The Islamia University of Bahawalpur - Pakistan Research ...

i

Lipid-Polymer Hybrid Nanoparticles for Potential

Delivery of Chemotherapeutic Agents: Formulation

and Characterization

A thesis submitted

in partial fulfillment of the requirement for the degree

of

Doctor of Philosophy

(Pharmaceutics)

By

Muhammad Muzamil Khan M.Phil. Pharmaceutics

Session: 2016-2019

Department of Pharmacy

Faculty of Pharmacy and Alternative Medicines,

The Islamia University of Bahawalpur

vi

Dedication

This thesis work is dedicated to my

Mother

Nasreen Akhtar (Late)

Who has supported me since the start of my studies and

helped me with her love, care and affection

vii

Acknowledgment

FIRST of all thanks to ALMIGHTY ALLAH (subhana wa taala) for giving me

power, courage and knowledge to conduct this study.

I had experienced difficult situations during my research but when I was in any

difficulty ALLAH was always there to help me. It was due to ALLAH’ blessing that

I have been able to overcome all the problem during my studies.

The sayings of our Holy Prophet Muhammad ( ) were also the continuous

source of guidance for me.

I have no words to pay my gratitude to my Research Supervisor, Dr. Muhammad

Asadullah Madni, Assistant Professor, Department of Pharmacy, Faculty of

Pharmacy and Alternative Medicine, The Islamia University of Bahawalpur (IUB),

for investing so much time and effort to teach me how to make scientifically sound

research, for always pushing me to do things. He has inculcated me the true spirit of

pharmaceutical research and it was due to his restlessness efforts that I have

completed my thesis. My research work would have been incomplete without the

guidance and support of Prof. Dr. Vladimir Torchilin, Head, Center of

Pharmaceutical Biotechnology and Nanomedicines (CPBN), Northeastern University,

Boston, USA I truly appreciate all the time and suggestions given by him. His kind

behavior and way of teaching novel ideas really helped me to complete my tasks in

due time.

I am exceedingly grateful to Prof. Dr. Naveed Akhtar (Dean), Faculty of Pharmacy

and Alternative Medicine, IUB, for providing me academic opportunities and

arranging intellectual inputs and necessary facilities to carry out my studies and

research work fruitfully.

I am thankful to my father Ghulam Muhammad Khan, for his continuous support and

encouragement and to my sister for their love and support, and my brother. I am

thankful to my wife for her support during my research stay at USA.

―Friend in need is a friend indeed‖ - my heartfelt thanks towards my dearest

colleagues Jiayi pan (PhD Candidate, CPBN), Nina Filipczak (Post-doc fellow,

CPBN), Dr. Mubashar Rehman (PhD), Dr. Nayab Tahir (PhD), Livia P Mendes (Post

http://upload.wikimedia.org/wikipedia/commons/c/c5/Mohamed_peace_be_upon_him.svg

viii

doc fellow, CPBN), Hassan Shah (PhD Scholar), Safi Ullah (M.Phil. Pharmaceutics),

Danish Saeed (M.Phil. Pharmaceutics), Farzana Parveen (PhD Scholar), Nadia Rai

(PhD Scholar), Abdul Raheem (PhD Scholar), Nasrulla Jan (PhD Scholar),

Muhammad Ahmad Mehmood (PhD Scholar), Abdul Jabbar (PhD Scholar),

Muhammad Shahzad Khan (PhD Scholar), Muhammad Umair Akram (M.Phil

Scholar), Assadullah Jan (M.Phil Scholar) who all shared their research and analytical

knowledge as well as helped me whenever I need them during my Lab work and stay

at University

I would like to express my sound gratitude to my departmental colleagues Dr. Attiq

Ur Rehman, Dr. Mudassir Shafiq, Khurram Mumtaz Khan, Shahzad Ali for their help

during my PhD.

In the end I want to express my sincere gratitude to everyone who in their own way,

helped me throughout the achievement of this destination.

