorovinyl acid (DCVA) [45]. The average half-life range of permethrin in water is

about 19-27 h. Permethrin can persist in sediments approximately for 1 year (1µg/mL of

120µg/mL after 1 year) [46]. Indoor studies of permethrin applied in a thin layer to a surface

showed that after 20 days of exposure to daylight 60% of the permethrin remained on the surface

[47]. In our studies (Figure 5b), a loss of the trans- and cis-permethrin concentration is detectable

after 3 years, but it is still active on the fabric. The trend of the permethrin content on the textile

after 3 years is linear and comparable with the permethrin concentration applied from the β-CD

nanogel dispersions. That means permethrin especially trans-permethrin is protected by

complexation in -CD nanogels against decomposition due to environmental influences. The

permethrin decomposition can be delayed but not prevented. The sensitive organic compound is

not permanently fixed to the textile and the release into the environment by evaporation, break

down or restructuration decrease the permethrin concentration on the textile by and by.

Interestingly, the loss of cis-permethrin concentration is in the same ratio as the one of the trans-

permethrin concentration. This leads to the conclusion that the trans-permethrin:-CD complex

and the cis-permethrin:-CD complex have the same stability in the nanogels.

225

9.3.4 Permanence of insecticides on wool applied after complexation in β-cyclodextrin

containing nanogels

Wool fabrics which were treated with permethrin loaded -CDcontaining nanogels are

investigated with regard to the fastness of the insecticides during washing (according to DIN EN

ISO 105-C10) or to light (according to DIN EN ISO 105-B02) (Figure 6).

Figure 6. Permethrin (Perm.) content (in wt.-%, trans-and cis-permethrin) on wool fabrics after

application, after a washing process and after 48 hours of irradiation of fabrics treated with

permethrin containing β-CD nanogels (a) and reference treated with permethrin containing

insecticides (b) - determined by HPLC analysis. The loss of permethrin from the fibers after

washing (c) and after UV irradiation (d) in direct comparison to permethrin complexed in -CD

nanogels and non-complexed permethrin.

Washing tests show that after one washing process about 40 % of the trans- and cis-

permethrin which were incorporated in -CD nanogels are removed from the wool. These losses

are similar to those determined for wool fabrics, which are treated with non-complexed

Perm. content Washing UV0.0

0.2

0.4

0.6

0.8

1.0

Pem

eth

rin o

n fib

ers

[w

t.-%

]

trans-Perm. in CD-Nanogels

cis-Perm. in CD-Nanogels b

d

a

c

Perm. content Washing UV0.0

0.2

0.4

0.6

0.8

Pem

eth

rin o

n fib

ers

[w

t.-%

]

trans-Perm. without Nanogels (Ref.)

cis-Perm. without Nanogels (Ref.)


10

20

30

40

50

60 trans-Perm.

cis-Perm.

Perm

. lo

ss o

n fib

ers

by U

V [w

t.-%

]


10

20

30

40

50

60

70

Perm

. lo

ss o

n fib

ers

by w

ashin

g [w

t.-%

]

trans-Perm.

cis-Perm.

40 %43 % 41 %

32 %

16 %20 %

43 % 38 %

226

permethrin by conventional bath exhaustion procedure (98 °C, 1 h). Due to the water insolubility

of permethrin, it can be concluded, that the physically self-bonded permethrin loaded nanogels

which are redispersible in water are removed from the fibers by washing. Thus, the treatment is

not permanent; however, permethrin loaded nanogels can be re-applied easily onto textiles or

other surfaces on demand.

Irradiation tests are made according to DIN EN ISO 105-B02. The results are also shown

in Figure 6. Here, the permethrin coated fabric is irradiated for 48 h, and the losses of permethrin

during irradiation are determined by RP-HPLC analysis. In case of wool fabrics which were

treated with non-complexed permethrin by conventional bath exhaustion procedure about 40 %

of cis- and trans-permethrin losses are found after 48 h of irradiation. However, in case of

permethrin which was complexed by β-CD nanogels markedly less permethrin is degraded during

light exposure. Here, losses of about 16 % of trans-permethrin and approx. 20 % of cis-

permethrin are determined for wool fabrics which were treated with permethrin loaded -CD

nanogels after 48 h of irradiation. Hence, β-CD domains in the nanogel form inclusion complexes

and act as protector for influences of the environment. These results show that permethrin, in

particular trans-permethrin, is protected from light degradation by complexation in β-CD

nanogels. The irreversible degradation of UV-sensitive permethrin by light exposure can be

decreased for about 47 % (cis) and 63 % (trans) in comparison to the non-complexed active

ingredients.

It is known that the photodegradation of permethrin proceeds via two major reactions, i.e.

isomerization of the cyclopropane ring and ester cleavage on photolysis [45]. The medium and

the environment (e.g. the solvent) of the permethrin influence the photodegradation. The

complexation of sensitive ingredients by cyclodextrin leads to changes of physical and chemical

properties due to supramolecular bonding of guests by the host molecules. Furthermore, the

complexation protects the guest molecule against environmental influences e.g. molecular

degradation due to light irradiation which is explained by sterical fixation and shielding against

the environment [48,49]. The complexation of cis- and trans-permethrin by the cyclodextrin

groups in the nanogels leads to such sterical stabilization and shielding of the molecule,

especially of the ester group by the β-CD cone so that the photodegradation due to

photoisomerization is suppressed.

227

Wool fabrics treated with permethrin containing -CD nanogels are also tested for color

fastness upon light exposure. The yellowness index (G-DIN 6167) documents that a more or less

pronounced lightening of the wool fabric was obtained upon exposure to light (here: negative

values for the yellowness ΔG-DIN 6167 are measured). In Figure 7, the color change of

permethrin loaded fabric samples are presented. In the diagram, the change in yellowness of the

untreated and the nanogel treated wool fabric are shown. The yellowness index of the fabric

treated with the pure nanogel does not decrease as strongly as the untreated wool fabric when

exposed to light. The fabric, which is treated with permethrin loaded β-CD nanogels shows in

comparison to the untreated fabric also a minimal decrease in the yellowness index after

irradiation. It shows that photobleaching of the fabric is reduced in case of nanogel containing

fabrics compared to the untreated wool sample.

Figure 7. Color change (as change in yellowness ΔG-DIN 6167) of wool fabrics treated with

permethrin loaded and unloaded β-CD containing nanogels in comparison to the untreated wool

fabric when exposed to light (DIN EN ISO 105-B02 (48 h)).

Obviously, the treatment with -CD containing nanogels or with permethrin loaded -CD

nanogels protect the wool fabrics from color changes when exposed to light. Merino wool

exhibits a slightly yellowish color. The characteristic color is due to the presence of yellow

chromophores which are prone to bleaching upon light exposure under special conditions (e.g.

short-time exposure, exposure behind window glass). It is known that photobleaching of wool

uncoated fibers I CD nanogels IPerm.CD nanogels0.0

-0.5

-1.0

-1.5

-2.0

-2.5

-3.0

-3.5

G

-DIN

61

67

228

depends on its yellowness, the more yellowish wool bleaches stronger than whiter wool [50].

