Design, Synthesis and Characterization of Targeted...

Design, Synthesis and Characterization of

Targeted Thiolated Nanocargoes in Cancer

Therapy

PhD Thesis

by

MUHAMMAD FARHAN SOHAIL

Department of Pharmacy

Faculty of Biological Sciences

Quaid-i-Azam University

Islamabad, Pakistan

2017

Design, Synthesis and Characterization of

Targeted Thiolated Nanocargoes in Cancer

Therapy

Thesis Submitted

by

MUHAMMAD FARHAN SOHAIL Registration No. 03331411002

to


In the partial fulfillment of the requirements for degree of

Doctor of Philosophy

in

Pharmacy (Pharmaceutics)


Faculty of Biological Sciences

Quaid-i-Azam University

Islamabad, Pakistan

2017

AUTHOR’S DECLARATION

I, Muhammad Farhan Sohail, hereby state that my PhD thesis titled “Design,

Synthesis and Characterization of Targeted Thiolated Nanocargoes in Cancer

Therapy” submitted to the Department of Pharmacy, Faculty of Biological Sciences,

Quaid-i-Azam University Islamabad, Pakistan for the award of degree of Doctor of

Philosophy in Pharmacy (Pharmaceutics) is the result of original research work carried

out by me. I further declare that the results presented in this thesis have not been

submitted for the award of any other degree from this University or anywhere else in the

country/world and the University has right to withdraw my PhD degree, If my statement

is found incorrect any time, even after my graduation.

__________________________


Date: November 11, 2017

PLAGIARISM UNDERTAKING

I, Muhammad Farhan Sohail, solemnly declare that research work presented in the

thesis titled “Design, Synthesis and Characterization of Targeted Thiolated

Nanocargoes in Cancer Therapy” is solely my research work with no significant

contribution from any other person. Small contribution/help wherever taken has been

duly acknowledged and that complete thesis has been written by me.

I understand zero tolerance policy of Quaid-i-Azam University, Islamabad and HEC

towards plagiarism. Therefore, I as an author of the above titled dissertation declare that

no portion of my thesis is plagiarized and every material used as reference is properly

referred/cited.

I undertake that if I am found guilty of committing any formal plagiarism in the above

titled thesis even after award of PhD degree, the University reserves the right to

withdraw/revoke my PhD degree and that HEC and University has the right to publish

my name on the HEC/University Website on which names of those students are placed

who submitted plagiarized thesis.

__________________________


APPROVAL CERTIFICATE

This is certified that the dissertation titled “Design, Synthesis and Characterization of

Targeted Thiolated Nanocargoes in Cancer Therapy.” submitted by Mr.

Muhammad Farhan Sohail to the Department of Pharmacy, Faculty of Biological

Sciences, Quaid-i-Azam University Islamabad, Pakistan is accepted in its present form

as it is satisfying the dissertation requirements for the degree of Doctor of Philosophy in

Pharmacy (Pharmaceutics).

Supervisor: ______________________

Dr. Gul Shahnaz

Assistant Professor

Department of Pharmacy,

QAU, Islamabad,

Co-Supervisor: ___________________

Dr. Irshad Hussain

Associate Professor

Department of Chemistry,

SBASSE, LUMS, Lahore.

External Examiner 1: ______________________

Dr. --------------

Assistant Professor


University

External Examiner 2: ______________________

Dr.----------------

Assistant Professor


University,

Chairman: ______________________

Prof. Dr. Gul Majid Khan


QAU, Islamabad

Date: ______________________

Dedicated

To

My parents

Who introduced me to the joy of reading from birth, taught me to believe in myself

and believe in hard working enabling such a study to take p lace today.

My Grandparents

Guided me to trust in Allah and that so much could be done with little

My Family

For encouraging and supporting throughout my educational career

My wife

For being there and supporting in completing the degree

My Friends

For sharing, enjoying and growing in every moment we spent together

i

ACKNOWLEDGEMENTS

First and foremost, all praises and thanks to Allah, the Almighty and the Creator of whole

Universe, for His countless blessings throughout my PhD to complete it successfully. I offer

my gratitude to the Holy Prophet MUHAMMAD ملسو هيلع هللا ىلص who preached us to seek knowledge

for the betterment of mankind in particular and other creatures in general.

This dissertation is the end of my journey in obtaining my PhD degree. This dissertation has

been kept on track and seen through to completion with the support and encouragement of

numerous people including my all teachers, friends, colleagues and various institutions.

At this moment of accomplishment, first of all I pay homage to my supervisor Dr. Gul

Shahnaz and co-supervisor Dr. Irshad Hussain. This work would have not been possible

without their guidance, support and encouragement. Under their guidance I successfully

overcame many difficulties and learned a lot. I am very much thankful for their valuable

advices, constructive criticism and extensive discussions around my work.

I am also extremely indebted to Prof Dr. Gul Majid Khan (Chairman), for providing

necessary support, infrastructure and resources to accomplish my research work.

I am also thankful to Dr. Tofeeq ur Rahman, Dr. Hussain Ali, Dr. Naveed Ahmed, Dr.

Ahmed Khan, Dr. Akhtar Nadhman, Dr. Abida Raza, Dr Sohail Akhtar, Dr. Khalid Tipu,

Dr. Bashir Ahmed and Dr. Hamid Saeed, for their constant support and guidance during this

journey. It’s my pleasure to acknowledge all the faculty members of department of

Pharmacy, QAU and Department of Chemistry SBASSE, LUMS for suggestions and

constant moral support.

I am indebted to my all colleagues especially Syed Zajif Hussain, Ibrahim Javed, Hafiz

Shoaib Sarwar, Ijaz Khan, Zil e Huma, Salma Batool, Hafiza Nosheen, Ahmed Mudassar,

Muhammad Abdullah, Mira Butt, Soneela Ali and Shazia Mumtaz, for providing a

stimulating and fun filled environment and many rounds of discussions in lab(s) that helped

me a lot.

I am also grateful to Prof. Ali Khademhosseini, Dr Basit Yameen, Dr Shabir Hasan, Dr

Shamsher Ali & family, Dr Ali & family, Dr Kamal Afridi & family, Fatemeh Sharifi,

Elisabeth Farah Hirth, Anne Metje van Genderen, Gyan Parkash, Wesley Wang, Maik

Schot, Vanessa Kappings, Muhammad Usman Khalid, Muhammad Usman Saleem,

ii

Muhammad Umair Khalid and Muhammad Mubashir for making my stay at Biomaterials

innovation research center at Harvard-MIT Health Sciences and Technology, Cambridge,

USA full of science and fun.

I would also like to extend warm thanks to all administrative staff, laboratory staff and

collaborators at Quaid-i-Azam University (QAU), Syed Baber Ali School of Science and

Engineering, Lahore University of Management Sciences (SBA-SSE LUMS), Higher

Education Commission HEC (IRSIP Program), Veterinary Research Institute (VRI),

Services Institute of Medical Sciences (SIMS) and University of Veterinary and Animal

Sciences (UVAS) for support and timely assistance in completing my bench work.

Last but not least, I would like to pay high regards to my parents, brothers, grandparents,

fiancé and all the family for their prayers, sincere encouragement and inspiration throughout

my research work and lifting me uphill this phase of life. I owe everything to them.


iii

TABLE OF CONTENTS

Title Page No.

Acknowledgements i

Table of contents iii

List of tables vii

List of figures ix

List of abbreviations xiii

Abstract xv

1. INTRODUCTION ___________________________________________________ 1

1.1. Breast Cancer ___________________________________________________ 1

1.2. Breast Cancer Treatment __________________________________________ 2

1.2.1. Chemotherapy __________________________________________________ 2

1.2.1.1. The Taxanes ____________________________________________________ 3

1.2.1.1.1. Docetaxel ______________________________________________________ 5

1.2.1.1.2. Mechanism of action _____________________________________________ 5

1.3. Challenges in Oral Delivery ________________________________________ 6

1.3.1. Physicochemical barriers for DTX __________________________________ 6

1.3.2. Physiological barriers for DTX _____________________________________ 7

1.3.3. Pre-systemic metabolism _________________________________________ 7

1.3.4. Transmembrane efflux of drugs ____________________________________ 7

1.4. Current Status of DTX Formulation _________________________________ 8

1.5. Emerging Trends in Oral Delivery __________________________________ 9

1.5.1. Nanocargoes based approaches ___________________________________ 10

1.5.1.1. Lipid based nanocargoes ________________________________________ 11

1.5.1.2. Metallic nanocargoes ___________________________________________ 13

1.6. Thiolated Polymers ______________________________________________ 14

1.6.1. In situ gelling _________________________________________________ 14

1.6.2. Permeation enhancement ________________________________________ 15

1.6.3. Mucoadhesion _________________________________________________ 15

1.6.4. Stabilizing and capping agent _____________________________________ 15

1.7. Folate Targeting ________________________________________________ 16

1.8. Aims and Objectives _____________________________________________ 17

2. MATERIALS AND METHOD ________________________________________ 18

2.1. Materials _______________________________________________________ 18

2.1.1. Chemicals _____________________________________________________ 18

2.1.2. Equipment/Instrument ___________________________________________ 20

2.1.3. Glass ware ____________________________________________________ 21

2.2. Methods _______________________________________________________ 22

2.2.1. Synthesis of thiolated chitosan (CS-TGA) ____________________________ 22

2.2.2. Synthesis of folate grafted thiolated chitosan (FA-CS-TGA) _____________ 22

2.2.3. Experimental design _____________________________________________ 22

iv

2.2.4. Synthesis of nanoliposomes (NLs) _________________________________ 23

2.2.5. Synthesis of enveloped nanoliposomes (ENLs) ________________________ 23

2.2.6. Characterization of formulations ___________________________________ 24

2.2.6.1. Quantification of stabilized thiol groups _____________________________ 24

2.2.6.2. Formation of disulfide bonds _____________________________________ 24

2.2.6.3. Mucoadhesion by rheological synergism ____________________________ 24

2.2.6.4. Surface morphology, particle size and zeta potential measurement ________ 25

2.2.6.5. FTIR, DSC, TGA and XRD analysis of formulations __________________ 25

2.2.7. HPLC Method development ______________________________________ 25

2.2.7.1. HPLC instrumentation and conditions ______________________________ 26

2.2.7.2. Chromatographic conditions ______________________________________ 26

2.2.7.3. Preparation of mobile phase ______________________________________ 26

2.2.7.4. Preparation of standard stock solution ______________________________ 26

2.2.7.5. Preparation of working standard solutions ___________________________ 26

2.2.8. Method validation ______________________________________________ 27

2.2.8.1. System suitability ______________________________________________ 27

2.2.8.2. Accuracy _____________________________________________________ 27

2.2.8.3. Precision _____________________________________________________ 27

2.2.8.4. Sensitivity of the method ________________________________________ 27

2.2.8.5. Linearity and range _____________________________________________ 28

2.2.8.6. Robustness ___________________________________________________ 28

2.2.9. Encapsulation Efficiency ________________________________________ 28

2.2.10. Swelling studies _______________________________________________ 28

2.2.11. In vitro drug release studies ______________________________________ 29

2.2.12. Ex vivo permeation enhancement and efflux pump inhibition analysis _____ 29

2.2.13. In vitro cytotoxicity studies _______________________________________ 30

2.2.14. In vivo relative bioavailability studies_______________________________ 31

2.2.15. Stability Studies. _______________________________________________ 31

2.2.16. In vitro toxicity against human macrophage __________________________ 31

2.2.17. In vitro hemolysis assay _________________________________________ 32

2.2.18. In vitro micronucleus assay _______________________________________ 32

2.2.19. Acute oral toxicity ______________________________________________ 33

2.2.19.1. Serum biochemistry analysis______________________________________ 34

2.2.19.2. Hematology analysis: ___________________________________________ 34

2.2.19.3. Organ to body ratio: ____________________________________________ 34

2.2.19.4. Histopathology of vital organs ____________________________________ 35

2.2.19.5. Tissue distribution analysis _______________________________________ 35

2.2.20. Synthesis of silver nanoclusters ___________________________________ 35

2.2.21. Preparation of nanocapsules (NCs) _________________________________ 35

2.2.22. Particle size and zeta potential measurement _________________________ 36

2.2.23. DSC, FTIR and XRD analysis ____________________________________ 36

2.2.24. SEM/EDX analysis _____________________________________________ 36

2.2.25. Optical evaluation and fluorescence intensity_________________________ 36

2.2.26. Encapsulation Efficiency ________________________________________ 37

2.2.27. In vitro drug release studies ______________________________________ 37

2.2.28. Biocompatibility _______________________________________________ 37

v

2.2.29. Cytotoxicity and imaging studies __________________________________ 37

2.2.30. Stability studies ________________________________________________ 38

2.2.31. Oral bioavailability _____________________________________________ 38


2.2.32.1. Serum biochemistry and hematology analysis ________________________ 39

2.2.32.2. Organ to body ratio _____________________________________________ 39

2.2.32.3. Histopathology of vital organs ____________________________________ 39

2.2.33. Statistical analysis ______________________________________________ 39

3. RESULTS _________________________________________________________ 40

3.1. Polymer Synthesis ______________________________________________ 40

3.1.1. Synthesis and characterization of thiolated chitosan __________________ 40

3.1.2. Synthesis and characterization of folic acid grafter thiolated chitosan ___ 40

3.2. FA-CS-TGA Enveloped Nanoliposomes with Enhanced Oral Relative

bioavailability and Anticancer Activity of Docetaxel __________________________ 42

3.2.1. Optimization of NLs synthesis through experimental design _____________ 43

3.2.2. Synthesis of nanoliposomes (NLs) and enveloped nanoliposomes (ENLs) __ 43

3.2.3. FTIR, DSC and XRD analysis of formulations _______________________ 45

3.2.4. Mucoadhesion by rheological synergism ____________________________ 48

3.2.5. Swelling studies _______________________________________________ 49

3.2.6. HPLC Method development and validation __________________________ 50

3.2.6.1. System suitability ______________________________________________ 50

3.2.6.2. Precision _____________________________________________________ 50

3.2.6.3. Accuracy _____________________________________________________ 50

3.2.6.4. Limit of detection (LOD) and Limit of Quantification (LOQ) ____________ 51

3.2.6.5. Linearity and range _____________________________________________ 51

3.2.6.6. Robustness ___________________________________________________ 54

3.2.7. In vitro release kinetics __________________________________________ 54

3.2.8. Ex vivo permeation enhancement __________________________________ 55

3.2.9. In vitro anticancer activity _______________________________________ 56

3.2.10. In vivo pharmacokinetics ________________________________________ 59


3.3. In vitro and in vivo toxicological evaluation __________________________ 62

3.3.1. In vitro hemolysis assay __________________________________________ 63

3.3.2. Biocompatibility with macrophages ________________________________ 63

3.3.3. Tissue drug distribution __________________________________________ 64


3.3.4.1. Organ to body index _____________________________________________ 65

3.3.4.2. Serum biochemistry _____________________________________________ 66

3.3.4.3. Tissue histology ________________________________________________ 66

3.3.5. Genotoxicity ___________________________________________________ 68

3.4. Thiolated Polymeric Nanocapsules Embedded with Fluorescent Silver

Nanoclusters for Breast Cancer Therapy ___________________________________ 69

3.4.1. Synthesis of AgNCs and NCs _____________________________________ 70

3.4.2. FTIR, DSC and XRD analysis _____________________________________ 71

vi

3.4.3. STEM/EDX analysis ____________________________________________ 72

3.4.4. Optical characterization __________________________________________ 72

3.4.5. Encapsulation efficiency and In vitro drug release _____________________ 74

3.4.6. Cytotoxicity and cell imaging studies _______________________________ 74

3.4.7. Biocompatibility studies__________________________________________ 76

3.4.8. Oral bioavailability______________________________________________ 77

3.4.9. Acute Oral Toxicity Evaluation ____________________________________ 78


4. DISCUSSION ______________________________________________________ 81

5. CONCLUSION _____________________________________________________ 97

6. FUTURE PERSPECTIVES ___________________________________________ 99

7. REFERENCES ____________________________________________________ 101

8. LIST OF PUBLICATIONS __________________________________________ 120

9. APPENDIX _______________________________________________________ 121

vii

LIST OF TABLES

Table 1.1: Chemotherapeutic agents against breast cancer with their mechanism of action

and known protein transporters involved in developing drug resistance. ............................. 3

Table 3.1: Coded values of independent factors (concentrations of ingredients) and

dependent responses (particle size, zeta potential, encapsulation efficiency and poly

dispersity) for optimization of NLs Synthesis obtained from CCD using Design Expert

Software. ............................................................................................................................. 45

Table 3.2: Characterization of particle size, PDI, zeta potential and encapsulation efficiency

of NLs and ENLs formulation synthesized. Results are shown as Mean ± S.D. of 3 different

experiments. ........................................................................................................................ 46

Table 3.3: Results of viscoelastic parameters i.e. storage modulus (G′) and loss modulus

(G′′) and apparent viscosity of the thiolated chitosan (CS-TGA), Folate grafted thiolated

chitosan (FA-CS-TGA), NLs and ENLs and their corresponding mucin (5%)/formulation

mixtures. .............................................................................................................................. 49

Table 3.4: System suitability and precision study of developed method by injecting 10 µL

from each of 6 samples in waters HPLC. ............................................................................ 51

Table 3.5: Recovery studies of developed method using spiked samples in aqueous

formulations (F) and rat plasma (A).................................................................................... 52

Table 3.6: Linearity and range of developed HPLC method ............................................. 52

Table 3.7: Robustness studies of developed HPLC method for Docetaxel. ...................... 54

Table 3.8: Dissolution data modeling based on in vitro drug release of various formulations

to determine drug release mechanism from NLs and ENLs. .............................................. 54

Table 3.9: Results showing ex vivo permeation enhancement from Apical to Basolateral and

Basolateral to Apical side of intestine, apparent permeability along with improvement ratios

of DTX in the presence of verapamil and synthesized NLs and ENLs. The findings are

shown as Mean ± S.D. of 3 different experiments. ............................................................. 56

Table 3.10: IC50 values of Pure DTX suspension, unmodified and modified liposomes

calculated from cytotoxicity data using Graphpad Prism software 6.0. ............................. 57

Table 3.11: Results of in vivo relative relative bioavailability and important

pharmacokinetic parameters obtained after oral administration of DTX suspension in

deionized water, NLs and MNLs to rabbit through oral gavage. ........................................ 60

Table 3.12: 3 months stability data of DTX loaded, NLs and ENLs based on changes in

particle size, PDI and encapsulation efficiency performed at different storage conditions i.e.,

viii

-20, 4 and 37 °C. The analysis was performed in triplicate and results are presented in terms

of Mean ± S.D. .................................................................................................................... 61

Table 3.13: The effect of DTX, NLs, ENLs and ENLs control on CBC of mice. The results

are presented as Mean ±S.D of triplicate. ........................................................................... 67

Table 3.14: Results showing in vitro MNs assay. The number of micronucleus counted in

1000 binucleated cells on slides are shown for three experiments as ± S.D. ...................... 68

Table 3.15: Physicochemical characterization of formulations synthesized showing particle

size, poly dispersity, zeta potential and encapsulation efficiency. The results are shown as

mean ± S.D of triplicate experiment. .................................................................................. 70

Table 3.16: EDX analysis showing percentage of various elements detected in NCs. ...... 73

Table 3.17: Different pharmacokinetic parameters calculated from plasma level-time curve

obtained after oral administration of DTX suspension and NCs to rabbits. ....................... 78

Table 3.18: Complete blood count (CBC) analysis of mice blood obtained after 14 days

acute oral toxicity analysis. The results from blood of 5 mice are shown as Mean ± SD. . 79

Table 3.19: 3-month stability studies data showing changes in particle size and PDI of B-

NCs and NCs stored in dark at 4 °C. The results are shown as Mean±S.D.. ...................... 80

ix

LIST OF FIGURES

Figure 1.1: Chemical structures of Paclitaxel and Docetaxel .............................................. 4

Figure 1.2: Mechanism of DTX showing inhibition of microtubule depolarization by

binding at β subunits of tubulin at +ive end. ......................................................................... 5

Figure 1.3: Absorption barriers for drugs followed by oral administration. ........................ 8

Figure 1.4: Emerging trends in permeation enhancement via oral drug delivery. ............. 10

Figure 1.5: Various mechanisms explored for oral permeation enhancement using nano

based drug delivery systems. .............................................................................................. 12

Figure 3.1: Schematic representation showing step wise synthesis of CS-TGA and folic FA-

CS-TGA via EDAC coupling chemistry. ............................................................................ 41

Figure 3.2: Graphical abstract ............................................................................................ 42

Figure 3.3: Schematic representation of nanoliposomes (NLs) synthesis via thin film

rehydration and subsequent electrostatic stabilization of folic acid grafted thiolated chitosan

resulting in enveloped nanoliposomes (ENLs). .................................................................. 44

Figure 3.4: RSM plot of nanoliposome synthesis showing effect of independent factors on

(a) particle size, (b) zeta potential, (c) encapsulation efficiency and (d) poly dispersity index

(PDI). ................................................................................................................................... 44

Figure 3.5: Scanning electron micrographs of (a) NLs, (b) NLs at higher magnification, (c)

ENLs and (d) ENLs at higher magnification. ..................................................................... 46

Figure 3.6: FTIR spectra of CS, TGA-CS, FA-CS-TGA, DTX, physical mixture of

polymers and DTX, NLs and ENLs showing presence of characteristic of substance during

and after synthesis of formulations. .................................................................................... 47

Figure 3.7: (a) DSC analysis and (b) TGA analysis of CS, CS-TGA, FA-CS-TGA, physical

mixture, NLs and ENLs. ..................................................................................................... 47

Figure 3.8: Powder X-ray diffraction studies (PXRD) of chitosan (CS), thiolated chitosan

(CS-TGA), folate grafted thiolated chitosan (FA-CS-TGA), nanoliposome (NLs) and

enveloped nanoliposome (ENLs). ....................................................................................... 48

Figure 3.9: Swelling studies of CS, CS-TGA, FA-CS-TGA, NLs and ENLs. The analysis

was done for 3 h in phosphate buffer (pH 7.4, 0.1 M) and results are shown as Mean ± SD

............................................................................................................................................. 50

Figure 3.10: (a) Typical chromatogram of DTX in formulation; (b) Chromatogram of DTX

in plasma using ACN, Methanol and Acetate buffer (10mM, pH=5) in (48:16:36; v/v/v)

x

respectively in isocratic mode at flow rate of 0.8 mL per and column oven temperature 25oC

and detection was monitored at 230 nm. ............................................................................. 53

Figure 3.11: Callibration curve of standarad DTX solution showing linearity of data over a

concentration range of 0.5-100 µg/mL. .............................................................................. 53

Figure 3.12: In vitro drug release of pure DTX from DTX suspension, NLs, ENLs,

performed using dialysis method in phosphate buffer (pH 2-7.4) for 12 h. The results are

presented as Mean ± SD of 3 analyses. ............................................................................... 55

Figure 3.13: Ex vivo studies (a) Apical to basolateral Permeation studies (b) Basolateral to

apical permeation studies of DTX alone, with verapamil, NLs and ENLs across rat intestine.

DTX transport expressed as cumulative transport. The results are shown as Mean ±S.D. 56

Figure 3.14: Scanning electron micrographs of rat intestine after permeation enhancement

studies (a) Rat intestine (b) Transverse section (TS) of Rat intestine, (c) Basal surface of

intestine and (d) Epical surface of intestine. ....................................................................... 57

Figure 3.15: In vitro cytotoxicity comparison of DTX suspension with NLs and ENLs

showing highly improved effect on MDA-MB-231 cell line using MTT assay. Both

modified and unmodified empty liposomes were used to compare the cytotoxic potential of

formulations. ....................................................................................................................... 58


showing improved effect on HCT-116 cell line using SRB assay. Both modified and

unmodified empty liposomes were used to compare the cytotoxic potential of formulations.

............................................................................................................................................. 58

Figure 3.17: Plasma concentration of DTX after oral administration of DTX suspension,

NLs and ENLs (Oral dose=10mg/kg). Blood samples were taken at predefined time till 96

hrs and analyzed through HPLC for DTX quantification. .................................................. 59

Figure 3.18: Graphical Abstract ......................................................................................... 62

Figure 3.19: In vitro biocompatibility studies of NLs and ENLs at different concentration

to determine toxicity against red blood cells via hemolysis assay. ..................................... 63


to determine toxicity against macrophages isolated from fresh human blood via MTT assay.

The results are presented as Mean ±S.D of triplicate. ......................................................... 64

Figure 3.21: Quantification of DTX in liver, kidneys and heart after 14 days of oral

administration. ..................................................................................................................... 65

Figure 3.22: Organ to body index of vital organs compared with control, indicating toxicity

induced by treatment. .......................................................................................................... 65

xi

Figure 3.23: Serum biochemistry analysis of mice plasma after acute oral treatment with

DTX, NLs and ENLs compared with control to monitor changes on (a) LFTs; (b) RFTs; (c)

electrolytes and (d) glucose, cholesterol and total protein, induced after treatment due to

metabolism of formulations or drug. The results are presented as Mean ±S.D of triplicate.