Muhammad Muzamil Khan

ix

Table of Contents

Part I

Title Page i

Bismillah ii

Student Certificate iii

Supervisor Certificate iv

Approval Certificate v

Dedication vi

Acknowledgment vii

Table of contents ix

List of Figure xvii

List of Tables xxi

List of Abbreviations xxii

Appendences Xxiv

Abstract Xxv

Part II

Chapter 1. Introduction

1.1 Introduction 1

1.2 Main objectives of study 8

Chapter 2. Literature Review

2.1 Cancer 11

2.2 Ovarian cancer 12

x

2.3 Classification of ovarian cancer 12

2.3.1 Epithelial tumors 12

2.3.2 Stromal cell tumors 13

2.3.3 Germ cell tumors 13

2.4 Risk factors of ovarian cancer 14

2.4.1 Hormonal 14

2.4.2 Pregnancy and infertility 14

2.4.3 Lactation 14

2.4.4 Use of oral contraceptives 14

2.5 Treatment of ovarian cancer 15

2.5.1 Surgery 15

2.5.2 Chemotherapy 15

2.6 Cisplatin 15

2.6.1 Chemical structure 16

2.6.2 Mechanism of action 16

2.6.3 Pharmacokinetic properties 16

2.6.4 Therapeutic uses 17

2.6.4.1 Cisplatin and ovarian cancer 17

2.6.4.2 Cisplatin and lung cancer 17

2.6.4.3 Cisplatin and breast cancer 17

2.6.4.4 Cisplatin and brain cancer 17

2.6.5 Side effects of cisplatin 18

2.6.5.1 Hepatoxicity 18

2.6.5.2 Nephrotoxicity 18

xi

2.6.5.3 Cardiotoxicity 18

2.7 Novel drug delivery system 19

2.8 Nanotechnology 20

2.9 Nanotechnology and cancer 21

2.10 Nanoparticles 23

2.11 Polymeric nanoparticles 24

2.12 Chitosan 26

2.13 Liposomes 28

2.14 Lipid-polymer hybrid nanoparticles 30

2.15 Types of lipid-polymer hybrid nanoparticles 33

2.15.1 Lipid-polymer hybrid core-shell structure 33

2.15.2 Polymer caged lipid hybrid nanoparticles 34

2.15.3 Mixed lipid-polymer hybrid nanoparticles 35

2.15.4 Lipid-polymer-lipid hybrid nanoparticles 36

2.16 Factor affecting hybrid nanoparticles 36

2.16.1 Lipid to polymer (L/P) ratio 37

2.16.2 Lipid coating 37

2.16.3 Pegylation 37

2.16.4 Nature of Polymer 37

2.17 Targeted Drug Delivery 38

2.17.1 Passive targeting 38

2.17.1.1 Enhanced permeability retention (EPR) effect 39

2.17.2 Active targeting 40

2.17.3 Folate targeting 43

xii

Chapter 3. Synthesis and physicochemical characterization of LPHNPs

3.1 Background 48

3.2 Materials and Methods 50

3.2.1 Materials 50

3.2.2 Method of preparation 50

3.2.3 Size and Zeta Potential of hybrid nanoparticles 51

3.2.4 Determination of drug loading and entrapment efficiency 51

3.2.5 Morphology of hybrid nanoparticles 52

3.2.6 Fourier transform infrared spectroscopy (FTIR) 52

3.2.7 Powdered X-Ray diffraction analysis (X-RD) 52

3.2.8 Differential scanning calorimeter (DSC) 52

3.2.9 Thermo-gravimetric analysis (TGA) 52

3.2.10 In vitro drug release studies 53

3.3 Results 54

3.3.1 Size and surface charge 54

3.3.2 Morphology of nanoparticles 56

3.3.3 Entrapment efficiency and drug loading 56

3.3.4 FTIR spectroscopy 57

3.3.5 XRD Studies 58

3.3.6 Differential scanning calorimetery (DSC) 59

3.3.7 Thermogravimetric analysis (TGA) 61

3.3.8 In vitro dissolution studies 63

3.5 Discussion 66

3.6 Conclusion 68

xiii

Chapter 4. Biological characterization of LPHNPs

4.1 Background 70


4.2.1 Materials 71

4.2.2 Cell viability 71

4.2.3 Florescence microscopy 71

4.2.4 Cellular uptake 72

4.2.5 In vivo toxicity 72

4.2.6 In vivo pharmacokinetics 72

4.3 Results 73

4.3.1 Cell viability studies 73

4.3.2 Cellular uptake studies 76

4.3.3 Cellular association 77

4.3.4 Toxicological studies in rats 79

4.3.4.1 Biochemical and blood analysis 80

4.3.4.2 Histopathological examination 82