Photobleaching of wool is caused by the action of radicals and oxygen active species which are

formed during exposure to light [50,51]. It can be assume that the β-CD containing nanogels

protect the wool from photobleaching, due to scavenging of radicals or oxygen active species.

This phenomenon is less pronounced in case of β-CD gels which contain complexed light-

sensitive permethrin.

9.3.5 Biological effectiveness of the insecticide treatment

Wool fabrics which were treated with in β-CD nanogels complexed and with non-

complexed permethrin are investigated with regard to their biological effectiveness against the

larvae of Tineola bisselliella (clothes moth) and of Anthrenocerus australis (Australian carpet

beetle). The tests are performed by AgResearch Ltd., Hamilton, New Zealand, according to the

Test Method Wools of New Zealand Method 25 which complies with the quality standard of ISO

3998-1977 (E). The effectiveness of the treatment is evaluated by the average mortality of the

larvae in %, the mean mass losses of the fabric in % (compared to a control) and by visual

assessment of the fabrics with regard to damages due to foraging.

Table 3 depicts the results of the biological tests against Tineola bisselliella (clothes

moth) obtained for wool fabrics that are treated with β-CD nanogels and permethrin containing

nanogels. For blank-treated fabrics and for wools that are treated with unloaded β-CD nanogels,

no killing of the larvae is achieved. In the fabric sample, which is treated with the nanogels shows

even an increase of the mass loss of 72 %. In these fabrics, large holes due to feeding damages

are observed. However, for wool fabrics which are treated with permethrin loaded β-CD nanogels

100 % mortality of the moth larvae is determined, while no mass losses and no visible damages

are found for the wool fabric after the test. From Table 3 it is apparent that the permethrin

containing wool fabric is also effective with a concentration of 0.4 % (wt.-%) with respect to the

mortality of moth larvae. The complexed permethrin is applied here in a high concentration of

0.94 wt.-% onto the wool fabric, thus the sample passed the biological test without any reduction

of the effectiveness.

229

Table 3. Results of the biological tests of wool fabrics which were treated with permethrin based

insecticides and with permethrin loaded -CD nanogels against larvae of Tineola bisselliella –

according to Test Method Wools of New Zealand Method 25.

Wool fabrics Content of

permethrin

(wt.-%)

Average

mortality

(%)

Mean mass loss

(% of the

control)

Visual assessment of

fiber damages due to

foraging

Control - 0.0 100 Strong damages

Blank treatment* - 0.0 86.7 Strong damages

β-CD-nanogels - 1.7 172.3 Strong damages

Perm.-β-CD-nanogels 0.94 100.0 -0.3 No visible damages

Perm.-based insecticide* 0.55 100.0 - 0.4 No visible damages

Perm.-based insecticide* 0.34 100.0 - 1.6 No visible damages

* Blank treatment and treatment with permethrin by bath exhaustion procedure (98 °C, 1 h, pH 6)

The results of the biological tests against larvae of Anthrenocerus australis (Australian

carpet beetle) are given in Table 4. Beside a control, a blank-treated and a wool fabric that is

treated with an unloaded β-CD containing nanogel, fabrics which are treated with permethrin or

with permethin loaded β-CD nanogels are tested. The blank-treated and the wool fabric which is

treated with unloaded β-CD nanogels show no effectiveness to prevent degradation by the larvae

of the Australian carpet beetle. Wool fabrics which are treated with permethrin by bath

exhaustion procedure and those which are treated with permethrin loaded -CD nanogels are

effective to kill the larvae of the beetles. As the concentration of permethrin on the wool fabric

which is coated with β-CD nanogels with 0.94 wt.-% permethrin is much higher than that on the

wool which is treated by the conventional bath exhaustion procedure (here: 0.34 wt.-%

permethrin), a markedly better effectiveness against the larvae of the beetles is found in the first

case. This is visible in the mortality of the larvae of 40 % and a mass loss of the wool fabric of

7.5 % relative to the control. Visual assessment of these wool fabrics treated with permethrin

demonstrates that no holes or foraging damages are detected on the wool, so that it can be

assumed that in future lower permethrin concentrations can be applied.

230

Table 4. Results of the biological tests of wool fabrics which were treated with uncomplexed

permethrin and with permethrin loaded β-CD nanogels against larvae of Anthrenocerus australis

– according to Test Method Wools of New Zealand Method 25.

Wool fabrics Content of

permethrin

(wt.-%)

Average

mortality (%)

Mean mass loss

(% of the control)

Visual assessment of

fiber damages due to

foraging

Control - 0.0 100 Moderate damage

Blank treatment* - 7.1 126.4 Moderate damage

β-CD-nanogels - 4.5 85.1 Moderate damage

Perm.-β-CD-nanogels 0.94 40.0 7.5 No visible damage

Perm.-based insecticide* 0.34 7.1 28.5 No visible damage

* Blank treatment and treatment with permethrin by bath exhaustion procedure (98 °C, 1 h, pH 6)

The biological tests show that permethrin containing β-CD nanogels as well as other

substances containing permethrin provide good protection against moth larvae and carpet beetle

larvae. Even in the presence of low concentrations of permethrin high efficiency is obtained. In

these studies, high concentrations of permethrin are tested in nanogels on wool fabrics. In future

work, permethrin concentrations of 0.2 - 0.5 wt.-% offer the same effectiveness in order to

optimize cost and performance.

231

9.4. Summary

In this work, it has been demonstrated that nanogels can be used for coating and

protection of keratin based fibers. Urethane cross-linked β-CD/NCO-terminated sP(EO-stat-PO)

nanogels that were developed and characterized regarding their ability to complex low molecular

weight hydrophobic guest molecules before were choosen for the uptake of permethrin, a

commonly used insecticide. Release tests show that the guest molecule can be extracted by the

use of a methanolic solvent which enables the quantitative analysis by HPLC. Permethrin

containing β-CD nanogels were applied onto wool or human hairs with homogeneous distribution

and particles sizes of 100-200 nm. By the use of higher concentrated nanogel dispersions for the

coating process a gel film was observed on the fibers. Keratin fibers can be treated with

controlled permethrin concentrations from aqueous nanogel dispersion. Washing tests showed

that the insecticide was released during washing and removed from the fibers. However,

irradiation experiments revealed that the complexation of permethrin by -CD nanogels results in

light protection of the light-sensitive ingredient. The irreversible photodegradation of permethrin

can be decreased for about 47 % (cis-permethrin) and 63 % (trans-permethrin) in comparison to

the non-complexed active ingredient. Furthermore, application of -CD nanogels onto wool

fabrics reduces photobleaching when exposed to light. The biological tests demonstrated that

permethrin-loaded β-CD nanogels provide protection of wool from damage by the larvae of

clothes moths or carpet beetles in the same way like non-complexed permethrin. The biological

effectiveness of the insecticide is not affected by the complexation by -CDs. Our method is

straightforward, robust and generic with respect to the active compound and thus opens an

attractive alternative for the treatment and protection of keratin based fibers as well as other

materials.