............................................................................................................................................. 66

Figure 3.24: Microscopic examination of tissue histology of vital organ (liver, kidney and

heart) to examine any necrosis or histological change as compare to control for these organs

after treatment with formulations; a) heart tissue of control; 1a) treated with NLs, 2a) treated

with ENLs and 3a) treated with ENLs-control; b) liver tissue of control, 1b) treated with

NLs, 2b) treated with ENLs and 3b) treated with ENLs-control; c) kidney tissue of control,

1c) treated with NLs, 2c) treated with ENLs and 3c) treated with ENLs-control. ............. 67

Figure 3.25: Pictures of representative slides stained with acridine orange showing results

of in vitro micronucleous assay performed on human peripheral blood; (a) treatment with

ENLs, (b) Positive control and (c) vehicle control. ............................................................ 68

Figure 3.26: Graphical Abstract ......................................................................................... 69

Figure 3.27: Synthesis of AgNCs and DTX-NCs (1a) before microwave treatment, (1b)

after microwave treatment followed by dialysis resulting formation of AgNCs, (2a) under

UV light before synthesis, (2b) AgNC formation with blue fluorescence, (3a, 3b, 3c) Control

and AgNCs in split channels blue, green and red respectively, (4a, 4b) Lyophilized B-NCs

and NCs under normal light, (5a) lyophilized B-NCs and NCs under UV light, (5a, 5b , 5c)

lyophilized B-NCs and NCs in split channels blue, green and red respectively. ................ 70

Figure 3.28: Compatibility analysis (a) FTIR spectra showing characteristic peaks for all

formulations, (b) XRD analysis of all the formulations representing specific peaks (c) DSC

thermogram showing temperature effect on all formulations. ............................................ 71

Figure 3.29: STEM/EDX analysis of NCs (a) STEM images of NCs, (b) spot EDX spectra

of NCs showing Ag and other metals in terms of percentage, (c) EDX analysis of NCs

showing different element within NCs. .............................................................................. 72

Figure 3.30: UV-vis absorbance spectra of NCs, B-NCs and AgNCs showing no plasmonic

response for AgNCs and B-NCs but appearance of bend in NCs because of absorbance by

DTX. ................................................................................................................................... 73

Figure 3.31: Fluorescence spectra of AgNCs and NCs showing slight decreased

fluorescence after DTX loading. ......................................................................................... 74

xii

Figure 3.32: In vitro drug release studies showing cumulative percentage drug release from

NCs and pure DTX suspension in 2M phosphate buffer at 37 °C against time over period of

12 h.. .................................................................................................................................... 75

Figure 3.33: In vitro cytotoxicity and imaging studies against human breast cancer cell line

(MDA-MB-231) using different concentrations of DTX suspension, NCs and B-NCs to

check anti-cancer activity and biocompatibility. ................................................................ 75


(MDA-MB-231) showing (a) bright field cellular image and (b) under UV-light showing

fluorescence and cell death after 24 hrs, and (c-f) MB-231 cells after 6 hrs incubation

stained with phalloidin green and DAPI showing cell uptake of NCs. ............................... 76

Figure 3.35: In vitro cytotoxicity against human macrophage using different concentrations

of DTX suspension, NCs and B-NCs to check anti-cancer activity and biocompatibility. 76

Figure 3.36: Oral relative bioavailability study of DTX suspension and NCs in rabbit (n=5)

showing the plasma drug concentration after oral administration of 10mg/kg of formulations

and blood withdrawn at predefined time interval was analyzed through HPLC. ............... 77

Figure 3.37: Serum biochemistry of mice blood determining acute oral toxicity (a) Liver

function tests, (b) Renal function tests, (c) serum biochemistry and (d) organ to body weight

analysis performed on Swiss albino mice, after DTX, DTX-NCs and B-NCs in accordance

with OECD 425 guidelines for acute oral toxicity. ............................................................. 79

Figure 3.38: Microscopic evaluation of tissue histology; (1) Control liver, (1a) treatment

with DTX, (1b) treatment with NCs, (1c) treatment with B-NCs and (2) Control kidney, (2a)

treatment with DTX, (2b) treatment with NCs, (2c) treatment with B-NCs obtained from

Swiss albino mice after being euthanized. .......................................................................... 80

xiii

LIST OF ABBREVIATIONS

ABC ATP Binding Cassette

ADME Absorption, Distribution, Metabolism and Excretion

AgNCs Silver Nanoclusters

AgNPs Silver Nanoparticles

ALP Alkaline Phosphatase

ANOVA Analysis of Variance

ATP Adenosine Triphosphate

BCL-2 B-Cell Lymphoma 2

BCS Biopharmaceutical Classification System

CBC Complete Blood Count

CT Computed Topography

CYP.450 Cytochrome P450

DTX Docetaxel

EDX Energy Dispersive X-ray Spectroscopy

ER Estrogen Receptor

FDA Food and Drug Administration

GIT Gastrointestinal Tract

HEC Hydroxyethyl Cellulose

HER-2 Human Epidermal Growth Factor Receptor 2

HPLC High Performance Liquid Chromatography

LFT Liver Function Test

LOD Limit of Detection

LOQ Limit of Quantification

xiv

MDR Multi Drug Resistant

MEC Minimum Effective Concentration

MNP Metal Nanoparticles

MRI Magnetic Resonance Imaging

MRT Mean Residence Time

NSCL Non-Small Cell Lungs Cancer

PDA Photo Diode Array

PEI Polyethylene Imine

PET Positron Emission Tomography

PGP P-glycoprotein

PR Progesterone Receptor

RFT Renal Function Test

RSD Relative Standard Deviation

RSM Response Surface Methodology

SD Standard Deviation

SEM Scanning Electron Microscopy

SGOT Serum Glutamic-Oxaloacetic Transaminase

SGPT Serum Glutamate Pyruvate Transaminase

SLN Solid Lipid Nanoparticles

TEM Transmission Electron Microscopy

TNBC Triple Negative Breas Cancer

UV Ultra Violet

WHO World Health Organization

XRD X-ray Diffraction

xv

ABSTRACT

The present study was designed to develop a folate grafted thiolated chitosan (FA-CS-TGA)

polymer as an enveloping and stabilizer biomaterial for targeting cancer cells overexpressed

with folate receptors focusing breast cancer. The stabilizing and targeting potential of the

FA-CS-TGA polymer was explored by manufacturing two different classes of nanocargoes

i.e. nanoliposomes (NLs) and silver nanoclusters (AgNCs) with docetaxel (DTX) as model

hydrophobic anticancer drug. The newly synthesized FA-CS-TGA polymer was

characterized to confirm grafting and changes in physicochemical properties as compared

.to chitosan, The FA-CS-TGA polymer enveloped nanoliposomes (ENLs) and silver

nanoclusters containing nanocapsules (DTX-Ag-NCPs) were characterized for their surface

chemistry, particle size, zeta potential, PDI, encapsulation efficiency, stability and release

profile. FTIR spectroscopic analysis, X-ray diffraction (XRD) and differential scanning

calorimeter (DSC) revealed the amorphous form of DTX inside ENLs and NCs. The

observed hydrodynamic diameter of ENLs and DTX-Ag-NCPs was to be 300 and 190 nm,

respectively. Furthermore, ENLs and DTX-Ag-NCPs showed homogeneity in synthesis

with low polydispersity and positive zeta potential due to stabilization with FA-CS-TGA

polymer. Over a period of 3 months, the ENLs and DTX-Ag-NCPs were found to be stable

in terms of particle size, PDI and encapsulation efficiency. In vitro release showed that FA-

CS-TGA polymer successfully controlled the release of DTX over a longer period because

of slow and gradual swelling and mucoadhesion owing to disulfide linkage developed by

thiol groups. In vitro cytotoxicity studies indicated that ENLs and DTX-Ag-NCPs can

efficiently kill folate positive breast cancer cells (MD-MB-231) and colon cancer cells

(HCT-116) as compared to the native DTX. The pharmacokinetic evaluation showed that

the ENLs and DTX-Ag-NCPs significantly improved the relative oral bioavailability of

docetaxel owing to permeation enhancement potential of FA-CS-TGA. Acute oral toxicity

of the ENLs and DTX-Ag-NCPs revealed no evidence of toxicity due to the

biocompatibility and biodegradability of FA-CS-TGA polymer. Long term stability was

greatly improved due to the presence of FA-CS-TGA envelope on ENLs and DTX-Ag-

NCPs. Based on these evidences, FA-CS-TGA polymer seems to be promising enveloping

stabilizer of diverse nanocargoes with strong targeting potential in cancer therapy.

Chapter 1

INTRODUCTION

Chapter 1: Introduction

1

1. INTRODUCTION

The cancer epidemic is increasing globally and shifted more towards developing countries

bearing 57 % of cases and 65% of deaths because of a steady increase in population growth

rate, repeated exposure to carcinogens, aging, and many other factors (Torre et al., 2015,

Vineis and Wild, 2014) .It is the primary cause of death in developed countries and second

in developing countries that accounts for about 8.2 million deaths in 2012 worldwide

(Thanki et al., 2013). According to world health organization (WHO) by 2035, the number

of cancer patient could increase to 24 million with 14.6 million deaths (Torre et al., 2016).

Among various types of cancer, lungs cancer in males and breast cancer in females is the

most frequently diagnosed and leading cause of death. The incidence rate of cancer is almost

twice in developing countries as compared to developed countries and mortality rate is 21%

higher in male and 2% higher in females. This dramatic difference in prevalence and

mortality is because of distribution of risk factors, detection practices and treatment

opportunities available (Lindsey et al., 2015). This dramatic increase in number of cancer

patients is in dire need of cheap and effective therapy to alleviate their suffering and

improving the quality of life (Fojo and Lo, 2016).

1.1.Breast Cancer

Breast cancer is the most frequently diagnosed cancer in females and ranks leading cause of

death from all types of cancers across the globe with higher mortality rate in developing

countries as compared to developed countries which have promising survival rates (Curado,

2011). In 2012, 1.7 million cases and 0.52 million deaths were reported (Torre et al., 2016).

Breast cancer, a malignant tumor, is highly heterogeneous disease in terms of its clinical

and molecular characteristics and divided into different classes like ductal carcinoma in situ,

invasive ductal carcinoma, invasive lobular carcinoma and inflammatory breast cancer

(Weigelt and Reis-Filho, 2009). Cancer is always given the name from the organ it started

despite of its spread in different parts of body e.g. breast cancer will always be called as

breast cancer though it spread in liver or any other organ. So, these types of cancer are highly

distinguishable in terms of their identification and treatment. Breast cancer is not a single

disease and can be diagnosed at different stage of development having different growth

(Weigelt and Reis-Filho, 2009). Notably triple negative breast cancer (TNBC) covers

approximately 15% of all cancer patients, which lack expression of estrogen receptors (ER),

progesterone receptors (PR) and human epidermal growth regulating factor 2 (HER-2)

receptors (Lehmann and Pietenpol, 2014). Major risk factors for breast cancer include


2

excess body weight, menopausal hormone therapy, alcoholism, physical inactivity and

reproductive and hormonal factors. TNBC patients have the major disadvantage that they

cannot be treated with currently available hormone targeted delivery of chemotherapeutics

(Pan et al., 2012). Cancer is a multifactorial disease caused by complex mixture of genetic

and environmental factors where comprehensive advances have led to a better

understanding of disease at molecular and cellular level revealing new targets and strategies

for therapy.

1.2.Breast Cancer Treatment

Current cancer treatment involves an intrusive process starting initially with chemotherapy

to reduce the tumor size (neo-adjuvant chemotherapy), if possible followed by surgical

procedures to remove the solid tumor, subsequently, another course of chemotherapy with

radiations ensuring the complete eradication of the cancer cells (Cojoc et al., 2013, Albain

et al., 2009). Breast cancer treatment and prognosis is based on the tumor node metastasis

staging. The chemotherapy along with adjuvant endocrine therapy before and after surgery

have proven to be highly effective in reducing the disease recurrence, preventing both local

and distant metastasis thus dropping rate of mortality (Maughan et al., 2010). Management

of breast cancer relies mainly on availability of appropriate pathological and clinical

prognostic and predictive factors that guide patients in selection of treatment options (Rakha

et al., 2010). Radiation therapy, typically performed on whole breast has significantly

reduce the five years local reoccurrence rate regardless of the adjuvant systemic therapy.

1.2.1. Chemotherapy

Breast cancer is treated with a wide variety of chemotherapeutic agents available, which

differ in their cellular target and mechanism of action. Chemotherapeutic agents commonly

used in combinations to treat early and advanced stage breast cancer are summarized in

Table 1.1.


3

Table 1.1: Chemotherapeutic agents against breast cancer with their mechanism of action

and known protein transporters involved in developing drug resistance.

Class Type of Agent Name Mechanism of

Action

Associated MDR

Transporters

Alkylating

agents

Nitrogen

Mustard

Cyclophosphamide DNA cross linkage ABCC2, ABCC4

Antimetabolites Pyrimidine

analogues

Fluorouracil (5-

FU)

DNA destabilization ABCC5, ABCC8

Gemcitabine DNA destabilization ABCC5

Natural drugs Taxanes Docetaxel

Paclitaxel

Albumin bound

Paclitaxel

Microtubulin-

targeted antimitotic

PGP, ABCC1 and

ABCC3, OATP1B3

Antibiotics Doxorubicin

Liposomal

Doxorubicin

Topoisomerase-II

inhibitor

PGP, ABCG2,

ABCC1

Miscellaneous Platinum

complexes

Carboplatin

Cisplatin

DNA cross linkage ABCC2, ATP7A,

ATP7B

Macrocyclic

analogue of

halichondrin B

Eribulin Micro-tubulin


ABCC2 and

ABCC3

Vinca alkaloid Vinorelbine Micro-tubulin


ABCC1 and

ABCC3

Epothilone B

analogue

Ixabepilone Micro-tubulin


ABCC1 and

ABCC3

Docetaxel and Paclitaxel are the prominent members of taxane family obtained from

European yew (Taxus baccata) and Pacific yew (Taxus brevifolia) respectively (Uoto et al.,

1997). They act by disrupting the microtubule network that blocks the cell cycle in the late

G2 and M phase thus inhibiting cell replication (Cortes and Pazdur, 1995). They also cause

BCL-2 phosphorylation resulting in cell apoptosis. Both are used effectively to treat wide

range of carcinomas. The chemical structure of Paclitaxel and Docetaxel (Fig. 1.1) is similar

but they show significant difference in pharmacology.


4

Figure 1.1: Chemical structures of Paclitaxel and Docetaxel


5

1.2.1.1.1. Docetaxel

Docetaxel (DTX) has proved its improved efficacy as compared to paclitaxel (Verweij et

al., 1994) in terms of better cellular uptake, slow efflux, better affinity for b-tubulin subunit

of microtubule, linear pharmacokinetics and no cardiotoxicity on co-administration with

anthracycline (Gligorov and Lotz, 2004). The drug has significant anti-tumor activity and is

approved for the treatment of breast cancer (Lwin and Leighl, 2009), ovarian cancer (Kaye,

2001), non-small cell lung cancer (NSCLC) (Belani and Eckardt, 2004), head and neck

cancer (Catimel et al., 1994), gastric cancer (Roth et al., 2000) and prostate cancer (Picus

and Schultz, 1999) at doses ranging from 60 to 100 mg/m2 administered as a 1-hr infusion

every 3 weeks (Engels et al., 2005).

1.2.1.1.2. Mechanism of action

DTX is a microtubule interfering agents (Fig 1.2) that blocks the cell in the late G2 and M

phase thus inhibiting cell replication (Cortes and Pazdur, 1995). These microtubules are

composed of tubulin molecules which have α and β sub units. β-sub units are the substrate

of DTX which inhibits depolymerization leading to apoptosis and cell death. Microtubules

have + ive end with rapid ability of tubulin assembly and a –ive end with slow assembly.

They are dynamically unstable and the assembly of tubulin unit is controlled by GTP and

magnesium.

Figure 1.2: Mechanism of DTX showing inhibition of microtubule depolarization by

binding at β subunits of tubulin at +ive end.


6

1.3.Challenges in Oral Delivery

Oral drug delivery is the most convenient way to administer cytotoxic medicines (Bedell,

2003). However, there is limited bioavailability of the drug due to extensive first pass effect

(Kato et al., 2003), poor solubility (Budha et al., 2012), efflux transport (Chidambaram et

al., 2011), and low intrinsic permeability (Aisner, 2007). Despite of afore mentioned

limitations the oral route still remains the preferred route of administration in terms of its

convenience in synthesis and administration, ease of designing, vast variety of formulations

and most importantly better patient compliance in chronic ailments (Yum et al., 2013,

Dharmadhikari et al., 2013, Jeanneret et al., 2011). These absorption barriers are shown in

Fig. 1.3 and could be divided into two main categories; 1) physicochemical properties of

drug molecule such as solubility, log P, dissolution, and stability, and 2) physiological

factors associated with gastrointestinal tract such as pH, gastric retention time, absorption

window, enzymatic degradation, hepatic first pass effect, and Permeability–glycoprotein

(PGP) efflux pumps (Stuurman et al., 2013, Baker et al., 2006). Most of the therapeutic

agents used for systemic and localized GIT effects are administered orally because of the

highly absorptive nature of the intestine that provides a large surface of around 300-400 m2,

for systemic absorption with varying conditions to facilitate to achieve different types of

outcomes (Masaoka et al., 2006, Helander and Fändriks, 2014).

1.3.1. Physicochemical barriers for DTX

The most important physicochemical properties of the drug include its aqueous solubility

and membrane permeability which are explained in Lipinski’s rule considering the Pka and

log P values of the drug (Lipinski, 2004). DTX has a Pka of 10.97 and a log P of 4.1 which

results in poor aqueous solubility (0.025 µg/mL) and low membrane permeability (1 cm/s x

10-6) (Fayad et al., 2011, Thanki et al., 2013). Biopharmaceutical classification system

(BCS) is another way of describing drugs on the bases of solubility and permeability. DTX

is classified as BCS class IV drug i.e. having low solubility and permeability (Moes et al.,

2011, Lim et al., 2015). The pharmacodynamics profile of a drug is fully dependent on its

pharmacokinetics such as absorption, distribution, metabolism, and excretion (ADME)

(Stangier, 2008). The absorption of a drug could be calculated by Fick’s law of diffusion

and drugs could be categorized as dissolution rate limited, permeation rate limited, or both

dissolution/permeability limited accordingly (Siepmann and Siepmann, 2012, Brouwers et

al., 2009). Poor dissolution and higher log P values place DTX in the

dissolution/permeability limited category.


7

1.3.2. Physiological barriers for DTX

Molecular bases of the physiological barriers faced by many anticancer drugs after oral

administration are still unknown and are constantly being investigated. However, the two

most important barriers faced by anticancer drugs, including DTX, are firstly the hepatic

first pass clearance by cytochrome P450 and secondly being a substrate for the PGP efflux

pump (Malingré et al., 2001a).

1.3.3. Pre-systemic metabolism

Oral bioavailability is the collective fraction of drug that is available systemically after; 1)

absorption from gastric mucosa, 2) absorption from entero-hepatic circulation and, 3) first

pass metabolism. The gastrointestinal absorption of the drug is affected by a number of

factors such as the metabolism by different metabolic enzymes (amylase, peptidase and

lipase etc.), normal flora of intestine, brush border metabolism (peptidase, alkaline

phosphatase and sucrose etc.), and intracellular metabolism carried out by extra hepatic

microsomal enzymes in the endoplasmic reticulum (Veber et al., 2002). CYP3A4 phase II

enzymes, like estrases and glutathione-s-transferases, are present in enterocytes and are

responsible for the metabolism at the gastrointestinal wall which could also serve as target

for amide or ester pro-drugs. The hepatic first pass effect by CYP450 family is another

major contributor in decreasing the oral bioavailability (Engels et al., 2004). DTX is an

extensively protein bound drug as > 98% of the systemic drug is bound to alpha-1 acidic

glycoproteins and albumin (Urien et al., 1996).

1.3.4. Transmembrane efflux of drugs

Clinically significant cellular transport systems like PGP, cytoplasmic transport, multi drug

resistant associated protein (MRP), breast cancer resistant proteins and flurochrome efflux

can decrease the oral bioavailability of many drugs which are substrate for these transporters

via efflux mechanisms (Eckford and Sharom, 2009).

PGP, a membrane associated protein belonging to ATP binding cassette (ABC) transporters,

is extensively distributed throughout the intestinal epithelium, hepatocytes, kidneys and

capillary endothelial cells resulting in the blood-brain and blood-testis barrier. PGP’s play

a major role in developing multi drug resistance against many anticancer drugs. PGP activity

is induced either by endogenous lipids and peptides, or by drugs which are substrate for this.

Depending on their ability to stimulate PGP, anticancer agents could be divided into three

categories. Class I, drugs stimulate in low concentrations and inhibit at higher concentration,


8

class II, produces dose dependent activation of ATPase and class III, can inhibit the activity

(Varma et al., 2003). DTX is a substrate for PGP and belongs to class II contributing to its

decreased oral bioavailability (Shirakawa et al., 1999). Development of drug resistance is a

major obstacle to the success of cancer chemotherapy. The abundance of drug efflux

transporters, the pharmacological outcome from non-invasive routes of chemotherapy are

at most moderate (Kunjachan et al., 2013).

Figure 1.3: (A) Absorption barriers for drugs followed by oral administration and (B)

various permeation enhancement strategies used to overcome these barriers.

1.4.Current Status of DTX Formulation

Taxotere (Sanofi-Aventis, Anthony Cedex, France) is the FDA approved intra venous (i.v)

administered formulation of DTX available in the market. Taxotere was approved by the

FDA for non-small lung cancer (December 1999), followed by prostate cancer (May 2004),

breast cancer (August 2004), gastric cancer (March 2006), and head and neck cancer

(October 2006) (Blagosklonny, 2004).

The main issue of poor aqueous solubility of DTX is successfully addressed in Taxotere by

the addition of tween-80, a nonionic surfactant from polyethylene glycol class. During

clinical trials, DTX was supplied as a sterile solution containing tween-80 and ethanol


9

(50:50) which thereafter was reduced. The commercially available formulation (Taxotere)

contains 26 mg tween-80 per mg of DTX which is further diluted with 13 % ethanol before

being injected into patients. The presence of tween-80 reported many cases of mild to severe

hypersensitivity along with peripheral edema, weight gain and pericardial effusion with

doses above 400 mg/m2 (Baker et al., 2004, Mazzaferro et al., 2013, Engels et al., 2007).

Tween-80, especially its metabolic products and oleic acid, accounts for the histamine

induced hypersensitivity associated with DTX formulations (Panday et al., 1997). Another,

recently suggested, mechanism for hypersensitivity includes pathogen induced vasoactive

substance release. Peripheral edema may be supported by the fact that vehicle increases the

membrane permeability. Lastly, tween-80 has been shown to induce changes in plasma

viscosity and red blood cell morphology resulting in cardiovascular side effects of DTX

therapy (Extra et al., 1993). Recent studies have also shown the antiangiogenic ability of

both DTX and tween-80 at low concentrations. However, the clinically achieved

concentration after DTX infusion abolish DTX anti-angiogenic potential (Engels et al.,

2007). Tween-80 also greatly influences the pharmacokinetics of DTX by increasing the

concentration of unbound DTX in plasma due to micelle formation by tween 80 which

interact with alpha acidic proteins and letting DTX unbound (Mark et al., 2001, Loos et al.,

2003). Furthermore, the higher plasma level of tween-80 decreases DTX plasma clearance

resulting in sever hematology toxicity due to unbound drug (Engels et al., 2007).

All these problems strongly demand the development of a tween-80 free formulation for

DTX with improved pharmacokinetics and pharmacodynamics of the drug. Despite of these

limitations, tween-80 has been reported for its synergistic anti-tumor activity. Oleic acid

plays an important role in this, as it has been reported for showing intrinsic cytotoxicity and

PGP inhibition activity to overcome multi drug resistance (MDR) in cancer therapeutics.

Most of the current anticancer agents are administered through intravenous route which

makes the treatment very expensive and requires proper supervision of a trained person

during course of therapy (Liu et al., 1997, Le Lay et al., 2007).

1.5.Emerging Trends in Oral Delivery

Provided that most of the anticancer drugs, including DTX, face challenges after oral

administration to achieve optimum bioavailability to achieve desired effects, yet promising

and encouraging results have been reported by various scientist after oral administration of

DTX. Also, newly developed tween-80 free formulations with improved pharmacokinetics,

pharmacodynamics and better tumor targeting have led to several new possibilities (Thanki


10

et al., 2013). One of the strategies employed to increase the dissolution and enhance

permeation is the co-administration of DTX with a surfactant that is more safe and

biocompatible. Various strategies to improve the oral administration of DTX are presented

in Fig.1.4 and discussed below.

1.5.1. Nanocargoes based approaches

The advent of nanotechnology has introduced new avenues of possibilities in every field

during recent past that none of the other technology can match its success. In medicine, it

has introduced so many possibilities in therapeutics and diagnostics that it could be named

the future of efficient personalized drug delivery (Farokhzad and Langer, 2009, Kompella,

2013). After decades of multidimensional research in nanotechnology, it has started

showing great potential to be developed as a drug carrier (Park, 2013). Engineered

Figure 1.4: Emerging trends in permeation enhancement via oral drug delivery.

nanocargoes can be tuned for their size ranging from 1-1000 nm, surface properties like

charge, and ligands which can be attached for specific cellular receptor and shape based

upon the features required for carrying a specific molecule to a specific site achieving


11

specific aims (Sun et al., 2014). Nanocargoes based drug delivery systems have significantly

improved cancer therapy and reshaped the landscape of the pharmaceutical industry (Mei et

al., 2013).