4.3.5 In vivo pharmacokinetics 84

4.4 Discussion 86

4.5 Conclusion 88

Chapter 5. Synthesis and Physicochemical characterization of Folate LPHNPs

5.1 Background 90


5.2.1 Materials 93

5.2.2 Preparation of folate-chitosan conjugates 93

xiv

5.2.3 Preparation of folate-chitosan conjugated lipid hybrid nanoparticles 95

5.2.4 Purity of folate-chitosan conjugate 98

5.2.5 Nuclear magnetic resonance 98

5.2.6 Size and zeta potential of hybrid nanoparticles 98

5.2.7 Determination of drug loading and entrapment efficiency 98

5.2.8 Morphology of hybrid nanoparticles 99

5.2.9 In vitro drug release studies 99

5.3 Results 100

5.3.1 Purity of the conjugate 100

5.3.2. Nuclear magnetic resonance spectroscopy 101

5.3.3 Size and zeta potential of hybrid nanoparticles 103

5.3.4 Drug entrapment and loading efficiency 104

5.3.5 Morphology of folate LPHNPs 105

5.3.6 In vitro drug release profile 106

5.4 Discussion 108

5.5 Conclusion 109

Chapter 6. Biological characterization of LPHNPs

6.1 Background 111


6.2.1 Materials 113

6.2.2 Cell viability studies 113


6.2.4 Cellular uptake 114

6.2.5 Cell apoptosis 114

xv

6.2.6 Cell cycle 114

6.2.7 Preparation of 3D spheroids 115

6.2.8 Cell viability towards 3D spheroids 115

6.2.9 Cell uptake studies towards 3D spheroids 115

6.2.10 Florescence microscopy images of 3D spheroids 115

6.3 Results 117

6.3.1 Cytotoxicity studies 117

6.3.2 Cell uptake studies 125


6.3.4 Cell cycle studies 127

6.3.5 Cell apoptosis studies 128

6.3.6 Cell Cytotoxicity towards cancer cell in 3D spheroids 134

6.3.7 Cell uptake studies towards cancer cell in 3D spheroids 137

6.3.8 Florescence microscopic images of cell uptake studies towards

cancer cell in 3D spheroids

138

6.4 Discussion 142

6.5 Conclusion 144

Chapter 7. Conclusions and Recommendation

7.1 Future prospects 148

Chapter 8. References

xvi

List of Figures

Figure 2.1: Chemical structure of Cisplatin 16

Figure 2.2: Areas of nanotechnology applications. 21

Figure 2.3: Structure of polymeric nanoparticles. 25

Figure 2.4: Chemical structure of chitosan 27

Figure 2.5: Structure of liposomes with PEG coating. 29

Figure 2.6: Core- shell lipid-polymer hybrid nanoparticles. 34

Figure 2.7: Lipid-polymer cage hybrid nanoparticle. 35

Figure 2.8: Mixed lipid-polymer hybrid system. 35

Figure 2.9: Lipid-polymer-lipid hybrid nanoparticles. 36

Figure 2.10: Advantage of targeted drug delivery system 38

Figure 2.11: Antibody targeted drug delivery system. 41

Figure 2.12: Folate receptor internalization mechanism. 44

Figure 3.1: Schematic diagram of lipid-chitosan hybrid nanoparticles. 51

Figure 3.2: Transmission electron microscopy images of lipid-chitosan

hybrid nanoparticles.

56

Figure 3.3: FTIR Spectra of individual components and lipid-polymer

hybrid formulation.

58

Figure 3.4: X-ray diffraction analysis of components and LPHNPs. 59

Figure 3.5: Differential Scanning calorimetry graph of LPHNPs and

components.

60

Figure 3.6: Thermo gravimetric analysis of cisplatin loaded lipid-chitosan


62

Figure 3.7: In vitro drug release profile of LPHNPs with various ratios of

lipid to polymer.

64

Figure 4.1: Cytotoxicity studies of cisplatin LPHNPs compared with

cisplatin solution and blank LPHNPs on A2780 cell lines after

24 hours of treatment.

74

Figure 4.2: Cytotoxicity studies of cisplatin LPHNPs compared with

cisplatin solution and blank LPHNPs on A2780 cell lines after

24 hours of treatment.

