Synthesis of Polymer Nanocomposites for Drug Delivery and...

Synthesis of Polymer Nanocomposites

for Drug Delivery and Bioimaging

Heba Asem

Licentiate Thesis in Physics

School of Information and Communication Technology

Royal Institute of Technology KTH

Stockholm, Sweden 2016

TRITA-ICT 2016:09 School of Information and

ISBN 978-91-7595-930-6 Communication Technology

SE-164 40 Kista

Sweden

Akademisk avhandling som med tillstånd av Kungliga Tekniska högskolan- KTH framlägges till

offentlig granskning för avläggande av teknologie licentiatexamen i fysik fredagen den 10 juni 2016

klockan 10:00 i Sal C, Electrum, KTH, Isafjordsgatan 26, Kista, Stockholm.

Cover image: schematic diagram showing the composition of multifunctional nanoparticle and its

multiple applications

© Heba Asem, June 2016

Universitetsservice US-AB, Stockholm 2016

This Thesis is Dedicated to my Family, my Lovely Husband

& In the Loving Memory of my Father

(1959-2013)

i

Abstract

Nanomaterials have gained great attention for biomedical applications due to their

extraordinary physico-chemical and biological properties. The current dissertation presents

the design and development of multifunctional nanoparticles for molecular imaging and

controlled drug delivery applications which include biodegradable polymeric nanoparticles,

superparamagnetic iron oxide nanoparticles (SPION)/polymeric nanocomposite for

magnetic resonance imaging (MRI) and drug delivery, manganese-doped zinc sulfide

(Mn:ZnS) quantum dots (QDs)/ SPION/ polymeric nanocomposites for fluorescence

imaging, MRI and drug delivery.

Bioimaging is an important function of multifunctional nanoparticles in this thesis.

Imaging probes were made of SPION and Mn:ZnS QDs for in vitro and in vivo imaging. The

SPION have been prepared through a high temperature decomposition method to be used

as MRI contrast agent. SPION and Mn:ZnS were encapsulated into poly (lactic-co-glycolic)

acid (PLGA) nanoparticles during the particles formation. The hydrophobic model drug,

busulphan, was loaded in the PLGA vesicles in the composite particles. T2*-weighted MRI of

SPION-Mn:ZnS-PLGA phantoms exhibited enhanced negative contrast with r2* relaxivity of

523 mM-1 s-1. SPION-Mn:ZnS-PLGA-NPs have been successfully applied to enhance the

contrast of liver in rat model.

The biodegradable and biocompatible poly (ethylene glycol)-co-poly (caprolactone)

(PEG-PCL) was used as matrix materials for polymeric nanoparticles -based drug delivery

system. The PEG-PCL nanoparticles have been constructed to encapsulate SPION and

therapeutic agent. The encapsulation efficiency of busulphan was found to be ~ 83 %. PEG-

PCL nanoparticles showed a sustained release of the loaded busulphan over a period of 10

h. The SPION-PEG-PCL phantoms showed contrast enhancement in T2*-weighted MRI.

Fluorescein-labeled PEG-PCL nanoparticles have been observed in the cytoplasm of the

murine macrophage cells (J774A) by fluorescence microscopy. Around 100 % cell viability

were noticed for PEG-PCL nanoparticles when incubated with HL60 cell line. The in vivo

biodistribution of fluorescent tagged PEG-PCL nanoparticles demonstrated accumulation of

PEG-PCL nanoparticles in different tissues including lungs, spleen, liver and kidneys after

intravenous administration.

Keywords: Biodegradable polymers, SPION, QDs, drug delivery, cytotoxicity, MRI, in

vivo fluorescence imaging, biodistribution

iii

Sammanfattning

Nanomaterial har fått stor uppmärksamhet för biomedicinska tillämpningar på grund av

deras extraordinära fysikalisk-kemiska och biologiska egenskaper. Denna avhandling

presenterar design och utveckling av multifunktionella nanopartiklar för molekylär

avbildning och kontrollerad drogleveransapplikationer som inkluderar bionedbrytbara

polymera nanopartiklar, superparamagnetiska järnoxidnanopartiklar (SPION)/ polymer

nanokomposit för magnetisk resonanstomografi (MRI) och drogleverans, mangan-dopade

zink sulfid (Mn:ZnS) kvantprickar (QDs) / SPION/polymera nanokompositer för

fluorescensavbildning, MRI och drogleverans.

Bioimaging är en viktig funktion av multifunktionella nanopartiklar i denna avhandling.

Avbildningssonder var gjorda av SPION och Mn: ZnS QDs för in vitro- och in vivo-

avbildning. SPION var tillverkad genom en hög temperatur sönderdelnings metod som skall

användas som kontrastmedel vid MRI. SPION och Mn:ZnS inkapslades i poly (mjölksyra-

Co-glykolsyra) (PLGA) nanopartiklar under partikelbildning. Den hydrofoba

modelläkemedel, busulphan, laddades i PLGA vesiklar i kompositpartiklar. T2* viktade MRI

av SPION-Mn:ZnS-PLGA fantomer uppvisade förstärkt negativ kontrast med r2*

relaxiviteten av 523 mM-1 s-1. SPION-Mn: ZnS-PLGA NPs har tillämpats med framgång för

att förbättra kontrast av levern i råttmodell.

Den bionedbrytbara och biokompatibla poly (etylenglykol) -Co-poly (kaprolakton)

(PEG-PCL) användes som matrismaterial för polymera nanopartikelbaserade

drogleveranssystem. De PEG-PCL-nanopartiklarna har konstruerats för att inkapsla SPION

och terapeutiskt medel. Inkapslingseffektiviteten av busulphan var ~ 83 %. PEG-PCL-

nanopartiklar visade en oavbruten frisättning av den laddade busulphan under en period av

10 timme. De SPION-PEG-PCL fantomerna visade kontrastförbättring i T2*- viktade MRI.

Fluorescein-märkt PEG-PCL-nanopartiklar har observerats i cytoplasman hos de murina

makrofagceller (J774A) genom fluoreszenzmikroskopie. Omkring 100 % cellviabilitet

märktes för PEG-PCL nanopartiklar när de inkuberas med HL60-cellinje. In vivo-

biofördelningen av fluorescerande-märkta PEG-PCL-nanopartiklarna visade ackumulering

av PEG-PCL-nanopartiklar i olika vävnader inkluderande lungorna, mjälten, lever och

njurar efter intravenös administrering.

Nyckelord: Bionedbrytbara polymerer, SPION, kvantprickar, drogleverans, cytotoxicitet,

MRI, in vivo fluorescens avbildning, biofördelningen.

iv

Papers included in the Thesis

This thesis is based on the following publications:

I. Fei Ye, Åsa Barrefelt*, Heba Asem*, Manuchehr Abedi-Valugerdi, Ibrahim El-

Serafi, Maryam Saghafian, Khalid Abu-Salah, Salman Alrokayan, Mamoun Muhammed,

Moustapha Hassan, “Biodegradable polymeric vesicles containing magnetic

nanoparticles, quantum dots and anticancer drugs for drug delivery and imaging”,

Biomaterials, 2014, 35, 3885–3894.

II. Heba Asem, Ahmed Abd El-Fattah, Noha Nafee, Ying Zhao, Labiba Khalil, Mamoun

Muhammed, Moustapha Hassan, Sherif Kandil, “Development and biodistribution of a

theranostic aluminum phthalocyanine nanophotosensitizer”, Photodiagnosis and

photodynamic therapy, 2016, 13, 48-57.

III. Heba Asem, Ying Zhao, Fei Ye, Åsa Barrefelt, Manuchehr Abedi-Valugerdi, Ramy EL-

Sayed, Ibrahim El-Serafi, Svetlana Pavlova, Khalid Abu-Salah, Salman A. Alrokayan,

Jörg Hamm, Mamoun Muhammed, Moustapha Hassan,“ Biodistribution of busulphan

loaded biodegradable nano-carrier system designed for multimodal imaging”,

Manuscript submitted to Molecular Pharmaceutics.

Other work not included in the Thesis

I. Åsa Barrefelt, Gaio Paradossi, Heba Asem, Silvia Margheritelli, Maryam Saghafian,

M., Letizia Oddo, Mamoun Muhammed, Peter Aspelin, Moustapha Hassan, Torkel

Brismar. “Dynamic MR imaging, biodistribution and pharmacokinetics of polymer

shelled micro bubbles containing SPION”, Nano, 2014, 9, 1450069.

II. Noha Nafee, Alaa Youssef, Hanan El-Gowelli, Heba Asem, Sherif Kandil, “Antibiotic-

free nanotherapeutics: Hypericin nanoparticles thereof for improved in vitro and in

vivo antimicrobial photodynamic therapy and wound healing”, International Journal

of pharmaceutics, 2013, 454, 249–258.

III. Ramy El-Sayed, Fei Ye, Heba Asem, Radwa Ashour, Wenyi Zheng, Mamoun

Muhammed, Moustapha Hassan. “Importance of the surface chemistry of nanoparticles

on peroxidase-like activity”, Manuscript.

*Equal contribution

http://www.sciencedirect.com/science/article/pii/S014296121400057X











http://www.sciencedirect.com/science/journal/01429612/35/12

http://www.scopus.com/authid/detail.url?authorId=55630953600&eid=2-s2.0-84905878066







http://www.sciencedirect.com/science/article/pii/S0378517313005875






v

Conference presentations

I. Heba Asem, Noha Nafee, Ahmed Abd El-Fattah, Labiba El-Khordagui, Mamoun

Muhammed, Moustapha Hassan, Sherif Kandil, “Biodegradable Polymeric

Nanoparticles for Photodynamic Therapy” (poster), Photodynamic and Nanomedicine

for Cancer Diagnosis and Therapy Workshop, 2014, 13-15 February, GUC (German

University in Cairo), Berlin, Germany.

II. Heba Asem, Ahmed Abd El-Fattah, Noha Nafee, Labiba Khalil, Mamoun muhammed,

Sherif Kandil, Moustapha Hassan “synthesis and characterization of biodegradable

aliphatic polyester for drug delivery applications” (poster), EUPOC: Bio-based Polymer

and Related Biomaterials, poster prize, 2011, 29 May- 3 June, Gargnano, Italy.

Contribution of the author

Paper I. Planning some experiments, preparing the materials, coordinating collaboration,

performing some of materials characterization, analyzing the results and writing part of the

article.

Paper II. Initiating the idea, planning of experiments, preparing the materials, carrying out

materials characterization, parsing the results and writing the article.