Nanocargoes are one of those potential future carriers that could led to highly effective

chemotherapeutics (Sinha et al., 2006). These nanocargoes can be designed with different

materials like polymers, lipids, inorganic materials, hybrid materials, metals, and proteins

to tune the nanocargoes for specific aims (Jiang et al., 2007). Drug delivery vehicles such

as liposomes, prodrugs, core-shell polymeric nanocargoes, metallic nanocargoes, solid-lipid

nanocargoes have been explored for several advantages. These advantages include: 1)

improved bioavailability by overcoming the solubility or permeability of the molecules; 2)

protection of the drug molecule from harsh environment e.g. against enzymatic degradation

by lysozymes, proteases in systemic circulation or highly acidic pH of stomach; 3) better

tumor targeting through a surface decorated with ligands for specific receptors or by using

a pH sensitive polymer which will release the drug in specific environment inside the tumor;

4) controlled drug release from the carrier in order to achieve site specific release and to

maintain the required plasma drug concentration; 5) co-delivery of drug combinations or

along with diagnostic agents for Magnetic Resonance Imaging (MRI), Commutated

Tomography (CT) or Positron Emission Tomography (PET) to achieve better therapeutic

outcomes and lastly improve patient compliance (Parveen et al., 2012). Several nano based

targeted liposomal and polymeric drug delivery systems against different cancer types are

approved by FDA for clinical trials (Xu et al., 2015).

Generally, nanocargoes with a particle size around 300 nm, cationic surface and

hydrophobic surface have improved uptake from enterocytes (Win and Feng, 2005). Many

research groups have explored these properties for oral administration of drugs. The

mechanisms followed by these nanocargoes for permeation enhancement are summarized

in Fig.1.5.

Lipid based materials have also proven their importance as drug carriers, including solid


12

lipid nanoparticles, liposomes, micro/nano emulsions, ethosomes, lipid based tablets, pro-

liposomes, lipo-polymeric hybrid nano carriers, and many more (Chime and Onyishi, 2013).

Out of these lipid based materials liposomes are the only successful carriers approved by

the FDA and a number of liposomal products are now available on the market (Meyerhoff,

1999). Liposomes are vesicles with an aqueous core surrounded by a phospholipid bilayer

resulting in amphiphilic and thermodynamically stabilized vesicles (Pattni et al., 2015,

Gregoriadis, 1995). The first anticancer liposomal doxorubicin was approved by the FDA

in 1995, and currently many liposomal drugs are in different phases of clinical trials

Figure 1.5: Various mechanisms explored for oral permeation enhancement using nano

based drug delivery systems.

(Pillai, 2014). Their most important feature is their ability to incorporate both hydrophilic

and hydrophobic moieties and delivering them to the site of action through extravascular


13

and vascular routes of administrations. Similarly, solid lipid nanoparticles (SLNs) and

nanostructured lipids have shown advantages in higher drug loading and stability upon

storage as compared to other lipid based nanocargoes for delivery of anticancer drugs (Sun

et al., 2016). Nano lipid carriers (NLC) and prodrug based on lipid-drug have been explored

for oral delivery of DTX and in vitro results have shown some good improvement in

permeation. The high pay load of hydrophobic drugs, better cellular internalization and

biocompatibility suggest a detailed exploration of liposome as a drug carrier for oral

delivery of hydrophobic anti-cancer drug. Currently, many liposomal formulations having

cytarabine and daunorubicin, oxaliplatin and siRNA are under clinical trials (Xu et al.,

2015).

Metal nanocargoes (MNCs) specially noble metal (gold, silver or combination of both)

based nanocroges are versatile carriers with different biomedical applications like; delicate

diagnostic assays and imaging (Selvan et al., 2009), radiotherapy enhancement and thermal

ablation (Hainfeld et al., 2008) and drug delivery potential (Bhattacharyya et al., 2011).

MNCs can be synthesized as small as less than 25 nm, presenting huge surface area for

different applications. MNCs present unique properties like wide optical properties, high

surface to volume ratios, facile surface chemistry, ease of synthesis and surface decoration

of these nanocaroges with different ligands are explored in various dimensions of drug

delivery including cancer (Yih and Al‐Fandi, 2006, Sau et al., 2010, Sperling et al., 2008).

These noble MNCs are highly tunable according to desired optical properties, shape

(clusters, rods, star shaped, particles), size (1-100 nm), composition (alloy, core/shell noble

metals) and surface modification (with peptide, DNA, polymer, enzymes) (Jain et al., 2008,

Nishiyama, 2007, Sperling and Parak, 2010). Among these metallic nanocargoes, silver

nanoparticles (AgNPs) have gained a lot of practical importance because of their biocidal

effects against microorganism and cancer cells. These biocidal activity of AgNPs is

dependent on size, shape and surface coating (Wei et al., 2015). These biocidal effects of

AgNPs have been explored against leukemia (Guo et al., 2014), breast cancer (Gurunathan

et al., 2013), hepatocellular cancer (Sahu et al., 2014), lung cancer (Foldbjerg et al., 2011)

and skin cancer (Austin et al., 2011) which have shown really good result. These results

raised an important cancer of toxicity which needs to be addressed via surface modification

or using some biocompatible capping agent to make it more targeted with least side effects.


14

On the other hand silver based nanoclusters (AgNCs) have also been reported with similar

biocidal potential with an advantage of improved compatibility, fluorescence emission and

stabilized structure enabling them to be used as theranostic agent (Sahoo et al., 2016).

1.6.Thiolated Polymers

Thiolated polymers or so called “thiolated chitosan” is a new class of biocompatible and

biodegradable polymers having thiol group immobilized on the polymeric back bone (Jiang

et al., 2013a). A number of natural and synthetic polymers like chitosan, dextran, poly

ethyleneimine (PEI), hydroxy ethyl cellulose (HEC) and poly acrylic acid have shown

increased mucoadhesion, PGP inhibition and improved para cellular transport once being

thiolated. Thiolated chitosan also provide better control of drug loading and release from

polymeric carrier system (Bonengel and Bernkop-Schnürch, 2014).

The unique properties of chitosan have encouraged its use in development of safe and

effective drug delivery systems. Chitosan is a naturally occurring nontoxic, semi-crystalline,

biocompatible and biodegradable polysaccharide having N-acetyl glucosamine units

randomly distributed throughout the molecule (Youling Yuan, 2011). It is referred as

biodegradable since it is metabolized by certain human enzymes such as lysozyme.

However, it is a derivative of chitin which happens to be the second most abundant

polysaccharide in nature; next to cellulose. Chitin is a major component of the exoskeleton

of Crustacean shells and insects. Moreover, it is also found in the cell wall of mushrooms

and fungi (Muzzarelli et al., 2012). Chitosan is chemically produced by deacetylation of N-

acetyl glucosamine units of chitin with various degree of deacetylation imparting different

properties. Thiol immobilization on chitosan backbone has been reported with numerous

advantages owing to the properties discussed below.

1.6.1. In situ gelling

Prompt clearance from the site of absorption is a significant reason that limits the

effectiveness of a drug administered to oral mucosa (Ensign et al., 2012). It is believed that

increasing the viscosity of the formulation will increase the retention time, thus increase the

bioavailability. Thiolated polymers provide a promising platform via in situ gelling at

physiological pH because of inter and intra disulfide linkages (Iqbal et al., 2012). The

viscosity and elastic modulus of the polymer is increased with increase in number of thiol

groups immobilized on polymer backbone (Shah et al., 2016).


15

1.6.2. Permeation enhancement

Permeation enhancers can facilitate the increased absorption of drugs through GI mucosa

by changing rheology of mucosal layer of increasing solubility of the substance (Thanou et

al., 2001). Usually two types of enhancers are used: low molecular mass like sodium

salicylate or medium chain glycerides and polymeric enhancers like thiolated

multifunctional polymers. Thiolated polymers have advantage over low molecular mass as

they can cross GI and reach systemic circulation producing toxic effects. The mechanism of

permeation enhancement is suggested to be based on mucoadhesion increasing their mucosa

contact time and inhibit the enzyme protein tyrosine phosphatase (PTP) which regulate the

tight junction, facilitating para cellular transport (Dünnhaupt et al., 2015, Grabovac et al.,

2015).

1.6.3. Mucoadhesion

Mucoadhesion is adopted as a successful option for oral drug delivery with a great control

over release. These mucoadhesive properties of polymers like: residence time of the drug

on mucosa is increased offering a sustained release at target site maximizing the therapeutic

effect; formulation can be localized to a certain area of absorption window that can pledge

a firm contact with the mucosa ensuring concentration gradient as driving force for drug

absorption, render them a beneficial tool in formulation development (Mansuri et al., 2016).

All these polymers adhere to mucosa via weak Wander Waal’s or ionic interaction resulting

in incomplete achievement of afore mentioned aims. However, thiol groups of thiolated

polymers can develop disulfide linkage with cysteine rich glycoprotein subunits of mucous

via thiol/disulfide exchange reaction and oxidation (Shah et al., 2016). Furthermore, these

thiolated polymers exhibit controllable and time dependent crosslinking which can avoid

adhesion bond failure with in polymer. Thiolated polymers can penetrate the mucus layer

more efficiently and develop disulfide linkages within the mucus network resulting in

stronger mucoadhesion to achieve the all the said objectives (Bonengel and Bernkop-

Schnürch, 2014).

1.6.4. Stabilizing and capping agent

Metals nanoparticles are synthesized by top-down approach or bottom-up approach. These

MNPs can be synthesized by using appropriate capping agents (organic, inorganic, DNA),

which can prevent the particle aggregation, resulting in stabilized nanostructure (Díez and

Ras, 2011). A large number of polymers like dextran, chitosan, polyethylene glycol and


16

acrylic acid are explored for their potential ability to stabilize nanoparticles. These

nanoparticles are reported for their successful ability in drug delivery, diagnosis or

theranostic potential (combined therapeutics and diagnostic). Thiolated chitosan has been

reported with a good metal stabilizing ability with iron resulting in (SPIONs) super

paramagnetic iron oxide nanoparticles for enhanced contrast agent for MRI (Shahnaz et al.,

2013), gold (Wang et al., 2011, Rezende et al., 2010) and silver (Sangsuwan et al., 2016)

nanoparticles. Anionic thiolated polymer (polyacrylic acid-cysteine) and cationic thiolated

polymer (Chitosan-thioglycolic acid) are explored in this regard. Anionic particles have

internalization limitation because of negatively charged cell surface which was reported to

be improved with cationic thiolated polymer.

1.7.Folate Targeting

Folate receptor, a glycophosphatidylinositol anchor cell surface receptor, targeting through

nanocargoes with folic acid on surface has exposed good potential to increase oral

bioavailability (Hamman et al., 2007). Active nanocargoes uptake by enterocytes is

mediated through various receptor mediated mechanism (Fig. 1.5) among these caveolate-

mediated endocytosis appears to be an important mechanism for transcellular transport

(Hillaireau and Couvreur, 2009). Several protein are known to initiate caveolate-mediated

endocytosis like autocrine motility factor receptor, folic acid receptor, interlukine-2

receptor, glycophosphatidylinositol anchor, GM1 gangliosides, platelet derived growth

factor receptor and CCK receptor (Roger et al., 2010). These folate binding proteins are also

over expressed in many tumors including breast cancer and highly restricted in normal cells

(Sudimack and Lee, 2000). Folate targeting presents several advantages like small size of

ligand with favorable pharmacokinetics, reduced probability of immunogenicity thus letting

repeated administration, low cost, simple conjugation chemistry with various materials, high

receptor affinity and specificity allowing better internalization into tumor cells (Low and

Antony, 2004, Esmaeili et al., 2008). Thus, targeting folate receptor via nanocargoes can

enhance relative oral bioavailability as well as tumor targeting towards breast cancer.


17

1.8.Aim and Objectives

The proficiency of multifunctional nanocargoes to combine targeting, therapeutic and

imaging modalities is a significant characteristic of their versatility and expected clinical

impact in cancer management. With such multifaceted compositions, the stability of all

constituents in nanocargoes is essential to their therapeutic function. The surface

modification of nanocargoes by enveloping inside polymer ligand has an important role in

endowing stability and specific targeting to cancer cells. The overall goal of the present

research was to design a folate grafted thiolated chitosan (FA-CS-TGA) polymer which

could improve stability of diverse targeted nanocargoes with extensive anti-cancer activity,

enhanced biocompatibility and relative bioavailability after oral administration. The

development of enveloped nanoliposomes (ENLs) stabilized by FA-CS-TGA polymer was

focused to explore the ability for preventing unexpected off-target and side effects,

enhancing intracellular penetration, and facilitating specific cancer targeting of model

hydrophobic docetaxel (DTX) drug. Whereas, DTX embedded silver nanoclusters (NCs)

stabilized by FA-CS-TGA polymer resulted in nanocapsules (DTX-Ag-NCPs) that was

investigated to generate superior fluorescence intensity for theranostic application in cancer

therapy along with improved stability.

The overall goal is supported by the following objectives:

• Design, synthesis and characterization of folate grafted thiolated chitosan (FA-CS-

TGA) polymer.

• Development of DTX loaded FA-CS-TGA polymer enveloped nanoliposomes

(ENLs) and DTX embedded silver nanoclusters stabilized by FA-CS-TGA resulting

in nanocapsules (DTX-Ag-NCPs).

• Evaluating the potential of FA-CS-TGA polymer for improving long term stability

and biocompatibility of diverse ENLs and DTX-Ag-NCPs.

• To investigate the ENLs and DTX-Ag-NCPs for improving intracellular penetration,

relative oral bioavailability, pharmacokinetics profile, acute oral cytotoxicity and

enhanced activity of DTX against cancer cells.

• To probe the DTX embedded florescent silver nanoclusters loaded DTX-Ag-NCPs

in terms of optical parameters and cellular imaging for theranostic potential.

Chapter 2

MATERIALS AND METHODS

Chapter 2: Materials and Methods

18

2. MATERIALS AND METHOD

2.1.Materials

2.1.1. Chemicals

1. Chitosan (low molecular weight, degree of deacetylation 75-85%)

2. Thioglycolic Acid (TGA 99%)

3. 5,5-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent)

4. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDAC)

5. Sodium tri polyphosphate (TPP)

6. Hydroxylamine

7. Hydrogen peroxide

8. Sodium hydroxide

9. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)

10. Egg yolk choline

11. Oleic acid

12. Cholesterol

13. Disodium di hydrogen phosphate

14. Sodium dihydrogen phosphate

15. Glucose

16. Sodium chloride

17. Sodium borohydride

18. Potassium chloride

19. Magnesium chloride

20. Trehalose

21. Fetal Bovine Serum (FBS)

22. Dulbecco’s Modified Eagle Medium (DMEM)

23. Dimethyl sulfoxide (DMSO)

24. 3-(4,5-Dimethylthiazolyl-2)-2,5-diphnyltetrazolium bromide (MTT)

25. Silver nitrate

26. Penicillin

27. Streptomycin

28. Sulforhodamine B (SRB)

29. Dialysis membrane (cutoff value 12KD)

30. Docetaxel


19

31. Deionized water

32. Methanol

33. Acetonitrile

34. Ammonium acetate

35. Glacial acetic acid

36. MilliQ water

37. Rosewell Park Memorial Institute (RPMI)

38. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES buffer)

39. Phytohemagglutanin (PHA)

40. Ficoll

41. Gastograffin

42. Cytochalasin-B (cyt-B)


20

2.1.2. Equipment/Instrument

1. Magnetic Hotplate Multi Stirrer (IKA, Germany)

2. FTIR (Bruker alph-P, USA).

3. DSC (TA Instruments, SD Q600, USA)

4. TGA (TA Instruments, SD Q600, USA)

5. XRD (Bruker, D2 Phaser, USA)

6. SEM/EDX (FEI Nova NanoSEM 450, USA)

7. HPLC (waters e2695, USA)

8. Multi-plate reader (Perkin-Elmer, EnSpire, USA)

9. CO2 incubator (Panasonic MC18AC-PE, Japan)

10. Inverted microscope (Olympus BX51M)

11. Fluorescent microscope (Optika B-383FL, Italy)

12. Rotary evaporator (Heidolph, Germany)

13. Bath sonicator (Elmasonic X-tra 70)

14. UV visible spectrophotometer (Shimadzu, UV-1800, Japan)

15. Freeze dryer (Scanvac; coolsafe 110, Denmark)


21

2.1.3. Glass ware

1. Reaction vials 10 mL

2. Reaction vials 20 mL

3. Round bottom flask 50 mL

4. Round bottom flask 100 mL

5. Beaker 50 mL

6. Beaker 100 mL

7. Beaker 250 mL

8. Beaker 1000mL

9. Microwave resistant glass tube

10. Pipette 10mL


22

2.2. Methods

All the synthesis and physicochemical characterization of the formulations was done

department of chemistry, SBASSE, LUMS, Lahore. The cytotoxicity assays were

performed in LUMS, NORI Islamabad. All the animal studies were performed in veterinary

research institute Lahore and Riphah international university, Lahore campus, Lahore.

2.2.1. Synthesis of thiolated chitosan (CS-TGA)

Thioglycolic acid (TGA) was initially coupled with chitosan (CS) via EDAC coupling (Iqbal

et al., 2012). Briefly, chitosan solution (1%, w/v) was dissolved in acetic acid (1% v/v). To

this solution, TGA (1%) and EDAC (50 mM) were added with stirring. Hydroxylamine (50

mM) was added to the reaction mixture to avoid oxidation during synthetic procedure. The

pH of the mixture was adjusted to 5.0 using 1M HCl solution and kept stirred for 4 hrs to

produce thiolated chitosan/thiolated chitosan (CS-TGA). To eliminate unreacted materials

and to obtain purified CS-TGA, the mixture was dialyzed under dark for 3 days in a

dialyzing membrane (Cutoff value 12KDa), at 10 ºC: 1 time with 5 mM HCl solution, 2

times again with the same medium having 1 % NaCl and finally 2 times with 1 mM HCl to

adjust the pH at 4.0. Thereafter, the CS-TGA solution was lyophilized and refrigerated until

further use.

2.2.2. Synthesis of folate grafted thiolated chitosan (FA-CS-TGA)

Folic acid (FA) was grafted to previously synthesized CS-TGA through EDAC coupling

(Wan et al., 2008). Briefly, 10 mg of FA and EDAC was dissolved in 5 mL DMSO and

added to 1 % (m/v) thiolated chitosan solution in deionized water. The pH of reaction

mixture was adjusted to 9.0 with 0.5 M NaOH and stirred for 16 h. Folate grafted thiolated

chitosan (FA-CS-TGA) was purified via dialysis against PBS (pH 7.4) and deionized water,

3 days each. Purified FA-CS-TGA was lyophilized and refrigerated until further use.

2.2.3. Experimental design

Formulation and process optimization is widely used and recommended by regulatory

authorities for the product design and development. Response surface methodology (RSM)

is one of those many statistical and analytical techniques widely used for the optimization

(Sharma et al., 2014, Koopaei et al., 2014). Under RSM there are many designs such as

Central Composite Design (CCD), One factor and D-optimal Design and Box-Behnken

Design (BBD), (Sharma et al., 2014). For 3 factors, CCD has been considered appropriate.


23

Therefore, in the present study, CCD was employed to optimize nanoliposome (NLs)

synthesis using DPCC, oleic acid, cholesterol and choline. For NLs formulations, the

dependent variables were the encapsulation efficiency particle size, zeta potential and

polydisperability index (PDI), DX® version 9.0.6 (Stat-Ease Inc., Minneapolis, MN)

generated design matrix was employed to prepare the respective formulations and data were

entered in the DX. Statistical analysis included stepwise linear regression and response

surface analysis. The data with p < 0.05 reflected significance and was included in the

model. The best mathematical model for each response was chosen based on the goodness

of fit statistics including the probability F value, noise level, lack of fit F-value, predicted

R-squared (Pred R-squared), adjusted R-square (Adj R-squared), and adequate precision

(Adeq Precision). In case Box-Cox plot suggested to transform the data, suitable

transformation was done accordingly and the appropriate model was selected again.

2.2.4. Synthesis of nanoliposomes (NLs)

All the nanoliposomes (NLs) were prepared by dry film rehydration technique (Jinturkar et

al., 2012, Gradauer et al., 2013). Briefly, 50 mg of lipid mixture containing choline, DPPC,

oleic acid and cholesterol were dissolved in 5 mL organic phase having chloroform and

methanol (9:1, v/v). The dried thin lipid film was obtained by removing organic phase in

rotary evaporator (Heidolph, Germany). The produced film was dried thoroughly under

vacuum, rehydrated with PBS (pH 7.4) and incubated at 60 °C (above phase transition

temperature) for 1 hr with repeated vortexing to produce multi-lamellar vesicles.

Nanoliposomal suspension was sonicated using bath type sonicator (Elmasonic X-tra 70)

for 10 min at 60 oC to further reduce the size of NLs. These empty NLs were used for in

vitro characterization of various parameters. DTX loaded NLs were produced in the same

manner except that DTX was dissolved with lipids mixture in organic phase, the remaining

procedure being the same. Free drug was separated from drug loaded liposomes by

centrifugation at 4000 rpm for 5 min. Liposomes were freeze dried and stored at -20 oC till

used for further studies.

2.2.5. Synthesis of enveloped nanoliposomes (ENLs)

For synthesis of blank (B-ENLs) and DTX loaded enveloped nanoliposomes (ENLs), the

weighed amount of lyophilized nanoliposome (NLs) was suspended in FA-CS-TGA

solution (1%, m/v) and stirred for 4 h for stabilized coating through electrostatic interaction


24

between anionic NLs and cationic FA-CS-TGA. Afterwards, ENLs were separated through

ultracentrifugation, freeze dried and stored at -20 oC until further use.

2.2.6. Characterization of formulations

Quantification of thiol group attached to the chitosan backbone was spectrophotometrically

determined using Ellman’s Reagent (Saremi et al., 2013). Briefly, 0.5 mg of each of CS,

thiolated chitosan and FA-CS-TGA was hydrated in 250 L of deionized water separately.

To this suspension, 250 L of phosphate buffer (pH 8.0, 0.5 M) and 500 L of freshly

prepared Ellman’s reagent was added. The samples were kept at room temperature for 3 h

and supernatant was carefully removed and transferred to a 96-well plate. The absorbance

was measured at 430 nm with a microtitre-plate reader (PerkinElmer, USA). TGA standards

were used for calculation of thiol groups on polymer graft.

Disulfide content was determined to quantify the total amount of available thiol groups

present on both thiolated chitosan and FA-thiomer (Bernkop-Schnürch et al., 1999). Briefly,

0.5 mg of polymer was hydrated in 350 L of deionized water and to that 650 L of

phosphate buffer (pH 6.8, 0.05 M) was added and left for 30 min. To this, sodium

borohydride solution (1 %, m/v) was added. The mixture was incubated for 1 h at 37 oC.

Afterwards, 200 L of HCl (5 M) was added to decompose the remaining sodium

borohydride followed by addition of phosphate buffer (pH 8.5, 1 M) and 100 L Ellman’s

Reagent (0.4 %, m/v) in phosphate buffer (pH 8.0). After 1 h of incubation, aliquot of 300

L was transferred to the microplate and absorbance was measured at 430 nm using

microtitre-plate reader (PerkinElmer, USA). The amount of free thiol group was determined

by subtracting the calculated thiol groups in earlier step from the total thiol groups

immobilized on the modified polymers.

The mucin (4 g, extracted from bovine stomach) was dissolved in 50 mL phosphate buffer

(0.1M, pH 7.4) and final pH was adjusted at 6.8. Lyophilized ENLs and NLs as control were

hydrated in deionized water to a final concentration (5 %, m/v). The formulations were

mixed with an equal volume of freshly prepared mucin solution having pH 7.5 adjusted with

0.1 M phosphate buffer. After 20 min, small amount of the mixture was placed on a cone-


25

plate viscometer (TA, AR2000ex) and allowed to equilibrate on the plate for 3 min at 37

°C. The storage modulus (G′) and loss modulus (G′′) along with apparent viscosity were

observed after. The rheological synergism parameter (Δη) was calculated at 50 s−1 shear rate

as under:

Δη=ηmix−(ηfor+ηmuc) [1]

Where ηmix is the apparent viscosity of the mucin-polymer mixture (Pas), ηfor is the apparent

viscosity of a formulation solution with same concentration as in the mixture (Pas) and ηmuc

is the apparent viscosity of a mucin dispersion with same concentration as that of mixture

(Pas).

Hydrodynamic diameter and surface zeta potential of NLs and ENLs, was measured through

zetasizer (Malvern, Nano ZSP, UK). Surface morphology of all formulations was studied

by scanning electron microscope (FEI Nova NanoSEM 450, USA) equipped with

transmission electron detector operated at 17.5 KV. Samples for STEM images were

carefully prepared by slow evaporation of a single dilute drop of formulation on carbon

coated copper grid followed by blotting with a drop of 1 % ammonium molybdate solution.

Drug stability, during and after the synthesis of different formulations, was studied through

different techniques performed on DTX, CS, CS-TGA, FA-CS-TGA, NLs and ENLs.

Amorphous nature of the drug was confirmed through Powder X-ray Diffractometer

(Bruker, D2 Phaser, USA). XRD patterns were collected by operating the instrument at

angle range 20o-70o with the step size of 0.0505 and Cu 1.54Ao. Differential Scanning

Calorimetry (DSC) and Thermogravimetric Analysis (TGA) was performed to access the

stability of the drug and all the ingredients at a temperature range of 25-350 ºC with heating

rate of 10 ºC per min under air purge of 10 mL per min using Differential Scanning

Calorimeter (TA Instruments, SD Q600, USA). The stability of drug and its functional

groups were studied through Fourier Transformed InfraRed (FTIR) Spectroscopy using

FTIR Spectrophotometer (Bruker alph-P, USA).

2.2.7. HPLC Method development

The development of HPLC method for DTX estimation in sample was carried out with

different mobile phase to buffer ratios, different flow rates and different temperature

conditions.


26

Chromatographic separation was achieved by using HPLC (Waters e2695, UK) with 10 L

injector (automatic) and UV-visible with PDA detector (Waters 2998) was attached along

with fraction collector. Waters spherisorb C18 (5 m, ODS2 4.6 x 250 mm) column was

used for the separation. Empower 5.0 software was used to evaluate output signals.