232

9.5 References


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2. Motornov, M.; Roiter, Y.; Tokarev, I.; Minko, S., Stimuli-responsive nanoparticles, nanogels and capsules for

integrated multifunctional intelligent systems, Prog. Polym. Sci. 2010, 35, 174-211.

3. Albrecht, K.; Moeller, M.; Groll, J., Nano- and microgels through addition reactions of functional oligomers

and polymers, Adv. Polym. Sci. 2011, 234, 65-93.

4. Pich, A.; Richtering, W., Microgels by Precipitation Polymerization: Synthesis, Characterization and

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235

9.6 Supporting Informations

Analytical data cf. Chapter 2.2: β-CD Containing Nanogels

FTIR (KBr): ν/cm-1

= 3356 (m, (OH)), 2870 (m), 1720 (m, sym(C=O), amide I band, urethane),

1677 (m, (N-H)), 1638 (m, (OH), 1458 (m), 1350 (m), 1301 (m), 1248 (m), 1147 (s), 1101 (s),

1081 (s), 1032 (s, (C-O-C)), 1004 (s), 945 (m, sym(C-O-C)), 863 (m).*

Raman: ν/cm-1

= 3190 (w, br, (O-H)), 2929 (s, asym(CH2)), 2873 (s, sym(CH2)), 1700.9 (w,

(C=O)), 1454 (m, asym(CH2)), 1373 (w, (CH2)), 1278.6 (m, (CH)), 1236 (w, (C-O)), 1124

(w, asym(C-O-C)), 1024 (w, (C-C)), 906 (w, (C-C)), 862 (w, sym(C-O-C)), 827 (w, (C-C)),

796.5 (w, (C-C)).*

1H-NMR (DMSO-d6, 400 MHz): (ppm): 1.036 (w, CH3, sP(EO-stat-PO)), 3.306 (m, CH-2, 4,

CD), 3.350 (w, sP(EO-stat-PO)), 3.508 (m, CH-5, 3, CH2-6, CD), 3.549 (s), 3.641 (s, CH2,

sP(EO-stat-PO)), 4.466 (w, C-OH-6, CD), 4.830, (w, CH-1, CD), 4.466 (w, C-OH-6, CD), 5.62-

5.66 (s, C-OH-2, 3) **.

13C-NMR (DMSO-d6, 400 MHz): (ppm): 17.113 (w, CH3 or CH2), 28.867 (w, CH3 or CH2 or

CH), 69.759 (m, CH2, sP(EO-stat-PO)), 72.380 (w, C5, CD), 74.208 (w, C-N, urethane), 81.497

(w, C4, CD), 102.149 (w, C1, CD) **.

Analytical data cf. Chapter 2.2: β-CD-Containing Nanogels after Complexation of Permethrin

UV: maximum at 278 nm (in water).

FTIR (KBr): /cm-1

= 3404 (s, (OH), 2921 (s, br, (C-H)), 1720 (w, (C=O), amide I band,

urethane), 1642 (w, (OH) or (C-N-H)), 1585 (w, (C=C)benz), 1535 (w, (C-N) + (N-H),

amide II band, urethane), 1487 (w, (C=C)benz and/or asym(CH3) + (CH2)), 1457 (m,

236

(C=C)benz), 1254 (m, asym (C-O-C)), 1156.5 (s, asym (C-O-C)), 1030 (s, (C-O-C), glucose),

945.5 (m, sym(C-O-C), 757.5 (w, (C-H)benz), 578 (w, (O-H)).*

Raman: /cm-1

= 3055 (s, (C-H)benz), 2954 (s, asym(C-H)), 2846 (m, sym(C-H)), 1732 (w,

(C=O)ester), 1617 (w, (C=C)), 1595 (w, (C-C) or (C=C)benz), 1456 (m, asym(CH3)), 1373 (w,

(CH2)), 1278.6 (w, (CH) or breath of cyclopropyl residue), 1238 (w, (C-O)), 1128 (w, (C-

C)), 1033 (w, (C-O)), 1003 (w, breath vibration of benzene ring), 921 (w, (C-H)benz), 837 (w,

breath vibration of glucose ring), 567 (w, (C-H)benz i.p. or (O-H)), 480 (out-of-plane

deformation of glucose ring or (C-C-C) of permethrin).*

1H-NMR (DMSO-d6, 400 MHz): (ppm): 1.036 (w, CH3, (sP(EO-stat-PO)), 1.164 (w, CH3,

cis/trans-permethrin), 2.042 (w, C-H, cyclopropane, permethrin), 3.306 (m, CH-2, 4, CD), 3.350

(w, sP(EO-stat-PO), 3.508 (m, CH-5, 3, CH2-6, CD), 3.549 (s), 3.641 (s, CH2, sP(EO-stat-PO)),

4.466 (w, C-OH-6, CD), 4.830 (w, CH-1, CD), 5.109 (w, COO-CH2-Ar, permethrin), 5.62-5.66

(s, C-OH-2, 3), 5.703 (w, HC=CCl2, permethrin), 7.005 (w, CHar, permethrin), 7.403 (w, CHar,

permethrin) **.

13C-NMR (DMSO-d6, 400 MHz): (ppm): 17.113 (w, CH3 or CH2), 28.867 (w, CH3 or CH2 or

CH, cyclopropane), 67.762 (s, CH-O or CH-C=), 69.759 (m, CH2, sP(EO-stat-PO)), 72.380 (w,

C5), 74.208 (w, C-N, urethane), 81.497 (w, C4), 102.149 (w, C1), 118.750 (w, C=C, olefinic or

aromatic, permethrin), 128.463 (w, C=C, olefinic or aromatic, permethrin), 130.059 (m, C=C,

olefinic or aromatic, permethrin).**

Remarks:

*IR or Raman: ν = stretching, δ =scissoring, = out-of-plane bending, br = broad, w = weak, m =

medium, s = strong, asym = asymmetric, sym = symmetric, i.p. = in-plane, o.o.p = out-of-plane,

benz = benzene.

** NMR: = chemical shift, s = strong, m = medium, w = weak, ar = aromatic.

237

Figure S1. Dispersion of cyclodextrin-containing sP(EO-stat-PO) nanogel (3) which contains

complexed permethrin after 3 days standing at ambient laboratory conditions.

Figure S2. FESEM images of uncoated human hairs.

3 µm

239

Chapter 10

β-Cyclodextrin based host-guest systems for safe application and

release of insecticides (Appendix to Chapter 9)

Abstract: In this work, a series of common and alternative active agents for insect-proofing were

selected to study the uptake and release properties of these agents by/from β-CDs. Here,

theoretical and preparative complexation studies of fourteen selected synthetic and natural

insecticides are made to evaluate the potential use in finishing of textiles for safe application with

maximum protection. Computer-based Monte Carlo force field simulations were performed to

calculate the energetically preferred -CD complexes of selected insecticides. The resulting

ranking list of all active agents complexed by β-CD is presented with the theoretically most

stable β-CD insecticide complexes. It shows that greater molecules form less stable complexes

with β-CD than smaller molecules. The real insecticide β-CD complexes were prepared in

aqueous solution based on the theoretical calculations, and they were characterized by different

analytical methods. Furthermore, release studies of active agents from the complexes are made by

head space GC/MS analysis. The results show that the release process depends on the guest

molecule and on the present water content.