75

xvii

Figure 4.3: Cellular uptake of LPHNPs by A2780 cell lines. 76

Figure 4.4: Uptake of chitosan-lipid hybrid nanoparticles loaded with Rh-PE

by A2780 cells.

78

Figure 4.5: Representative histopathological images of rat vital organs (A)

Control (B) Blank LPHNPs (C) Cisplatin loaded LPHNPs.

83

Figure 4.6: Concentration versus time profile curve of cisplatin LPHNPs

and cisplatin solution. (Mean± SD n=6)

85

Figure 5.1: Schematic chemistry of folate-chitosan conjugate. 94

Figure 5.2: Schematic diagram of folate targeted lipid-chitosan hybrid

nanoparticles.

95

Figure 5.3: Schematic chemistry of formation of folate lipid-chitosan hybrid

nanoparticles.

96

Figure 5.4: Mechanism of internalization of folate targeted lipid-chitosan

hybrid nanoparticles via endocytosis.

97

Figure 5.5: Thin layer chromatography of folic acid and folate-chitosan

conjugate.

100

Figure 5.6: Figure 5.6. 1H-NMR spectra of folate-chitosan conjugate,

chitosan and folic acid.

102

Figure 5.7: Transmission electron microscopy image of folate targeted lipid-

chitosan hybrid nanoparticles.

106

Figure 5.8: In vitro release profile of folate lipid-chitosan hybrid

nanoparticles and cisplatin drug solution

107

Figure 6.1: Cell viability study on A2780 cell lines after 24 hours of

incubation comparison of blank folate LPHNPS, folate LPHNPS

and LPHNPs.

119

Figure 6.2: Cell viability study on A2780 cell lines after 48 hours of


and LPHNPs.

120

Figure 6.3: Cell viability study on SKOV3 cell lines after 24 hours of


and LPHNPs.

121

Figure 6.4: Cell viability study on SKOV3 cell lines after 48 hours of


122

xviii

and LPHNPs.

Figure 6.5: Cell viability study on MCF-7 cell lines after 24 hours of


and LPHNPs.

123

Figure 6.6: Cell viability study on MCF-7 cell lines after 48 hours of


and LPHNPs.

124

Figure 6.7: Cell uptake studies using flow cytometry. Comparison of

LPHNPs and Folate LPHNPs loaded with rhodamie-123.

125

Figure 6.8: Fluorescence microscopy images of SKOV3 cells treated with

folate LPHNPs and folate LPHNPs loaded with Rh-123 and Rh-

PE.

126

Figure 6.9: Cell cycle studies after 24 hours of treatment. 127

Figure 6.10: Cell cycle studies after 48 hours of treatment. 128

Figure 6.11: Cell apoptosis after 24 and 48 hours of treatment comparison of

cisplatin solution, LPHNPs and folate LPHNPs.

129

Figure 6.12: Control group cell apoptosis showing early and late apoptosis. 130

Figure 6.13: Cell apoptosis of cisplatin drug solution showing early and late

apoptosis.

131

Figure 6.14: Cell apoptosis of lipid- chitosan hybrid nanoparticle loaded with

cisplatin.

132

Figure 6.15: Cell apoptosis of folate targeted lipid-chitosan hybrid

nanoparticles.

133

Figure 6.16: Cytotoxicity studies on 3D spheroids after 24 hours of treatment

comparison of folate LPHNPs, LPHNPs and cisplatin drug

solution.1

135


comparison of folate LPHNPs, LPHNPs and cisplatin drug

solution.

136

Figure 6.18:

Cell uptake studies using flow cytometry comparison of folate

LPHNPs, LPHNPs.

137

Figure 6.19: Cell uptake towards 3D spheroids using fluorescence

microscope comparison of folate LPHNPs with LPHNPs.

138

xix

Figure 6.20:

Z-stack images of 3D spheroids control group. 139

Figure 6.21: Z-stack image of lipid-chitosan hybrid nanoparticle group of 3D

spheroids.

140

Figure 6.22: Z-stack image of folate targeted lipid-chitosan hybrid

nanoparticle group of 3D spheroids.

141

xx

List of Tables

Table 2.1: Summary of clinical development of folate targeted ovarian

cancer drugs.