The mixture of Acetonitrile, methanol and acetate buffer was passed through 0.45

membrane filter and degassed by sonicating for 30 min. The solvent was pumped from

reservoir under isocratic condition (100 %) into C18 column at flow rate of 0.8 mL per min.

The column and sample temperature was set at 25 + 0.5 oC. Detection was done at 230 nm

and sample run time was 10 min.

Mobile phase comprising of acetonitrile, methanol and acetate buffer (10 mM, pH 5.0) in a

ratio of (48:16:36, v/v/v) respectively was prepared and filtered through 0.45 membrane

filter. The mixture was degassed by sonication for 30 min in bath sonicator. Mobile phase

was used as diluent as well.

1 mg/mL DTX standard solution was prepared by carefully weighing 10 mg DTX and

transferred it to 10 mL volumetric flask further diluted to give final concentration of 1

mg/mL.

1 mL of the stock solution was added to volumetric flask (10 mL) and volume was made up

using diluent resulting final concentration of 100 g/mL. The similar procedure was

repeated to prepare solutions of 50 g, 10 g, 5 g, 1 g and 0.5 g/mL. Single solvent

extraction technique was used to extract DTX from plasma samples for HPLC analysis.

Briefly, 200 L of plasma was taken in 2 mL Eppendorf and to this 1.5 mL of diluent was

added. The mixture was rocked on vortex mixture for 10 min to achieve complete extraction

of DTX from plasma in diluent. The mixture was then centrifuged at 4000 G for 10 min and

clear supernatant was carefully transferred to glass vial. The solution was dried using rotary


27

evaporator at 60 oC. The dried residue was reconstituted in 500 L of diluent, sonicated for

2 min and injected into HPLC for analysis.

2.2.8. Method validation

International Council for Harmonization (ICH) guidelines were followed to validate the

developed method covering system suitability, linearity, robustness, precision, accuracy,

limit of detection (LOD), and limit of quantification (LOQ) (Rao and Abbaraju, 2016).

Six replicate injections of DTX were analyzed to develop the method. The acceptance

criteria of theoretical plate count above 3000, % RSD of peak area and retention time less

than 2 %, and tailing less than 1.5 % was set for the procedure.

Three injections with known amount of DTX i.e. (50 %, 100 % and 150 %) were added to

pre-analyzed samples and their recovery was calculated using developed method. The

percentage recovery within 100 ± 2 % and RSD less than 2 % were set as acceptance criteria

and was calculate using equation below.

𝑅𝑒𝑐𝑜𝑣𝑒𝑟𝑦 (%) =peak area of extracted sample

𝑃𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑢𝑛𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒 [2]

Three replicate injections of DTX were analyzed using developed method with short period

of time to check intraday variability and same method as repeated after 24 h to check

variability. Tailing, assay and % RSD of peak area, less than 2 % was set as acceptance

criteria.

Sensitivity in terms of limit of detection (LOD) and limit of quantification (LOQ) were

calculated based on calibration curves obtained in linearity studies. LOD and LOQ were

calculated statistically using equations (3.3 x ) / m and (10 x ) / m respectively where

is the standard deviation of y-intercept of three regression lines and m is the mean of slopes

of three calibration curves. Signal to noise ratio was used to calculate LOD and LOQ. Signal


28

to noise ratio 3:1 and 10:1 was set as acceptance criteria for LOD and LOQ respectively

(Reddy et al., 2014).

Linearity was calculated through regression analysis of six different concentrations i.e. 0.5,

1, 5, 10, 50 and 100 g/mL of target assay concentration of DTX plotted against drug

concentration. The highest and lowest concentrations of analyte where used to calculate

analytical range by the acceptable values of linearity, precision and accuracy.

The robustness refers to the capacity of method to withstand small but deliberate change in

conditions that indicates its reliability during normal use. The samples were studied at slight

different column temperature and pH of mobile phase.

2.2.9. Encapsulation Efficiency

The encapsulation efficiency of all the formulations developed was calculated by re-

suspending 2 mg of lyophilized formulation in 2 mL of deionized water. The suspension

was then sonicated for 30 min in water bath to rupture the particles and setting drug free in

the water. To this mixture, 1 mL of mobile phase (acetonitrile : methanol : buffer) was added

and sonicated for another 15 min to dissolve the drug and extract further traces entrapped in

the formulations. The solution was filtered through 0.22 syringe filter and transferred to

HPLC vial (Saboktakin et al., 2011). The quantity of DTX loaded in 2 mg of formulation

was estimated through the HPLC-PDA method developed and mentioned earlier. Same

method was repeated in triplicate and average was calculated to determine encapsulation

efficiency from all the formulations by using the formula:

𝐸𝑛𝑐𝑎𝑝𝑠𝑢𝑙𝑎𝑡𝑖𝑜𝑛 𝐸𝑓𝑓𝑒𝑐𝑖𝑒𝑛𝑐𝑦 = 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑖𝑛 𝑓𝑜𝑟𝑚𝑢𝑙𝑎𝑡𝑖𝑜𝑛

𝑡𝑜𝑡𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑋 100 [3]

2.2.10. Swelling studies

The water absorbing capacity was calculated to estimate the muco-adhesive properties of

the CS, thiolated chitosan, FA-CS-TGA, NLs and ENLs synthesized as described previously

(Shahnaz et al., 2010). The weighed quantity (25 mg) of each formulation was processed in

to a thin tablet of 5 mm diameter and was gently fixed on the tip of needle. The needle was


29

immersed in phosphate buffer (pH 7.0, 0.1 M) at 37 ºC. At definite time intervals tablet was

taken out and excess water was carefully removed by tissue paper. Tablet was weighed again

and amount of water absorbed was calculated gravimetrically using formula given below.

𝑊𝑎𝑡𝑒𝑟 𝑈𝑝𝑡𝑎𝑘𝑒 (%) = 𝑊𝑓 − 𝑊𝑜

𝑊𝑜 𝑋 100 [4]

Where Wo is the initial weight and Wf is the weight of a hydrated tablet at given time

interval. Same method was repeated for all the formulations.

2.2.11. In vitro drug release studies

The dialysis membrane diffusion method was applied to study the in vitro drug release from

NLs and ENLs (Javed et al., 2015). The weighed quantity of formulations containing drug

equivalent to 5 mg was re-suspended in deionized water and placed in a dialysis membrane

(Cutoff value 12 KDa) sealed and immersed in 30 mL of phosphate buffer (pH 2-7.4, 0.1

M) containing tween-80 (1% m/v) to maintain the sink conditions as DTX has less solubility

in buffer solution. Pure DTX was used as a standard to compare the drug release from the

formulations. The system was maintained at 37 ºC ± 0.5 and 100 rpm. Samples were

collected at predefined intervals, filtered through 0.22 syringe filter and analyzed through

HPLC (waters e2695, USA) using the same method developed discussed above. The release

data was then analyzed with DDSolver, a free Microsoft Excel Add-in, to study the release

kinetics from NC’s (Muhammad Farhan Sohail et al., 2014).

2.2.12. Ex vivo permeation enhancement and efflux pump inhibition analysis

Ex vivo permeation enhancement was analyzed using everted sac method by comparing the

synthesized formulations with DTX dispersion (Ibrahim et al., 2014). Briefly, the study was

conducted on intestine of healthy rats weighing between 200-250 g and were used for the

first time for experiment. The rats were anesthetized with chloroform and abdomen was

opened with middle incision. The intestine was immediately removed, thoroughly washed

with Krebs ringer solution (pH 6.5) and was cut into pieces of 4-5 cm. The intestine was

everted by carefully passing a narrow glass rod from one end of the intestine and then gently

rolling it on a glass rod. All the pieces were stored in oxygenated Krebs ringer solution at 4

oC till further use. 1% Tween-80 was added to enhance the wettability of DTX. Each

segment was tied at one end with silk suture and 1 mL of sample (1 mg/mL) was carefully

filled in the sac using hypodermic syringe and the other end was tied with silk suture.

Verapamil (100 μg/mL), a PGP inhibitor, was filled in one sac to compare the apparent


30

permeation enhancement with NCs. All filled sacs were immersed in tubes filled with 10

mL of oxygenated Krebs Solution and incubated at 37 oC under gentle mechanical shaking.

The samples were collected from the surrounding medium at definite time and replaced with

the same amount of fresh solution. The samples were analyzed using HPLC and apparent

permeability was calculated using following equation:

𝐴𝑝𝑝𝑎𝑟𝑒𝑛𝑡 𝑃𝑒𝑟𝑚𝑒𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (µ𝑔/𝑐𝑚2) = 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 × 𝑉𝑜𝑙𝑢𝑚𝑒

𝑀𝑢𝑐𝑜𝑠𝑎𝑙 𝑆𝑢𝑟𝑓𝑎𝑐𝑒 𝐴𝑟𝑒𝑎 [5]

Mucosal surface area was calculated by assuming intestine a cylinder and using formula:

𝑀𝑢𝑐𝑜𝑠𝑎𝑙 𝑆𝑢𝑟𝑓𝑎𝑐𝑒 𝐴𝑟𝑒𝑎 (𝑐𝑚2) = 𝐶𝑖𝑟𝑐𝑢𝑚𝑓𝑒𝑟𝑒𝑛𝑐𝑒 (𝜋 × 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟) × 𝐿𝑒𝑛𝑔𝑡ℎ [6]

2.2.13. In vitro cytotoxicity studies

In vitro cytotoxicity of pure DTX and all the formulations were screened through MTT

assay using breast cancer (MDA-MB-231) cell line (Jiang et al., 2013a). Briefly, MB-231

cell line was seeded in 96-well optiplate at a density of 6000 cells per well in DMEM with

10 % FBS and incubated for 24 h in 5 % CO2. The cells were incubated with 5 g, 2.5 g,

1.25 g, 0.625 g, 0.312 g, 0.156 g, 0.05 g and 0.001 g DTX and formulations

containing equivalent drug concentration and blank nanocarriers for 72 h. After incubation,

the medium was replaced with fresh DMEM and 10 L of MTT reagent was added to each

well and incubated for another 4 h. After 4 h, MTT-containing media was aspired off and

100 L of DMSO was added in each well to dissolve the formazan crystals formed by living

cells. Then the absorbance was measured at 570 nm using multi plate reader (Perkin-Elmer,

USA). Untreated cells with 100% viability were taken as control and the cells without MTT

served as blank to calibrate the instrument. IC50 values for each formulation was calculated

using Graphpad Prism 6.02 software. The results were presented as mean ± SD of three

independent experiments (Jain et al., 2014).

In vitro cytotoxicity of NLs and ENLs was also screened against colon cancer (HCT-116)

through sulforhodamine B (SRB) assay (Huang et al., 2012). Briefly, the cells were seeded

in 96-well optiplate at a density of 3000 cells per well, suspended in 10 % FBS and incubated

for 24 h in CO2 incubator. Thereafter, cells were fixed with 10 % trichloroacetic acid

representing cell population at the time of treatment (To). The cells were treated with vehicle

control (0.1% DMSO), DTX suspension and different concentrations of NLs and ENLs


31

equivalent to 10 µg, 5 µg, 2.5 µg, 1.25 µg, 0.625 µg, 0.312 µg and 0.156 µg of DTX for 48

h. Blank NLs and ENLs served as control. After incubation, the cells were again fixed with

10% trichloroacetic acid followed by staining with sulforhodamine B (0.4 %, w/v) in 1 %

acetic acid solution. Excess SRB was removed by 1 % acetic acid solution and dye

containing cell were lysed with 10 mmol Trizma base. The absorbance was measured at 490

nm using multi plate reader (Perkin-Elmer, USA). Untreated cells with 100% viability were

taken as control and the cells without addition of SRB were used as blank to calibrate the

instrument. IC50 values for each formulation was calculated using Graphpad Prism 6.02

software. The results are expressed as mean ± S.D. of three experiments (Jain et al., 2014).

2.2.14. In vivo oral bioavailability studies

The animal investigations were conducted following the protocol approved by the Bio-

Ethical Committee of Quaid-i-Azam University Islamabad, Pakistan (Protocol No.

DFBS/216-266 / BEC-FBS-QAU-21). Healthy rabbits weighing 1800 ± 200 g were selected

and kept in animal house with free access to food and water a day prior to experiment. The

rabbits were divided into 4 groups, having 5 rabbits each (n=5). Group 1 was treated with

pure DTX suspension, Group 2 was given DTX loaded NLs and Group 3 was treated with

ENLs using oral gavage. Group 4 served as a control. Blood samples were withdrawn from

ear marginal vein of each rabbit at predefined time interval (0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 10,

12, 24, 48 and 96 h) using sterile syringe each time. The samples were transferred to 1.5 mL

Eppendorf containing 100 L anticoagulant (11% sodium citrate). The samples were

centrifuged at 4000 rpm for 15 min to separate plasma. The plasma was stored at -20 ºC till

further used for analysis (Venkatesh et al., 2015, Jiao et al., 2002). The drug was extracted

from plasma samples and was analyzed using HPLC method developed and used for the

measurement of encapsulation efficiency.

2.2.15. Stability Studies.

Stability of formulations was analyzed for change in particle size and encapsulation

efficiency over a period of 3 months while keeping them at varying stress conditions of -20

ºC, 4 ºC and 37 ºC (Jain et al., 2014).

2.2.16. In vitro toxicity against human macrophage

Macrophages, from fresh human blood (with volunteer consent), were separated using

Ficoll-percoll purification technique (De Almeida et al., 2000, Nadhman et al., 2014).

Briefly, macrophage isolation was achieved using ficoll-gastrografin gradient (density 1.070


32

g/ml). Ficoll solution was prepared by dissolving 5.6 g ficoll in 9.5 mL deionized water and

5 mL gastrografin to achieve density 1.070 g/mL. The fresh human blood (5 mL) was diluted

three times with Hank’s buffer salt solution (HBSS) and carefully layered over ficoll-

gastrografin solution and centrifuged for 5 min at 400 G to separate macrophage layer.

Percoll density (1.064 g/mL) was achieved through deionized water and 10x HBSS. The

separated cells were suspended in RPMI medium (10 % FBS, 100 U/mL penicillin, 0.1

mg/mL streptomycin and 25 mM HEPES) and incubated in 5% CO2. Viable cells were

seeded to 96-well plate (1 x 105 per well) and treated with different concentrations of NLs,

ENLs and vehicle control. Blank NLs and ENLs served as negative control and Triton X

(1%) served as positive control to check cytotoxicity of treatment. After 24 h incubation,

the cell viability was checked through trypan blue. The IC50 was calculated for viable cells

using graph pad prism software (version 6.02).

2.2.17. In vitro hemolysis assay

Fresh human blood was used for in vitro hemolysis assay as reported (Malagoli, 2007). The

fresh blood (with volunteer consent) withdrawn and washed thrice with sterile normal saline

(0.9 % NaCl). The RBCs were pelleted out after each washing at 150 G for 5 min and

supernatant was discarded. The final pellet was diluted 9 time (v/v) with sterile normal saline

and finally suspended in Dulbecco phosphate buffer saline (DPBS). Afterwards, 100 µL of

RBC suspension per well was seeded to 96-well plate. RBCs were treated with different

concentrations of NLs and ENLs. Blank NLs and ENLs served as negative control and

Triton X (1%) was used as positive control to check hemolysis induced by the formulations.

After 24 h incubation, the absorbance was measured at 404 nm using multiplate reader

(Perkin-Elmer, USA). Hemolysis percent induced was calculated using formula:

% 𝐻𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠 =absorbance of sample−absorbance of negative control

𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙−𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 [7]

2.2.18. In vitro micronucleus assay

Fresh peripheral blood (with volunteer consent) was collected in heparinized sterile vials

(BD Vacutainer). Triplicate Blood cultures were set up by diluting 0.6 mL blood in 9.4 mL

RPMI media containing Fetal Bovine Serum (FBS, 10 %), penicillin, streptomycin and

HEPES buffer solution. Phytohaemagglutinin (PHA) solution (4 %) was added to culture

and incubated for 48 h at 37 °C with gentle shaking to stimulate lymphocyte growth. After


33

incubation of 48 h, cultures were treated with NLs and ENLs (500 µg/mL) followed by 24

h incubation. Cytokinesis in binucleated lymphocytes were arrested by replacing

supernatant with fresh RPMI media having cytochalasin-B (cyt-B) at final concentration 6

µg/mL. After 4 h incubation, the culture was centrifuged at 300 G for 10 min and supernatant

was carefully replace with pre-warmed (37 °C) mild hypotonic solution (0.075% KCl) and

incubated for 4 min to allow swelling to occur. Thereafter, the lymphocytes were harvested

using ice-cold Carony’s fixative (methanol: glacial acetic acid; 3:1). The culture was

centrifuged at 300 G and pellet was resuspended in Carnoy’s medium and gently mixed.

The process was repeated (1250 G, 2-3 min) until a clear pellet is obtained and suspension

was refrigerated for 3 h prior to slide preparation. The slides were prepared by placing single

drop of suspension and air dried for 1 h, followed by staining with Giemsa (4% in PBS) for

10 min. After that washed with PBS and air dried (Huerta et al., 2014). Negative control

was treated with sterile water for injection.

The prepared slides were scored for in vitro micronucleus by observing 1000 bi-nucleated

cells per treatment (500 cells per slide) blindly.

When cyt B is used, evaluation should be based on replication index (RI) which indicates

average number of cell cycles per cell has undergone during exposure to cyt.B and may be

used to calculate cell proliferation.

𝑅𝐼 = ((𝑛𝑜.𝑜𝑓 𝑏𝑖𝑛𝑢𝑐𝑙𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠)+(2∗𝑛𝑜.𝑜𝑓 𝑏𝑖𝑛𝑢𝑐𝑙𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠)/(𝑡𝑜𝑡𝑎𝑙 𝑛𝑜.𝑜𝑓 𝑐𝑒𝑙𝑙𝑠))𝑇

((𝑛𝑜.𝑜𝑓 𝑏𝑖𝑛𝑢𝑐𝑙𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠)+(2∗𝑛𝑜.𝑜𝑓 𝑏𝑖𝑛𝑢𝑐𝑙𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠)/(𝑡𝑜𝑡𝑎𝑙 𝑛𝑜.𝑜𝑓 𝑐𝑒𝑙𝑙𝑠))𝐶 x 100 [8]

Where T is test sample and C is control.

2.2.19. Acute oral toxicity

Acute oral toxicity of NLs and ENLs was evaluated in mice for 14 days following OECD

425 guidelines. The in vivo studies were proceeded as per the approved guide lines of Bio-

Ethical Committee of Quaid-i-Azam University Islamabad, Pakistan (Protocol No.

DFBS/216-266 / BEC-FBS-QAU-21). Healthy, female Swiss albino mice, weighing 30 ± 5

g and 8-10 weeks aged, were obtained from animal house. Mice were divided into 5 groups

(n = 6) and kept under standard condition of food and water at controlled environment. The

Group 1 was administered DTX suspension, group 2 NLs, group 3 ENLs, group 4 B-ENLs

and group 5 served as control and given NS. The dose (10 mg/kg) was administered orally

through gavage. The mice were kept under observation for 24 h for change in weight and

visual observations for mortality, behavior pattern (fur and skin, consistency of feces,


34

urination color, salivation, eyes, respiration, sleep pattern, mucous membrane, convulsions,

and coma), physical appearance changes and signs of illness were conducted daily

throughout the week. (Saleem et al., 2015). After 14 days, the mice were sacrificed for

serum biochemistry and tissue histology studies (Singh et al., 2013).

After 14 days, the serum biochemistry was performed to check the toxicity induced by the

NLs and ENLs. The blood from each mice was drawn through cardiac puncture into sterile

vial. The blood was centrifuged at 1200 G for 10 min to separate plasma. The clear

supernatant was carefully removed and stored at -20 °C. Liver function tests (LFT’s)

including (ALP, SGPT, SGOT and bilirubin), Renal functions tests (RFTs) including (Urea

and creatinine), serum electrolytes (Na, Mg, Ca and P), glucose, total protein were analyzed

using the serum.

Hematology analysis was performed on the other part of blood collected in heparinized vial

through cardiac puncture from each mice. Hematology parameters i.e. red blood cells

(RBCs), packed cell volume (PCV), red cell distribution width (RDW), mean corpuscular

hemoglobin concentration (MCHC), hemoglobin distribution width (HDW), mean

corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), hemoglobin

concentration (Hb), hematocrit (HCT), platelet count (PLT), & mean platelet volume

(MPV). In addition, number and percentage of neutrophils, monocytes, lymphocytes,

eosinophils, and basophils were also measured using a hematology Autoanalyzer (Mindrey,

BC 2800VET) (Vandebriel et al., 2014).

Change in organ weight is measured for toxicity evaluation of test formulation after

exposure to a definite time. The vital organs (Heart, kidneys and liver) were removed from

mice after being sacrificed washed with normal saline and weighed individually. The

weights of organs from treated groups were compared with control group and body mass

index was calculated using formula (Venkatasubbu et al., 2015, Saleem et al., 2015).

𝑂𝑟𝑔𝑎𝑛 − 𝑏𝑜𝑑𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑖𝑛𝑑𝑒𝑥 (%) = 𝑂𝑟𝑔𝑎𝑛 𝑤𝑒𝑖𝑔ℎ𝑡

𝑏𝑜𝑑𝑦 𝑤𝑒𝑖𝑔ℎ𝑡∗ 100 [8]


35

The washed vital organs (liver, kidney and heat) were macroscopically examined for any

abnormality or lesions against control. After that the organs were stored in 10 % formalin

solution. The organs were fixed in paraffin blocks and sections (0.5 µm) were cut carefully

using rotary microtome and fixed on glass slide followed by staining with hematoxylin and

eosin periodic acid Schiff (PAS). The sections were microscopically examined for any toxic

effect produced by NPs.

Tissue distribution of DTX loaded NLs and ENLs was analyzed using tissue homogenate

analysis. Briefly, weighed amount of chopped organ (liver, Kidneys and heart) was mixed

with 1 mL NS (0.9 % w/v) and homogenized. To this 1 mL mobile phase was added to

extract drug from tissues and the mixture was further sonicated for 15 min followed by

centrifugation at 5000 G for 10 min. The supernatant was carefully separated and analyzed

using HPLC method previously developed for DTX quantification in plasma samples.

2.2.20. Synthesis of silver nanoclusters (NCs)

50 mg of FA-CS-TGA was dissolved in 5 mL of deionized water along with 20 mg of

EDTA. To this, solution silver nitrate (0.5 mM;1mL) was mixed under stirring for 15 mins.

Afterwards, the reaction mixture was transferred to microwave resistant reaction vial and

irradiated with microwaves for 2 min using microwave reactor (CEM; Discoverer, UK)

operating at power of 100 W. The resulting solution was then purified to remove any excess

or unreacted materials, using dialysis membrane (2,000 MWCO) for 24 h with deionized

water exchanged at regular intervals of 6 h. The yellowish-brown colloidal dispersion at the

end, indicated the formation of silver nanoclusters. The solution was afterwards stored under

dark conditions in refrigerator until further used (Tang et al., 2013).

2.2.21. Preparation of nanocapsules (DTX-Ag-NCPs)

DTX was loaded in NCs containing FA-CS-TGA polymer to produce nanocapsules (DTX-

Ag-NCPs) containing both DTX and NCs through ionic gelation technique (Iqbal et al.,

2012). DTX (1 mg) was added to solution of NCs (5 mL) under continuous stirring and then,

1 % solution of tween-80 was added to increase wettability of DTX. After 15 min, TPP

solution (1%) was added dropwise to above mixture for crosslinking the polymer to

synthesize NCs. The solution was left under stirring for 4 h followed by dialysis to remove


36

any unreacted materials. Same method was used to synthesize blank nanocapsules (Ag-

NCPs i.e. without DTX) to serve as control in different experiments. Purified Ag-NCPs

were further divided in two parts: one portion was lyophilized and other was left as solution.

Both were stored in refrigerator under dark conditions till further use.

2.2.22. Particle size and zeta potential measurement

Hydrodynamic radius and zeta potential of NCs, DTX-Ag-NCPs and Ag-NCPs were

measured by DLS using zetasizer (Malvern, NanoZSP).

2.2.23. DSC, FTIR and XRD analysis

Physicochemical integrity of DTX, during synthesis and after loading in DTX-Ag-NCPs,

was studied through DSC, FTIR and XRD on DTX, NCs, DTX-Ag-NCPs and Ag-NCPs

following same conditions and protocol mentioned above.

2.2.24. SEM/EDX analysis

Surface morphology and elemental analysis of DTX-Ag-NCPs were studied through SEM

(FEI Nova NanoSEM 450) equipped with transmission electron detector and energy

dispersive x-ray (EDX) detector operating between 15-25 kV with working distance of 5

mm. The samples for STEM/EDX analysis were prepared by evaporating a single droplet

of DTX-Ag-NCPs formulation (~10 µL) on carbon coated coper grid followed by blotting

a drop of 1% ammonium molybdate solution. For better contrast, the dried sample was

further coated with gold, using sputter coater (Denton, Desk V HP) operating at 40 mA for

15 sec under vacuum. Afterwards, the sample was analyzed for STEM and EDX results.

The EDX analysis was conducted for qualitative measurement of elements specially Ag in

DTX-Ag-NCPs (Sahoo et al., 2016).

2.2.25. Optical evaluation and fluorescence intensity

Fluorescence and absorption spectra of NCs and DTX-Ag-NCPs was analyzed using

multiplate reader (Perkin-Elmer, EnSpire Multimode Plate Reader and UV visible

spectrophotometer (Shimadzu, UV-1800) respectively. The fluorescence was measured at

emission peak (λem) 430 nm, when excited (λex) at 365 nm.