Paper III. Initiating the idea, planning of experiments, preparing the materials, performing

materials characterization, analyzing the results and writing the manuscript.

vi

List of abbreviations and symbols

APTMS (3-aminopropyl) trimethoxysilane

CMC critical micelle concentration

CT computed X-ray tomography

DCM dichloromethane

DDS drug delivery systems

DI deionized water

DMEM dulbecco’s modified Eagle medium

DMF dimethylformamide

DNA deoxyribonucleic acid

DSC differential scanning calorimetry

ε-CL epsilon-caprolactone

EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

FDA food and drug administration

FBS fetal bovine serum

FeCl3· 6H2O iron chloride hexahydrate

FE-TEM field emission transmission electron microscopy

FTIR Fourier transform infrared spectroscopy

HPLC high-performance liquid chromatography

IV intravenous

IVIS In vivo imaging system

Mn:ZnS manganese-doped zinc sulfide

MnCl2 manganese chloride

MNPs magnetic nanoparticles

MnSt2 manganese stearate

MPS mononuclear phagocyte system

MRI magnetic resonance imaging

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Mw weight average molecular weight

N2 nitrogen gas

NHS N- Hydroxysuccinimide

NIR near-infrared

NMR nuclear magnetic resonance

NPs nanoparticles

ODE octadecene

http://www.sigmaaldrich.com/catalog/product/aldrich/281778

https://en.wikipedia.org/wiki/Di-

https://en.wikipedia.org/wiki/Di-

https://en.wikipedia.org/wiki/Thiazole

https://en.wikipedia.org/wiki/Phenyl

https://www.google.se/url?sa=t&rct=j&q=&esrc=s&source=web&cd=13&cad=rja&uact=8&ved=0CD0QFjAMahUKEwjnr46o-IDHAhUilHIKHcYeDDs&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F21106329&ei=YRe5Vaf7HqKoygPGvbDYAw&usg=AFQjCNFDAuA2inUv-3y5a0bdDZh8SOS3qw&sig2=IzC2rbz0pmRvXp8uLQMqpA&bvm=bv.99028883,d.bGQ

vii

OLA oleylamine

O/W oil-in-water emulsion

PBS phosphate buffer saline

PEG-PCL poly(ethylene glycol)-co- poly(caprolactone)

PEG polyethylene glycol

PEO polyethylene oxide

PCL poly(caprolactone)

PGA poly(glycolic acid)

PICM polyion complex micelles

PLA poly(lactic acid)

PLGA poly(lactic-co-glycolic) acid

PS photosensitizer

PVA polyvinyl alcohol

QDs quantum dots

RF radio frequency

ROI region of interest

ROP ring opening polymerization

RPMI Roswell Park Memorial Institute medium

SA stearic acid

SEC size exclusion chromatography

SPECT single-photon emission computed tomography

SPION superparamagnetic iron oxide nanoparticles

TE echo time

TEM transmission electron microscopy

TGA thermogravimetic analysis

THF tetrahydrofuran

TMAOH tetramethylammonium hydroxide

UV/Vis ultraviolet-visible

ZnAc2 zinc acetate dehydrate

σ standard deviation

ix

Table of contents

ABSTRACT.......................................................................................................... i

SAMMANFATTNING .......................................................................................... iii

Papers included in the Thesis .....................................................................................................iv

Other work not included in the Thesis..........................................................................................iv

Conference presentations .......................................................................................................... v

Contribution of the author ........................................................................................................... v

List of abbreviations and symbols ...............................................................................................vi

1 INTRODUCTION ............................................................................................. 1

1.1 Objectives ...................................................................................................................... 1

1.2 Outlines ......................................................................................................................... 2

1.3 Drug delivery systems (DDS)........................................................................................... 3

1.3.1 Polymeric matrices ........................................................................................................................ 4

1.3.2 Micellar nanostructures................................................................................................................. 5

1.3.3 Magnetic nanoparticles ................................................................................................................. 6

1.3.4 Quantum dots ................................................................................................................................. 6

1.4 Multifunctionality of drug carriers...................................................................................... 7

1.4.1 Drug loading and release ............................................................................................................. 7

1.4.2 Targeting ......................................................................................................................................... 8

1.4.3 Visualization ................................................................................................................................... 9

1.4.3.1 Magnetic resonance imaging ............................................................................................... 9

1.4.3.2 Fluorescence imaging ......................................................................................................... 11

1.5 Preparation methods of nanoparticles ............................................................................. 11

1.5.1 Preparation of polymeric nanoparticles .................................................................................... 11

1.5.2 Preparation of magnetic nanoparticles ..................................................................................... 12

2 EXPERIMENTAL .......................................................................................... 13

2.1 Synthesis of PEG-PCL drug carrier ............................................................................... 13

2.2 Fabrication of nanoparticles for imaging ........................................................................ 13

2.2.1 Synthesis of SPION ................................................................................................................... 13

2.2.3 Preparation of SPION-polymeric NPs ..................................................................................... 14

2.2.4 Fluorescent PEG-PCL-NPs ...................................................................................................... 14

x

2.3 Characterization ........................................................................................................... 14

2.4 Drug release ................................................................................................................ 15

2.5 Degradation of polymer matrix ....................................................................................... 16

2.6 Cell uptake and biocompatibility evaluation..................................................................... 17

2.6.1 Cell lines and culture conditions ................................................................................................ 17

2.6.2 Cellular uptake.............................................................................................................................. 17

2.6.3 Cell viability (MTT) assay............................................................................................................ 17

2.7 MRI and fluorescence imaging....................................................................................... 18

2.7.1 In vitro magnetic resonance imaging ........................................................................................ 18

2.7.2 In vivo fluorescence imaging/ computed tomography (CT) ................................................... 18

2.7.3 Necropsy and histology ............................................................................................................... 19

3 RESULTS AND DISCUSSION .......................................................................20

3.1 Synthesis and characterization of PEG-PCL drug carrier .................................................. 20

3.2 Inorganic imaging probes ............................................................................................. 20

3.2.1 Microscopic studies of SPION and QDs .................................................................... 20

3.2.2 Magnetic and optical properties ................................................................................................. 22

3.3 Multifunctional DDS ...................................................................................................... 22

3.3.1 Microscopic studies and size control ........................................................................................ 22

3.3.1.1 SPION-Mn:ZnS-PLGA NPs ................................................................................................ 22

3.3.1.2 SPION-PEG-PCL-NPs ......................................................................................................... 23

3.3.2 Biocompatibility evaluation of polymeric NPs .......................................................................... 24

3.3.3 Cell uptake assessment of polymeric NPs .............................................................................. 24

3.4 Drug release................................................................................................................. 25

3.5 MRI and fluorescence imaging....................................................................................... 27

3.5.1 MRI phantoms of SPION-Mn:ZnS-PLGA and SPION-PEG-PCL-NPs ................................ 27

3.5.2 In vivo MRI .................................................................................................................................... 28

3.5.3 In vivo fluorescence imaging/ Computed Tomography (CT) ................................................ 29

3.5.4 Histological analysis .................................................................................................................... 29

4 CONCLUSIONS ............................................................................................32

FUTURE WORK ................................................................................................33

ACKNOWLEDGEMENTS ..................................................................................34

REFERENCES...................................................................................................35

1

1 Introduction

Nanotechnology refers to an interdisciplinary area of research such as chemistry, physics,

biology, and medicine that have been achieved tremendous progress in the past several

decades. Due to the size reduction to nanometer, the nanomaterials gained extraordinary

chemical, physical, optical and biological properties.1,2 These unique properties offer great

opportunities and potential benefits in a wide spectrum of applications involving energy

storage3, 4, water remediation5, 6, food processing7, 8, health monitoring and diseases

therapy.9 The interaction between the nanotechnology and medicine will support the next

generation of nanomedicine and facilitate a personalized and efficient therapy with

minimized side effects.10 Nanotechnology has demonstrated a tremendous potential in

improving both biomedical research and clinical applications because the nanoobjects are

generally in the similar size range with biological entities, e.g. cells, organelles, DNA, and

proteins. Therefore, nanomaterials have potentials to tune the bioactivity in the molecular

level. Fabricating nanomaterials such as nanospheres, nanotubes, nanorods, porous

nanoparticles and nanocomposites have been designed for diverse biomedical applications

especially on cell separation11, 12, controlled drug delivery, gene delivery13-15, non-invasive

imaging16-21, and cancer therapy.22-25 The advantages of nanostructures such as controlled

size and shape, design flexibility, surface modification with multivalent ligands are suitable

for multifunction applications including bioimaging and targeted drug delivery. This will

facilitate the improvement of drug pharmacokinetics and greatly increase the efficiency of

medical therapy at the level of single molecule or molecular assemblies.

1.1 Objectives

Two drug delivery systems are investigated in the present work: polymeric nanoparticles and

polymer/inorganic nanocomposites for imaging and controlled drug delivery:

A series of synthesized poly (ethylene glycol)-co-poly (caprolactone) (PEG-PCL)

copolymers are used to prepare nanophotosensitizer with different particles size. PEG-PCL

nanoparticles (NPs) are prepared to study the loading of a hydrophobic drug model

(photosensitizer) for photodynamic therapy with a sustained drug release. The

biodistribution of the free drug and drug-loaded PEG-PCL-NPs are studied in animal model

in different organs.

2

Multifunctional nanoparticles composed of PEG-PCL loaded with SPION are developed to

be used as a drug carrier to deliver anticancer drug such as busulphan. The drug release from

the NPs is studied over period of 10 h. The multifunctional drug carrier are examined as a

contrast agent for T2*-weighted MR imaging. The biocompatibility and cell uptake of PEG-

PCL-NPs are assessed in cell lines. For simultaneous biodistribution evaluation, the PEG-

PCL was modified with amino group and conjugated with VivoTag 680XL for in vivo

fluorescence imaging and monitoring the PEG-PCL-NPs in different organs.

Furthermore, nanocomposite system is designed for multimodal imaging and

controlled anticancer drug release. PLGA vesicles encapsulating two kind of inorganic

nanocrystals, SPION as contrast probe for MRI and Mn: ZnS QDs for fluorescence imaging.

SPION-Mn: ZnS-PLGA NPs are examined as drug delivery system for cytostatic drugs and

as T2 contrast agent for MRI. The SPION-Mn: ZnS-PLGA NPs are employed to study the cell

uptake by fluorescence microscopy. In addition, these nanocomposites are served to image

some organs in a rat model under MRI.

1.2 Outlines

This thesis addresses the development of multifunctional NPs such as drug carrier for

various biomedical applications including controlled drug release, MRI and photodynamic

therapy.

Chapter 1.3 briefly introduces different categories of drug delivery systems, e.g.

biodegradable polymers, liposomes, denderimers and smart polymers, contrast agents for

MRI and fluorescence imaging e.g. SPION and QDs. Chapter 1.4 introduces biomedical

application of multifunctional drug carriers such as drug loading of cytostatic drugs, drug

release, targeted drug delivery and bioimaging. Different imaging techniques such as MRI

and fluorescence imaging were mentioned in this chapter. A brief explanation of physical

background of MRI and superparamagnetism are discussed in this chapter as well. Chapter

1.5 briefly introduces different techniques for preparation of polymeric nanoparticles and

magnetic nanoparticles.