240

10.1 Introduction

Many potential active agents find application for insect-proofing of humans, crops or

materials. Permethrin (pyrethroid ester), sulcofuron (urea insecticide) and chlorfenapyr (pyrrole

insecticide) are such insecticides which are used to prevent the attack of moths and beetles to

wool textiles [1]. Furthermore, pyrethroid esters show activity against mosquitos as well. This led

to the development of different types of textiles which are treated with e.g. permethrin containing

products. Other insecticides like chlorpyrifos (pyridine organothiophosphate), propoxur

(phenylmethyl carbamate) and mosquito repellents like icaridin and DEET are used in different

areas against many species of insects.

Naturally occurring insecticides find more and more acceptance, but their activity is

controversially discussed and is not proofed sufficiently. A great many of terpenes are interesting

for insect-proofing especially for moth-proofing. Azadirachtin A, the main component of neem

oil, shows activity against many insects, e.g. against moths and carpet beetles [2]. Cedrol, the

main active ingredient of cedar wood oil, terpineol, menthone, limonene und camphor are also

used against insects and as alternative to synthetic insecticides [3]. In any case, the right

concentration must be applied to sustain the activity against insects, keep up the permanence and

protect people from contact with high concentrations of toxic insecticides.

The complexation of insecticides by cyclodextrins (CDs) presents an interesting approach

for both safe application and controlled release [4]. Cyclodextrins are well known to create host-

guest systems with amphiphilic and hydrophobic compounds because of their conical structure

providing a hydrophobic interior space and a hydrophilic outer shell. Hence, sensitive active

agents in different applications can be protected by such inclusion complexation from

degradation caused by various environmental factors e.g. UV-irradiation and from uncontrolled

release in the near environment [5]. Nowadays, different applications of CDs in commercial

products are used in wide range and especially the combination of CD with textiles show focus of

interest. On textiles, CDs have been fixed covalently using reactive groups e.g.

monochlorotriazinyl groups or physically by self-bonding of CD containing nano- and microgels

almost for the uptake of bad odors or release of perfumes [6,7].

In this study, seven synthetic and seven natural active agents against insects were selected

on basis of their effectiveness, availability, structure, and agent class. The aim of the work is the

241

investigation of the complexation and release properties of these agents by β-CD for safe

application on textiles with maximum protection.

10.2 Experimental

10.2.1 Computer based simulations of insecticide-β-CD complexes

Theoretical complexation studies were made by means of Macromodel 8.0, using the

OPLS force-field [8]. The solvent environment (water) was simulated as dielectric continuum,

parameterized in OPLS. Two guest molecules and one host molecule were positioned together

and in respect to their orientations and their energetically favored positions a sufficient large

number of configurations was simulated, a mode-integration procedure (MINTA [9]) was used to

estimate the Gibbs free energy of the system.

10.2.2 Preparation of insecticide-β-CD complexes

Practical complexation of the hydrophobic guest molecules into the β-CD core were made

in water. An oversaturated aqueous solution of β-CD (Wacker, Burghausen) (8.81 x 10-4

mol/L)

was heated up to 50 °C under stirring. The guest molecules (Aldrich, Taufkirchen) were added in

an equimolar ratio to the clear solution and the mixture was stirred over 6 h at 50 °C. After

cooling to room temperature the host-guest complexes precipitate as solid. The precipitated was

filtered off and washed with small amounts of cold water and acetone. The β-CD insecticide

complex was dried gently at 40 °C for two hours.

10.2.3 Characterization of β-CD complexes

1H-NMR and

13C-NMR spectra were recorded with the help of a Bruker 400 NMR

spectrometer (Bruker, Rheinstetten) operating at 400 MHz. The samples were dissolved in

deuterated dimethylsulfoxide (DMSO-d6). IR measurements were made using a FTIR

spectrometer: Nexus 470 (Thermo Nicolet, Offenbach) (spectral disintegration: 8 cm-1

). -CD

complexes were dried and pressed in KBr-pellets.

UV-vis absorption spectra were recorded with a Varian Cary 100 UV-VIS

spectrophotometer (Varian, Darmstadt) using quartz cuvettes with an optical path length of 1 cm.

Water was used as reference.

242

Isothermal titration microcalorimetry studies were performed with the help of Microcal

VP-ITC Isothermal Titration Calorimeter (MicroCal, LLC, Northampton, MA). Measurements of

the enthalpy changes associated with the addition of successive aliquots of a solution of guest

compounds (i.e. 4.8 mM camphor, 0.1 mM sulcofuron) to 0.1mM host (β-CD) solution at 25 °C

were made. Thus, for all the systems, the sample cell of the microcalorimeter (1.4 mL) was filled

with 0.1 mM solution of respective host molecules, and after baseline equilibration, i. e. 4.8 mM

of camphor solution was injected in 34 × 8 µL aliquots using the default injection rate. It was

found that adding guest solution from the syringe to the host system solution in the cell gives a

very good representation of the saturation process of the β-CD cavities by guest. ITC software

supplied by MicroCal, LLC was used. Experimental parameters for titration experiments were:

number of injections: 34; cell temperature: 25 °C; stirring speed: 290 rpm; cell volume: 1.43 mL;

injection volume: 8 µL; injection duration: 16 s; spacing: 300 s; and filter speed: 2 s. Figure 6

(left) shows a typical ITC result of the camphor titration to the host system. Considerable strong

interaction occurs when camphor was titrated with such β-CD, as indicated by the heat flow.

signal (in µcal/sec in Figure 5).

Headspace (HS) solid phase micro extraction (SPME) gas liquid chromatography mass

spectrometry studies (GC-MS) were performed by Varian GC: 3800, MS: 2000 Saturn (Varian

Deutschland GmbH, Darmstadt) using Optima 5-MS-Separation Column (30 m x 0.25 mm),

(Macherey & Nagel, Düren). Helium was used as carrier gas. The process of release was

followed by headspace SPME-GC-MS. 25 mg of each β-CD complex were gently dried and

weighed in to headspace vials under standard conditions (constant atmosphere: 65 % r. h., 20 °C).

A second series of samples was prepared in the same way and 30 µL of water were added. All

samples were kept at RT for 12 h and, afterwards, the samples were heated for 2 h at 100 °C to

establish equilibrium conditions. Finally, samples from the gas phase were taken and analyzed by

headspace-SPME-GC-MS.

243


10.3.1 Theoretical studies of the stability of insecticide β-CD complexes

Since force-field calculations do not easily permit to determine absolute values of

thermodynamic functions, a series of mutually comparative calculations was performed that

allowed to generate a relative sequence of host-guest stabilities. In aqueous solvent environment

which was simulated as dielectric continuum, (parameterized in OPLS) two different guest

molecules were positioned in the vicinity of one β-CD molecule. Subsequently, relative positions

and orientations of the three molecules were randomly altered during a Monte Carlo stochastic

search, followed by minimizing the force field energy after each Monte Carlo step. After a

sufficient large number of configurations were simulated, a mode-integration procedure

(MINTA) was used to estimate the Gibbs free energy of the system. The most stable

configurations contained a host-guest complex with the more suitable of the two competing

molecules located in the β-CDs cavity (Figure 1). Pair-wise combinations of 14 different guest

molecules were treated in this procedure, allowing the setting up of a rank list of relative

stabilities according to (i) calculated free enthalpies, and (ii) the ability of a certain guest to

occupy the host’s cavity in the presence of a competitor (cf. Table 1).

