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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
Department of Pharmacy
In the partial fulfillment of the requirements for degree of
Doctor of Philosophy
in
Pharmacy (Pharmaceutics)
Department of Pharmacy
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.
__________________________
MUHAMMAD FARHAN SOHAIL
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.
__________________________
MUHAMMAD FARHAN SOHAIL
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
Department of Pharmacy,
University
External Examiner 2: ______________________
Dr.----------------
Assistant Professor
Department of Pharmacy,
University,
Chairman: ______________________
Prof. Dr. Gul Majid Khan
Department of Pharmacy,
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.
MUHAMMAD FARHAN SOHAIL
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. Acute oral toxicity ______________________________________________ 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
3.2.11. Stability studies ________________________________________________ 60
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. Acute oral toxicity ______________________________________________ 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
3.4.10. Stability studies ________________________________________________ 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
Figure 3.16: In vitro cytotoxicity comparison of DTX suspension with NLs and ENLs
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
Figure 3.20: In vitro biocompatibility studies of NLs and ENLs at different concentration
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
Figure 3.34: In vitro cytotoxicity and imaging studies against human breast cancer cell line
(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
Chapter 1: Introduction
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.
Chapter 1: Introduction
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
targeted antimitotic
ABCC2 and
ABCC3
Vinca alkaloid Vinorelbine Micro-tubulin
targeted antimitotic
ABCC1 and
ABCC3
Epothilone B
analogue
Ixabepilone Micro-tubulin
targeted antimitotic
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.
Chapter 1: Introduction
4
Figure 1.1: Chemical structures of Paclitaxel and Docetaxel
Chapter 1: Introduction
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.
Chapter 1: Introduction
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.
Chapter 1: Introduction
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,
Chapter 1: Introduction
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
Chapter 1: Introduction
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
Chapter 1: Introduction
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
Chapter 1: Introduction
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
Chapter 1: Introduction
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
Chapter 1: Introduction
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.
Chapter 1: Introduction
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).
Chapter 1: Introduction
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
Chapter 1: Introduction
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.
Chapter 1: Introduction
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
Chapter 2: Materials and Methods
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)
Chapter 2: Materials and Methods
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)
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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.
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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-
Chapter 2: Materials and Methods
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.
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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,
Chapter 2: Materials and Methods
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]
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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.
Chapter 2: Materials and Methods
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.
Chapter 2: Materials and Methods
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.
2.2.32. Acute oral toxicity
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).
Chapter 2: Materials and Methods
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
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. The results are shown as Mean ± S.D.
Figure 3.16: In vitro cytotoxicity comparison of DTX suspension with NLs and ENLs
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.
3.2.11. Stability studies
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.
Figure 3.19: In vitro biocompatibility studies of NLs and ENLs at different concentration
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.
Figure 3.20: In vitro biocompatibility studies of NLs and ENLs at different concentration
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.
3.3.4. Acute oral toxicity
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)
Polydispersity Index
(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.
Figure 3.33: In vitro cytotoxicity and imaging studies against human breast cancer cell line
(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
Figure 3.34: In vitro cytotoxicity and imaging studies against human breast cancer cell line
(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.
3.4.10. Stability studies
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)
Polydispersity Index
(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
Chapter 4: Discussion
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.
Chapter 4: Discussion
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.
Chapter 4: Discussion
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
Chapter 4: Discussion
85
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
Chapter 4: Discussion
86
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
Chapter 4: Discussion
87
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
Chapter 4: Discussion
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
Chapter 4: Discussion
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
Chapter 4: Discussion
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
Chapter 4: Discussion
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
Chapter 4: Discussion
92
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
Chapter 4: Discussion
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
Chapter 4: Discussion
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.
Chapter 4: Discussion
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
Chapter 4: Discussion
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
Ready for submission
Appendix
Appendix
121
9. APPENDIX
Appendix
122