37

2.2.26. Encapsulation Efficiency

The quantity of encapsulated DTX in DTX-Ag-NCPs was calculated using method reported

earlier for NLs and ENLs through HPLC analysis (Saboktakin et al., 2011, Sohail et al.,

2016).

2.2.27. In vitro drug release studies

In vitro release of DTX from DTX-Ag-NCPs was studied through dialysis membrane

following diffusion technique as previously described for DTX release from NLs and ENLs.

2.2.28. Biocompatibility

The biocompatibility of the NCs and DTX-Ag-NCPs was assessed against the fresh human

macrophages. The same protocol was followed as for ENLs.

2.2.29. Cytotoxicity and imaging studies

In vitro theranostic potential of DTX-Ag-NCPs was explored and compared with NCs and

DTX through MTT assay and cellular imaging using breast cancer (MDA-MB-231) cell line

(Jiang et al., 2013a, Wang and Huang, 2014). MB-231 cells were seeded in 96-well optiplate

having density of 6000 cells in well-prepared solution of DMEM and FBS. The cells were

incubated with different concentrations of formulations of DTX, NCs and DTX-Ag-NCPs

containing equivalent amount of DTX and Ag-NCPs as internal control for 24 h. After

incubation, the medium was replaced with fresh solution of DMEM and MTT (10 L) in

each well and incubated for another 4 h.

After 4 h, the media was removed and DMSO (100 L) was added in each well to dissolve

the formazan crystals made by living cells. Then the absorbance was measured at 570 nm

using multi plate reader (Perkin-Elmer, USA). Untreated cells with 100% viability served

as positive control and the cells without MTT were used as blank to calibrate the instrument.

IC50 values for each formulation was calculated using Graphpad Prism 6.02 software (Jain

et al., 2014). For cellular imaging, the cells were transferred to 8 chamber slide and treated

with DTX-Ag-NCPs suspended in DPBS followed by 12 h incubation in CO2 chamber. The

cells were fixed on slide and excess DTX-Ag-NCPs were removed by washing twice with

warm DPBS, stained and examined under fluorescent microscope.


38

2.2.30. Stability studies

Stability of NCs and DTX-Ag-NCPs was analyzed for change in physical appearance by

examining the change in particle size, PDI and zeta potential over a period of 3 months

while keeping them refrigerated at 4 ºC under dark conditions (Jain et al., 2014).

2.2.31. Oral bioavailability

All the animal studies were conducted in compliance to the approved protocol of Bio-Ethical

Committee of Quaid-i-Azam University Islamabad, Pakistan (Protocol No. BEC-FBS-

QAU-20). Relative oral bioavailability studies of DTX-Ag-NCPs were conducted in rabbits.

The rabbits were divided into 3 groups (n = 5) and kept in the animal house with free access

to food and water. Group 1 was given DTX-Ag-NCPs, group 2 was given DTX suspension

and group 3 was given NS to serve as a control. The samples (10 mg/kg) were orally

administered through gavage needle. Blood samples were withdrawn from ear marginal vein

of each rabbit at predefined time interval using 1 mL sterile syringe each time. The separated

plasma was stored at -20 ºC till further used for analysis (Venkatesh et al., 2015, Jiao et al.,

2002). The drug was extracted from plasma samples and was analyzed using HPLC method

described earlier.


Acute oral toxicity of NCs was evaluated in mice following OECD 425 guidelines. The

studies were proceeded as per the approved guidelines of Bio-ethical Committee of Quaid-

i-Azam University, Islamabad, Pakistan (Protocol No. DFBS/216-266 / BEC-FBS-QAU-

21). Female Swiss albino mice weighing 32 ± 5 g, were obtained from animal house. Mice

were divided into 4 groups (n = 5) and were kept to free access of food and water at

controlled environment. The group 1 was given DTX suspension, group 2 was given DTX-

Ag-NCPs and group 3 was given Ag-NCPs, whereas group 4 was given NS to serve as

control. The dose (10 mg/kg) was administered orally through gavage. The mice were kept

under observation for 24 h for change in weight and visual observations for mortality,

behavior pattern (fur and skin, consistency of feces, urination color, salivation, eyes,

respiration, sleep pattern, mucous membrane, convulsions, and coma), physical appearance

changes and sign of illness were conducted daily throughout the week (Saleem et al., 2015).

After 14 days, the mice were sacrificed for serum biochemistry and tissue histology studies

(Singh et al., 2013).


39

The treatment effect was evaluated on different parameters including liver function tests

(LFTs), renal function tests (RFTs), complete blood count (CBC), serum glucose,

cholesterol and total protein as discussed earlier.

The toxic effects of formulations on vital organs could serve as an indicator for induced

toxicity followed by the treatment. Same steps were repeated as described in detail earlier.

The previously removed and washed organs were macroscopically examined for any

abnormalities or lesions against control. Later on the organs were fixed in paraffin blocks

and sections (0.5 µm) were cut carefully using rotary microtome and fixed on glass slide

followed by staining with hematoxylin and eosin. The sections were microscopically

examined using Olympus (Olympus BX51M) for any evident sign of toxicity induced by

NCs.

2.2.33. Statistical analysis

All the experiments were performed in triplicates and repeated 3 times to reduce the chances

of error and to establish the significant correlation between the data obtained. All the results

were generated using two-way analysis of variance (ANOVA) to compare the results of

different treatments with NLs, ENLs, DTX-Ag-NCPs and DTX. Data is presented as mean

± SD using SPSS 21 and Graphpad Prism 6.1. The p value less than 0.05 (*p<0.05) was

considered to indicate the significant difference (Jiang et al., 2013a).

Chapter 3

RESULTS

Chapter 3: Results

40

3. RESULTS

The synthesized folic acid grafted chitosan was expected to improve the oral permeation

enhancement and relative oral bioavailability of hydrophobic anticancer agents enclosed in

different multifunctional nanocargoes. The FA-CS-TGA was expected to improve the

targeting potential towards folate positive tumors via folate targeting and stability of

different nanocarogoes on long term storage. A number of experiments were designed and

conducted to evaluate afore mentioned potentials and results are presented below to support

the objectives achieved during the study.

3.1. Polymer Synthesis

3.1.1. Synthesis and characterization of thiolated chitosan

Thiolated chitosan (CS-TGA) was synthesized by modifying the chitosan (CS) backbone

via covalent linkage with thioglycolic acid (TGA) resulting in thiolated chitosan (Fig.3.1).

Quantification of the thiol groups immobilized on thiolated chitosan (CS-TGA) revealed an

average 845 ± 67 µM of thiol moieties per gram of the polymer. In addition, 128 ± 73 µM

disulfide bonds and 596 ± 14 µM primary amino groups were present per gram of CS-TGA.

The obtained lyophilized CS-TGA appeared as white, odorless powder of fibrous structure.

The lyophilized polymer was stored at 4 oC and found stable towards oxidation throughout

the course of the study. The FTIR spectra of CS and CS-TGA in Fig. 3.6 clearly showed

absorbance bands at 1654 cm-1 (amide I), 1604 cm-1 (NH2) bending and 1382 cm-1 (amide

III). The band at 1156 cm-1 (asymmetric stretching of COOOC bridge), 1072 cm-1 and 1023

cm-1 (skeletal vibration because of -COO stretching) are important features of its saccharin

structure.

3.1.2. Synthesis and characterization of folic acid grafter thiolated chitosan

The lyophilized thiolated chitosan was grafted with folic acid in the next phase following

the same EDAC coupling mechanism resulting in folic acid conjugated thiolated chitosan

(FA-CS-TGA) as shown in Fig. 3 1. The quantity of disulfide linkage and primary amino

groups were found to be 158 ± 47 µM and 361 ± 22 µM per gram of polymer. The

attachment of folic acid to CS-TGA was confirmed by FTIR spectroscopy shown in Fig.

3.6. FTIR spectrum of folic acid grafted CS-TGA showed characteristic peak at 3372 cm-1

and 3274 cm-1. The appearance of two characteristic peak at 1662 cm-1 and 1585 cm-1and a

new sharp band at 1314 cm-1.

Chapter 3: Results

41

Figure 3.1: Schematic representation showing step wise synthesis of CS-TGA and folic FA-

CS-TGA via EDAC coupling chemistry.

Chapter 3: Results

42

3.2. FA-CS-TGA Enveloped Nanoliposomes with Enhanced Oral bioavailability and

Anticancer Activity of Docetaxel

Figure 3.2: Graphical abstract

Chapter 3: Results

43

3.2.1. Optimization of nanoliposomes (NLs) synthesis through experimental design

Formulations were theoretically optimized via Design Expert Software simulation based

RSM plots for most suitable concentrations of ingredients by central composite design

(CCD). The ingredients were taken as mentioned in Table 3.1 with coded values given by

the software. For every parameter, an equation in terms of coded values is generated by the

software for particle size, zeta potential, PDI and encapsulation efficiency are mentioned

below.

Particle Size = 168.42*11.50A+11.00B-4.60C-11.50D-

8.00AB+5.50AC*9.50AD+2.75BC-1.75BD-8.25CD-4.27A236.23B2-10.77C2-3.27D2 [1]

Zeta Potential = 22.13+3.05A-6.75B-1.04C+4.65D+3.88AB-2.60AC*8.47AD-1.23BC*

0.050BD-2.27CD+ 3.29A2+0.39B2+2.94C2-2.21D2 [2]

Encapsulation efficiency =70.35+1.50A-1.85B+3.04C+9.05D*8.66AB+0.46AC-4.99AD

5.14BC+4.58BD+0.21CD+2.98A2+2.23B2-9.37C2+2.63D2 [3]

PDI = 0.24-0.040A*5.500E-003B-9.40E-003C-0.049D*3.25E-003AB-

0.014AC+0.025AD* 0.043BC-0.055BD-0.032CD-0.018A2+0.017B2*0.038C2-0.010D2 [4]

Based on ANOVA results, predictive analysis and numerical optimization was done using

Design Expert Software that produced various formulations with varying ratio of

ingredients. First three formulations were selected and reproduced to confirm the prediction.

The predictions were made using equations for each factors and are given below. The

various RSM graphs showing link between dependent and independent factors are shown in

Fig. 3.4.

3.2.2. Synthesis of nanoliposomes (NLs) and enveloped nanoliposomes (ENLs)

NLs, both empty and loaded with DTX, were successfully synthesized using thin film

rehydration technique (Fig. 3.3) with ingredient ratio obtained from Design Expert Software

(Table 3.1). Lyophilized NLs were successfully coated with FA-CS-TGA through ionic

interaction between positively charged polymer and negatively charged lipid bilayer of

liposomes.

The successful synthesis of ENLs was confirmed by the change in zeta potential which

turned to positive (ELNs) from negative (NLs) (Table 3.2). Particle size, zeta potential and

Chapter 3: Results

44

polydispersity of all formulations are shown in Table 3.2. Surface morphology was

observed to be smooth and liposomes appeared fairly spherical and bi-layered in STEM

images (Fig. 3.5).

Figure 3.3: Schematic representation of nanoliposomes (NLs) synthesis via thin film

rehydration and subsequent electrostatic stabilization of folic acid grafted thiolated chitosan

resulting in enveloped nanoliposomes (ENLs).

Figure 3.4: RSM plot of nanoliposome synthesis showing effect of independent factors on

(a) particle size, (b) zeta potential, (c) encapsulation efficiency and (d) poly dispersity index

(PDI).

Chapter 3: Results

45

Table 3.1: Coded values of independent factors (concentrations of ingredients) and

dependent responses (particle size, zeta potential, encapsulation efficiency and poly

dispersity) for optimization of NLs Synthesis obtained from CCD using Design Expert

Software.

3.2.3. FTIR, DSC and XRD analysis of formulations

The FTIR spectra (Fig. 3.6) of DTX, physical mixture, NLs and ENLs showed the

characteristic peaks of drug which confirmed the presence of drug in chemically unmodified

form in formulations.

STD Run Factor 1

A:DPPC

Mg

Factor 2

B:Choline

Mg

Factor

3

C: OA

mg

Factor 4

Cholesterol7

Mg

Response1

Particle

Size

Nm

Response

2

PDI

Response

3

EE

%

Response

4

ZP

eV

8 1 -1.000 -1.000 -1.000 -1.000 180 0.324 60.5 15.4

17 2 0.000 0.000 0.000 0.000 165 0.213 69.2 23.6

20 3 0.000 0.000 0.000 0.000 170 0.241 68.3 23.5

15 4 0.000 0.000 -1.000 0.000 177 0.281 63.5 15.3

5 5 -1.000 1.000 1.000 -1.000 205 0.342 53.8 32.7

1 6 1.000 1.000 -1.000 1.000 216 0.351 67.2 28.5

12 7 0.000 0.000 0.000 1.000 216 0.265 70.3 15.8

14 8 1.000 0.000 0.000 0.000 155 0.291 61.8 21.7

10 9 0.000 1.000 0.000 0.000 176 0.185 74.4 28.5

13 10 -1.000 0.000 0.000 0.000 161 0.271 59.3 28.5

11 11 0.000 0.000 0.000 -1.000 194 0.254 74 29.3

3 12 1.000 1.000 1.000 -1.000 184 0.143 72.4 24.5

2 13 -1.000 1.000 -1.000 1.000 193 0.264 70 32.5

6 14 1.000 -1.000 -1.000 -1.000 170 0.306 76.4 26.7

16 15 0.000 0.000 1.000 0.000 154 0.184 81.6 24.6

18 16 0.000 0.000 0.000 0.000 165 0.214 72.7 21.4

9 17 0.000 -1.000 0.000 0.000 153 0.264 71.4 22.4

4 18 -1.000 -1.000 1.000 1.000 187 0.165 77.4 27.4

7 19 1.000 -1.000 1.000 1.000 155 0.181 73.6 24.7

21 20 0.000 0.000 0.000 0.000 174 0.212 72.8 21.6

19 21 0.000 0.000 0.000 0.000 166 0.255 71.3 20.4

Chapter 3: Results

46

Table 3.2: Characterization of particle size, PDI, zeta potential and encapsulation efficiency

of NLs and ENLs formulation synthesized. Results are shown as Mean ± S.D. of 3 different

experiments.

Figure 3.5: Scanning electron micrographs of (a) NLs, (b) NLs at higher magnification, (c)

ENLs and (d) ENLs at higher magnification.

DSC thermogram (Fig. 3.7a) showed an endothermic peak of crystalline DTX at 169 °C.

Thermogravimetric analysis (TGA) showed highest thermal decomposition (58 %) of FA-

CS-TGA occurred at 330 °C. DTX showed the highest decomposition of 60 % at 280 °C.

(Fig. 3.7b). Furthermore, the XRD pattern showed characteristic peaks of DTX which were

diminished in physical mixture and further diminished in formulations (Fig. 3.8). This again

indicated that during formulation development, drug lost its crystallinity and was present

inside NLs in amorphous form.

Formulation Particle size

(nm)

Polydispersity

Index (PDI)

Zeta potential

(mV)

Encapsulation

Efficiency (%)

NLs-Blank 132.50 ± 2.34 0.22 ± 0.01 - 43.10 ± 0.34 -

NLs 246.50 ± 1.39 0.32 ± 0.05 - 22.60± 0.18 71.49 ± 3.82

ENLs 328.50 ± 0.36 0.36 ± 0.01 + 18.30 ± 2.52 83.47 ± 5.62

Chapter 3: Results

47

Figure 3.6: FTIR spectra of CS, TGA-CS, FA-CS-TGA, DTX, physical mixture of

polymers and DTX, NLs and ENLs showing presence of characteristic of substance during

and after synthesis of formulations.

Figure 3.7: (a) Differential Scanning Calorimetry (DSC) analysis and (b) Thermo

Gravimetric Analysis (TGA) of CS, CS-TGA, FA-CS-TGA, physical mixture, NLs and

ENLs.

Chapter 3: Results

48

Figure 3.8: Powder X-ray diffraction studies (PXRD) of chitosan (CS), thiolated chitosan

(CS-TGA), folate grafted thiolated chitosan (FA-CS-TGA), nanoliposome (NLs) and

enveloped nanoliposome (ENLs).

3.2.4. Mucoadhesion by rheological synergism

The viscoelastic parameters G′ and G′′ were measured for 5% (w/v) mixture of control i.e.

NLs or ENLs with mucin (5% mucin per formulation) and results are shown in Table 3.3.

There was non-significant deviation between the time-dependent rheological changes of

various mucus in formulation mixtures at various pH levels. For ENLs, G′ and G′′ were

higher for the mucin-formulation mixtures than those for the ENLs solutions alone. Ten-

fold higher storage modulus (G′) values were obtained for mucin- ENLs mixtures than for

the ENLs solutions alone within 2 h. However, no considerable increase in the viscoelastic

parameters was observed for NLs and their corresponding mucin mixture (Table 3.3). These

findings demonstrated the lack of conformational changes between NLs and mucin and

demonstrates the advantage of thiolated chitosan coating for better oral bioavailability.

Chapter 3: Results

49

Table 3.3: Results of viscoelastic parameters i.e. storage modulus (G′) and loss modulus

(G′′) and apparent viscosity of the thiolated chitosan (CS-TGA), Folate grafted thiolated

chitosan (FA-CS-TGA), NLs and ENLs and their corresponding mucin (5%)/formulation

mixtures. The results are shown as Mean ± S.D.

3.2.5. Swelling studies

The water uptake studies for the thiolated chitosan, NLs and ENLs was performed on 25 mg

sample compressed to thin tablet and immersed in phosphate buffer (pH 7.4, 0.1 M). Among

the polymers, CS-TGA showed the highest swelling as compared to FA-CS-TGA (Fig. 3.9).

ENLs showed better water uptake compared to NLs. It was also observed that ENLs showed

slow and gradual swelling that is necessary for strong mucoadhesion. On the other hand,

NLs showed very slow and least swelling because of hydrophobic nature. ENLs showed

relatively better swelling that resulted in better mucoadhesion.

Formulation

Time

1h 6h 12h

G'(Pa)

G''(Pa

)

V(Pa.

S) G'(Pa)

G''(Pa

)

V(Pa.

S) G'(Pa)

G''(Pa

)

V(Pa.S

)

CS-TGA

18.31 ±

3.22

12.52 ±

4.18

0.08 ±

2.14

56.34 ±

4.56

42.67 ±

4.30

1.14 ±

1.38

113.44

± 23.58

68.34 ±

7.52

3.53 ±

1.21

CS-TGA

with Mucin

28.41 ±

4.25

17.35 ±

3.50

0.18 ±

2.45

69.27 ±

3.35

54.75 ±

5.40

3.33 ±

1.67

82.32 ±

8.31

77.64 ±

5.27

7.44 ±

1.43

FA-CS-

TGA

13.45 ±

2.67

19.44 ±

2.87

0.04±

2.36

51.37 ±

5.13

42.10 ±

6.32

0.09 ±

1.45

105.50

± 11.37

64.89 ±

8.76

2.23 ±

1.21

FA-CS-

TGA with

Mucin

20.44 ±

5.32

16.43 ±

3.64

0.35 ±

3.34

89.29 ±

7.55

65.21 ±

7.39

3.12 ±

1.57

195.43

± 8.55

134.11

± 13.45

5.83 ±

1.46

NLs

7.71 ±

4.47

6.95 ±

3.67

0.02 ±

1.62

29.76 ±

2.47

21.94 ±

3.27

0.06 ±

1.13

48.13 ±

7.45

38.51 ±

7.40

0.94 ±

1.26

NLs with

Mucin

9.31 ±

6.65

8.41±

4.58

0.06 ±

1.44

37.40 ±

23.26

31.43 ±

27.57

0.15 ±

1.19

63.59 ±

15.38

57.25 ±

21.52

1.28 ±

1.15

ENLs

63.42 ±

6.44

57.35 ±

6.37

0.03 ±

1.36

95.42 ±

4.56

71.34 ±

7.89

0.07 ±

1.22

178.91

± 12.40

161.44

± 11.76

3.31 ±

1.10

ENLs with

Mucin

80.43 ±

8.78

64.31 ±

4.33

0.45 ±

1.67

645.16

± 65.87

479.44

± 16

2.75 ±

1.45

3542.82

± 47.64

2978.4

3 ± 54

17.93 ±

1.64

Chapter 3: Results

50

Figure 3.9: Swelling studies of CS, CS-TGA, FA-CS-TGA, NLs and ENLs. The analysis

was done for 3 h in phosphate buffer (pH 7.4, 0.1 M) and results are shown as Mean ± SD

3.2.6. HPLC Method development and validation

Six samples were injected to check the system suitability and the results obtained for

different factors are summarized in Table 3 4, showing the number of theoretical plate count

that was 6552.52 ± 1.76, tailing factor of 1.32 ± 0.26 and RSD % peak area of 0.062 ± 0.03.

Six samples of known concentrations were injected and analyzed using the developed

method. The results are shown in Table 3.4 that summarizes RSD % of peak area, assay

and tailing factor.

Three replicate injections each of known concentrations i.e. 50%, 100% and 150% were

added to pre-analyzed samples containing 100 g/mL of DTX and analyzed using the

developed method. The results of recovery studies are shown in Table 3.5.

Chapter 3: Results

51

Table 3.4: System suitability and precision study of developed method by injecting 10 µL

from each of 6 samples in waters HPLC.

Sample Peak Area Assay

% Tailing

Retention

Time (min)

Theoretical Plate

Count

A1 750619 99.98 1.31 5.909 6547.172

A2 750587 100.51 1.32 5.938 6579.304

A3 751663 100.23 1.32 5.908 6546.064

A4 750742 100.32 1.32 5.911 6549.388

A5 751626 99.87 1.33 5.908 6546.064

A6 750692 100.05 1.33 5.909 6547.172

Average 750988.16 100.16 1.32 5.91 6552.527

Standard Deviation 466.86 0.21 0.0068 0.01 12.02

RSD % 0.062 0.22 0.52 0.18 0.18

LOD and LOQ were calculated statistically from calibration curve using linearity data. The

LOD was found to be 0.00215 g/mL and LOQ was 0.00652 g/mL.

Standard calibration curve was plotted by using area under curve against different

concentrations of standard reference solutions analyzed using the developed method. The

results are shown in Table 3.6. A sample HPLC chromatogram showing characteristic peaks

is shown in Fig. 3.10 and calibration curve is shown in Fig. 3.11.

Chapter 3: Results

52

Table 3.5: Recovery studies of developed method using spiked samples in aqueous

formulations (F) and rat plasma (A).

Drug Sample Level Spiked

(g/ml)

Recovered

(g/ml) Recovery %

R.S.D

%

Docetaxel

F1

50

50 49.83 99.66

0.99

F2 50 49.71 99.42

F3 50 50.82 101.64

F1

100

100 100.23 100.17

0.23 F2 100 99.86 99.86

F3 100 100.42 100.42

F1

150

150 150.44 100.2933

0.23 F2 150 149.93 99.95333

F3 150 150.81 100.54

A1

50

50 49.78 99.56 0.098

A2 50 49.83 99.66

A3 50 49.71 99.42

Table 3.6: Linearity and range of developed HPLC method

Concentration (g/mL) Area

100 1569824

50 750619

10 160873

5 76908

1 31832

0.5 18940

Slope 15507

Intercept 4509.3

Linearity Equation Y=15507x + 4509.3

R2 0.9993

Range 0.5-100 g/mL

Chapter 3: Results

53

Figure 3.10: (a) Typical chromatogram of DTX in formulation; (b) Chromatogram of DTX

in plasma using ACN, Methanol and Acetate buffer (10mM, pH=5) in (48:16:36; v/v/v)

respectively in isocratic mode at flow rate of 0.8 mL per and column oven temperature 25oC

and detection was monitored at 230 nm.

Figure 3.11: Callibration curve of standarad DTX solution showing linearity of data over

a concentration range of 0.5-100 µg/mL.

y = 15507x + 4509.3

R² = 0.9993

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

0 20 40 60 80 100 120

Are

a u

nd

er c

urv

e

Conc. µg/mL

Chapter 3: Results

54

As a part of the robustness, a deliberate change in the column temperature, injection volume

and pH of the buffer solution was studied applying the above developed method and results

are shown in Table 3.7.

Table 3.7: Robustness studies of developed HPLC method for Docetaxel.

3.2.7. In vitro release kinetics

In this study, in vitro DTX release profiles from NLs and ENLs was evaluated for four days

(96 h) at physiological pH of 7.4 by sink condition dialysis. The percentage of DTX released

from the formulations was evaluated in a time dependent manner (Fig. 3.12).

To study the mechanism of drug release, various release kinetics models were applied to

release data. The results shown in Table 3.8 indicate that drug released from formulations

followed Korsmeyer-peppas model based on R square value. The release of DTX from

ENLs continued for up to 12 h while NLs released >75 % of drug in 12 h which can be

explained on the basis of poor stability of NLs to retain the drug.

Table 3.8: Dissolution data modeling based on in vitro drug release of various formulations

to determine drug release mechanism from NLs and ENLs.

Parameter Level Assay % RSD %

Temp 25oC 100.02 0.17772

30oC 99.92 0.09298

pH 4.5 100.11 0.10804

5.0 100.08 0.13856

Injection volume 100 L 100.14 0.04925

10 L 99.03 0.64025

Formulation Zero Order Korsmeyer-peppas Higuchi Hixon-Crowell

R2 Ko R2 N R2 KH R2 KHC

DTX 0.46 1.31 0.98 0.39 0.94 9.15 0.70 0.05

NLs 0.24 2.01 0.95 0.41 0.86 14.36 0.92 0.02

ENLs 0.58 1.65 0.98 0.43 0.96 11.40 0.88 0.01

Chapter 3: Results

55

Figure 3.12: In vitro drug release of DTX from DTX suspension, NLs, ENLs, performed

using dialysis method in phosphate buffer (pH 2-7.4) for 12 h. The results are presented as

Mean ± SD of 3 analyses.