Chapter 2.1 starts with the synthetic methodology of the PEG-PCL copolymer and the

polymerization conditions. Chapter 2.2 presents the synthesis of SPION by thermal

decomposition method and Mn: ZnS QDs using nucleation-doping method. The loading of

different materials into the PEG-PCL and PLGA NPs such as SPION, QDs, busulphan and

aluminum phthalocyanine chloride (AlPc) drug are studied to form multifunctional

nanoparticles. Chapter 2.3 mentions various characterization tools to measure the physico-

3

chemical properties of the prepared polymers and nanoparticles. Chapter 2.4 and 2.5 study

the drug release from nanocarriers by dialysis method and the degradation of the polymer

matrix, respectively. The cell uptake of the nanoparticles in different cell lines is described

in chapter 2.6. Studies of in vitro and in vivo MRI and fluorescence imaging are summarized

in chapter 2.7.

Chapter 3 presents and discusses the results of synthesis and properties of

nanomaterials for biomedical applications. In chapter 3.1 analysis of PEG-PCL copolymer

are discussed using FTIR, 1H-NMR, and TGA. Chapter 3.2 demonstrates the morphologies

of SPION and QDs as well as the magnetic and optical properties of the nanocrystals. In

chapter 3.3 the properties of multifunctional drug delivery system comprised of SPION-

Mn:ZnS-PLGA-NPs and SPION-PEG-PCL-NPs are studied. In this chapter the cytotoxicity

evaluation and cellular uptake of the PEG-PCL-NPs reveals that these particles are

biocompatible at the studied concentrations and can be taken by different cell lines. In

chapter 3.4, the drug release of busulphan from PLGA and PEG-PCL polymeric matrices

show sustain release profiles in period of 5 h and 10 h respectively. The imaging applications

of the nanoparticles as a contrast agent are investigated in chapter 3.5 in which the SPION-

Mn:ZnS-PLGA phantoms show high relaxivity in vitro and in vivo MRI. Furthermore the

VivoTag 680XL-PEG-PCL-NPs are presented in various organs such as lungs, spleen, liver

and kidneys.

1.3 Drug delivery systems (DDS)

DDS have received great attention and implementations in development of novel medical

devices. DDS enable the introduction of therapeutic agents to human body, improve their

efficacy and safety compared with using unmodified drugs by controlling the rate, time, and

place of release of drugs in the body.26-28 The ideal drug carrier should regulate and optimize

the drug release rate during the therapy in a controlled manner. Minimizing the drug

fluctuation in plasma level and extending its duration of action are the robust functions of

ideal drug carriers. Therefore the frequency of dosing of drug can be reduced and the patient

compliance increased.

Several biocompatible nanomaterials have been utilized to deliver different biological

molecules and therapeutic substances. Liposomes, polymeric nanoparticles, dendrimers,

carbon nanotubes, mesoporous silica and gold NPs (Figure 1.1) are usually used as drug

carriers.29 Liposomes and polymeric NPs are the most extensively investigated drug carrier.

Doxorubicin-loaded liposome (Doxil®) has been approved from FDA as first liposomal drug

delivery formulation for human cancer.30 The hydrophilic drugs can be encapsulated in the

4

inner aqueous core of the liposome, while the lipid bilayer is used to deliver the lipophilic

drugs.31 The particulate drug carriers must be hidden from immune system especially from

the phagocytes uptake and glomerular filtration, as long as liver and kidney are not the target

organs.

Figure 1.1 Different types of nanocarrier systems for drug delivery 29, 32, 33

Phagocytic uptake of the drug carriers is influenced by particles size, surface charge and

hydrophobicity.34 The particles size from 200 nm to several microns showed higher

phagocytic uptake and adsorption of opsonins on the surface of the particle. However,

modification the particles surface with hydrophilic polymer (e.g. poly (ethylene glycol), PEG)

impedes interaction of the particle towards environmental proteins including opsonins.35-37

PEGylation of polymeric nanoparticles can be achieved by adhering surfactants like

poloxamers or polysorbates onto nanoparticles, or by formulating nanoparticles with

copolymers comprising PEG and a biodegradable moiety.

1.3.1 Polymeric matrices

The most attractive class of polymers for drug delivery system is biodegradable polymers.

Biodegradable polymers are one type of biomaterials that can decompose into nontoxic

products and normal metabolites.38 Biodegradable polymers are classified into two

categories: natural polymer, such as cellulose, chitosan, polypeptides and proteins, and

synthetic polymers including poly (glycolic-co-lactic) acid (PLGA), poly (lactic acid) (PLA),

poly (glycolic acid) (PGA), poly (caprolactone) (PCL), aliphatic polycarbonates and

polyphosphazenes.39 Biodegradable polymers such as poly esters are produced by two

general routs from a variety of monomer via ring opening polymerization (ROP) of lactones

and lactides (e.g. ROP of ε-caprolactone monomer (Figure 1.2)) or condensation polymer-

5

ization between diols and diacids.40-42 The active linkage (ester, amine, urethane and enol-

ketone) along the main polymer chain has great influence on the biodegradability of

polymers, which can be hydrolyzed and/or oxidized to small compartments under hydrolysis

or enzymatic action.43 Lactic acid and glycolic acid are the main degradation products of PLA

and PGA, respectively which are biocompatible and are easily eliminated from the body.44

Additionally, the degradation rate of bulk polymer is influence by different factors including

the hydrophilicity, molecular weight, crystallinity, surface area, type of repeating units, and

polymer compositions.45

Biodegradable polymers-based drug carriers have been developed for controlled release

of drug over prolonged period. Polymeric nanospheres protect drugs, particularly

hydrophobic drugs, from degradation by physiological environment. Further protection of

the drug is achieved by PEGylation of the polymeric nanoparticles.46 The drug can be

entrapped in the polymeric NPs in different ways; the payload disperses in the matrix space

of the nanoparticles forming nanospheres or the drug accumulates in the particle core

producing nanocapsule or core-shell nanostructure. Additionally, bioactive agents including

proteins, enzymes and antibodies can be chemically conjugated to polymers chain via

covalent bond.47Another type of polymer-based drug carrier is environment responsive

polymers which demonstrate physicochemical changes in response to environmental

stimuli, such as change in pH, temperature, redox potential, ionic strength, biochemical

agents, and ultrasound48, 49. Hydrogels are three-dimensional, cross-linked networks of

water-soluble polymers which use as carriers for controlled drug delivery due to their

biocompatibility and inertness toward various drugs, especially proteins.50, 51

1.3.2 Micellar nanostructures

Micelles and core-shell structures are class of self-assembly materials in nanoscale which

enclose active constituents in their core. Block copolymers are well established building

blocks for preparation of micelles drug delivery.52, 53 Block copolymer micelles can be

classified depending on the type of intermolecular force between the core and the aqueous

milieu54, 55 ; a) amphiphilic block micelles which formed by hydrophobic interaction between

Figure 1.2 Scheme of ring opening polymerization of ε-caprolactone (ε -CL) monomer

m + catalyst

ɛ-CL monomer PEG PCL-PEG

6

the hydrophobic blocks producing core; b) polyion complex micelles (PICM) which resulting

from electrostatic interaction between polycation and polyanion such as plasmid DNA56, 57;

c) micelles produced by metal complexation.58 The hydrophilic block such as polyethylene

oxide (PEO) can form hydrogen bond with the aqueous environment which creates tight

shell around the micelles core. The amphiphilic block copolymer can form stable micelles

at/or above the critical micelle concentration (CMC). The polymer chains association is

driven by entropy via the expulsion of water molecules into the bulk aqueous phase.59

Further stability of micelles can be achieved by crosslinking of the micelles core as well as

the micelles shell. The cross linking can be affected on the permeability of micelles corona

which in turn control the drug release rate from the core.60 Several methods of drug loading

into micelles have been developed include direct dissolution61, dialysis62, oil/water

emulsion63, chemical conjugation64 and various evaporation procedures.

1.3.3 Magnetic nanoparticles

Magnetic NPs (MNPs) have been applied in various clinical purposes such as targeted drug

delivery systems65-67, hyperthermia68 and contrast agents for MRI.69, 70 MNPs made of

superparamagnetic magnetite (Fe3O4) and maghemite (γ-Fe2O3) are employed for

biomedical applications. One of the major advantages of MNPs is miniaturized particles size

which would strongly enhance their magnetic properties to be superparamagnetic. The ease

surface functionalization of MNPs would increase the chance of attaching various ligands

and cargos with particles surface. MNPs can be coated with polymers e.g. dextran71,

polysaccharides72 , PEG73, as well as mesoporous silica74 for delivering several drugs and

biomolecules. The coating layer protects and shields the particles from environment

conditions. The particles surfaces can be also attached to the function groups such as

carboxyl group, amino group, biotin and carbodi-imide. The drug carrier containing MNPs

inside are magnetically guided and delivers the active agent such as DNA, PDT-agents and

antineoplastic drugs to the site of action. Superparamagnetic iron oxide NPs and gadolinium

oxide are extensively used as MRI markers for tumor visualization in DDS.

1.3.4 Quantum dots

Early detection of tumor is an important prerequisite and crucial stage in cancer therapy.

Some inorganic NPs possess unique optical, electrical or magnetic properties which make it

possible to monitor the neoplastic tissues and the drug carrier with different imaging

modalities. Semiconductor nanocrystals, also called as QDs reveal high fluorescence

quantum yield for biological imaging75-78, higher brightness and photostability than conven-

7

tional organic dyes. The small size of QDs (≤10 nm) has great influence on their absorption

and emission in visible region due to quantum confinement effect. Biocompatible

semiconductors with strong and stable photoluminescence have been fabricated. Different

strategies of surface modification of QDs were used to achieve biocompatibility. Surface

functionalization of QDs with biomolecules such as antibodies, nucleic acids and peptides is

important to minimize their toxicity and greatly improve their use in biomedical

applications, particularly in vivo imaging. These biomolecules can be attached covalently or

non-covalently to the surface of QDs.79, 80 QDs bearing surface functional groups such as

carboxylic, primary amine and thiol can be conjugated with antibodies and peptides by

exploiting cross-linking chemistry of carbodiimide, maleimide and succinimide. Recently,

doped semiconductor nanocrystals are developed presenting additional advantages than the

undoped nanocrystals including larger stockes shift, enhanced thermal and environmental

stabilities and reduced toxicity.81 By introducing transition metal dopant into semiconductor

host, a new class of materials are generated with unique optical, electronic and magnetic

properties.82, 83 ZnS and ZnSe nanocrystals have usually been doped with Mn2+ ion which act

as efficient luminescence center and interact strongly with the s-p electronic state of the host

nanocrystals.84, 85

1.4 Multifunctionality of drug carriers

Conventional drugs administration routes are mostly through intravenous injection causing

general systemic distribution resulting in exposure of drug to physiological degradation by

specific proteins in plasma and losing its therapeutic functions. Prolonged retention time of

drug in blood provides regular distribution to the desired tissue, leading to an increase in

duration of their pharmacological activities.86 Successful combination of different

components of functional nanostructure materials in one entity will enable to integrate

therapeutic and diagnostic in synergistic way. Multifunctional drug carriers-based

nanoparticles will allow delivery of bioactive molecules to target tissues selectively via

passive targeting derived from their nanosize or adsorption of bioactive ligands on the

particles surface for active targeting. Additionally, real time non-invasive monitoring of

biological responses to the therapy can be achieved by these nanosystems which can provide

vital feedback in the treatment of the pathological diseases.