Figure 1. Force-field simulation of cedrol, azadirachtin A and β-CD and Monte Carlo stochastic

search of the most stable configuration.

Azadirachtin

Cedrolenergetically preferred

constitution of the molecules

All energetically stable constitutions of

Azadirachtin, Cedrol and β-Cyclodextrin in Waterβ-Cyclodextrin

244

Menthone and terpineol were found to form the most stable complexes with β-CD. Cedrol,

limonene and the large azadirachtin A formed energetically unfavorable β-CD complexes; hence

the investigated natural insecticides created the two best, but also the complexes of lowest

stability (limonene, azadirachtin) with β-CD. All synthetic insecticides formed β-CD inclusion

complexes of intermediate stability and were ranked in the upper middle field of Table 1.

Icaridin, sulcofuron und permethrin formed the most stable β-CD complexes of the synthetic

insecticides.

Table 1. Ranking list of selected active agents for insect-proofing complexed by β-CD and the

included Figure 2. energetically preferred positions of two guest and one β-CD molecule as host

after Monte Carlo stochastic search.

energetically preferred

β-cyclodextrin complex

energetically preferred

β-cyclodextrin complex

No. Name Agent class

1 (-) Menthone natural material/ monoterpene

2 Terpineol natural material/ monoterpene

3 Icaridin repellent/ piperidinecarboxylate

4 Sulcofuron urea insecticide

5 Permethrin pyrethroidester

6 Chlorfenapyr pyrrol insecticide

7 Diethyltoluamide/DEET repellent/ diethyltoluamide

8 Chlorpyrifos pyridine organothiophosphate

9 Propoxur phenylmethyl-carbamate

10 Quassin natural material/ triterpene-lactone

11 L-(-) Camphor natural material/ monoterpene

12 R-(+) Cedrol natural material/sesquiterpene

13 R-(+) Limonene natural material/ monoterpene

14 Azadirachtin A natural material / tetranortriterpenoid

No. Name Agent class

1 (-) Menthone natural material/ monoterpene

2 Terpineol natural material/ monoterpene

3 Icaridin repellent/ piperidinecarboxylate

4 Sulcofuron urea insecticide

5 Permethrin pyrethroidester

6 Chlorfenapyr pyrrol insecticide

7 Diethyltoluamide/DEET repellent/ diethyltoluamide

8 Chlorpyrifos pyridine organothiophosphate

9 Propoxur phenylmethyl-carbamate

10 Quassin natural material/ triterpene-lactone

11 L-(-) Camphor natural material/ monoterpene

12 R-(+) Cedrol natural material/sesquiterpene

13 R-(+) Limonene natural material/ monoterpene

14 Azadirachtin A natural material / tetranortriterpenoid

Figure 2: Sample of force-field

simulation of menthone, sulcofuron

und β-cyclodextrin

sulcofuron

menthone

245

10.3.2 Experimental studies of the complexation of synthetic and natural active agents by β-CD

The preparation of solid β-CD complexes is a simple procedure which can be performed

in one synthesis step. The proof of the formation of host-guest systems was required for all

selected guests. Complexations of guest molecules into the β-CD cone take place in water. Water

insoluble organic agents were added in an equimolar ratio to a clear oversaturated β-CD solution

and the mixture was stirred over 6 h at 50 °C. Water molecules act as means of transportation and

conduct the hydrophobic guests to the hydrophobic inner cavity of the β-CD cone. Trapped water

molecules are pushed out by the hydrophobic guests and the energetically preferred host-guest

complexes are formed. After cooling to room temperature, the host-guest complexes precipitate

as solid. Depending on the molecule size and on the bearing functional groups the inclusion takes

place as 1:1, but also as 2:1 or 1:2 host-guest complexations. The complexes were characterized

by different analytical methods depending on the guest molecule. IR, UV-vis and headspace

GC/MS analysis was made to prove that the complexation occurred. NMR studies show how

many guest molecules can be incorporated in one β-CD ring. Thermodynamic data characterize

the complexation in situ. For molecules with high vapor pressure headspace-GC/MS analysis

characterizes the complex and the release behavior of the insecticide from the β-CD.

Preparative investigations

The obtained yield after reaction and purification is also a good parameter to characterize

the complexation process itself and furthermore, the hydrophobicity and the resulting solubility

properties of the complex in water. β-CD complexes loaded with amphiphilic guest molecules

show good solubility behavior, in most cases a better solubility than the pure uncomplexed guest

molecule. Some highly structured hydrophobic guests complexed in the β-CD cavity show bad

solubility in water and precipitate directly after complexation. Working in oversaturate β-CD

solution promotes the precipitation of the complex after cooling.

Hence, high yield of precipitated complexes results in good uptake of water insoluble guest

molecules. On the other hand, a low yield of the complex indicates the complexation of an

amphiphilic, water soluble guest, of completely encapsulated small hydrophobic molecules or of

the occurrence of a bad complexation process (cf. Table 2).

246

Cedrol and camphor containing β-CD complexes precipitate in a high amount with yields of 70

%. This leads to the conclusion that the complexation of molecules or just parts of the high

structured molecules takes place. Here, further hydrophobic groups of the large molecule stick

out of the β-CD molecule and the hydrophobicity of the whole complex increases, this effect

results in high water insolubility of the complex.

DEET and icaridin are rather water insoluble molecules as well and their corresponding

complexes have been obtained in yields of around 20 %. These molecules are smaller and the

structure exhibits a better complexation than in case of cedrol and camphor. Furthermore, a better

solubility of the complex than of the guest molecules themselves is expected which results in a

lower yield of the complexation.

Complexes of β-CD with smaller guest molecules like menthone could be obtained in a very

small yield. Here, in conformity with the theoretical simulations, the complextion should be

formed, but the small size of the molecules and the tendency for the formation of a fully

encapsulated 1:1 complex led to better solubility of the complex than of the highly structured

guest.

The complex of terpineol with β-CD on the other side could be obtained in yields of 50 %. It is

theoretically ranked together with menthone on top of the theoretically calculated ranking list of

complexes (Table 1). The structure is also relatively similar, but the yield of the corresponding

complex is different due to its solubility. It occurs that the carbonyl group of menthone influences

the complexation more than the hydroxy group of terpineol. It can be concluded that the

formation of hydrogen bonds of terpineol and β-CD between its hydroxy groups and hydroxy

groups of the β-CD ring are more handicapped than hydrogen bonds between the carbonyl group

of menthone and β-CD. These hydrogen bond formations between host and guest can determine

the inner circle of the hydrogen bonds linked between all glucose units within the β-CD molecule

and results in better solubility of the host and the guest. Hence, menthone β-CD complexes do not

precipitate in such a large amount in water as terpineol β-CD complexes.