3.2.8. Ex vivo permeation enhancement

Results of permeation studies with DTX in the presence or absence of verapamil on everted

rat intestinal sac are represented in Fig. 3.13a and Papp values with enhancement ratios are

summarized in Table 3.9. Our results demonstrated that due to PGP inhibitor, verapamil

(100 μg/mL), DTX absorption into the sac contents was markedly increased by 5.87-folds

(p < 0.05) as compared to the buffer control. However, DTX absorption in case of ENLs

and NLs was highly significant. The Papp enhancement ratio was 13.62-folds higher for the

ENLs formulation and 8.8-folds higher for the NLs formulation. The SEM results of rat

intestine (Fig. 3.14) also showed the presence of increased concentration of ENLs on

basolateral surface.

To further investigate the possible involvement of intestinal efflux pumps in the

permeability process, DTX, NLs and ENLs were evaluated for its apparent permeability

coefficients (Papp) in the reverse basal to apical direction (secretory transport). The results

shown in Fig 3.13b and Table 3.9 indicate the secretory Papp of DTX across rat mucosa

was 5.8-folds of the absorptive (apical to basal) Papp suggesting that the movement of DTX

across rat mucosa is secretory-oriented.

0

20

40

60

80

100

0 2 4 6 8 10 12 14Cu

mu

lati

ve

Dru

g R

elea

se (

%)

Time (h)

DTX NLs ENLs

Chapter 3: Results

56

Figure 3.13: Ex vivo studies (a) Apical to basolateral permeation studies (b) Basolateral to

apical permeation studies of DTX alone, with verapamil, NLs and ENLs across rat intestine.

DTX transport expressed as cumulative transport. The results are shown as Mean ± S.D.

3.2.9. In vitro anticancer activity

The anti-proliferative effect of NLs, ENLs and DTX was investigated against MDA-MB-

231 breast cancer cells and HCT-116 colon cancer cells. All of the DTX formulations

provided time and concentration dependent inhibiting effect on MD-MB-231 cells (Fig.

3.15). As shown in Table 3.10, the IC50 value was 13.6, 0.18 and 0.065 µg/mL for DTX,

NLs and ENLs, respectively. DTX-loaded ENLs were the most effective among all the DTX

formulations for cell growth inhibition. Nearly 200-folds higher cytotoxicity with ENLs,

compared to pure DTX, might be attributed to the synergistic effect of thiol groups (-SH)

Table 3.9: Results showing ex vivo permeation enhancement from Apical to Basolateral and

Basolateral to Apical side of intestine, apparent permeability along with improvement ratios

of DTX in the presence of verapamil and synthesized NLs and ENLs. The findings are

shown as Mean ± S.D.

Formulation Papp (A-B)

(cm/s)x 10-6

Improve-

ment ratio

Papp (B-A)

(cm/s)x 10-6

Improve-

ment ratio

Efflux

ratio=

B-A/A-B

DTX in Buffer 0.08 ± 0.01 - 0.48 ± 0.7 - 5.78

DTX-Verapamil 0.47 ± 0.1 5.87 0.65± 0.2 0.06 1.38

NLs 0.77* ± 0.1 9.62 1.82* ± 0.1 6.36 2.36

ENLs 1.05* ± 0.1 13.12 1.04* ± 0.8 0.61 1.0

Chapter 3: Results

57

incorporated on NLs and folate receptor ligand. ENLs can be adsorbed on the cellular

surface and increase cellular transport by improving para-cellular and transcellular

movement of DTX.

The in vitro cytotoxicity of NLs and ENLs was also tested against Human colon cancer

cells. SRB assay was performed using HCT-116 and results are shown in Fig. 3.16. Pure

DTX being hydrophobic in nature that has difficulties in crossing cell membrane and

showed relatively higher IC50 value of 2.38 µg/mL as shown in Table 3.10. NLs and ENLs

showed improved cell interaction and showed lower IC50 i.e. 0.532 and 0.148 µg/mL

respectively. ENLs controlled remained unreacted towards HCT-116 and didn’t produce

any cytotoxicity at almost every concentration.

Figure 3.14: Scanning electron micrographs of rat intestine after permeation enhancement

studies (a) Rat intestine (b) Transverse section (TS) of Rat intestine, (c) Basal surface of

intestine and (d) Epical surface of intestine.

Table 3.10: IC50 values of Pure DTX suspension, unmodified and modified liposomes

calculated from cytotoxicity data using Graphpad Prism software 6.0. The results are shown

as Mean ± S.D.

Formulation IC50 Value (µg/mL)

MB-231

IC50 Value (µg/mL)

HCT-116

DTX 13.62 ± 3.31 2.38

NLs 0.18* ± 0.11 0.532

ENLs 0.06* ± 0.14 0.148

Chapter 3: Results

58


showing highly improved effect on MDA-MB-231 cell line using MTT assay. Both

modified and unmodified empty liposomes were used to compare the cytotoxic potential of

formulations. The results are shown as Mean ± S.D.


showing improved effect on HCT-116 cell line using SRB assay. Both modified and

unmodified empty liposomes were used to compare the cytotoxic potential of formulations.

The results are shown as Mean ± S.D.

Chapter 3: Results

59

3.2.10. In vivo pharmacokinetics

Plasma drug concentration of NLs and ENLs, administered orally are shown in Fig. 3.17.

Plasma pharmacokinetic parameters are summarized in Table 3.11. It was observed that

after oral administration, pure DTX reached the Cmax after 3 h and remained above the

minimum effective concentration MEC (35 ng/mL) for 3 h only. On the other hand, the

modified liposomes attained the MEC levels after 15 min and remained within therapeutic

window till 96 h. Half-life (t1/2) of ENLs was 86.31 h, which was around 3-folds higher than

that of pure drug. Cmax was increased 10-fold with ENLs as compared to the pure DTX,

however, 4-fold increase was observed with NLs.

This study presents 13.60-folds increase in AUC0-96 of DTX with ENLs as compared to DTX

aqueous dispersion.

Figure 3.17: Plasma concentration of DTX after oral administration of DTX suspension,

NLs and ENLs (Oral dose=10mg/kg). Blood samples were taken at predefined time till 96

h and analyzed through HPLC for DTX quantification. The results are shown as Mean ±

S.D.

0

100

200

300

400

500

0 20 40 60 80 100

Pla

sma

Dru

g C

on

c. (

ng

/mL

)

Time (hrs)

DTX NLS ENLs

Chapter 3: Results

60

Table 3.11: Results of in vivo relative oral bioavailability and important pharmacokinetic

parameters obtained after oral administration of DTX suspension in deionized water, NLs

and MNLs to rabbit through oral gavage.


NLs and ENLs formulation might increase the surface area by many folds but also face

aggregation of particles during long term storage. Table 3.12 represents the 3 months’

stability data of drug loaded NLs and ENLs formulations, kept under different storage

temperatures i.e. -20, 4 and 37 °C. After 3 months, the ENLs were found to be stable in

terms of particle size, PDI and encapsulation efficiency. There were no significant changes

except for a slight increase in particle size. However, after 2 months statistically significant

increase (p ≤ 0.05) in average size and PDI (approximately 2 and 1.5-fold increase in size

and PDI, respectively) were observed for NLs.

Pk Parameter Unit Formulations

DTX NLs ENLs

Dose mg/mL 20 20 20

Cmax ng/mL 41.78 ± 2.43 174.59 ± 6.71 430.14 ± 5.81

Tmax H 3.03 ± 1.82 2.08 ± 1.34

AUC0-96 H.(ng/mL) 963.30 ± 14.31 5956.98 ± 38.54 9428.42 ± 24.31

AUMC0-96 H.(ng/mL) 30410.01 ± 15.44 225353.19 ± 28.86 342848.91 ± 21.75

MRT0-96 H 31.57 ± 5.32 37.83 ± 5.32 36.36 ± 7.522

T1/2 H 33.04 ± 3.89 72.18 ± 6.51 86.31 ± 4.83

F % 1 6.2 13.6

Chapter 3: Results

61

Table 3.12: 3 months stability data of DTX loaded, NLs and ENLs based on changes in

particle size, PDI and encapsulation efficiency performed at different storage conditions i.e.,

-20, 4 and 37 °C. The analysis was performed in triplicate and results are presented in terms

of Mean ± S.D.

Formula

tion

Tem

p

(°C)

Particle size

(nm)

Polydispersity Index

(PDI)

Encapsulation Efficiency

(%)

1

month

2

month

3

Month

1

month

2

Mont

h

3

Mont

h

1

month

2

month

3

month

NLs

-20

246.45

± 0.32*

267.81

± 0.66*

283.74

± 0.83*

0.33 ±

0.12*

0.36 ±

0.72*

0.38 ±

0.19*

71.49

± 3.83

69.87

± 6.45

66.43

± 5.84

ENLs 328.53

± 0.24*

332.16

± 0.48*

345.58

± 0.47*

0.37 ±

0.31*

0.37 ±

0.21*

0.38 ±

0.14*

78.47

± 6.73

76.45

± 8.46

77.39

± 7.13

NLs

4

276.57

± 0.31*

297.38

± 0.62*

313.24

± 0.89*

0.31 ±

0.21*

0.34 ±

0.73*

0.41 ±

0.15*

71.49

± 3.85

67.87

± 6.58

62.73

± 6.36

ENLs 317.77

± 0.33*

348.32

± 0.47*

383.46

± 0.61*

0.33 ±

0.13*

0.36 ±

0.17*

0.36 ±

0.71*

78.47

± 6.75

74.76

± 5.89

71.94

± 8.42

NLs

37

246.45

± 0.36*

347.86

± 0.64*

463.54

± 0.78*

0.34 ±

0.51*

0.36 ±

0.44*

0.48 ±

0.76*

69.93

± 3.82

67.74

± 8.54

59.67

± 6.76

ENLs 341.37

± 0.61*

368.91

± 0.57*

387.80

± 0.54

0.37 ±

0.14*

0.38 ±

0.37*

0.39 ±

0.29*

78.47

± 6.76

74.54

± 5.36

71.52

± 4.62

Chapter 3: Results

62

3.3. In vitro and in vivo toxicological evaluation

Figure 3.18: Graphical Abstract

Chapter 3: Results

63

3.3.1. In vitro hemolysis assay

In vitro hemolysis assay was performed on human blood to check the hemolytic profile of

NLs and ENLs. Fresh human RBCs were treated with different concentrations of DTX

suspension, NLs, ENLs and ENLs control (without drug) to examine concentration

dependent response on percentage hemolysis. The results in Fig 3.19 indicted that pure drug

was highly toxic even at lowest concentration used. Contrary to that, the ENLs reduced the

hemolytic effect of DTX at all concentrations indicating the improved biocompatibility with

RBCs resulting in decreased hemolysis. ENLs control showed that formulations along with

all ingredients were biocompatible. IC50 for ENLs and ENLs control was observed to be

164.2 and 487.3 µg/mL respectively which demonstrated the high therapeutic window with

these NLs.


to determine toxicity against red blood cells via hemolysis assay. The results are shown as

Mean ± S.D.

3.3.2. Biocompatibility with macrophages

To assess the compatibility and potential toxicity of DTX loaded NLs and ENLs an in vitro

assay with human macrophage was performed with different concentrations of DTX loaded

formulations and pure DTX suspension. The results (Fig. 3.20) demonstrated that at higher

concentrations, the nanocargoes were cytotoxic to macrophages. However, this cytotoxicity

0

50

100

0 100 200

RB

Cs

Via

bil

ity %

Concentration (µg/mL)

DTX

NLs

ENLs

ENLs Control

Chapter 3: Results

64

was significantly low at all concentrations as compared to DTX. The results in Fig. 3.20

showed that at highest concentrations, the cell viability remained above 65 % indicating the

biocompatibility of all the materials used and hybrid ENLs itself. The LD50 of ENLs and

hybrid ENLs control were 113.4 and 341.2 µg/mL respectively. This high IC50 of hybrid

ENLs showed its higher level of biocompatibility and safety.


to determine toxicity against macrophages isolated from fresh human blood via MTT assay.

The results are presented as Mean ±S.D.

3.3.3. Tissue drug distribution

The DTX was quantified in vital organs using HPLC and results shown in Fig. 3.21

presented the least amount of drug in liver and kidney with hybrid ENLs as compared to

NLs and also pure DTX which showed maximum drug.


The in-vivo toxic potential of these orally administered NLs and hybrid ENLs was evaluated

in female Swiss albino mice to establish the safety profile of formulations and ingredients.

After 14 days, the blood was collected from all mice in sterilized vials depending upon the

analysis to be performed and the mice were euthanized to collect different organs for further

studies.

0

50

100

0 50 100 150 200 250

Macr

op

hag

e V

iab

ilit

y %

Concentration (µg/mL)

DTX

NLs

ENLs

ENLs Control

Chapter 3: Results

65

Figure 3.21: Quantification of DTX in liver, kidneys and heart after 14 days of oral

administration. The results are shown as Mean ± S.D.

After 14 days treatment, organ to body index was calculated for vital organs including

kidney, liver and heart. The organs were carefully removed from euthanized mice and

washed with normal saline. The relative organ to body index of each organ was compared

with control (Fig. 3.22).

Figure 3.22: Organ to body index of vital organs compared with control, indicating toxicity

induced by treatment. The results are shown as Mean ± S.D.

0

15

30

Liver Kidney Heart

Org

an

-Bod

y I

nd

ex

Control

DTX

NLs

ENLs

ENLs Control

0

100

200

Liver Kidney Heart

Con

cen

trati

on

(µ

g/m

L)

DTX NLs ENLs

Chapter 3: Results

66

The effect of DTX suspension and DTX loaded NLs and hybrid ENLs and hybrid ENLs

control on serum biochemistry and hematology was assessed on mice blood. Liver function

tests (LFTs) shown in Fig. 3.23a gives an idea about liver’s state, effect on kidney was

assessed through RFTs in Fig. 3.23b, Serum electrolytes in Fig. 3.23c showed effect on Na,

Ca, Mg and P, and serum glucose and cholesterol in Fig. 3.23d. To check the

biocompatibility of NLs and hybrid ENLs with blood and its component, complete blood

count CBC was performed and results are shown in Table 3.13.

Figure 3.23: Serum biochemistry analysis of mice plasma after acute oral treatment with

DTX, NLs and ENLs compared with control to monitor changes on (a) LFTs; (b) RFTs; (c)

electrolytes and (d) glucose, cholesterol and total protein, induced after treatment due to

metabolism of formulations or drug. The results are presented as Mean ± S.D of triplicate.

The histological slides of heart, liver and kidney were carefully prepared through microtome

and stained slides were examined for structural changes and lesions in tissues. The images

in Fig. 3.24 show the histology comparison of cardiac tissue in Fig. 3.24a(1-3), liver

histology as compared to control as shown in Fig. 3.24b(1-3) and of kidneys in Fig. 3.24c(1-

3).

Chapter 3: Results

67

Table 3.13: The effect of DTX, NLs, ENLs and ENLs control on CBC of mice. The results

are presented as Mean ± S.D of triplicate.

Blood Parameter Control DTX NLs

ENLs-

Control ENLs

RBC (1012/L) 9.02±2.13 7.42±2.75 8.35±2.54 7.95±2.79 8.,9s6±2.51

MCV(fL) 56.41±5.21 53.57±5.94 55.46±6.76 54.59±5.83 54.84±4.76

MCH (pg) 15.53±2.65 15.54±2.76 15.44±3.65 18.69±4.33 15.94±3.65

PCV (%) 48.02±7.53 47.71±3.76 47.88±8.32 48.17±6.72 47.93±5.77

Hb (g/dL) 14.15±3.65 12.87±2.65 14.63±3.52 13.66±3.65 13.94±3.65

WBC (109/L) 15.56±4.21 14.45±3.65 14.49±3.76 15.01±2.54 14.85±4.23

Platelets 109/L 753.33±34.4 702.66±65.87 733.66±56.29 737.33±65.82 741.66±68.66

RDW (%) 16.07±3.12 17.58±3.54 16.69±4.28 17.46±3.65 17.34±3.55

MPV (fL) 6.75±1.67 7.45±1.43 6.63±1.69 7.49±1.65 7.33±2.43

Figure 3.24: Microscopic examination of tissue histology of vital organ (liver, kidney and

heart) to examine any necrosis or histological change as compare to control for these organs

after treatment with formulations; a) heart tissue of control; 1a) treated with NLs, 2a) treated

with ENLs and 3a) treated with ENLs-control; b) liver tissue of control, 1b) treated with

NLs, 2b) treated with ENLs and 3b) treated with ENLs-control; c) kidney tissue of control,

1c) treated with NLs, 2c) treated with ENLs and 3c) treated with ENLs-control.

Chapter 3: Results

68

3.3.5. Genotoxicity

In vitro MN assay was performed in triplicate to check the genotoxic potential of the ENLs

control and compared with positive and vehicle control. Treatment with test samples

resulted in micro-nucleated binucleate cells that were similar to concurrently vehicle

control. Genotoxicity is expressed in percent of micronuclei per 1000 binucleated cells and

calculated replication index (RI) was 1.0315. The results are tabulated in Table 3.14. Fig

3.25 represents fluorescent spectroscopy images of acridine orange stained slides of cells.

Table 3.14: Results showing in vitro MNs assay. The number of micronucleus counted in

1000 binucleated cells on slides. The results are shown as Mean ± S.D.

Formulation Cells with 1 MNs Cells with 2 MNs Cells with 3 MNs

Vehicle Control 4±2 0 0

ENLs 143±8 38±5 14±1

ENLs-Control 12±3 3±1 0

Positive Control 82±5 27±3 9±1

Figure 3.25: Pictures of representative slides stained with acridine orange showing results

of in vitro micronucleous assay performed on human peripheral blood; (a) treatment with

ENLs, (b) Positive control and (c) vehicle control.

Chapter 3: Results

69

3.4. Thiolated Polymeric Nanocapsules Embedded with Fluorescent Silver

Nanoclusters for Breast Cancer Therapy

Figure 3.26: Graphical Abstract

Chapter 3: Results

70

3.4.1. Synthesis of AgNCs and NCs

FA-CS-TGA stabilized silver nanoclusters (NCs) with blue fluorescence were synthesized

via microwave assisted method. Nanocapsules (DTX-Ag-NCPs) containing both the DTX

and NCs were further prepared using TPP as a cross-linking agent. The NCs retained their

fluorescence in solution and lyophilized state as shown in Fig. 3.27. The particle size, PDI

and zeta potential of the DTX-Ag-NCPs are shown in the Table 3.15. Moreover, the amount

of elemental silver in DTX-Ag-NCPs was determined to be 16.58 µg/g of the formulation

using inductively coupled plasma mass spectrometry (ICP-MS).

Figure 3.27: Synthesis of NCs and DTX-Ag-NCPs (1a) before microwave treatment, (1b)

after microwave treatment followed by dialysis resulting formation of NCs, (2a) under UV

light before synthesis, (2b) NCs formation with blue fluorescence, (3a, 3b, 3c) Control and

NCs in split channels blue, green and red respectively, (4a, 4b) Lyophilized Ag-NCPs and

DTX-Ag-NCPs under normal light, (5a) lyophilized Ag-NCPs and DTX-Ag-NCPs under

UV light, (5a, 5b , 5c) lyophilized Ag-NCPs and DTX-Ag-NCPs in split channels blue,

green and red respectively.

Table 3.15: Physicochemical characterization of formulations synthesized showing particle

size, poly dispersity, zeta potential and encapsulation efficiency. The results are shown as

mean ± S.D of triplicate experiment.

Formulation Particle size

(nm)


(PDI)

Zeta potential

(mV)

Encapsulation

Efficiency (%)

NCs 42.50 ± 3.61 0.21 ± 0.15 +3.10 ± 1.84 -

Ag-NCPs 112.48 ± 5.87 0.18 ± 0.17 +18.43± 3.22 -

DTX-Ag-NCPs 190.72 ± 2.19 0.13 ± 0.12 + 22.70 ± 2.22 73.65 ± 6.5

Chapter 3: Results

71

3.4.2. FTIR, DSC and XRD analysis

FTIR spectra of CS, FA-CS-TGA, NCs, DTX, DTX-Ag-NCPs are shown in Fig.3 28a. The

CS spectra represented the characteristic peaks at 1656 1590, and 1256 cm-1. The presence

of NCs in FA-CS-TGA resulted in significant shifting in the stretching peaks of amide band

at 1656 and 1590 cm-1. The presence of NCs also shifted the –OH stretches from 1424 to

1410 cm-1. The vibrations of NH2 and O-H respectively on CS-TGA, shifted to 3349 cm-1.

The chemical integrity of DTX was confirmed by the characteristic stretching peaks

appearing in FTIR spectra (Fig. 3.28a) at 3449, 3351 and 1713 cm-1. Whereas, the peaks

appearing between 2810 and 3070 cm-1 are attributed to aliphatic and aromatic C-H

stretches. The XRD pattern in (Fig. 3.28b) showed characteristic reflection of NCs capped

with FA-CS-TGA at 34° and 44°, which slightly diminished in DTX-Ag-NCPs in the

presence of DTX.

The DTX showed melting point at around 169 °C (Fig. 3.28c), which was not observed in

DTX-Ag-NCPs showing presence of DTX in amorphous form inside the DTX-Ag-NCPs.

Figure 3.28: Compatibility analysis (a) FTIR spectra showing characteristic peaks for all

formulations, (b) XRD analysis of all the formulations representing specific peaks (c) DSC

thermogram showing temperature effect on all formulations.

Chapter 3: Results

72

3.4.3. STEM/EDX analysis

STEM analysis revealed spherical appearance of DTX-Ag-NCPs having smooth surface

with particle having diameter around 175 nm as shown in Fig. 3.29a. The elemental

composition by EDX spectra of DTX-Ag-NCPs is shown in Fig. 3.29(b-c) and Table 3.16.

EDX analysis confirmed the presence of NCs distribute with in DTX-Ag-NCPs.

Figure 3.29: STEM/EDX analysis of DTX-Ag-NCPs (a) STEM images of DTX-Ag-NCPs

(b) spot EDX spectra of NCs showing Ag and other metals in terms of percentage, (c) EDX

analysis showing different element within DTX-Ag-NCPs.

3.4.4. Optical characterization

Optical characterization of prepared DTX-Ag-NCPs, was performed using UV-vis

spectrophotometer having fluorescence. The synthesized NCs did not show any plasmonic

response as shown in Fig. 3.30. The fluorescence emission spectra of NCs at emission peak

(λem) at 430 nm, when excited (λex) at 365 nm is shown if Fig. 3.31. The fluorescent images

of dried DTX-Ag-NCPs before and after drug loading in split channels i.e. blue, green and

red clearly indicating the maximum fluorescence in blue channel are shown in Fig. 3.27.

Chapter 3: Results

73

Table 3.16: EDX analysis showing percentage of various elements detected in DTX-Ag-

NCPs.

Element Line

Type

Apparent

Concentration

k Ratio Wt% Wt%

Sigma

Standard

Label

Factory

Standard

C K series 2.93 0.02935 16.96 21.08 C Vit Yes

O K series 42.37 0.14259 44.92 11.41 SiO2 Yes

Na K series 9.89 0.04176 9.81 2.5 Albite Yes

Mg K series 0.46 0.00308 0.62 0.17 MgO Yes

Al K series 16.29 0.117 19.58 5 Al2O3 Yes

P K series 2.42 0.01356 2.12 0.54 GaP Yes

Cl K series 0.84 0.00735 0.02 0.26 NaCl Yes

K K series 0.24 0.002 0.26 0.08 KBr Yes

Ca K series 0.16 0.00143 0.18 0.06 Wollastonite Yes

Pd L series 1.09 0.01092 1.47 0.43 Pd Yes

Ag L series 0.18 0.00162 2.23 0.05 Ag Yes

Au M series 1.85 0.01846 1.83 0.76 Au Yes

Total:

100

Figure 3.30: UV-vis absorbance spectra of DTX-Ag-NCPs, Ag-NCPs and NCs showing no

plasmonic response for NCs and Ag-NCPs but appearance of bend in DTX-Ag-NCPs

because of absorbance by DTX.

Chapter 3: Results

74

Figure 3.31: Fluorescence spectra of NCs and DTX-Ag-NCPs showing slight decreased

fluorescence after DTX loading.

3.4.5. Encapsulation efficiency and In vitro drug release

The encapsulation efficiency is an important factor to be determined for developing a good

formulation. The encapsulation efficiency was observed to be 73.65 % which was

considered to be very good for a hydrophobic drug. The in vitro DTX release from DTX-

Ag-NCPs was studied for 12 h and cumulative drug release against time was plotted in Fig.

3.32 showing a sustained release form DTX-Ag-NCPs. The release mechanism from DTX-

Ag-NCPs was observed to be following Korsmeyer-Peppas model based upon R2 values.

3.4.6. Cytotoxicity and cell imaging studies

The cytotoxic effect of these DTX-Ag-NCPs was assessed against MB-231, human breast

cancer cell line. The results of treatment with different concentrations of DTX suspension,

NCs and DTX-Ag-NCPs are shown in Fig. 3.33. The IC50 values for DTX and DTX-Ag-

NCPs was calculated as 14.32 µg/mL and 0.0427 µg/mL respectively. The uptake study of

DTX-Ag-NCPs by the cells was done for 3 h using 8 chambered slide. The fluorescent

images in Fig. 3.34(a-f) present the successful internalization and imaging ability of the

DTX-Ag-NCPs after staining with phalloidin green and DAPI.

Chapter 3: Results

75

Figure 3.32: In vitro drug release studies showing cumulative percentage drug release from

DTX-Ag-NCPs and pure DTX suspension in phosphate buffer (pH 2-7.4) at 37 °C against

time over period of 12 h.