1.4.1 Drug loading and release

Enclosing the drugs and bioactive agents in delivery devices is vital to protect the drug from

Degradation and achieve the maximum therapeutic effect. The advantages of protected

8

devices encompass delaying the dissolution of drug molecules, inhibiting the diffusion of the

drug out of the device and controlling the flow of drug solutions.87 The drug can be loaded

into DDS by different means. In polymer-based DDS, drug can form covalent bond with

polymer chain generating polymer-drug conjugate systems. Another loading system that the

drug dissolves or disperses in the polymer matrix forming monolithic matrix system.88

Likewise the drug core can be enveloped by polymer coating and the drug release rate is

controlled by polymer composition, molecular weight and the thickness of the polymer layer.

Most of therapeutic drugs tend to be released rapidly after the administration which

may cause an increase of drug concentration leading to toxicity in the body. Controlled DDS

can prohibit the systemic toxicity of drug with relatively high drug concentration at the site

of treatment. Controlled drug release from polymer matrix can be explained generally

through two main mechanisms, diffusion and dissolution-based release systems. Drug

release can be defined as transfer of drug solutes from the initial position in drug carrier to

the outer of carrier surface and then to the release medium89. For instance, the drug is

uniformly mixed with a polymeric matrix and is present either in a dissolved or dispersed

structure. The release model of dissolved drug follows Fick’s laws of diffusion from devices

where the drug is enveloped. When the drug is dispersed in the polymeric matrix, the rate of

release follows square root of time kinetics until the concentration of the drug decreases

below the saturation value. 90 The desired releasing profiles of the drug in bulk degrading

systems can be manipulated by adjusting the molecular weight of the polymer, copolymer

composition, crystallinity, the loading amounts of the drug, interaction between polymer

and drug, etc. In addition, the physicochemical properties of the drug, such as solubility,

drug particle size and molecular weight can also affect the release rate.88, 91

1.4.2 Targeting

The main goal of DDS is to deliver the drug molecules to the desired pathological site with

sufficient amount and in selective manner. Two strategies of targeting can be applied, active

or passive targeting to the site of action. Active targeting requires the carrier system attach

covalently with the cell-specific ligand. While in passive targeting, the drug encapsulated in

carrier system reach passively the tumor via enhanced permeability and retention effect

(EPR). 92, 93 Varieties of target ligands can be attached to the drug carrier such as antibodies,

peptides, folate, sugar moieties and other ligands. Drug nanocarriers attached with

glucosamine saccharide moieties on the surface can target liver cells carcinoma.94 Folate-

modified nanocarriers are widely used to target tumors because folate receptor is highly

overexpressed in the majority of human tumor cells.95-97 many nanocarriers systems has

https://www.google.se/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&sqi=2&ved=0CCAQFjAA&url=https%3A%2F%2Fen.wikipedia.org%2Fwiki%2FEnhanced_permeability_and_retention_effect&ei=UFieVZXiI4fpywOLtqroCg&usg=AFQjCNGi6xdoRnTScMsmOgiqyFpVyroCNg&bvm=bv.96952980,d.bGQ

9

been developed with folate tagging on the surface including polymeric NPs98, 99, iron oxide

NPs100, 101, QDs102, mesoporous silica NPs.103

1.4.3 Visualization

Real-time imaging of body internal organs as well as biomolecules such as DNA, peptides

and proteins is of great interest in clinical trials. Various imaging modalities have been

involved including MRI, CT, SPECT and ultrasonography.104 Particularly, contrast probes

such as iron oxide NPs and QDs are used for MRI and fluorescence imaging respectively.

Among others, MRI is a potent imaging modality due to its high spatial resolution, excellent

soft tissue contrast, and non-invasive nature.105, 106 Recently, MRI markers-based drug

carriers have gained remarkable research interest. Polymeric nanoparticles encapsulating

SPION are synthesized as MRI-contrast agent.107 Mesoporous silica coated SPION are

fabricated for theranostic DDS incorporating chemotherapeutics and MRI.21, 108

1.4.3.1 Magnetic resonance imaging

Magnetic resonance imaging has become one of the most important clinical imaging

modality which is widely used in cancer diagnosis. MRI is non-invasive techniques, it

produces high-definition images and detailed visualize the cellular structures and

functions.106, 107, 109 MRI is based on the nuclear magnetic resonance (NMR) which was

discovered in 1946 by two research groups Purcell et al110 and Bloch et al.111 NMR is an

interaction between magnetic field and proton. Under a static magnetic field (B₀), a torque

is produced which results in a precessional movement around the B₀ field. The frequency of

precession, also called the Larmor frequency112, is proportional to B₀, and the constant called

gyromagnetic ratio (γ) as shown in Figure 1.3. All the protons in the magnetic field precess

at the same Larmor frequency, which is a resonance condition. Depending on the internal

energy state, proton will align along the B₀ field which corresponds to a lower energy state.

𝜔₀ = 𝛾𝐵₀

Figure 1.3 Larmor precession of a spinning nucleus about the axis of an applied magnetic field.

10

When a resonant radio frequency (RF) transverse pulse is perpendicularly applied to B₀, it

causes resonant excitation of the magnetic moment precession into the perpendicular plane.

Upon removal of the RF, the magnetic moment gradually relaxes to equilibrium by

realigning to B₀. Such relaxation processes involve two pathways, a) longitudinal or T1

relaxation associated with energy transfer from the excited state to its surroundings (lattice)

and recovery of decreased net magnetization to the initial state, and b) transverse or T2

relaxation from the disappearance of induced magnetization on the perpendicular plane by

the dephasing of the spins.113

An important requirement for useful T2 contrast agents is the large transverse and

longitudinal relaxivities (r2/r1) ratio, in combination with a high absolute value of r2.

Contrast agents that satisfy these conditions are SPION. Another important parameter used

to characterize ferri- or ferromagnetic materials are superparamagnetism.

Superparamagnetic state has been shown in ferromagnetic material when the size is reduced

below critical size (Dc), dependent on the material, which is typically 10-20 nm.114, 115 The

magnetic nanoparticles contain a single magnetic domain with no domain boundary116, upon

applied magnetic field, the magnetic moment is aligned along the direction of easy

magnetization. In the absence of an external magnetic field the magnetic nanoparticles

exhibit no net magnetization due to the rapid reverse of magnetic moment.117 Additionally,

the magnetic anisotropy energy (Ea) is proportional to the particle volume118 which is

expressed as follows:

Ea = 𝐾𝑉 sin2 𝜃 (1.1)

Where V is the particle’s volume, K is anisotropy constant and θ is the angle between

magnetization and the easy axis. When the volume is small enough, the magnetic anisotropy

energy of the particle exceeds its thermal energy KBT, where KB is Boltzmann constant and

T is the absolute temperature, i.e. KBT < Ea, and the magnetization is easily flipped. The

average time to perform such a flip is given by the relaxation time (τ) which is determined

by Néel-Brown relaxation law119:

𝜏 = 𝜏0(𝐸𝑎 𝐾𝐵𝑇⁄ ) (1.2)

Where τ0 is typically in the range of 10-13-10-9 s. If the average time of flip is much smaller

than the measurement time (τm), τ << τm, the system is in supermagnetic state which implies

that the relaxation happens so fast that a time average of the magnetization orientation is

observed within the experimental time. SPION can cause remarkable shortening of T2

relaxation times with signal loss in the diseased tissue and generate contrast to other tissues.

11

1.4.3.2 Fluorescence imaging

Tissue observation with light is probably the most common imaging practice in medicine

and biomedical research ranging from the simple visual inspection of a patient to advance

in vitro and in vivo spectroscopic and microscopy techniques. Fluorescence imaging is

widely used to provide spatial information by measuring the illumination emitted from the

intended organ.120, 121 Traditional optical imaging approaches have been utilized for in vivo

surface and subsurface fluorescence imaging using confocal imaging, multiphoton imaging

microscopic and fluorescence microscopy. Recently, NIR range is used in fluorescence

imaging for in vivo visualization which can penetrate for several centimeters into tissues and

enables disease diagnosis and imaging of treatment response. Various fluorophores have

been developed such as organic dye21, QDs122 and lanthanide-doped upconversion

nanocrystals.123 Conventional organic dyes and fluorescent protein usually require specific

wavelength for excitation and have broad emission spectra, as well as affected by fast

photobleaching and subsequent short fluorescence lifetime.124 However, biocompatible QDs

arise with strong and stable photoluminescence distributed in the visible and NIR region.

Their unique optical properties confer the QDs great attractive for biological applications

due to broad absorption bands, size tunable light emission, large two-photon absorption

cross-section.79, 125

1.5 Preparation methods of nanoparticles

1.5.1 Preparation of polymeric nanoparticles

The properties of polymeric nanoparticles such as hydrodynamic diameter, morphology,

and zeta potential can be optimized depending on the intended application. The preparation

techniques of nanoparticles play a vital role to achieve the desired properties. Many methods

have been developed for preparing polymeric nanoparticles which can be broadly classified

into two main categories, a) the nanoparticles formed through a polymerization reaction or,

b) it is achieved directly from an already prepared polymer. Nanoparticles can be prepared

directly from the dispersion of the preformed synthetic or natural polymers by various

techniques including emulsification-solvent evaporation method, nanoprecipitation method

and salting-out method.126 Emulsification-solvent evaporation method is commonly used for

the preparation of polymeric nanoparticles. This method is characterized by two main steps,

emulsion formation and solvent evaporation. The polymers and drug are dissolved in

organic solvent forming oil phase. Subsequently, the emulsion formed by mixing the oil

phase with an aqueous phase with surfactant. Homogenization or sonication is used to

12

produce nanosized polymer droplets. The organic solvent is then evaporated and the

nanoparticles are usually collected by centrifugation and lyophilization. This method has

been used for encapsulating hydrophobic drugs, however, the drugs with hydrophilic nature

have poor incorporation into the nanoparticles by emulsion method.127, 128 The particle size

can be controlled by adjusting the stirring rate, type and amount of dispersing agent,

viscosity of organic and aqueous phases, and temperature.