45

Table 3.1: Composition of cisplatin loaded lecithin-chitosan hybrid

nanoparticles

55

Table 3.2: Effect of lipid to polymer ratio on particle size and surface

charge

55

Table 3.3: Effect of Lipid to Polymer ratio on entrapment efficiency

and Drug Loading

57

Table 3.1: Kinetic Modeling of Drug Release profile of Cisplatin

loaded Lecithin-Chitosan Hybrid Nanoparticles

65

Table 4.1: Body weight and food, water intake of rats of different group 79

Table 4.2: Comparative hematological parameters in rats. 81

Table 4.3: Comparative liver and renal function parameters in rats. 82

Table 5.2: Composition of cisplatin loaded lecithin-chitosan hybrid

nanoparticles

103

Table 5.3: Effect of lipid to polymer ratio on particle size and surface

charge.

104

Table 5.4: Effect of Lipid to polymer ratio on entrapment efficiency

and drug loading

105

xxi

List of abbreviations

Term Description

CTB Cell titer blue

CTG Cell titer glo

CBC Complete blood count

DMEM Dulbecco’s Modified Eagle’s Medium

DMSO Dimethyl sulfoxide

DLS Dynamic light scattering

DSC Differential scanning calorimetry

DDS Drug delivery system(s)

DL Drug loading

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

EE Encapsulation efficiency

FBS Fetal bovine serum

FTIR Fourier transform infrared spectroscopy

Folate LPHNPs Folate lipid-polymer hybrid nanoparticles

LPHNPs Lipid-polymer hybrid nanoparticles

LFT Liver function test

MRT Mean residence time

PBS Phosphate buffer saline

PI Propidium iodide

PDI Polydispersity index

xxii

RPMI Roswell Park Memorial Institute medium

Rh-123 Rhodamine-123

Rh-PE Rhodamine phosphatidylehtanolamine

RFT Renal function test

TLC Thin layer chromatography

TEM Transmission electron microscope

UV Ultraviolet

X-RD X-ray diffraction

xxiii

List of Publications

1. Lipid-chitosan hybrid nanoparticles for controlled delivery of cisplatin

(Drug Delivery) (IF: 3.89) …171

Submitted

1. Lipid-chitosan hybrid nanoparticles for co-delivery of cisplatin and curcumin

with enhanced therapeutic efficacy

2. Folate targeted lipid-chitosan hybrid nanoparticles for enhanced therapeutic

efficacy

xxiv

Abstract

Nanotechnology has emerged as a hope to deliver drugs at targeted site to obtain

maximum therapeutic benefits. Lipid-polymer hybrid nanoparticles have provided the

platform to deliver drugs in a controlled manner with enhanced stability and

biocompatibility. LPHNPs have the dual advantages of polymeric and liposomal drug

delivery system and can encapsulate both hydrophilic and hydrophobic drugs. The

objective of this dissertation was to develop the lipid-chitosan hybrid nanoparticles

for potential delivery of chemotherapeutic agents at tumor site to achieve maximum

therapeutic benefits and decrease side effects associated with chemotherapeutic

agents.

Lipid-chitosan hybrid nanoparticles were prepared by using chitosan as a polymer and

LIPOID S75 as a lipid using modified ionic gelation method. The prepared

nanoparticles have size in range of 200-300nm with PDI values less than 0.3 and

surface charge showed good stability in suspension form. The transmission electron

microscopy images showed spherical nanoparticles having lipoplex like structure. All

prepared nanoparticles showed high entrapment efficiency (>80%) and good drug

loading. The FTIR analysis confirmed the compatibility among the excipients and X-

RD analysis showed no sharp peaks of cisplatin in formulation and cisplatin is

converted into amorphous form inside lipid-chitosan hybrid nanoparticles system.

Thermal studies using differential scanning calorimetry and thermogravimetric

analysis confirmed the excellent stability of prepared hybrid nanoparticles. The in

vitro release showed that controlled release of drug over prolong period of time.

Kinetic modeling showed that the release pattern follows super case II transport

mechanism. The physicochemical evaluation confirmed the excellent stability and

controlled release profile.