(MDA-MB-231) using different concentrations of DTX suspension, DTX-Ag-NCPs and

Ag-NCPs to check anti-cancer activity and biocompatibility. The results are shown as Mean

± S.D.

0

20

40

60

80

100

0 4 8 12 16

Cu

mu

lati

ve

Dru

g R

elea

se (

%)

Time (h)

DTX DTX-Ag-NCPs

Chapter 3: Results

76


(MDA-MB-231) showing (a) bright field cellular image and (b) under UV-light showing

fluorescence and cell death after 24 h, and (c-f) MB-231 cells after 6 h incubation stained

with phalloidin green and DAPI showing cell uptake of DTX-Ag-NCPs.

3.4.7. Biocompatibility studies

The biocompatibility of the formulation was checked in vitro against fresh human

macrophages. The results showed concentration dependent cytotoxicity of all the treatment

(Fig. 3.35). DTX-Ag-NCPs showed more than 80 % viability at lower concentration as

compared to DTX at the same concentration.

Figure 3.35: In vitro cytotoxicity against human macrophage using different concentrations

of DTX suspension, DTX-Ag-NCPs and Ag-NCPs to check anti-cancer activity and

biocompatibility. The results are shown as Mean ± S.D.

Chapter 3: Results

77

3.4.8. Oral bioavailability

Relative oral bioavailability and pharmacokinetics were studied in healthy rabbits of either

sex. Plasma level was measured at predefined intervals for 24 h after single dose oral

administration (Fig. 3.36). From this plasma level-concentration data, pharmacokinetic

parameters including Cmax, Tmax, T1/2, Cl, AUC0-24, and MRT were calculated (Table 3.17).

It was observed that after oral administration, DTX suspension reached to Cmax after 5 h and

remained above the minimum effective concentration (35 ng/mL) for 3 h only. On the other

hand, the DTX-Ag-NCPs attained the minimum effective concentration (MEC) levels after

3 h and remained within therapeutic window for 24 hrs. Plasma half-life (t1/2) of DTX-NCs

was 123.5 h, which is around 5-folds higher than that of pure DTX i.e. 18.03. Cmax was also

increased 6-folds with DTX-Ag-NCPs as compared to that with pure DTX. The AUC0-24 of

DTX-Ag-NCPs showed high increase in relative oral bioavailability i.e. 8.89-folds as

compared to pure drug.

Figure 3.36: Relative oral bioavailability study of DTX suspension and DTX-Ag-NCPs in

rabbit (n=5) showing the plasma drug concentration after oral administration of 10mg/kg of

formulations and blood withdrawn at predefined time interval was analyzed through HPLC.

The results are shown as Mean ± S.D.

Chapter 3: Results

78

3.4.9. Acute Oral Toxicity Evaluation

No mortality and significant change in body weights was observed for 14 days. Serum

biochemistry was performed on the plasma isolated from pooled blood from each group

after 14 days.

Table 3.17: Different pharmacokinetic parameters calculated from plasma level-time curve

obtained after oral administration of DTX suspension and DTX-Ag-NCPs to rabbits.

Pk Parameter Unit DTX DTX-Ag-NCPs

Tmax H 5 3

Cmax ng/ml 41.78 297.39

t1/2 H 18.039 123.46

AUC 0-24 ng/ml*h 440.70 3921.46

AUC 0-inf_obs ng/ml*h 730.25 26794.91

AUMC 0-inf_obs ng/ml*h^2 19100.51 4664644.30

MRT 0-inf_obs H 26.16 174.08

Cl/F_obs (mg)/(ng/ml)/h 0.014 0.00037

F

1 8.89

The effect of treatment on liver was assessed by LFTs and results in Fig. 3.37a showed a

decrease in level of SGOT, ALP and bilirubin as compared to control, except for SGPT

which was increased with DTX. The results for RFTs in Fig. 3.37b showed increased urea

level with DTX as compared to control and DTX-Ag-NCPs. However, no effect was

observed on creatinine. A decrease in level of cholesterol, total protein and glucose was

observed as compared to control with all the treatment as shown in Fig. 3.37c. The organ to

body ratio was calculated for all three treatments against control and results are summarized

in Fig. 3.37d showing now significant effect on organ weights. The effect of formulation

was evaluated on blood and its components through complete blood count (CBC) and

detailed evaluation is presented in Table 3.18. The CBC analysis showed more

compatibility and safety of DTX-Ag-NCPs as compared to DTX in terms of destruction of

blood cells. The stained slides were microscopically examined and images shown in Fig.

3.38 appeared normal as compared to control group.


The 3 months stability studies of formulation at 4 °C in Table 3.19 showed no significant

changes in particle size, PDI and zeta potential of DTX-Ag-NCPs.

Chapter 3: Results

79

Figure 3.37: Serum biochemistry of mice blood determining acute oral toxicity (a) Liver

function tests, (b) Renal function tests, (c) serum biochemistry and (d) organ to body weight

analysis performed on Swiss albino mice, after DTX, DTX-Ag-NCPs and Ag-NCPs in

accordance with OECD 425 guidelines for acute oral toxicity. The results are shown as

Mean ± S.D.

Table 3.18: Complete blood count (CBC) analysis of mice blood obtained after 14 days’

acute oral toxicity analysis. The results are shown as Mean ± S.D.

DTX DTX-Ag-NCPs Ag-NCPs Control

RBC 6.86 ± 4.91 8.01 ± 5.67 7.83 ± 4.15 8.22 ± 4.92

MCV 57.57 ± 5.52 55.46 ± 5.36 54.59 ± 8.32 56.84 ± 2.28

MCH 15.54 ± 4.35 15.44 ± 7.41 18.69 ± 6.24 16.56 ± 4.61

PCV 54.37 ± 11.93 73.88 ± 8.49 78.17 ± 12.63 50.92 ± 6.82

Hb 13.20 ± 6.37 14.97 ± 2.18 14.19 ± 5.25 15.44 ± 5.14

WBC 12.45 ± 7.63 13.73 ± 7.07 13.69 ± 6.08 14.28 ± 7.37

Platelets 632 ± 87.57 707 ± 78.54 677.33 ± 64.04 723.66 ± 89.23

RDW % 16.92 ± 6.63 16.69 ± 5.31 17.46 ± 5.29 17.05 ± 6.70

MPV 7.11 ± 4.91 6.63 ± 6.43 7.49 ± 4.33 6.76 ± 7.17

Chapter 3: Results

80

Figure 3.38: Microscopic evaluation of tissue histology; (1) Control liver, (1a) treatment

with DTX, (1b) treatment with DTX-Ag-NCPs, (1c) treatment with Ag-NCPs and (2)

Control kidney, (2a) treatment with DTX, (2b) treatment with DTX-Ag-NCPs, (2c)

treatment with Ag-NCPs obtained from Swiss albino mice after being euthanized.

Table 3.19: 3-month stability studies data showing changes in particle size and PDI of B-

NCs and NCs stored in dark at 4 °C. The results are shown as Mean ± S.D.

Formulat

ion

Tem

p

(°C)

Particle size

(nm)


(PDI)

Zeta Potential

(meV)

1

month

2

month

3

month

1

month

2

month

3

month

1

month

2

month

3

month

Ag-NCPs 4 112.48

± 5.87

123.50

± 4.84

128.30

± 4.72

0.18 ±

0.17*

0.20 ±

0.18

0.24 ±

0.15

17.13±

4.17

17.20

± 2.45

15.36

± 2.76

DTX-Ag-

NCPs

190.72

± 2.19

196.40

± 3.20

211.60

± 4.75

0.13 ±

0.12*

0.16 ±

0.14

0.17 ±

0.19*

21.88

± 3.42

20.71

±4.38

18.40

± 3.56

Chapter 4

DISCUSSION

Chapter 4: Discussion

81

4. DISCUSSION

Chemotherapy, a major platform for cancer therapy, devoid the specificity to confine the

cancer remedies in the tumor site, thus influencing normal healthy tissues and encouraging

toxic adverse effects. Nanocargoes based intervention has significantly revolutionized the

treatment of cancer by surmounting the existing limitations in traditional chemotherapy,

which comprise of undesirable bio-distribution, drug resistance, and severe systemic

adverse effects. But in most cases lack of stability and incompetence to cross the barriers of

tumor microenvironment is hard to overcome. Nanocargoes can achieve preferential

accumulation in the tumor, owing to their ligand-based active targeting.

The comprehensive opportunity for chemically modifying the polymer as stabilizer and an

enveloping biomaterial into desired ligand-based targeting construct makes it a versatile

delivery system. The folate receptor is the most widely sought tumor marker to bind with

folate-anchored nanocargoes with a great affinity and internalizes into the cells via receptor-

mediated endocytosis. Functional folate receptors are overexpressed in wide range of tumors

including breast cancer and confined to the apical surfaces of polarized epithelia. The

pronounced utility of these folate ligands stems from the fact that they are economical,

biocompatible and non-immunogenic. The folate receptors also have high binding capacity,

stability on storage and in circulation, and are straightforwardly grafted to nanocargoes.

Hence, to accomplish the main objective of the study, i.e. development of a folate grafted

thiolated chitosan (FA-CS-TGA) polymer as enveloping stabilizer for diverse targeted

nanocargoes with promising chemotherapeutic potential was achieved in two steps via

EDAC coupling mechanism. In the first step, thiolated chitosan (CS-TGA) was successfully

synthesized by modifying the low molecular weight chitosan (CS) backbone via covalent

linkage with thioglycolic acid (TGA) through amide bond formation between amino

moieties of chitosan and carboxylic acid groups of TGA. The FTIR spectrum (Fig.3.3) of

CS clearly showed absorbance bands at 1654 cm-1 (amide I), 1604 cm-1 (NH2) bending and

1382 cm-1 (amide III). However, in the CS-TGA spectrum peaks at 3351 cm-1 and 3209 cm-

1 represented O-H and N-H stretching and peak observed at 1630 cm-1 was assigned to

acylamino group. Also the intensity of peak around 1607 cm-1 decreased, indicating that

amino groups were partly conjugated to TGA (Saboktakin et al., 2010). In the second step,

folic acid (FA) was subsequently attached to this thiolated chitosan (CS-TGA) via same

carbodiimide chemistry. The carboxylic acid groups of folic acid were activated by EDAC

to generate an amine reactive O-acylisourea intermediate. Afterwards, this intermediate


82

reacted with the free amino groups of CS-TGA to form folate grafted thiolated chitosan

(FA-CS-TGA) polymer as shown in Fig. 3.2. FTIR spectrum of FA CS-TGA showed

characteristic peak at 3372 cm-1 and 3274 cm-1 corresponding to primary and secondary

amine (N-H stretching) respectively. The appearance of two characteristic peak at 1662 cm-

1 and 1585 cm-1 representing carbonyl (C=O) stretching and NH associated (N-H) bending

in secondary amine. In addition, a new sharp band at 1314 cm-1 corresponded to C-N

stretching of secondary amine. Therefore, the analysis suggested that available NH2 groups

of CS-TGA were converted to NH groups and folic acid was attached to the thiolated

chitosan (Wan et al., 2008). The newly synthesized FA-CS-TGA was assessed for its

potential by enveloping docetaxel (DTX) loaded nanoliposome as model carrier for breast

cancer treatment.

For quantification of DTX in formulation and plasma samples, a reproducible validated

reverse phase HPLC-PDA method was developed and validated according to ICH

guidelines. System suitability was carried out by varying experimental conditions of

temperature, mobile phase ratios and two different columns. Interestingly, C18 showed

higher theoretical plate count and comparatively lesser tailing. Similarly, different ratios of

acetate buffer to organic solvents was used for the separation of DTX from samples and

showed that most suitable ratio was (48:16:36, v/v/v) that produced a characteristic sharp

peak at retention time of 5.9 min. Initially, 1 mL flow rate was applied but later 0.8 mL per

minutes was found more suitable for the appropriate separation. Similarly, isocratic mode

showed better detection as compared to gradient mode. After extensive preliminary trials,

the most suitable chromatographic conditions to obtain DTX characteristic peak were

achieved from column C18 using mobile phase consisting of methanol, acetonitrile and

acetate buffer (10mM, pH 5.0) in (48:16:36, v/v/v) respectively in isocratic mode at a flow

rate of 0.8 mL per minute under column and sample temperature at 25oC. The retention time

for DTX was 5.9 min using PDA detector at 230 nm.

The results for system suitability clearly demonstrated the justification of the acceptance

criteria set for theoretical plate count above 3000, tailing factor below 1.5 and RSD less than

2 %. Moreover, the recovery studies, precision and accuracy performed using different

formulations in vitro and in vivo, satisfied the acceptance criteria and ensured precision and

accuracy of the developed method as RSD % of peak area, assay and tailing factor obtained

were observed to be within the limits. The LOD was found 2.15 ng/mL and LOQ of 6.52

ng/mL. The method was applied throughout research for quantification of DTX in various

in vitro and in vivo analyses.


83

Folate grafted thiolated chitosan (FA-CS-TGA) polymer enveloped nanoliposomes (ENLs)

were theoretically optimized via Design Expert Software simulation based RSM plots for

most suitable components. Central composite design (CCD) was selected for the

optimization of formulations. Based on ANOVA results, predictive analysis and numerical

optimization was done using Design Expert Software that produced various formulations

with varying ratio of ingredients. First three formulations were selected and reproduced to

confirm the prediction. Based on the impact of selected factors of particle size, PDI and

encapsulation efficiency one point was selected at the optimal area where particle size and

PDI was minimum and encapsulation efficiency was maximum. Predicted and experimental

values of the confirmation for formulations were very closely related to each other showing

good predictability and application of CCD in formulation optimization at nano-scale

(Cheng et al., 2014).

The associative interactions between nanoliposomes (NLs) or FA-CS-TGA polymer

enveloped nanoliposomes (ENLs) with mucin, like electrostatic interactions, mechanical

chain interlocking, conformational changes and chemical interactions are expected to

change the rheology of the two species. In this perspective, the viscosity of molecular

dispersion of completely hydrated NLs, ENLs with mucin may reflect the strength of the

mucoadhesive joints. Rheological synergism has been suggested as an in vitro parameter to

measure the mucoadhesive properties of polymeric formulation: the higher the rheological

synergism, the stronger the polymer interaction with mucin which predict better gastric

absorption of nanoparticle.(Shahnaz et al., 2010) The increase was presumably not due to

the contribution of mucin, but due to physico-chemical interactions between the mucin and

the ENLs. Ten-fold higher storage modulus (G′) values were obtained for mucin- ENLs

mixtures than for the ENLs solutions alone within 2 hrs. This can also be explained by the

liberated free thiol groups and/or thiolate anions from ENLs reacting with disulfide bond

within mucin, leading to the formation of new disulfide bonds between surface thiol

moieties of ENLs and mucin as suggested by (Iqbal et al., 2012). The formation of new

disulfide bonds strengthened the adhesive joints leading to time-dependent rheological

changes of various mucus-ENLs. However, no considerable increase in the viscoelastic

parameters was observed for NLs and their corresponding mucin mixture (Table 3.3). These

findings demonstrated the lack of conformational changes between NLs and mucin and

demonstrate the advantage of FA-CS-TGA coating for better oral bioavailability.


84

The swelling behavior of polymer-lipids mixtures greatly influence their stability,

mucoadhesive properties and drug release (Brazel and Peppas, 2000). Once attached to the

mucus membrane, the particle swells up by absorbing water from the underlying mucosal

tissues via capillary action and diffusion, strengthening the adhesion. Contrary to this,

excess absorption can result in over swelling thus weakening the mucoadhesion.(Roldo et

al., 2004) Therefore moderate swelling is necessary for developing strong mucoadhesion

between particles and mucus membrane (De Robertis et al., 2015). It was also observed that

ENLs showed slow and gradual swelling due to surface thiol groups and disulfide bonds

which controlled the water uptake that is necessary for developing strong mucoadhesion and

retention of ENLs in mucosa for longer time. On the other hand, NLs showed very slow

swelling because of hydrophobic nature. This swelling behaviour affected the drug released

from formulations, which clearly showed that ENLs were swelled gradually and released

drug in a controlled manner over a longer period as compared to NLs.

Smooth plasma levels of drug over a longer period of time and controlled drug release

systems can diminish side effects, improve efficacy and reduce dose frequency to

maximizes patient compliance (Feng, 2014). The in vitro release showed good control over

DTX release from ENLs as compared to NLs and pure drug suspension indicating a

sustained effect which could help in decreasing dose related side effects. A possible

explanation for the observed sustained drug release from ENLs may be due to covalent

cross-linking of disulfide bonds formed within the ENLs matrix during swelling process

(Dash et al., 2010). The mechanism of drug release was investigated through various release

kinetics models applied to release data and observed to be Fickian diffusion, which refer to

a process in which material relaxation time is much greater than the solvent diffusion time

into matrix system (Fu and Kao, 2010).

Intestinal absorption is a prime factor to improve the bioavailability of drugs administered

via oral route. It is highly recognized that influx and efflux transporters for instance P-

glycoprotein (PGP) available on the intestinal epithelial cells membrane have a considerable

influence on drug absorption.(Fang et al., 2015) Influx transporters promote absorptive

transport, while efflux transporters antagonize it. PGP transporters modify intestinal

permeation of DTX by preventing the influx into cells and encourage DTX efflux from

intestinal epithelium back into the lumen (Gaikwad and Bhatia, 2013). Thiolated chitosans

have intrinsic property of PGP inhibition thus ENL’s ability to transport the drug across


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intestinal membrane and keep the PGP efflux function inhibited, was compared with DTX

absorption alone and in the presence of verapamil (PGP Inhibitor).

To further investigate the possible involvement of intestinal efflux pumps in the

permeability process, DTX, NLs and ENLs were evaluated for its apparent permeability

coefficients (Papp) in the reverse basal to apical direction (secretory transport). Given that

efflux pumps is preferentially located on the apical side of membrane (Hunter et al., 1993),

clearly suggest that the secretory transport of DTX occurs via predominantly transcellular

pathway in rat mucosa. Tested DTX in the presence of verapamil showed small dominancy

of basal to apical permeation over apical to basal permeation across rat mucosa with

decreased efflux ratios as compared to DTX alone. However, the permeability rate for ENLs

was found to be identical for both basal to apical and apical to basal directions suggesting

the completed PGP efflux pump inhibition due to intrinsic inhibitory property of ENLs. It

was reported that the primary mechanism of permeation improvement by ENLs is based on

the inhibition of protein tyrosine phosphatase (PTP) because of surface thiol groups (Iqbal

et al., 2012). The inhibition of PTP can be accomplished by a disulfide bond (S-S)

generation between thiol group and cysteine site of the PTP. Consequently, a higher degree

of tyrosine phosphorylation of the membrane protein, contributes to the opening of tight

junctions. Hence, an appreciably improved permeability through tight junctions was

examined. As DTX has a low bioavailability due to its reduced permeability and efflux

transporters, therefore the inhibition of PGP transporters could be a promising strategy to

enhance permeation in absorptive direction.

The target specific cytotoxic potential of ENLs was investigated with two different cell lines

using two different assays. The overexpression of folate receptors on epithelial tumors of

breast, lungs and colon has led to enormous attention in using the folate receptors as tumor

target. Upon attachment of the targeting moiety, the moiety-receptor complex is internalized

via receptor mediated endocytosis. It has been revealed that folate grafted liposomes, in

acute malignant leukemia, has ability of escaping P-glycoprotein (PGP) mediated expulsion

of drug from cell. DTX-loaded ENLs were the most effective among all the DTX

formulations for cell growth inhibition. Nearly 200-folds higher cytotoxicity with ENLs,

compared to pure DTX, might be attributed to the synergistic effect of thiol groups (-SH)

incorporated on NLs and folate receptor ligand. In comparison, NLs and ENLs were used

against Human colon cancer cell line HCT-116, which is over expressed with folate

receptors (Jaszewski et al., 1999) and could be successfully targeted through folic acid on

ENLs. About 17-folds increased IC50 with ENLs was observed as compared to pure DTX


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being hydrophobic in nature that has difficulties in crossing cell membrane. ENLs possessed

highest toxicity owing to the grafting of surface folic acid which facilitated particles

attachment, subsequent internalization and triggered other mechanism like swelling of

mitochondria, interaction with cellular proteins or lysosomal damage leading to cell death

(Fröhlich, 2012). ENLs controlled remained unreacted towards HCT-116 and didn’t

produce any cytotoxicity at almost every concentration. ENLs can be adsorbed on the

cellular surface and increase cellular transport by improving para-cellular and transcellular

movement of DTX. Cancerous cells exhibit enhanced endocytic activity and internalization

of NCs inside the cells leading to increased intracellular DTX concentration. The efflux

pumps inhibitory effect of thiol groups on ENLs might have resulted in retaining the DTX

inside cell. Thus, enhanced cytotoxic effect of ENLs was observed as compared to NLs and

DTX alone (Panyam and Labhasetwar, 2003, Jain et al., 2014).

The FA-CS-TGA was also aimed to check the oral permeation potential to increase relative

oral bioavailability. Thus ENLs were expected to enhance the oral permeation and thus oral

bioavailability of DTX (Javed et al., 2016). Oral absorption through GIT describes that

villus tips can take up particles of size range 5-150 µm, while intestinal macrophages up to

1 µM and enterocytes can allow transport of particles ranging in size of 300-400 nm through

transcellular route (Javed et al., 2015). Folate receptors are among various cellular receptors

which facilitate caveolate-mediated endocytosis of materials from enterocytes (Hillaireau

and Couvreur, 2009). This provided a better size window for sub-micron sized particles.

Drug solubility is the rate limiting step for orally administered drugs to cross gastric barrier.

Plasma drug concentration of NLs and ENLs, administered orally are shown in Fig. 3.17.

Plasma pharmacokinetic parameters revealed improvement in AUC and Cmax, with ENLs

which were below the minimum toxic concentration (2700 ng/mL) as observed after

intravenous administration of DTX as reported earlier.(Saremi et al., 2013). 3-folds increase

in Half-life (t1/2) and 13-folds increase in relative oral bioavailability was observed which

was better than of 10-folds increased reported with co-administration of DTX with different

PGP inhibitors and permeation enhancement (Yan et al., 2010, Malingré et al., 2001b,

Oostendorp et al., 2009). This 13.60-folds increased relative oral bioavailability of DTX

from ENLs may be due to combination of mechanisms involving increased muco-adhesion,

inhibition of PGP and enhanced para-cellular transport attributed to thiolated polymer,

grafted on the surface of liposomes. The particle size i.e., ~300 nm might have also

facilitated the paracellular transport through gastric mucosa. Moreover, the enhanced oral


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bioavailability for ENLs may be because of oleic acid present in lipid bilayer of liposomes

which also imparted permeation enhancement effect by opening of tight junctions (Babu et

al., 2015).

Nanotoxicology is an emerging area of science focusing of toxic effects produced by various

types of nanoparticles owing to their extremely small size and physicochemical properties

(Shvedova et al., 2016). It was expected that FA-CS-TGA will increase the biocompatibility

when nanoparticles are enveloped by the FA-CS-TGA. To investigate this, in vitro and in

vivo characterization was done. Charge density and charger polarity play an important role

in inducing cytotoxicity (Schaeublin et al., 2011). Cationic particles induce cytotoxicity via

membrane damage whereas anionic particles cause intracellular damage (Asati et al., 2010).

Generally cationic particles are considered to be more toxic as compared to anionic particles

having same size and chemistry. Phagocytic cells preferentially interact with anionic

particles and engulf them assuming them as bacteria that have negative charge. This results

in higher cytotoxicity of anionic particles as compared to cationic particles (Tomita et al.,

2011). Free amino groups of polymers play a vital role in magnitude of toxicity produced

by cationic nanoparticle (Naha et al., 2010). Primary amino groups of chitosan were

neutralized due to covalently attached thioglycolic acid and folic acid which reduced the

toxicity of cationic ENLs (Fröhlich, 2012).

To further investigate the toxic potential of ENLs, compatibility with fresh human blood is

of prime concern because of their initial interaction with blood components and blood cells

resulting toxic hemolytic effects (Fornaguera et al., 2015). Red blood cells (RBCs) have no

phagocytic receptors or actin-myocin system so they can be used to study the nanoparticle

internalization and cytotoxicity induced by these nanoparticles (Rothen-Rutishauser et al.,

2006). In vitro hemolysis assay was performed on human blood to check the hemolytic

profile of NLs and ENLs. The data suggested higher cytotoxicity of DTX and NLs as

compared to the ENLs indicating the improved biocompatibility with RBCs resulting in

decreased hemolysis. ENLs control showed that formulations along with all ingredients

were biocompatible.

Nanoparticles, having plasma proteins adsorbed on them, are readily engulfed and cleared

by immune system as they reach systemic circulation. However, this clearance is highly

dependent on certain properties like particle size, surface charge and hydrophilicity/

hydrophobicity (Ma et al., 2015). These characteristics of nanoparticles influence the


88

interaction with plasma proteins which after being adsorbed on the surface, form bridges

between particles and monocytes. Thus, the particles with high surface potential and polarity

have long circulating life and least interaction with macrophages (Laine et al., 2014)..To

assess the compatibility and potential toxicity of DTX loaded NLs and ENLs, an in vitro

assay with human macrophages was performed with different concentrations of DTX and

DTX loaded NLs and ENLs. At higher concentrations (< 150 µg), NLs and ENLs showed

toxicity towards macrophages due to their engulfment and increased DTX internalization in

macrophages. ENLs showed least cytotoxicity, owning to surface modification with FA-

CS-TGA. ENLs control was used to evaluate the carrier only induced cytotoxicity. This

could be due to terminal thiol groups with polymers that can improve in vivo stability by

avoiding RES uptake (Manson et al., 2011).