The nanoprecipitation method was developed by Fessi et al.129 which based on the

interfacial deposition of a polymer following displacement of a semi-polar solvent miscible

with water from a lipophilic solution in the presence or absence of a surfactant. This method

has been applied to various polymeric materials such as PLA, PLGA, PCL, and poly (methyl

vinyl ether-co-maleic anhydride) (PVM/MA). High loading efficiencies are achieved for

lipophilic drugs when nanocapsules are prepared by nanoprecipitation method.130

1.5.2 Preparation of magnetic nanoparticles

Several chemical methods have been developed to describe the efficient synthesis

approaches of iron oxide NPs with controlled size and shape. Biocompatible and

monodisperse iron oxide NPs are prerequisites for biomedical applications not to induce the

immunogenicity. The most common synthetic approaches of SPION are coprecipitation,

thermal decomposition, hydrothermal synthesis, microemulsion, sonochemical synthesis

which are directed to the synthesis of high quality of iron oxide NPs. The chemical reaction

of Fe3O4 formation can be written as:

OHOFeOHFeFe 243

32 482 (1.3)

Coprecipitation method is the most conventional method for obtaining Fe3O4 or α-

Fe2O3. This method consists of mixing Fe+2/Fe+3 ions in highly basic solutions at room

temperature or at elevated temperature.131 The size and shape of the iron oxide NPs are

dependent on type of salts used (e.g. chlorides, sulfates, nitrates) and several other factors

such as Fe+2/Fe+3 ions ratio, reaction temperature, pH and ionic strength of the media. In

addition, Fe3O4 NPs are not very stable under ambient conditions and are easily oxidized to

Fe2O3 or dissolved in an acidic medium. In order to avoid the possible oxidation in the air,

the synthesis of Fe3O4 NPs must be done in inert condition (oxygen free environment). The

reaction parameters such as stirring rate, dropping speed of basic solution are also effect on

the quality of the nanoparticles. The advantages of the coprecipitation method are high

reproducibility of NPs and large amount of magnetic NPs that can be produced per batch

however, difficulties in controlling the shape and size distribution of the NPs are well

13

identified. Efforts have been devoted to achieve monodisperse magnetic NPs.132 Surfactant,

temperature, pH and ions concentrations are reaction parameters which have been altered

to accomplish small size and morphology control.133-135

Thermal decomposition method has been widely used to synthesize monodisperse iron

oxide NPs.136, 137 Organometallic precursors are decomposed in organic solution at relatively

high temperature in presence of capping agent.138, 139 The organometallic precursors include

metal-oleate complex136, acetylacetonates137, or metal carbonyls.140 Fatty acids141, oleic

acid142, and hexadecylamine143 are often used as surfactants. Other methods, including

microemulsion144, hydrothermal145, and sol-gel146, are also investigated for synthesis of

magnetic NPs.

2 Experimental

2.1 Synthesis of PEG-PCL drug carrier

Amphiphilic copolymer PEG-PCL was synthesized by ring opening polymerization of ε-CL

monomer using PEG as a macroinitiator. An appropriate amount of distilled ε-CL was added

into previously dried PEG in presence of tin (II) 2-ethylhexanoate as a catalyst. The mixture

was stirred under nitrogen atmosphere at 160 °C for 4h. The resulting copolymer was

precipitated in cold diethyl ether, filtered and dried under vacuum at 40 °C for 48 h.

2.2 Fabrication of nanoparticles for imaging

2.2.1 Synthesis of SPION

Monodisperse SPION was synthesized via thermal decomposition method according to

previously reported method.136 Iron-oleate complex was synthesized from sodium oleate and

FeCl3.6H2O followed by refluxing the complex in octyl ether and oleic acid at approximately

297 °C for 1.5 h.

2.2.2 Synthesis of Mn:ZnS QDs

Mn:ZnS QDs were synthesized by a nucleation-doping method. Briefly, manganese stearate

(MnSt2) was prepared by dropwise addition of MnCl2/methanol solution into a mixture of

SA and TMAOH in methanol. A mixture of MnSt2 and 1-dodecanethiol in ODE was then

degassed at 100 °C for 15 min, followed by the addition of sulfur and ZnAc2 at 250 °C.

14

The Mn:ZnS nanoparticles obtained were washed by acetone and re-dispersed in

dichloromethane.

2.2.3 Preparation of SPION-polymeric NPs

SPION-PEG-PCL and SPION-PLGA nanoparticles were prepared by O/W emulsion solvent

evaporation technique. Briefly, an appropriate amount of polymer and SPION were

dissolved in organic solvent (DCM). The organic solution was mixed with aqueous PVA

solution (1:20 oil to water ratio) under probe type sonication for 15 min to form emulsion.

The resulting brownish emulsion was stirred overnight to evaporate organic solvent at room

temperature. The nanoparticles were washed three times against de-ionized (DI) water

(15 MΩ cm) to collect SPION-PEG-PCL and SPION-PLGA-NPs.

2.2.4 Fluorescent PEG-PCL-NPs

The fluorescent nanoparticles were prepared for further biological examination such as

cellar uptake. Fluorescein-labeled PEG-PCL-NPs have been prepared in one step

conjugation reaction. First, chain modification of hydroxyl terminated PEG-PCL copolymer

into amino group by adding 2mmol APTMS to 0.3mmol hydroxyl-PEG-PCL copolymer in

THF, and reflux under N2 overnight. The copolymer was collected by precipitation in excess

diethyl ether, filtered and dried. The carboxy fluorescein was then conjugated with amino

copolymer by adding proper amount of EDC and NHS. The nanoparticles were prepared

from fluorescein-PEG-PCL in DCM by emulsifying in PVA aqueous solution as described

above.

In order to study the biodistribution of the nanoparticles, the VivoTag 680XL labeled-

PEG-PCL were prepared as follows, a solution of VivoTag 680 XL in dimethyl sulfoxide (0.37

mg/ml) was mixed with amino terminated PEG-PCL copolymer solution under stirring for

1h. The labeled copolymer was collected by precipitation in cold diethyl ether and dried. The

non-conjugated VivoTag 680XL was removed by dialysis (MWCO 10kDa) against PBS at

room temperature. The VivoTag 680XL-labeled PEG-PCL-NPs were prepared by previously

described emulsion solvent evaporation method.

2.3 Characterization

The prepared PEG-PCL polymer was characterized using proton nuclear magnetic

resonance (1H-NMR) with Bruker AM 400 at 400 MHz and deuterated chloroform (CDCl3)

as solvent. Fourier transform infrared (FTIR) spectroscopy was performed using a Thermo

15

Scientific Nicolet iS10 spectrometer in the attenuated total reflection (ATR) mode, with a

ZnSe focusing optic in ATR crystal plates. The molecular weight and polydispersity index

(PdI) of polymers were determined by size exclusion chromatography (SEC) with

dimethylformamide (DMF) as mobile phase. The analysis were performed on a TOSOH

EcoSEC HLC-8320GPC system equipped with an EcoSEC RI detector and three columns

(PSS PFG 5µm, Microguard, 100 Å, and 300 Å) (Mw resolving range: 100-300,000 g mol-1)

from PSS GmbH, using DMF (0.2 ml min-1). Differential scanning calorimetry (DSC-Q2000

TA Instruments) was used to measure the thermal behavior of polymers. The samples

heating range was from 25 to 100°C with a constant rate (10°C/min). Thermogravimetic

analysis (TGA-Q500 TA Instruments.) was performed to study the changes of polymer

sample weight with regard to increase the temperature. The temperature was increased from

room temperature to 700°C with constant heating rate of 10°C/min under nitrogen gas.

The morphology of nanoparticles was investigated by JEM-2100 (JEOL Ltd., Tokyo,

Japan) field emission transmission electron microscopy (FE-TEM) at an accelerating voltage

of 200KV. The hydrodynamic diameter of nanoparticles in deionized water was measured

using Beckman Coulter DelsaTM Nano particle size analyzer. The concentration of elements

in suspension was measured by inductively coupled plasma-optical emission spectrometry

(ICP-OES) (Thermo Scientific iCAP 6000 series). The absorbance spectra of the particles

were evaluated by Perkin Elmer 750UV/Vis spectrophotometer. The fluorescence intensity

and images were examined using Perkin Elmer Ls55 Fluorescence spectrophotometer and

IVIS optical imaging system. The amount of lactic acid was measured by high-performance

liquid chromatography (HPLC). The HPLC system consisted of a Gilson autoinjector (100 μl

loop), an LKB HPLC pump 2150 (Pharmacia Inc., Sweden), an LDC analytical

spectromonitor 3200 UV detector (Riviera Beach, FL, USA) and a CSW 32 chromatography

station integrator. Separation was performed on a Zorbax SB-CN column

(4.6 mm × 150 mm, 5 μm) from Agilent Technologies (Santa Clara, CA, USA), and the

column was maintained at room temperature during analysis. The mobile phase was

composed of NH4H2PO4 (0.1 M, pH 2.5) and flow rate was set at 0.8 ml/min with a running

time of 6 min. Injection volume was 50 μl. Detection of lactic acid was performed at 210 nm

compared to a calibration curve for lactic and glycolic acids. Lactic acid eluted after 4 min.

2.4 Drug release

The release profile of busulphan (Figure 2.1) and/or AlPc from two nanocarrier systems,

SPION-PEG-PCL-NPs and SPION-Mn:ZnS-PLGA-NPs, was evaluated using dialysis

method. The drug loaded-NPs were redispersed in PBS and placed in dialysis tube (MWCO

16

10kDa). The dialysis tube was immersed in appropriate amount of PBS pH 7.4 at 37± 0.4°C

under regular shaking (Shaker InFors AGCH-4103 BOTTMNGEN) at 80 rpm. An aliquot of

the buffer was taken from the dissolution medium at different time points. The

concentration of busulphan was measured by gas chromatography (SCION 436-GC, Bruker)

with electron capture detector (ECD). The encapsulation efficacy of the drug was calculated

as amount of the drug released from the nanoparticles and the residual drug in the dialysis

membrane divided by the total amount of the drug. The concentration of the released AlPc

was measured by UV-VIS- spectrophotometer at 670 nm.

2.5 Degradation of polymer matrix

The PLGA polymer is degraded by hydrolytic cleavage of the ester linkage into lactic acid

and glycolic acid (Figure 2.2). The degradation of PLGA-NPs was determined by measuring

the concentration of lactic acid as a degradation product. The prepared PLGA-NPs were

dialyzed against 1 L PBS (pH 7.4) at 37 °C. At predetermined time intervals, a sample of PBS

solution was taken and fresh PBS was added into the dissolution medium. The

concentration of lactic acid released in PBS was measured by HPLC.