According to the theoretical calculations, the complexation of limonene should be

disadvantageous but in combination with a fully interior encapsulation by the β-CD cone the

solubility should increase. Thus, a low yield of the complex formation is expected.

Quassin has also a voluminous high structure and thus it is more handicapped with regard to the

formation of a complex with β-CD. On the other hand the complex should have a decrease in

247

solubility because β-CD cannot include the molecule completely and the part which sticks out

promotes the formation of a more hydrophobic complex. These are indications for a rather poor

complexation process.

Table 2 List of all active agents which were complexed by β-CD. Data of the complexes, the

achieved yields and the characterization are presented.

No Complex Empirical formula Results/

Yield

Analysis

1 (-)Menthone-β-CD C10H18O – C42H70O35 white solid;

8 %

IR and NMR

spectroscopy,

headspace GC/MS

2 Terpineol-β-CD C10H18O – C42H70O35 white solid;

56 %

NMR spectroscopy,

headspace GC/MS

3 Icaridin-β-CD C10H18O – C42H70O35 white solid;

21 %

headspace GC/MS

4 Sulcofuron-β-CD C19H11Cl4N2Na O5S –C42H70O35 white solid

34 %

NMR spectroscopy,

ITC

5 Permethrin-β-CD C21H20Cl2O3 – C42H70O35 white solid

30 %

HPLC and

headspace GC/MS

6 Chlorfenapyr-β-CD C15H11BrClF3N2O – C42H70O35 white solid

28 %

headspace GC/MS

7 DEET-β-CD C12H17NO – C42H70O35 white solid;

20 %

headspace GC/MS

8 Chlorpyrifos-β-CD C9H11Cl3NO3PS – C42H70O35 white solid

24%

headspace GC/MS

9 Propoxur-β-CD C11H15NO3 – C42H70O35 white solid

16 %

headspace GC/MS

10 Quassin-β-CD C20H26O7 – C42H70O35 yellow solid;

9 %

headspace GC/MS

11 L-Camphor-β-CD C10H16O – C42H70O35 white solid;

76 %

IR, UV-Vis and

NMR spectroscopy,

headspace GC/MS

ITC

12 R-(+)Cedrol-β-CD C15H26O – C42H70O35 white solid;

74 %

NMR spectroscopy,

headspace GC/MS

13 R-(+)Limonene-β-CD C10H16 – C42H70O35 white solid;

7 %

UV-Vis and

NMR spectroscopy,

headspace GC/MS

14 Azadirachtin-β-CD C35H44O16 – C42H70O35 yellow solid

6 %

HPLC analysis

248

IR spectroscopy

If these guests contain a typical functional group, e. g. a carbonyl group, the purified

complex can be studied by IR spectroscopy. Here, a typical IR signal of specific functional

groups characterizes the presence of the guest molecule trapped by the host molecule. Camphor

and menthone contain carbonyl groups. The typical bands at 1709 and 1730 cm-1

in the IR spectra

of the respective complexes prove that the complexation occurred (Figure 3). The band at 1032

cm-1

belongs to β-CD and characterizes the C-O-C stretching vibration of the glucose units.

Figure 3. IR spectra of menthone (left) and camphor (right) show the typical bands of the

carbonyl groups at 1709 and 1730 cm-1

.

4000 3500 3000 2500 2000 1500 1000 500

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

Menthone-CD Complex

Wavenumbers [cm-1]

Absorb

ance

1709

4000 3500 3000 2500 2000 1500 1000 500

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Ab

so

rba

nce

Wavenumbers [cm-1]

Camphor-CD Complex

1730

12

3

456

7

H3C8 CH3

9

CH3

10

O 11

1

23

4

5

6

CH3

7

8

CH3 9H3C10

O 11

249

UV-vis spectroscopy

UV-vis spectroscopy is a good analytical method as well to study the complexation in

solution. β-CD does not show any absorbance in the UV-vis spectrum. Hence, UV-vis active

guest molecules can be studied without any disturbance by the host molecules. Hypsochromic or

bathochromic shift or increase or decreas of absorbance without change in λmax have been

considered as evidence for the interaction between β-CD and the guest in the formation of the

complex. Hydrogen bonding can be considered as the main force behind the inclusion complex

formation. In Figure 4, UV-vis spectra of camphor and limonene are shown with the typical

absorbance of the guest molecule in DMSO. For investigation of the complex in solution DMSO

was chosen because the two components, guest as well as host are soluble in the same solvent. It

occurs, that after dilution the complex was stable and the guest was not decomplexed by DMSO.

Cleavage or change of the existing hydrogen bonds in the compound can lead to a bathochromic

shift (red shift) due to complexation. Here, bathochromic shifts were observed in complexation of

limonene and in complexation of camphor in β-CD.

Figure 4. UV-Vis spectra of limonene (left) and camphor (right) in DMSO show the typical

absorbance, but with a lower absorbance and a bathochromic shift of the corresponding

complexes.

250 275 300 325 3500,0

0,1

0,2

0,3

0,4

0,5

Limonene CD Complex

Limonene

Ab

sorb

ance

Wavelength [nm]

275 300 325 3500,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

Ab

so

rba

nce

Wavelength [nm]

Camphor CD Complex

Camphor

12

3

456

7

H3C8 CH3

9

CH3

10

O 11

1

23

4

5

6

CH3

7

8

H3C9 CH2 11

250

NMR spectroscopy

By NMR spectroscopy the structure of the obtained β-CD complex can be determined. In

Figure 5, 1H-NMR and

13C-NMR spectra of 1:1 β-CD complexes of cedrol and terpineol are

given. The integrals of the peaks belonging to the guest molecule can be compared with the peak

integrals belonging to the host. The results show that the integral of the C1-proton at each of the 7

glucose units standing for 1 β-CD molecule, is equivalent to all protons of the guest molecule

which appear in the low field of the 1H-NMR spectrum. The obtained solid terpene β-CD

complexes are characterized as simple 1:1 complex. Here, cyclic structure in combination with

the molecular size favors a 1:1 complexation with β-CD. Cyclohexane and phenol rings are

suitable for the β-CD cone.