Tissue distribution and drug accumulation in organs is a key factor in producing off-target

effects. NPs have ability to penetrate deep into tissues and this distribution is highly

dependent on particle size and surface properties (Costa and Fadeel, 2016). The similar

distribution patterns for both formulations demonstrated that thiolated chitosan coating did

not alter the tissue distribution behavior of DTX in mice significantly. The DTX was

quantified in vital organs using HPLC and the least amount of drug in liver and kidney with

ENLs as compared to NLs and also pure DTX which showed maximum drug.

The in vivo toxic potential of NLs and ENLs was evaluated in female Swiss albino mice

following OECD 425 guidelines for acute oral toxicity evaluation of materials

(Maneewattanapinyo et al., 2011). Formulations were orally administered at relatively

higher concentrations of 50 mg/kg. The mice showed normal signs for skin, fur, behavioral

patterns and digestion during first 24 h of administration that prevailed throughout the week.

No mortality was observed during the course of study. No significant change in body weight

was observed during study. After 14 days, the blood was collected from all mice in sterilized

vials depending upon the analysis to be performed and the mice were euthanized to collect

different organs for further studies. Firstly, organ to body index was calculated for vital

organs including kidney, liver and heart. The organs were carefully removed from

euthanized mice and washed with normal saline. The relative organ to body index of each

organ was compared with control (Fig. 3a). The decrease in liver weight was observed with

all the treatments in which DTX showed slight increasing effect as compared to NLs and

ENLs. Whereas ENLs control formulations showed minor change in weight of liver. The

kidneys showed no significant weight change after treatments. The heart remained


89

unaffected with all the treatments showing no toxic effects of formulations towards cardiac

muscles. The blood from mice was collected via cardiac puncture to study the effect NLs

and ENLs different biochemistry parameters were evaluated to study the effect of DTX

suspension, DTX loaded NLs, ENLs and ENLs control on serum biochemistry and

hematology. Liver function tests (LFTs) describes its functionality through albumin level

which was not influenced by treatment. Cellular integrity of liver is depicted in

transaminases (SGPT) which is produced within liver cells (Agbaje et al., 2009). The higher

blood level of SGPT indicates cell damage or necrosis leading to leakage of this enzyme in

blood. An increased level of SGPT was observed with DTX and DTX loaded ENLs as

compared to control yet remained within the acceptable limits. However, it was lest affected

by ENLs control. Cellular integrity and its link with bile duct is represented by ALP which

is characteristic finding of cholestatic liver disease. ALP level was increased with all

treatments suggesting some obstruction in bile duct. Hybrid ENLs decreased ALP level as

compared to control. Bilirubin level also did not show any significant changes. Liver is the

prime source of all serum proteins and any changes in total protein is an indicator of liver

abnormality. The total protein content remained unaffected with NLs and ENLs. The effect

of ENLs and ENLs control on LFTs was insignificant (P<0.005) as compared to DTX

showing compatibility of ENLs (Ozer et al., 2008, Thapa and Walia, 2007).

The effect on kidney was assessed through RFTs. The results in Fig 3.23b showed no

significant deviation from reference values of RFTs. Creatinine level remained unaffected

with all treatments. However, BUN was increased with NLs as compared to control yet

remained within the reference ranges. Serum electrolytes (Na, Ca, Mg and P) were assessed

to investigate any other toxicity induced during the treatment. The results shown in Fig

3.23c revealed slight increase in Na level with maximum increase with NLs as compared to

control but the values were below the reference range (140-160 mEq/L). The level of Ca,

Mg and Phosphate were slightly decreased as compared to control yet were within the limits.

The influence on serum glucose and cholesterol was assessed. The results in Fig. 3.23d

showed decrease in cholesterol and glucose level in all group as compared to control.

The critical task in nano drug delivery systems is to ensure its biocompatibility once it comes

in contact with blood. Inflammatory response is most likely to occur, depending upon the

level of incompatibility (Simak, 2009). To check the biocompatibility of NLs and ENLs

with blood and its component, complete blood count CBC was performed and results are

shown in Table 3.13. The results revealed that DTX suspension destroyed RBCs resulting


90

in decreased RBC count which in turn led to a decreased Hb level. DTX loaded NLs and

ENLs showed hemolysis but that was less as compared to pure DTX. Moreover, ENLs-

control showed negligible hemolysis. Neutropenia is side effect of DTX that was mitigated

by encapsulating DTX inside NLs and ENLs. ENLs control didn’t show significant effect

on WBCs. The other parameters (MCV, MCH, PCV, PDW %) were also monitored.

Previously, similar results were observed in case of lipid emulsified DTX (Zhao et al.,

2010).There were slight changes in the values but no significant change was observed

declaring the safety of DTX loaded ENLs. This supported the in vitro results of safety

against RBCs and macrophages.

The functional biochemical analysis was coupled with tissue histological studies as they are

helpful in anatomical localization of toxicity induced by the treatment. The histological

slides of heart, liver and kidney were prepared through microtome and stained slides were

examined for structural changes and lesions in tissues.

Genotoxicity can be assessed very well with end point of chromosomal anomaly using

rodent micronucleus (MN) assay. In vitro MN assay was performed in triplicate to check

the genotoxic potential of the ENLs control as compared to pure ENLs-DTX and positive

control. Genotoxicity is expressed in percent of micronuclei per 1000 binucleated cells. For

the cytokinesis blocking MN assay, 1000 bi-nucleated cell per slide with well-preserved

cytoplasm were examined against each treatment. The results showed that means of MN

produced by ENLs as compared to vehicle control and positive control were very low and

the results are statistically significant in revealing that the ENLs or any of the ingredients of

the ENLs is non-genotoxic in nature.

The other aim of the study was to explore folate grafted thiolated chitosan (FA-CS-TGA)

polymer as capping/stabilizing agent for synthesis of highly stable docetaxel (DTX)

embedded florescent silver nanoclusters (NCs) and their subsequent cellular imaging for

theranostic potential in cancer therapy.

Various organic scaffolds have been reported as very good capping/stabilizing agents with

different metals (Moon et al., 2013). Silver based nanoparticles and nanoclusters have

shown promising results as theranostic agent against various tumors (Palama et al., 2016).

FA-CS-TGA polymer stabilized sliver nanoclusters (NCs) with subsequent loading of DTX

resulted in nanocapslues (DTX-Ag-NCPs) via ionic gelation method. This positive charge


91

could facilitate intestinal uptake of DTX-Ag-NCPs because of anionic nature of mucous

layer (Jiang et al., 2013b).

The compatibility and stability of ingredients in formulations was assessed by FTIR

analysis. All the peaks in FA-CS-TGA were found with some shift in position and stretching

indicating the chemical bonding between folic acid and thiol moieties with amino group of

CS. The presence of NCs in FA-CS-TGA resulted in significant shifting in the stretching

peaks of amide band at 1656 and 1590 cm-1. The presence of NCs also shifted the –OH

stretches from 1424 to 1410 cm-1. The chemical integrity of DTX was confirmed by the

characteristic stretching peaks appearing in FTIR spectra at 3449, 3351 and 1713 cm-1

indicating the presence of functionalities like N-H, O-H and C=O respectively. The

comparison of FTIR spectrum of DTX-Ag-NCPs with that of DTX and NCs revealed the

presence of chemically unmodified DTX in DTX-Ag-NCPs (Jain et al., 2014). Furthermore,

XRD analysis of all the samples was performed to explore any crystalline changes in the

formulations particularly with drug after the formation of DTX-Ag-NCPs. The

characteristic XRD patterns of pure DTX disappearing in XRD spectra of DTX-Ag-NCPs

indicated its presence in amorphous state (Yadav et al., 2015). The DSC analysis was

performed to check any change in physical state of pure DTX and encapsulated DTX-Ag-

NCPs in the presence of NCs. These results suggested the presence of DTX in amorphous

form within DTX-Ag-NCPs which is supportive towards aqueous solubility and oral

bioavailability of hydrophobic crystalline drug (Kulhari et al., 2014).

The elemental composition of formulation was checked by SEM-EDX analysis, which

confirmed the presence of Ag inside the DTX-Ag-NCPs, which is in the form of NCs. This

was also confirmed with the elemental peaks observed in point and ID scan of its EDX

analysis.

The nanoclusters do not show any plasmonic properties as reported due to their extremely

small size, they behave like molecules having discontinuous and discrete energy levels

(Díez and Ras, 2011). The synthesized Ag nanoclusters did not show any plasmonic

response and no change was observed in absorbance pattern once the DTX was loaded to

produce DTX-Ag-NCPs except for the appearance of drug peak in the spectrum.

Additionally, the DTX-Ag-NCPs were found to be fluorescent in dark but it was decreased

slightly upon the encapsulation of DTX in DTX-Ag-NCPs


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The 12 h in vitro release showed a sustained release pattern from the DTX-Ag-NCPs this

was similar to the effect observed with ENLs that gradual swelling of polymer governs the

release profile of entrapped drug. DTX being hydrophobic in nature, was available about 53

% from pure DTX suspension that was facilitated by the presence of tween 80 which

increased its wettability. Whereas, a sustained and consistent release of DTX (>80 %) from

DTX-Ag-NCPs was calculated for 12 h To further probe into the release mechanism,

different mathematical models were applied to the dissolution data. The results based upon

R2 values the release mechanism from DTX-Ag-NCPs followed Korsmeyer-Peppas model

and the value of release point n (0.67) suggested the mechanism as anomalous transport

which describes the release was mainly governed by erosion and diffusion from polymer

(Murtaza et al., 2012).

On account of good physicochemical and in vitro biological properties, the DTX-Ag-NCPs

were investigated for their imaging potential. Epithelial tumors of lungs, breast and colon

have shown overexpressed folate receptors, presenting potential tumor targeting site for

these types (Kim, 1999). Once, attached to the folate receptor, the carrier-receptor complex

is internalized and initiates the effects. The cytotoxic effect of these DTX-Ag-NCPs was

assessed against MB-231, human breast cancer cell line. The cells were treated with

different concentrations of DTX suspension, NCs and DTX-Ag-NCPs. This significantly

low IC50 value might attribute to the synergistic cytotoxic effect of DTX and NCs within

the DTX-Ag-NCPs and the presence of thiol group on their surface may also have resulted

in their improved internalization through efflux pump inhibition (Saremi et al., 2011). The

uptake of DTX-Ag-NCPs was done for 3 h using 8 chambered slide using MBA-231 cells.

After 12 h, less than 10 % of the cell viability was observed showing maximum cell death.

The DTX-Ag-NCPs in the sample retained fluorescence showing their cellular imaging

potential along with high degree of cytotoxicity proving their theranostic potential.

Biocompatibility assessment is an important parameter to study body response towards the

formulation once it is inside the body either for shorter or longer durations. Silver is known

to have toxic potential and could lead to severe damage to liver, kidney, lungs or spleen

depending upon their exposed concentration (Levard et al., 2012). These toxic effects could

be minimized or avoided by surface modification of these metal-based formulations or

capping them with some biocompatible moieties. In the present study, silver nanoclusters

were stabilized by a biocompatible and biodegradable scaffold FA-CS-TGA. The amount

of elemental silver in DTX-Ag-NCPs was found to be 16.58 µg/g using ICP-MS. The


93

biocompatibility of the formulation was checked in vitro using fresh human macrophages.

The cytotoxicity of anticancer drugs is assessed with tumor cells in culture but, such

methods are justified for free drugs, not appropriate for encapsulated anticancer drugs.

Anticancer drug loaded nanoparticles are primarily captured by macrophages, which makes

them a suitable candidate for cytotoxicity evaluations (Soma et al., 2000). DTX-Ag-NCPs

showed more than 80 % viability at 50 µg/mL as compared to DTX at the same

concentration. However, the toxicity of DTX-Ag-NCPs was increased at higher

concentrations, which may be attributed to their increased internalization as compared to

pure DTX. Ag-NCPs showed more than 80 % viability even at higher concentrations

indicating that the amount of silver present in the biocompatible scaffold DTX-Ag-NCPs is

safe to human cells.

Relative oral bioavailability and pharmacokinetics were studied in healthy rabbits of either

sex. DTX suspension and DTX-Ag-NCPs containing DTX equivalent to 20 mg/kg were

administered orally to rabbits (n=5) per group. Plasma level was measured at predefined

intervals for 24 h after single dose oral administration. It was observed that after oral

administration, DTX suspension reached to Cmax after 5 h and remained above the minimum

effective concentration (35 ng/mL) for 3 h only. Plasma half-life (t1/2) of DTX-Ag-NCPs

increased around 5 folds than that of pure DTX. This prolonged half-life in plasma may be

due to stronger mucoadhesion of thiol modified chitosan that resulted in prolonged retention

in gut (Saremi et al., 2013). Cmax was also increased 6-folds with DTX-Ag-NCPs as

compared to that with pure DTX. The AUC0-24 of DTX-Ag-NCPs showed high increase in

relative oral bioavailability i.e. 8.89-folds as compared to pure drug. This enhanced relative

oral bioavailability of DTX from DTX-Ag-NCPs might be due to combination of factors

supported by increased mucoadhesion, mixing of PGP inhibitors in formulations and

enhanced paracellular transport because of thiolated polymer on the surface of nanocarriers.

The particle size ~300 nm might have also facilitated the paracellular transport through

gastric mucosa (Javed et al., 2015).

The safety of the DTX-Ag-NCPs was assessed following OECD 425 guidelines by

monitoring various biochemical indicators. The LFTs showed no significant changes in

Bilirubin level as compared to control. The level was observed slightly higher for DTX-Ag-

NCPs as compared to Ag-NCPs and DTX, but all within the limits (Javed et al., 2016).

SGPT was observed at the highest level in DTX treatment as compared to that with Ag-

NCPs and DTX-Ag-NCPs treatment. SGOT level was decreased with all treatments and the


94

maximum decrease was found with DTX-Ag-NCPs among the three treatments. ALP level

was significantly increased with all treatments, being highest with DTX-Ag-NCPs as

compared to control indicating the liver activity against detoxification of foreign materials.

However, the ALP level was decreased with DTX-Ag-NCPs. Except for the ALP, the other

parameters did not produce significant changes in serum level indicating the fairly safe use

of DTX-Ag-NCPs in the presence of NCs and DTX. The effect on kidneys was assessed by

measuring RFTs. The BUN level was slightly increased with the DTX and NCs but it

remained within the limits. However, this level was slightly decreased with Ag-NCPs as

compared to that for control. The creatinine level was slightly raised with DTX-Ag-NCPs

when compared to control yet again was found within limits. Contrary to that, a slight

decrease in creatinine level with DTX and Ag-NCPs was observed which again was within

the acceptable limits. The effect of the formulation was also investigated on serum glucose,

cholesterol and total protein level. The DTX-Ag-NCPs significantly decreased the serum

glucose and cholesterol level as compared to the other two treatment groups, which also

decreased the serum glucose and cholesterol level. Total protein remained unaffected with

all the treatments. The effect of formulation was evaluated on blood and its components

through complete blood count (CBC) showing prominent effects on different blood

components which markedly decreased by NCs as compared to pure DTX, showing more

toxic potential against RBCs and platelets. The Ag-NCPs blank did not produce any

significant effect on the CBC results and remained closer to the results for control group.

Organ weights are described as a good parameter for in vivo toxicity studies. Any change in

organ to body ratio indicates the treatment associated effects (Sellers et al., 2007). The

results showed no significant changes for any of the three organs i.e. liver, kidney and heart.

However, there was a minute change observed within liver only with NCs, which was not

significant as compared to control. Over all, there was no significant effect on organs as

observed by the treatment during acute oral toxicity testing.

The macroscopic examination of liver and kidney did not show any visible changes or

lesions on these organs. To explore further, histological investigations were performed on

the slides prepared from liver and kidney. The microscopic examination of the slides did

not show any changes like necrosis or fatty changes in the liver and similarly, kidney

appeared to be normal as compared to control. Thus, DTX-Ag-NCPs did not produce any

toxicity on liver and kidney and support the results obtained from LFTs and RFTs indicating

the safety of these DTX-Ag-NCPs.


95

The establishment of appropriate storage conditions for nanoparticles is very important to

determine their shelf-life. Lipid based formulations require special attention to remain intact

and stable physically and chemically upon long term storage. Their chemical stability is

effected by hydrolysis and oxidation of lipids and physical stability is effected by particle

aggregation or sedimentation (Grit and Crommelin, 1993). Similarly, stability of metal

nanoparticles and nanoclusters can be greatly improved by polymeric scaffolds like chitosan

and PVP, which obstruct particle-particle contact through steric repulsion (Masoudi et al.,

2012, Tejamaya et al., 2012). Nanoparticles might not only increase the surface area by

many folds but also face aggregation of particles during long term storage. This poor long

term stability due to fluctuations in temperature and humidity may affect physicochemical

properties of drug and formulations.(Morris et al., 2011). Generally, stability is ensured by

freezing or lyophilizing the formulation in the presence of cryoprotectant that prevents

aggregation or structural deformation upon long term storage (Yang et al., 2007). The

stability of nanoparticles has been evaluated using different conditions following ICH

guidelines Q1A (R2) and lyophilized state by different research groups out of which 4°C

and 37°C have been reported most suitable for polymeric nanoparticles (Muthu and Feng,

2009).

Keeping all these in view, the effect of FA-CS-TGA on long term stability of NLs, ENLs

and DTX-Ag-NCPs at different storage conditions was evaluated. Lyophilized formulations

were stored at -20 °C and aqueous dispersion of lyophilized formulations was stored at 4 °C

and 37 °C over a period of three months. ENLs and DTX-Ag-NCPs showed more stability

as compared to NLs and NC respectively, in terms of any changes in their particle size, PDI,

surface potential and fluorescence intensity. Formulations were more stable in lyophilized

form as compared to aqueous environment. However, in aqueous media, formulations

showed relatively less change in evaluation parameters as compared to 37 °C. It was

probably due to cool environment which restrict the particle movement and surface erosion

or degradation of polymer coat, resulting in the surface charge and particle size least

effected. This suggested that stability could be improved if nanoparticles are lyophilized and

stored at cool temperature. This could help in overcoming the particle aggregation and

change in surface charge once stored as suspension in cool places (Katas et al., 2013). The

fluorescence intensity of NCs was slightly decreased as compared to DTX-Ag-NCPs in

relevance to their initial fluorescence. The formulation retained their fluorescence owing to

its greater stability on storage. The results clearly reflected the influence of polymer coating


96

in improving the stability of nanoparticulate formulations on storage in aqueous media as

well as in lyophilized state.

Conclusion

97

5. CONCLUSION

The application of nano based delivery carriers have opened new avenues of advancement

in cancer therapy, therefore, designing new polymer with improved biocompatibility and

tumor targeting ability, can help in overcoming various limitations of conventional delivery

systems. Chitosan is an exceptional cationic polymer having amino groups which enable it

to develop covalent linkage with different moieties resulting in polymeric graft with

improved properties. The present study successfully highlighted the potential of folic acid

grafted thiolated chitosan (FA-CS-TGA) polymer in terms of increased mucoadhesion,

tumor targeting, oral permeation enhancement and increased stability via enveloping

nanoliposomes and sliver nanoclusters.

Enveloped nanoliposomes loaded with docetaxel designed for oral delivery, showed better

control over drug release from the carrier over 12 h and most importantly, the system

showed a promising potential by enhancing the oral bioavailability of DTX by 13.6-folds as

compared to nanoliposomes. Moreover, the pharmacokinetics of the docetaxel was

significantly improved than that of native drug. Tumor targeting against folate positive

breast cancer cells showed greater cytotoxicity (⁓200 folds) with enveloped nanoliposomes

showing the better tumor targeting ability of FA-CS-TGA polymer as compared to

nanoliposomes. In vitro and in vivo toxicity profile ENLS and NLs showed limited cellular

toxicity at lower doses as compared to higher doses. In rodent animal model, vital organs

including kidney and heart remained significantly unaffected with all formulations, but

ENLs control showed very slight effect on liver. The functional biochemical and

hematology analysis data also confirmed biocompatible potential of ENLs.

The other part of the study folic acid grafted thiolated chitosan (FA-CS-TGA) polymer was

explored as capping and stabilizing agent for silver nanoclusters because of it known

antimicrobial and anticancer potential. Fluorescent silver nanoclusters were synthesized

using FA-CS-TGA polymer and characterized for various physicochemical parameters to

evaluate their theranostic potential for cancer therapy. Fluorescent nanoclusters embedded

nanocapsules (DTX-Ag-NCPs) containing docetaxel (DTX) were synthesized. These DTX-

Ag-NCPs successfully increased oral delivery of docetaxel with improved

pharmacokinetics. The great increase in cytotoxicity against MDA-MB-231cell line was a

result of synergistic effect of docetaxel (DTX) and 16.58 µg/g of elemental silver inside the

Conclusion

98

DTX-Ag-NCPs. The acute oral toxicity studies showed no significant toxicity of the

formulation.

The stability of the ENLs and NCs was also significantly improved by FA-CS-TGA polymer

which prevented aggregation and on long term storage in refrigerator. Thus, all studied

parameters for both nanocaroges i.e. ENLs and DTX-Ag-NCPs with FA-CS-TGA

suggested that the polymer could serve as a safe and efficient carrier for oral delivery of

hydrophobic drugs and targeting potential against tumors with over expressed folate

receptors. Also, FA-CS-TGA might turn to be a valuable system in term of improved

stability, enhanced intestinal permeation and safety profile.

Future Perspectives

99

6. FUTURE PERSPECTIVES

The findings of the thesis suggest a promising platform for further studies on application of

FA-CS-TGA polymer for nanocargoes as drug delivery systems. However, several future

studies could be designed and conducted to obtain detailed understanding of various

mechanisms influenced by the FA-CS-TGA polymer in drug delivery and tumor targeting.

• Nearly 40% of newly developed drugs are hydrophobic, so these ENLs and DTX-

Ag-NCPs could serve as potential carrier for oral delivery of other hydrophobic

molecules.

• It would thus be interesting to study the influence of formulation variables on

physicochemical properties of the nanoparticles and their interaction with GIT

mucosa in improving oral bioavailability in the presence of FA-CS-TGA polymer

coat on nanocargoes.

• FA-CS-TGA polymer could be used to envelope other nano formulations like

micelles, niosomes having stability problem. This might increase their stability, oral

permeation potential and tumor targeting.

• Tumor targeting could be further improved by replacing folic acid with some

antibody or monoclonal antibody resulting in highly specific carriers with decreased

side effects or by replacing with some other ligand oriented towards other diseases.

• The currently synthesized ENLs and DTX-Ag-NCPs should not be restricted only

to cancer or else they should be used on other infectious diseases especially the one

to which the available drugs are getting resistant. This is because of the high activity

at lower concentrations, the biocompatibility and delivery of these nanoparticles

with drug or even metals to the cell.

• Further investigations are required to study the effect of these nanoparticles on

tissues mainly the tissue distribution study, pharmacokinetics, the biochemical

analysis and removal of these nanoparticles.

• The investigation should also be excelled long term toxicity evaluation including the

effect on immune cells response, especially the release of particular immune

chemicals (cytokines and interleukins) upon exposure to these nanoparticles and also

the elicitation of particular immune response.

Future Perspectives

100

• Advance investigations are suggested to evaluate the fate and the effect of these

nanoparticles on tissues mainly the tissue distribution study, pharmacokinetics, the

biochemical analysis and removal of these nanoparticles.

• The theranostic potential of DTX-Ag-NCPs could be further investigated as these

DTX-Ag-NCPs could turn to be a potential nanocargoes having combined

therapeutic and diagnostic ability.

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List of Publications

120

8. LIST OF PUBLICATIONS

Folate grafted thiolated chitosan enveloped nanoliposomes with enhanced oral relative

bioavailability and anticancer activity

Muhammad Farhan Sohail, Ibrahim Javed, Syed Zajif Hussain, Shoaib Sarwar, Sohail

Akhtar, Akhtar Nadhman, Salma Batool, Nadeem Irfan Bukhari, Rahman Shah Zaib

Saleem, Irshad Hussain, Gul Shahnaz

Published in Journal of Materials Chemistry B, 2016. 4(37): p 6240-6248.

Cell to Rodent: Toxicological Profiling of Folate Grafted Thiomer Enveloped

Nanoliposomes

Muhammad Farhan Sohail, Hafiz Shoaib Sarwar, Ibrahim Javed, Akhtar Nadhman, Syed

Zajif Hussain, Hamid Saeed, Abida Raza, Nadeem Irfan Bukhari, Irshad Hussain, Gul

Shahnaz

Published in Toxicology Research, 2017, 6, 814-821

Evolution and Clinical Translation of Drug Delivery Nanomaterials

Shabir Hassan, Gyan Prakash, AYCA BAL OZTURK, Saghi Saghazadeh, Muhammad Farhan

Sohail, Jungmok Seo, Mehmet Dockmeci Yu Shrike Zhang, and Ali Khademhosseini

Published in Nano Today. 2017;15:91-106.

Pharmacological Enhancement of Thiolated Chitosan Nanocapsules Loaded with

Fluorescent Silver Nanoclusters for Anti-Cancer Drug Delivery

Muhammad Farhan Sohail, Syed Zajif Hussain, Shoaib Sarwar, Ibrahim Javed, Akhtar

Nadhman, Anees ur rehman, Zil e huma, Sarwat Jahan, Irshad Hussain, Gul Shahnaz

Under review in Journal of Materials Chemistry B

Oral delivery of Docetaxel: Current Status, Challenge and Future Opportunities

Muhammad Farhan Sohail, Hafiz Shoaib Sarwar, Anne Metje van Geijtenbeek, Akhtar

Nadhman, Anees ur Rehman, Irshad Hussain, Gul Shahnaz

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Appendix

Appendix

121

9. APPENDIX

Appendix

122

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