Figure 2.1 Chemical structure of: (a) busulphan drug and (b) Aluminum phthalocyanine

chloride (AlPc).

(a) (b)

Figure 2.2 In vitro degradation of PLGA by hydrolysis.

hydrolysis +

PLGA D,L Lactic acid Glycolic acid

17

2.6 Cell uptake and biocompatibility evaluation

2.6.1 Cell lines and culture conditions

The murine macrophage cell line (J774A, the European Type Tissue Culture Collection

(CAMR, Salisbury, UK) was a kind gift from Professor Carmen Fernandez (Department of

Immunology, Wenner-Gren Institute, Stockholm University, Stockholm, Sweden). J774A

cell line was cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with

10 % heat-inactivated FBS (Invitrogen), penicillin (100 µg /ml) (Invitrogen), and

streptomycin (100 µg/ml) (Invitrogen) in 50 cm2 tissue culture flasks (Costar, Corning, NY,

USA). The cultures were maintained at 37°C in a humidified atmosphere containing 5 % CO2.

J774A with electron capture detector (ECD). The encapsulation efficacy of the drug was

calculated as amount of the drug released from the nanoparticles and the residual drug in

the dialysis cells were cultured in 8-chamber polystyrene vessel tissue culture treated glasses

at the density of 5 × 105 cells/chamber at 37°C for 12 h in a 5 % CO2 atmosphere.

The myeloid HL60 leukemia cell line was line was purchased from DSMZ

(Braunschweig, Germany). The cells were cultured in RPMI 1640 medium supplemented

with 90 % GlutaMAX™ and 10% heat-inactivated FBS at 37°C in 95% humidified 5 % CO2

atmosphere. HL60 cells were seeded in 96-well plate at the density of 105 cell/well.

2.6.2 Cellular uptake

The uptake of the SPION-PEG-PCL-NPs by J774A cells was investigated. The nanoparticles

were labeled with fluorescein dye and QDs in order to visualize the nanoparticles by

fluorescence microscopy. The fluorescent nanoparticles were dispersed in medium and

incubated with the cells at concentrations of 1000, 100, 50, 25, 12.5, 6.25 and/or 3μg/ml.

The cell culture medium was aspirated from each chamber and substituted with the medium

alone (control). And then, chambers were incubated at 37°C for 2 h, 4 h, and 24 h in an

atmosphere containing 5 % CO2. The cell uptake experiment was terminated at each time

point by aspirating the test samples, removing the chamber and washing the cell mono layers

with ice-cold PBS three times. Each slide was fixed with methanol-acetone (1:1, v/v),

followed by examination the uptake of fluorescein-PEG-PCL-NPs by employing Eclipse i80

(Nikon, Tokyo, Japan) fluorescent microscope at a wavelength of 520 nm.

2.6.3 Cell viability (MTT) assay

The biocompatibility of the PEG-PCL-NPs was studied by evaluating the cytotoxic effect of

the nanoparticles on HL60 cells using MTT assay. PEG-PCL-NPs were incubated with HL60

18

cells at different concentrations of in 96-well plate with medium (RPMI 1640 with

GlutaMAX™ and 10% FBS) for 48 h at 37°C, 5 % CO2. After the incubation period, MTT

reagent was added to each well. The cells were further incubated for 4 h at 37°C. The

solubilizing agent was added to each well and crystals were solubilized by pipetting up and

down. The absorbance was measured at 570 nm and 690 nm as reference. PBS was used as

blank and cells in medium as control. The cells viability percentage was calculated as (cells

treated with the nanoparticles/ non treated control cells)*100%.

2.7 MRI and fluorescence imaging

2.7.1 In vitro magnetic resonance imaging

MRI phantoms of SPION-PEG-PCL-NPs and SPION-Mn:ZnS-PLGA-NPs were made with

concentrations of (0.1, 0.3, 0.5,1mM) and (0.05, 0.1, 0.2, 0.3, 0.4 , 0.5, 1mM), respectively.

The phantoms were prepared by mixing the nanoparticles suspension with agarose in water

(3 w/v %), heating and then drying in 50 ml falcon tubes at slow rate over night to become a

gel. The phantoms were placed in the extremity coil of a 3 T MR scanner (Siemens, Erlangen,

Germany) and a gradient echo T2* sequence was applied. The repetition time was 2000 ms

and six stepwise increasing echo times (TEs) of 2-17.2 ms for SPION-PEG-PCL-NPs and 12

TEs of 2–22.9 ms for SPION-Mn:ZnS-PLGA-NPs were used to obtain the T2*-weighted

images of the phantoms. Regions of interest (ROIs) were manually placed on the images.

The relaxation time, T2*, was then calculated as the slope of a semi-log plot of the signal

intensity in the ROIs versus the TEs. R2* was calculated as 1/ T2

*. All R2* values for the

phantoms were subtracted by R2* value for the control sample (plain agarose gel). A

standard curve was plotted with R2* (s-1, y-axis) versus concentration (mM, x-axis).

2.7.2 In vivo fluorescence imaging/ computed tomography (CT)

Animal studies were approved by the Stockholm Southern Ethical Committee and

performed in accordance with Swedish Animal Welfare law. The distribution of fluorescence

was observed by an IVIS® Spectrum (PerkinElmer, Waltham, MA, USA). Quantum FX

(Perkin Elmer, Waltham, MA, USA) was also used to co-register functional optical signals

with anatomical μCT. Balb/C mice (22 ± 2 g) were purchased from Charles River (Charles

River Laboratories, Sulzfeld, Germany) and kept for one week in the animal facility to

acclimatize before the experiments. The animals had free access to food and water, ad

libitum, and were kept in a 12 h light/dark cycle under controlled humidity (55 % ± 5 %) and

temperature (21°C ± 2°C). Prior to all experiments, mice were fed for three weeks on a

19

synthetic diet free of unrefined chlorophyll-containing ingredients (alfalfa free, Research

diets, Inc., USA) to minimize the fluorescence noise signal from the gastrointestinal tract. A

suspension of VivoTag 680XL-labeled PEG-PCL-NPs (0.2 ml) was intravenously injected

into the lateral tail vein of the mice. The mice (n= 2 per time point) were anaesthetized using

2-3 % isoflurane (Baxter Medical AB, Kista, Sweden) during the whole imaging procedure.

The 2D/3D fluorescence imaging and μCT scans were performed at 1 h, 4 h, 24 h and 48 h

post injection. The Mouse Imaging Shuttle (MIS, 25 mm high, PerkinElmer) was used to

transfer the mice from the IVIS Spectrum to the Quantum FX-μCT while maintaining their

positions. Mice were firstly imaged by 2D epi-illumination fluorescent imaging in a ventral

position. Subsequently, the mice were imaged in the MIS using the 3D Fluorescent Imaging

Tomography (FLIT) with trans-illumination in a dorsal position. The 3D FLIT imaging

sequence was set up and images were acquired at excitation 675 nm and emission 720 nm.

The mouse in the MIS was then transferred to the Quantum FX-μCT and subjected to a fast,

low dose CT scan with a field of view (FOV) at 60 mm and 17 second scan-time. All images

were generated using the Living Image® 4.3.1 software (PerkinElmer, Waltham, MA, USA).

2.7.3 Necropsy and histology

The mice were sacrificed at pre-determined time points immediately after the imaging

procedure, and histological analysis of lungs, liver, spleen and kidneys was performed using

phase contrast and fluorescence microscopy. To verify the observations from the in vivo live

imaging, the organs were removed from the mice, fixed in paraformaldehyde (4 %) for 24 h,

then transferred to ethanol (70 %), routinely processed and embedded in paraffin. Later,

tissue sections (4 μm) were mounted on super frost glass slides. Slides were routinely stained

with 4',6-diamidino-2-phenylindole (DAPI, 300 nM) to produce nuclear counter stain for

fluorescence microscopic evaluation.

20

3 Results and Discussion

3.1 Synthesis and characterization of PEG-PCL drug carrier

An interesting method to synthesize poly (ester–ether) di-block copolymers is the ring

opening polymerization of lactones using tin (II) 2-ethylhexanoate as a catalyst.147, 148 Ring

opening polymerization is utilized to produce polyesters with controlled molecular weight

under mild reaction conditions and no byproducts.

FTIR spectra of PEG-PCL copolymers are shown in Figure 3.1a. A strong sharp

absorption band appears at 1721 cm-1 is characteristic to the stretching vibration of ester

carbonyl group (C=O) of PCL. PEG-PCL copolymers exhibit characteristic peaks of both PEG

and PCL. The (C-H) stretching bands in PEG and PCL are vibrating at 2863 and 2942 cm ־¹ ,

respectively. PEG-PCL copolymers with different Mw have been prepared (paper II) and the

FTIR shows that the intensity of the band of the carbonyl group (C=O) has increased by

increasing the length of PCL block in the prepared copolymer (Figure 1A, paper II). The

intensity of aliphatic (C-H) stretching band of PCL in the copolymer has increased gradually

by increasing molar feed ratio of monomer/ initiator. The FTIR signals of prepared PEG-

PCL are similar to the results reported by Gyun Shin et.al149 which indicate that the

copolymerization of PCL and PEG is successfully formed. Figure 3.1b shows the ¹H-NMR

spectrum of PEG-PCL copolymer exhibits sharp singlet peak at chemical shift δ ~ 3.63 ppm

which is corresponding to the methylene protons of the PEG blocks unit in PEG-PCL

copolymer. It is clearly seen from the spectrum that there are two equally intense triplet

peaks at peak δ ~ 2.26 and 4.01 ppm, assigned to the methylene protons in PCL chain.

Additionally, the structure of PEG-PCL copolymer is confirmed by thermogravimetric

analysis as shown in Figure 3.1c. PEG-PCL was thermally decomposed at two weight loss

events. These two stages of copolymer weight loss are equivalent to the hydrophilic and

hydrophobic polymers chains in the PEG-PCL copolymer.

3.2 Inorganic imaging probes

3.2.1 Microscopic studies of SPION and QDs

The morphology and crystal structure of SPION and Mn:ZnS QDs were investigated by

transmission electron microscopy (TEM). Figure 3.2a shows the average diameter of SPION

21

Figure 3.2 Field emission transmission electron microscope (FE-TEM) images of hydrophobic SPION (a),

Mn: ZnS QDs (b) with inset of electron diffraction patterns, and high resolution image of a single SPION(c)

and Mn: ZnS QDs (d).

Figure 3.1 FTIR spectra of PEG-PCL copolymers (a), 1H-NMR spectrum (b) and thermogravimetic

analysis of PEG-PCL copolymer (c).