Figure 5. 1H-NMR (left) and

13C-NMR spectra (right) of terpineol (above) and cedrol (below) in

DMSO-d6 show the typical H and C signals of the guests and the host with an integral ratio of

1:1.

ppm (t1)

1.02.03.04.05.06.0

0

100000000

200000000

300000000

400000000

5.7

37

5.7

20

5.6

78

5.6

72

4.8

32

4.8

24

4.4

58

3.9

39

3.6

61

3.6

56

3.6

34

3.6

15

3.3

73

3.3

63

3.3

28

3.3

03

2.5

05

2.5

01

1.8

35

1.8

20

1.7

22

1.7

01

1.5

15

1.4

59

1.4

46

1.3

22

1.3

10

1.2

80

1.2

31

1.1

46

0.9

22

0.8

12

0.7

94

7.0

0

14.1

6

7.0

0

28.4

0

29.3

4

1.8

2

0.9

8

26.8

7

Cedrol beta CD KomplexCedrol-β-CD Complex

C15H26O - C42H70O35

prim OHCH 12 sek OH

11

12 13

9,5,1,2,10,7,3,14,

9,5,1,10,14

15

CH 4,2,3 CH 5

CH2 6

β-CD

1

2

3

4

5

67

8

9

10

CH3

11

H3C12

CH3

13

14

CH3

15

OH 16

ppm (t1)

50100

0

50000000

100000000

150000000

200000000

250000000

300000000

10

1.9

03

81

.50

3

73

.00

2

72

.56

4

72

.37

0

71

.99

4

60

.20

6

59

.87

0

55

.93

4

53

.66

0

43

.02

7

41

.18

1

40

.88

5

36

.44

0

34

.74

6

30

.98

9

30

.54

7

28

.90

8

28

.00

6

24

.90

8

15

.50

0

Cedrol beta CD Komplex

Cedrol-β-CD Complex

C15H26O - C42H70O35

5, 6,98 7 4 3 14 1 10,2,15,12 13 11C1 C4 C2,3,5 C6

1

2

3

4

5

67

8

9

10

CH3

11

H3C12

CH3

13

14

CH3

15

OH 16

ppm (t1)

1.02.03.04.05.06.0

0

100000000

200000000

300000000

400000000

5.6

99

5.3

48

4.8

32

4.8

23

4.4

56

4.4

52

4.4

41

3.6

56

3.6

34

3.6

19

3.6

12

3.3

74

3.3

51

2.5

04

1.6

07

1.5

98

1.0

87

1.0

32

1.0

19

13

.14

7.0

0

6.1

6

29

.91

35

.39

2.0

4

0.7

9

16

.55

Terpinol beta CD Komplex

1

23

4

5

6

CH3

7

8

H3C9

OH10

CH3 11

5

9, 11

7

14 3 2

CH 1

prim OH

2 sek OH

Terpineol-β-CD Complex

C10H18O - C42H70O35

CH 4,2,3

CH2 6

CH 5

β-CD

ppm (t1)

50100

0

50000000

100000000

133.0

03

130.9

77

120.9

96

101.9

02

81.4

96

73.0

02

72.3

68

71.9

87

70.4

21

67.5

16

59.8

65

44.4

99

30.6

64

29.5

57

27.2

96

26.5

08

26.4

36

26.2

80

25.3

66

23.5

50

23.2

04

19.8

25

Terpinol beta CD Komplex1

23

4

5

6

CH3

7

8

H3C9

OH10

CH3 11

6

35

8

1, 9, 11, 4, 7, 2

Terpineol-β-CD Complex

C10H18O - C42H70O35

C1

C6

C2,3,5C4

251

Isothermal titration microcalorimetry (ITC)

Stability constants of the complex and the change in thermodynamic data of the host-

guest system serve as good parameter of the complexation and stability of the complex. In case of

slightly water soluble molecules with solubility constants higher than 10-3

-10-4

mol/L, the

complexation can just be investigated by isothermal titration microcalorimetry (ITC). Here, the

temperature change generated during the reaction can be measured and the changes in enthalpy

can be determined. With the determination of the complexation constants, the free Gibbs enthalpy

can be calculated and furthermore, the free entropy of the complexation. Changes in enthalpy and

entropy are associated with the alteration of the water structure within the cavity and the removal

of the water from the cavity as well as restructuring of water around the guest molecule and

release of water into the bulk. Other contributions to the overall energies of reaction are due to

the restriction in rotation around the glycosidal linkages of the β-CD when the guest molecule

enters the cavity [10].

Sulcofuron and camphor are slightly water soluble, thus complexation studies via isothermal

titration microcalorimetry (ITC) experiments were performed with β-CD in aqueous solution at

25 °C to allow the investigation of the complexation in more detail. Measurements of the

enthalpy changes associated with the addition of successive aliquots of a solution of guest

compound (i.e. 4.8 mM camphor, 0.1 mM sulcofuron) to 0.1 mM host (β-CD) solution at a fixed

temperature were made.

The thermograms of the complexation and the calculated thermodynamic data are shown for

camphor (Figure 6) and sulcofuron (Figure 7). From these studies, the stoichiometry of binding

interaction between the guests and the hosts are confirmed to be 1:1. The calorimetric data for the

interaction of sulcofuron and camphor with β-CD indicated extremely strong binding as the

association constants are in the order 103-10

4/M, which is in correlation with the established 10

2-

104/M range quoted for other β-CD complexes [11].

252

Figure 6. Calorimetric titration of the host with L-camphor (a) and sulcofuron (b) in water at

298.15 K. Raw data for 34 sequential injections of camphor solution to β-CD solution (left

above). Reaction heat obtained from the integration of the calorimetric traces (left below). “Net”

heat effects of the host-guest interaction for each titration step, fitted by computer simulation

using the “one set of binding sites model” for calculation of complexation data (right).

253

10.3.3 Release studies of the insecticides from the β-CD complexes

Four natural insecticides were selected for release studies of the active agents from β-CD.

Cedrol, L-camphor, menthone and terpineol exhibit a high vapor pressure and are suitable for

release under different conditions. The process of release was followed by headspace (HS) solid

phase micro extraction (SPME) gas liquid chromatography mass spectrometry studies (GC-MS).

25 mg of each terpene-β-CD complex were gently dried and weighed into headspace vials under

standard conditions (constant atmosphere: 65 % r. h., 20 °C). A second series of samples was

prepared in the same way and 30 µL of water were added. All samples were kept at RT for 12 h

and, afterwards, the samples were heated for 2 h at 100 °C to establish equilibrium conditions.

Finally, samples from the gas phase were taken and analyzed by headspace-SPME-GC-MS.The

obtained chromatograms with the corresponding mass spectra of each released active agent are

shown in Figure 7. Different amounts of the guest molecule are released from the terpene β-CD

complexes. For each type of active molecule the same conditions for preparation of the

complexes and of the samples for the GC-MS were ensured; the only difference for the release of

the guest molecule was the controlled contingent of water. The first result is that the release of

the guest molecules from the complexes is readily supported by moisture in the ambient air.

Furthermore, the chromatograms show an increase in the water content of the gas phase going

along with a higher release of the active agent. The experiment shows the influence of water on

the release properties of the host-guest complexes. Higher water contents of the environment

favor the complex dissociation, and accelerate the guest release.

In comparison with the theoretical calculations, menthone and terpineol complexes form

stable complexes with β-CD but on the other side these complexes need more water for the

release of the agents than host-guest complexes ranked in the lower part of the Table 1. Hence,

stable complexes in the dry state which are rather suitable for complexation in β-CD needs more

water for the release than complexes with guest molecules theoretically ranked in the lower part

of (Table 1).

254

Figure 7. Mass spectrum (above) and the release of the terpenes cedrol (a), camphor (b),

menthone (c) and terpineol (d) from their β-CD complexes in absence (middle) or presence

(bottom) of additional water (determined by headspace-SPME-GC-MS). The prozentual

difference of the release in presence and absence additional water is calculated.