The therapeutic efficacy of cisplatin loaded lipid-chitosan hybrid nanoparticles was

evaluated by using A2780 ovarian cell lines. The results confirmed the enhanced

cytotoxicity of cisplatin loaded lipid-chitosan hybrid nanoparticles as compared to

cisplatin solution. Cellular interaction and cell uptake showed 8 times greater uptake

as compared to control. Further in vivo pharmacokinetic studies confirmed enhanced

mean residence time of LPHNPS inside the biological system. Toxicology studies

confirmed the safety profile of lipid-chitosan hybrid nanoparticles.

xxv

The folic acid was conjugated with chitosan for folate targeting to achieve maximum

therapeutic benefits at the tumor site. The TLC analysis confirmed the purity of

conjugate and absence of free folic acid while nuclear magnetic resonance

spectroscopy confirmed the successful conjugation of folic acid with chitosan. The

folate-chitosan conjugate was then used to prepare nanoparticle by ionic gelation

method with anionic lipids. The prepared nanoparticles have particle size in range of

200nm and low polydispersity index and surface charge of greater than +20. The

folate LPHNPs showed greater than 75% encapsulation with excellent drug loading.

The prepared nanoparticles are spherical in shape with lipoplex like structure having

folate on the outer side of nanoparticles. In vitro release profile shows sustained

release of cisplatin over a period of 48 hours.

The therapeutic efficacy of folate lipid-chitosan hybrid nanoparticles was evaluated

on ovarian and breast cell lines. The A2780 and SKOV3 were used as ovarian cell

lines and treated with folate targeted LPHNPs and untargeted LPHNPs and cisplatin

solution. The results confirmed the enhanced cytotoxic effect of folate LPHNPs as

compared to untargeted LPHNPs and cisplatin solution. Similar enhanced cytotoxic

effect of folate LPHNPs was observed on MCF-7 breast cancer cell lines. The cell

uptake studies showed two times more uptake of folate LPHNPs are compared to

untargeted LPHNPs that is due to folate receptor mediated endocytosis and leads to

enhanced therapeutic efficacy. The therapeutic efficacy of folate LPHNPs was further

evaluated on 3D spheroids in vivo model to check the response of nanoparticles in in

vivo environment. The cell viability studies on 3D spheroids confirmed the enhanced

cytotoxic effect of folate LPHNPs as compared to untargeted and much more

significant cytotoxic effect as compared to cisplatin solution. The fluorescence

microscopy images, and flow cytometry analysis confirmed the enhanced cellular

uptake of folate LPHNPs in 3D spheroids that leads to enhanced therapeutic efficacy.

In conclusion, lipid-chitosan hybrid nanoparticles are suitable platform for controlled

delivery of chemotherapeutic agents. Folate targeted LPHNPs with added advantage

of lipid coating is suitable for active targeting with enhanced therapeutic efficacy and

minimum side effects.

0

CHAPTER 1

Introduction

1

1.1. Introduction

Nanotechnology has multiple applications including agriculture, space , forensic and

medical therapeutics (Lai et al., 2006). The term nanotechnology was first coined by

an American scientist Richar Fenman in 1959, that result in conceptual building of

nanomaterial (Lyshevski et al., 2007). Nanotechnology has played wonders in the

area of food technology in identification of bacteria and monitoring quality of food.

Biosensors carbon nanotubes are being used in packaging of food to improve its

mechanical properties (Kang et al., 2007). Nanoparticles have also found applications

in cosmetics development of moisturizers with prolong skin contact, sun block and

anti-aging. Liposomes provide sustained skin contact and delivery of vitamins and

regeneration of epidermis with improved efficiency (Thong et al., 2007).

Pharmaceutical nanotechnology has provided a breakthrough in the delivery of drugs.

Nanotechnology is helpful in targeting drugs to the tissue and cellular level, crossing

the endothelial and blood brain barrier and co-delivery in combinatorial therapy

(Farokhzad and Langer, 2009). The emergence of nanotechnology has a great

influence on different research industries and particularly pharmaceutical research

industry. Nanotechnology tools enable drug to be delivered at a specific site and being

utilize for drug development and biological screening at nanoscale (Kharb et al.,

2006). Nanotechnology has significantly increased the bioavailability of drugs and

also play role in diagnosis of disease by interacting at cellular level and detoxification

of drugs from body is another application of nanotechnology that has been

successfully tested in rats. Nanotechnology can deliver the drug into the cell

cytoplasm, thus reducing side effect and enhance therapeutic efficacy. Nanoparticle

can serve as a diagnostic sensor and also can protect the drug for prolong time and

release upon specific signals (Nikalje, 2015). Delivery of genes such as siRNA and

DNA has proved to be fundamental in treatment of ailments, but their oral and

intravenous delivery is limited due to enzymatic cleavage, uptake by RES, and kidney

filtration, however nanotechnology provided the way for the delivery of siRNA and

DNA (Anderson et al., 2004). Pharmaceutical nanotechnology plays a vital role in the

formulation of new chemical entities with poor solubility and permeability and helps

in achieving desired pharmacokinetic properties. Size of nanoparticle play crucial role

2

in achieving the desired properties (Devalapally et al., 2007). Pharmaceutical

nanotechnology has also played the role to revolutionize the herbal medicines.