22

which is 10.7 nm (standards deviation σ≈ 8 %) while average diameter of Mn:ZnS QDs is

3.1 nm (σ ≈ 10 %) (Figure 3.2b). High resolution TEM images (Figure 3.2c, d) show the single

crystalline nature of SPION and Mn: ZnS QDs, respectively.

3.2.2 Magnetic and optical properties

The magnetic property of PION was measured on field-dependent magnetization and no

hysteresis was observed (Figure 3.3a) demonstrating the superparamagnetic property

desired for T2*

-MRI application. The saturation magnetization of SPION 10.7 nm is 52 A m2

kg-1. Figure 3.3b shows the UV-Visible absorbance and fluorescence spectra of Mn: ZnS QDs.

The characteristic peak of fluorescence emission at 594 nm is due to doping Mn2+ ions into

the ZnS lattice (3 wt % of Mn to Zn according to ICP analysis).

3.3 Multifunctional DDS

3.3.1 Microscopic studies and size control

3.3.1.1 SPION-Mn:ZnS-PLGA NPs

Two multifunctional DDS have been designed consisting of SPION-Mn:ZnS-PLGA and

SPION-PEG-PCL-NPs. The polymeric nanoparticles were prepared by O/W emulsion

solvent evaporation technique. The oil phase containing polymer (PLGA or PEG-PCL) and

inorganic gradients (SPION and QDs) were emulsified into aqueous phase containing poly

(vinyl alcohol) (PVA) as a stabilizing agent.

The morphology of polymeric nanoparticles and SPION-Mn:ZnS-PLGA-NPs was

investigated by TEM. The loading of these particles in PLGA vesicles can be clearly seen by

TEM. The images of unstained and stained SPION-Mn:ZnS-PLGA vesicles with

phosphotungstic acid are shown in Figure 3.4 a and b, respectively. The mean hydrodynamic

Figure 3.3 Field-dependent magnetization measurement of SPION at ambient temperature (a) and

UV-Vis absorbance and fluorescence intensity for Mn: ZnS QDs (b).

http://www.sciencedirect.com/science/article/pii/S014296121400057X#fig2




23

hydrodynamic diameter of vesicles in volume distribution is 93 nm (σ ≈ 20 %) as shown in

Figure 3.4 c.

3.3.1.2 SPION-PEG-PCL-NPs

Figure 3.5 shows the TEM micrograph of PEG-PCL-NPs and SPION-PEG-PCL-NPs

dispersed in deionized water, in which spheres are formed and particles aggregation is not

observed (Figure 3.5 a and b respectively). According to the TEM micrograph of PEG-PCL-

NPs (Figure 3.5 a), the average diameter of the particles is 212 nm (σ ~ 30 %). The loading

of SPION into PEG-PCL nanocarriers is clearly seen in Figure 3.5 b. The contrast agent,

SPION, was entrapped into the polymeric NPs during nanoparticles formation. The

hydrodynamic size of SPION-PEG-PCL is shown in Figure S3 paper III. It has a negative zeta

potential of approximately -2.8 mV at neutral pH. We also prepared small nanoparticles

from PEG-PCL copolymer using nanoprecipitation method (paper II). SEM images showed

uniform size distribution of the nanoparticles (Figure 7, paper II). Hydrodynamic diameter

of PEG-PCL-NPs prepared by nanoprecipitation method is lower than 100 nm. AlPc, a model

of photosensitizer drug for PDT, was encapsulated into these NPs. The loading of AlPc into

these particles led to an increase in the mean particle diameter (66.5- 127.4 nm) (Table 2,

paper II).

Figure 3.4 Field emission transmission electron microscope (FE-TEM) of unstained (a), stained

PLGA-SPION-Mn:ZnS NPs (b) and dynamic light scattering (c).

Figure 3.5 Field emission transmission electron microscope (FE-TEM) images of PEG-PCL

nanoparticles. Stained PEG-PCL nanoparticles (a), stained SPION-PEG-PCL nanoparticles (b).

24

3.3.2 Biocompatibility evaluation of polymeric NPs

The biocompatibility and cytotoxicity of the PEG-PCL-NPs was assessed on HL60 cell line.

The cell viability was determined by MTT assay after incubation with PEG-PCL-NPs for 48

h and the results are plotted in Figure 3.6. The viability of the cells was calculated as the

percentage of living cells treated with nanoparticles to that of the non-treated cells. PEG-

PCL-NPs exhibited 100 % cells viability and didn’t induce major cytotoxicity up to 25 µg/ml.

Cell viability of 80 % was observed at PEG-PCL-NPs concentrations of 50 and 100 µg/ml.

The synthesized PEG-PCL nanoparticles showed no cytotoxicity which implied that the

PEG-PCL-NPs is safe for biomedical applications at the experimental concentrations.

3.3.3 Cell uptake assessment of polymeric NPs

Cellular uptake and cellular distribution of PEG-PCL-NPs were investigated by fluorescence

microscopy using fluorescein-labeled PEG-PCL-NPs. The carboxy-fluorescein was

conjugated with the amino-terminated PEG-PCL copolymer via APTMS linker using the

carbodiimide chemistry. Fluorescein conjugation was confirmed by measuring the

fluorescence intensity of the PEG-PCL-NPs after removing excess dye by dialysis. The

maximum absorbance and fluorescence intensity of the PEG-PCL-NPs are observed at 460

nm and 518 nm, respectively (Figure 5, paper III). Internalization of PEG-PCL-NPs is

examined in murine macrophage cell line J774A as shown in Figure 3.7. Figure 3.7 a and b

show the superimposed optical and fluorescence imaging of non-treated J774A cells,

respectively. Internalization of fluorescein-PEG-PCL-NPs was observed at 4 h and 24 h co-

culture as shown in Figure 3.7 c and e, respectively. It is worth mentioning that nanoparticles

were seen in the cell cytosol. The fluorescence images of the uptake of fluorescein-PEG-PCL-

NPs by the J774A cells at 4 h (Figure 3.7 d) and at 24 h (Figure 3.7 f) show initial and

Figure 3.6 Cytotoxicity of PEG-PCL-NPs in HL60 cell line.

25

maximum fluorescence intensity of the cells treated with fluorescein-PEG-PCL-NPs

compared with non-treated cells. Incubation 24 h of SPION-Mn:ZnS-PLGA-NPs with J774A

cell line (Figure 5, paper I), showed strong fluorescence intensity in the cell plasma

indicating high uptake efficacy of the SPION-Mn:ZnS-PLGA-NPs.

3.4 Drug release

Lipophilic drug model has been entrapped into nanocarrier system for specific cancer

treatment. In this study, busulphan as anticancer drug was successfully encapsulated in two

polymeric drug carriers PEG-PCL and PLGA with high entrapment efficiency 83 % and 89

% respectively. The drug was loaded in to the hydrophobic core of nanoparticles during the

nanoparticles formulation using O/W emulsion solvent evaporation technique. The drug

release behavior of busulphan was studied from SPION-PEG-PCL and SPION-Mn:ZnS-

PLGA-NPs by dialysis method.

In Figure 3.8 the percentage of drug released, represent on the vertical axis, was

calculated by dividing the amount of busulphan diffused into dialysis media by the total

Figure 3.7 Superimposed fluorescence and light microscopy images of J774A incubated with

fluorescein-labeled PEG-PCL-NPs. Non-treated control J774A cells, overlay images and

fluorescence images, respectively (a, b). Overlay images of J774A incubated with

nanoparticles for 4 h and 24 h, respectively (c, e). Fluorescence images of J774A incubated

with nanoparticles for 4 h and 24 h, respectively (d, f).

26

amount of busulphan loaded into the nanoparticles. Figure 3.8a shows a sustained drug

release pattern from PEG-PCL-NPs during 10 h and around 98 % drug was released. In PEG-

PCL-NPs, we control the burst effect by increase the initial concentration of the monomer

(105 mM of ε-caprolactone) during the polymerization reaction to produce long hydrophobic

PCL chain. It resulted in reducing the initial penetration of water into the copolymer, and

hence we were able to depress burst release. Another drug model, photosensitizer, AlPc was

loaded into the PEG-PCL-NPs during the particles formation by nanoprecipitation method.

The release profile of AlPc from PEG-PCL-NPs showed a sustained release pattern up to 7

days (Figure 3, paper II).

Unlike other drug carrier systems, where the drug release shows a burst model at the

beginning of the release process, our results showed a relatively constant release rate from

PLGA particles (Figure 3.8 b) during the first 2 h, after which drug release continued at a

lower rate for 5–6 h to release a total of 70–80 % of the loaded drugs. The burst effect is

attributed to diffusion of the drug located at the interface between the nanoparticle core and

the surface in to the dissolution medium as well as the hydrophobicity of the drug carriers.

The degradation of the drug carrier in PBS was also tested. Figure 7, paper I, shows the

percentage of lactic acid degraded from PLGA nanoparticles at pH 7.4 and 37 °C for a period

of 5 weeks. At the second week, about 12 % lactic acid was degraded and released. The

degradation was found to follow a quasi-linear trend and around 32 % lactic acid was

degraded after 5 weeks.

Figure 3.8 Profile of busulphan release from SPION-PEG-PCL-NPs (a) and from SPION–

Mn:ZnS- PLGA-NPs (b) in PBS solution at pH 7.4.

27

3.5 MRI and fluorescence imaging

3.5.1 MRI phantoms of SPION-Mn:ZnS-PLGA and SPION-PEG-PCL-NPs

The T2*-weighted MRI was measured on two different phantoms containing SPION-

Mn:ZnS-PLGA-NPs and SPION-PEG-PCL-NPs with different iron concentrations different

echo time (TEs). For PLGA vesicles phantoms, with increasing concentrations of PLGA

vesicles, the signal intensity of MRI decreased owing to the increase in loaded SPION

(Figure 3.9 a). It is noted that the signal intensity decreases more rapidly with increasing TE

for phantoms containing higher concentrations of iron oxide. These characteristics allow the

application of SPION-Mn: ZnS-PLGA as a negative contrast agent for MRI. The transverse

relaxivity (r2*) is obtained as the slope of linear fitting for the relaxation rate at different iron

concentrations. The calculated relaxivity for SPION-Mn:ZnS-PLGA vesicles (Figure 3.9 b)

and SPION-PEG-PCL phantoms (Figure 4, paper III) are 523 mM-1 s-1 and 103.3 mM-1 s-1

respectively which is much more higher than SPION alone, demonstrating the high

efficiency of SPION-Mn:ZnS-PLGA and SPION-PEG-PCL-NPs for MR T2*- imaging and

utilized in further in vivo MRI studies.