C h ro m a t o g r a m P lo ts

P l o t 1 : d : \ h a u s \ s i l ly \ s p m e \ k e t t e l \ k 1 a t e r p i n e o l . s m s R I C a l l

P l o t 2 : d : \ h a u s \ s i l ly \ s p m e \ k e t t e l \ k 1 b t e r p i n e o l . s m s R I C a l l

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0m /z

0 %

2 5 %

5 0 %

7 5 %

1 0 0 % 8 1

1 3 6

2 2 7

S p e c t 1

8 . 8 3 2 m in . S c a n : 5 4 9 C h a n : 1 I o n : 1 4 9 u s R IC : 1 6 6 5 2 2 8B P 1 3 6 ( 1 9 0 6 0 4 = 1 0 0 % ) k 1 a t e r p i n e o l .s m s

6 7 8 9 1 0 1 1 1 2m in u te s

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

M C o u n t s

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

M C o u n t s

R IC a l l k 1 a te r p i n e o l .s m s

R IC a l l k 1 b te r p i n e o l .s m s


P l o t 1 : d : \ h a u s \ s i l ly \ s p m e \ k e t t e l \ k 2 a m e n t h o n . s m s R I C a l l

P l o t 2 : d : \ h a u s \ s i l ly \ s p m e \ k e t t e l \ k 2 b m e n t h o n . s m s R I C a l l

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0m /z

0 %

2 5 %

5 0 %

7 5 %

1 0 0 %

3 9 6 9

1 1 2

1 3 9

2 1 1 2 8 1

S p e c t 1

8 . 2 7 1 m i n . S c a n : 5 1 5 C h a n : 1 I o n : 1 4 7 2 u s R IC : 2 2 9 8 6 0B P 1 1 2 ( 2 1 3 3 1 = 1 0 0 % ) k 2 a m e n t h o n .s m s

6 7 8 9 1 0 1 1 1 2m i n u te s

0

1

2

3

4

5

6

M C o u n t s

0

1

2

3

4

5

6

M C o u n t s

R IC a l l k 2 a m e n th o n .s m s

R IC a l l k 2 b m e n th o n .s m s

Mass spectrum

of Terpineol

Mass spectrum

of Menthone

ambient

conditions

ambient

conditions

Plus

additional

water

Plus

additional

water

209 %

more release

21 %

more release


P l o t 1 : d : \ h a u s \ s i l ly \ s p m e \ k e t t e l \ k 4 a c e d ro l. s m s R I C a ll

P l o t 2 : d : \ h a u s \ s i l ly \ s p m e \ k e t t e l \ k 4 b c e d ro l. s m s R I C a ll

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0m /z

0 %

2 5 %

5 0 %

7 5 %

1 0 0 %

4 3

9 5

1 3 5

2 0 7

S p e c t 1

1 7 . 1 0 4 m i n . S c a n : 1 0 7 5 C h a n : 1 Io n : 2 0 5 u s R IC : 1 6 0 0 5 2 5B P 9 5 ( 1 2 3 5 3 6 = 1 0 0 % ) k 4 a c e d r o l. s m s

1 5 1 6 1 7 1 8 1 9 2 0m i n u te s

0

5

1 0

1 5

2 0

M C o u n t s

0

5

1 0

1 5

2 0

M C o u n t s

R IC a l l k 4 a c e d r o l . s m s

R IC a l l k 4 b c e d r o l . s m s

C h ro m a t o g r a m P l o ts

P l o t 1 : d : \ h a u s \ s i l ly \ s p m e \ k e t t e l \ k 5 a c a m p h e r . s m s R I C a l l

P l o t 2 : d : \ h a u s \ s i l ly \ s p m e \ k e t t e l \ k 5 b c a m p h e r . s m s R I C a l l

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0m /z

0 %

2 5 %

5 0 %

7 5 %

1 0 0 %

3 9

9 5

1 5 2

2 1 1 2 8 1

S p e c t 1

8 . 2 7 3 m i n . S c a n : 5 1 7 C h a n : 1 I o n : 6 4 0 u s R IC : 4 7 5 7 9 4B P 9 5 ( 6 2 0 7 0 = 1 0 0 % ) k 5 a c a m p h e r .s m s

6 7 8 9 1 0m i n u te s

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

M C o u n t s

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

M C o u n t s

R IC a l l k 5 a c a m p h e r . s m s

R IC a l l k 5 b c a m p h e r . s m s

Mass spectrum

of Camphor

Mass spectrum

of Cedrol

ambient

conditions

ambient

conditions

Plus

additional

water

Plus

additional

water

93 %

more release

11 %

more release

c d

a b

HS-SPEME-GC HS-SPEME-GC

HS-SPEME-GC HS-SPEME-GC

255

10.4 Summary

Fourteen selected synthetic insecticides and natural active agents were investigated in

respect to their complexation behavior with β-CD. Theoretical, computer based studies of the

stability and the complexation possibilities in β-CDs were performed. All selected agents were

calculated against each other in the presence of β-CD and the most energetically preferred host-

guest complexes were obtained by Monte Carlo-Force Field-Simulations. The constructed

ranking list of all insecticides showed that smaller molecules like menthone and terpineol are

complexed more preferentially to bigger molecules like azadirachtin A. β-CD complexes

containing synthetic active agents like permethrin and chlorfenapyr are arranged in the middle

field of the ranking list. Complexes from the selected insecticides and β-CD were successfully

prepared in the laboratory using water as solvent. Different analytic methods like IR, UV-Vis,

NMR spectroscopy, Headspace-GC/MS and ITC experiments were used for specific

characterization of host-guest systems and for the investigation of the complexation process in

more detail. Release experiments with four selected natural active agent-β-CD complexes (cedrol,

terpineol, menthone, camphor) showed that the release depends on the guest molecule and on the

water concentration in the environment. The release is preferred by addition of water which

accelerates the decomplexation by displacement with water.

256

10.5 References

1. Schaefer K.; Kettel M. J., Moeller M., New Concepts for the Insect Proofing of Textile Materials (AiF 15123 N),

Proceedings of the 2nd

Aachen-Dresden International Textile Conference 2008, ed. Doerfel A., ITB/TU Dresden

(ISSN 1867-6405), Dresden/Germany, Poster P83.

2. Schaefer K., Neem-Extrakte zur Parasitenschutzausrüstung von Wolle (AiF 15123 N), Proceedings 3rd

Aachen-

Dresden International Textile Conference 2009, ed. Kueppers B., DWI an der RWTH Aachen e.V. (ISSN 1867-

6405), Aachen/Germany, Poster P43.

3. Skórka M.; Asztemborska M.; Zukowski J., Thermodynamic studies of complexation and enantiorecognition

processes of monoterpenoids by α- and β-cyclodextrin in gas chromatography, J. Chromatography A 2005,

1078, 136-143.

4. Szente L.; Szejtli J., Cyclodextrins in Pesticides, Compr. Supramol. Chem. 1996, 3, 503-514.

5. Dodziuk, H., Applications other than in pharmaceutical industry, Cyclodextrins and Their Complexes, Dodziuk,

H., Eds.; WILEY-VCH: Weinheim, 2006; pp 450-466.

6. Buschmann, H. J.; Knittel, D.; Beermann, K.; Schollmeyer, E., Cyclodextrine und Textilien, Nachrichten aus der

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