Figure 3.9 T2* -weighted MR phantom images of SPION-Mn:ZnS-PLGA at different TEs

(TR = 1200 ms; TE = 2 ms, 3.9 ms, 5.8 ms)(a). Proton transverse relaxation rate R2* = 1/ T2* of

phantom samples versus iron concentration (b).



28

3.5.2 In vivo MRI

We further investigated the effects of SPION-Mn:ZnS-PLGA-NPs on in vivo MR imaging

using a rat model. Figure 3.10 shows a coronal view of liver slices of T2*-weighted images at

different time points after intravenous tail injection of PLGA vesicles. The liver appears

white on T2*-weighted images before injection of PLGA vesicles. A rapid signal decrease was

observed in the liver and spleen post-injection and the liver become darker after 7 min post-

injection, demonstrating the fast accumulation of iron-containing PLGA vesicles in the liver.

The signal continues to decrease and the imaged liver becomes very dark, especially on the

images taken with longer TEs. Minimal signal intensity appears at 4 h post-injection and

remains low until 24 h post-injection. SPION-Mn:ZnS-PLGA-NPs show high contrast

efficiency, which provided higher relaxation rate by using a dose of iron 20 times lower than

the previously reported dextran-coated SPION.150 The rapid decrease of signal intensity or

faster relaxation in the liver compared to other organs is attributed to the SPION-Mn:ZnS-

PLGA-NPs being taken up by hepatic macrophages. Other organs had fewer macrophage

cells able to take up the nanoparticles, and the signal intensity therefore exhibited less or no

change.

Figure 3.10 T2*-weighted in vivo MR images of rat before and after intravenous injection of

SPION-Mn: ZnS-PLGA-NPs at a dose of 0.42 mg Fe/kg. Images were taken pre-injection and at

7 min, 2 h 40 min, 4 h, and 24 h post-injection with different TEs (2 ms, 3.9 ms, and 5.8 ms). The

regions of liver in the coronal planes are encircled by white dashed lines.

t= 0 (pre) 7min 2h 40min 4h 24h

TE= 2 ms

TE= 3.9 ms

TE= 5.8 ms


29

3.5.3 In vivo fluorescence imaging/ Computed Tomography (CT)

The PEG-PCL-NPs were further functionalized for in vivo fluorescence imaging using a near

infrared (NIR) fluorescence probe, VivoTag 680XL. The near infra-red (NIR) property of

VivoTag 680XL-PEG-PCL-NPs permits in vivo fluorescence imaging due to its high

penetration in tissue at activation wavelength (Ex/Em 675/720 nm) allowing organ

resolution. After IV administration of PEG-PCL-NPs, the biodistribution was followed up to

48 h. Figure 3.11 shows whole body 3D fluorescence imaging. Images were acquired at 1 h, 4

h, 24 h and 48 h post injection. During the first hour, we noticed that NPs distributed

primarily into the lungs, liver and spleen. Despite the fact that lungs are the first capillary

organ, higher distribution was observed in the spleen and liver 1h post injection due to the

sequestration effect by the mononuclear phagocyte system (MPS). At 4 h post injection, the

fluorescence intensity was reduced in the lungs, and increased in the spleen and liver. After

24 h, fluorescence intensity in the lungs was further reduced, and signals with equal strength

were observed in the liver and spleen. At 48 h, almost no fluorescence signal was observed

in the lungs and high signal accumulation in the spleen and liver.

3.5.4 Histological analysis

The histological examination of lungs, liver, spleen and kidneys removed from the sacrificed

mice was performed at 1 h, 4 h, 24 h and 48 h respectively. Microscopic analysis showed the

presence of PEG-PCL-NPs in the lungs, liver and spleen as shown in Figure 3.12.

Fluorescence microscopy showed tissue structure in addition to a red signal for VivoTag

680XL-labeled PEG-PCL-NPs and a blue signal from DAPI stained nuclei. At early

investigation time points (1 h and 4 h), NPs are clearly present in the lungs as shown in

Figure 3.12. Moreover, single NPs are found in septal capillaries and as small endocytosed

clusters in alveolar/interstitial macrophages. Notably, at 24 h post injection, relatively larger

aggregates of NPs appear in larger arterioles without any sign of emboli/fibrillary material

(eosinophilic intravascular occlusive material causing fibrin thrombo-emboli). At 48 h post

injection, very few NPs were observed in the lungs. There was no sign of increased cellularity

in the pulmonary septa and no macrophage accumulation in any of the mice, nor any sign of

inflammation. To a higher extent, we noticed NPs presence in the liver. Histological

investigation shows NPs association with sinusoidal Kupffer cells. There is no remarkable

change in the amount of NPs in liver over the time. The spleen seems to be another major

target organ, which can be seen in histological examinations as well as in the fluorescence

imaging. Nanoparticles were observed mainly in association with macrophages, most

30

commonly in the marginal zone. Slight signs of clearance of NPs in the spleen and liver were

observed at 48 h. Very few NPs were observed in the kidneys (Figure S5, paper III).

Figure 3.11 3D fluorescence imaging-CT imaging co-registration of Balb/c mice at 1 h, 4

h, 24 h and 48 h after IV administration of VivoTag XL680-PEG-PCL-NPs , dorsal side

and left side as shown. Heat map represents the area with fluorescence signal and color

represents the intensity.

31

Figure 3.12 Fluorescence microscopy overlaid phase contrast images of lung, liver and

spleen harvested at 1 h, 4 h, 24 h and 48 h. Fluorescence images of VivoTag 680XL

PEG-PCL-NPs are shown in red (CY5) while nuclei are stained with (DAPI) using 4',6-

diamidino-2-phenylindole. Scale bar = 100 μm.

32

4 Conclusions

The current thesis presents the essential ingredients to develop multifunctional drug

delivery system (DDS) including biocompatible and biodegradable polymers,

chemotherapeutic agents, and high performance MRI T2 contrast agent and fluorescent

probes through different methodologies.

Monodisperse superparamagnetic iron oxide nanoparticles (SPION) were synthesized

via high temperature decomposition of iron-oleate precursor in an organic solvent. The

nanoparticles showed uniform size distribution and no hysteresis were observed which

demonstrated the desired superparamagnetism for T2 MRI application. Mn:ZnS

nanocrystals showed strong fluorescence emission peak at 594 nm of 3.1 nm QDs size.

SPION and Mn:ZnS were encapsulated into a matrix of biodegradable polymer (PLGA) via

O/W emulsion method. The MRI phantom of PLGA vesicles displays enhancement of the

T2* MRI contrast in vivo as it showed in liver of animal model. The drug delivery capability

of PLGA vesicles was achieved by loading busulphan as antineoplastic agent into the polymer

matrix. Sustained drug release rate was achieved for 5-6 h with low burst effect.

Biodegradable amphiphilic polymers were selected as mainstay to form the nanocarrier

system. Multifunctional drug carrier has been prepared by introducing SPION and

anticancer drug to the hydrophobic layer of PEG-PCL-NPs. The drug carrier showed slow

drug release rate during 10 h. SPION-PEG-PCL phantoms were successfully enhanced the

T2* MRI contrast. The cytotoxicity studies showed that PEG-PCL-NPs didn’t cause severe

damage in the treated cells providing the safe use of PEG-PCL-NPs for biomedical

application. The in vivo fluorescence imaging of PEG-PCL-NPs showed uptake and

distribution of NPs in various tissues mostly in liver and spleen as target tissues.

33

Future work

Extensive studies have been devoted to design ideal therapeutic device for clinical

applications. It should be in a form of minimum toxicity with low administration frequency.

A multi-in-single agent with simultaneous diagnostic and therapeutic functions is required

to achieve maximum therapeutic efficacy with low dosage and minimal side effects. This

agent should deliver the payloads to the pathological sites and release them in a controlled

manner. Further biological studies on busulphan loaded-PEG-PCL and PLGA nanocarriers

for in vivo and preclinical applications have to be assessed as follows

The fast drug diffusion from the polymeric nanocarriers can be overcome by covalently

linking of the drug molecules with the building units of polymer chains. Sustain drug release

from the polymeric nanoparticles with limited burst effect can be achieved by developing

nanocarrier system based on non-biodegradable polymers including polyvinyl alcohol

(PVA) and ethylene vinyl acetate (EVA) and/or mesoporous silica coated the drug carrier

which are utilized as a controlled elution membrane around the release area of the drug to

minimize the burst effect.

The therapeutic efficacy of busulphan loaded -PEG-PCL and PLGA NPs will be

evaluated in vivo using tumor models such myeloid leukemia, lymphatic leukemia, etc.

Moreover, the photodynamic activity of the developed AlPc loaded-PEG-PCL-NPs as a

nanophotosensitizer is essential to be investigated in vitro and in vivo by measuring the

generated singlet oxygen species after light irradiation of the nanophotosensitizer.

For early detection of diseases and targeted drug delivery, SPION-PEG-PCL-NPs and

SPION-PLGA-NPs can be functionalized with targeting moieties. It can be used to load

anticancer drug and reach to the site of action which recognized by the specific receptors on

the surface of cancer cells.

34

Acknowledgements

First of all, I would like to thank my main supervisor, Prof. Joydeep Dutta for his supervision,

great help and scientific discussion. My deepest gratitude to Prof. Mamoun Muhammed, for

giving me the precious opportunity to join the Division of Functional Materials and leading

me to the world of nanotechnology. His enlightening ideas and kindness are always strong

supports during my study.

I would like to thank my co-supervisor Prof. Moustapha Hassan from Karolinska

Institute for his great help from research to everyday life, especially for his kind help in

animal studies. I wish him a great success in the scientific career.

Thanks to my co-supervisor Dr. Fei Ye for his great patience, scientific discussion and

professional guidance on my first step of research. My appreciation is extended to Prof.

Muhammet Toprak and Dr. Abdusalam Uheida for their help and precious advices. Many

thanks to the members in the Division of Functional Materials, Adrine, Carmen, Venkatesh,

Mohsen, Noroozi, Nader, Yichen, Adem, Mohsin, Bejan, Natalia, Wei, Tanjim, Felipe, Aqib

and Lorenzo for their friendship and support. I appreciate Wubeshet for his great work of

maintaining and helping on use of electron microscopes.

I am grateful to my coworkers from the department of Laboratory Medicine, Karolinska

Institutet for the fruitful collaborations: Assoc. Prof. Manuchehr Abedi-Valugerdi, Ibrahim,

Maria, Mona, Fadwa, Shiva, Svetlana, Ying and Wenyi I wish all of them a splendid future

for their career.

I would also like to thank Prof. Sherif Kandil and Prof. Moataz Soliman from Alexandria

University in Egypt for their productive collaboration, support and encouragement.

Last but not least, I would like to thank my family for their endless support and

encouragement and the most important person, my beloved husband Raul Molines, for his

deep love and strong support during my research studies.

https://www.kth.se/profile/121011/

35

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