Cancer nanotechnology is an emerging field, and bringing wonders in the diagnosis

and treatment of cancer (Bharali and Mousa, 2010). Traditional drugs delivery system

in cancer treatment has several disadvantages, such as non-specific delivery and toxic

effects on normal cells. Cancer nanotechnology has several advantages and in

combination with biodegradable polymer, idea of polymer-drug conjugate in

nanotechnology was first introduced by Ringdorf in 1975 (Ringsdorf, 1975).

Nanotechnology is also overcoming the challenges of tumor diagnosis by using

quantum dots and high quality imaging (Ehdaie, 2007). The main concern in cancer

treatment is to achieve desired therapeutic concentration of drug at the tumor site and

to avoid the side effects on the normal body tissues, thus nanotechnology has served

as a tool for the targeted delivery by conjugating with various targeting moieties

including aptamers and antibodies (Misra et al., 2010).

First ever nanotechnology system discovered was of lipid vesicles in 1960 and later

on it was called as Liposomes (Bangham et al., 1965). Specific delivery of liposomes

at targeted site was achieved in 1980 (Leserman et al., 1980). Liposomes are formed

by incorporating phospholipids in aqueous phase, forming bilayer structure. They can

incorporate both hydrophilic and hydrophobic drugs (Israelachvili et al., 1980). First

generation of nanotherapeutics was non-targeted but still provide advantage over

conventional drug delivery system (DDS) such as increase in half life and enhanced

solubility and enabling sustained release (Riehemann et al., 2009). Liposomes have

superior biocompatible properties and have ability to load the drug as well as RNA,

DNA. Liposome has long circulating properties and ability to specifically target the

cells by attaching to various targeting moieties. Liposomes has the ability to deliver

their load into the cytoplasm and also utilized as a vehicle in photodynamic therapy

(Torchilin, 2005). Liposomes has provided breakthrough in gene therapy by

successful delivery of nucleic acid with increase transfection efficiency. Cationic

lipids are particularly used for the nucleic acid delivery. High biocompatible and

biodegradable properties also make liposome ideal system for delivery of drug to

brain (Samad et al., 2007). Lipids can adopt different structure including gel, sol and

micelle formation. Liposomes prepared from the saturated lipids have the better

stability as compared to unsaturated lipids. Liposome surface can be modified by

3

using anionic, cationic and neutral lipids according to the target of action. Liposomes

can permeate through the leaky microvasculature and reside for longer time, enabling

targeted delivery at the tumor site (Maurer et al., 2001).

The major drawbacks of liposomes are rapid clearance from the body and are

captured by RES, mainly in Liver. Liposomes have biomimetic properties but are

cleared by macrophages, thus suffer the problem of shorter half-life. Liposomes have

low encapsulation efficiency for hydrophilic drugs because of limited aqueous phase

and volume of hydration is greater on the outer side. Sterilization of liposome is

challenging because of their composition, phospholipids can undergo phase transition

after exposure to the surrounding high temperature, which may result in loss of

product. The lipid component of phospholipid may also undergo oxidation and result

in degradation (Pattni et al., 2015). The rapid elimination of liposomes is due to

opsonization by the plasma components, uptake by reticuloendothelial system in

spleen (Hua and Wu, 2013).

Polymeric nanoparticles (PNPs) are biodegradable, stable and can effectively deliver

the drug at the target site with controlled manner. PNPs have excellent entrapment

efficiency because drug is confined inside the polymer matrix that provide stability

and controlled release (Soppimath et al., 2001). PNPs can respond to both internal and

external stimuli, thus delivering the drug to targeted tissue and ensuring maximum

therapeutic efficacy and minimizing side effects (Cheng et al., 2013). Polymers used

for nanoparticle-based drug delivery are biocompatible, they deliver drug at the

targeted site, then degraded and released out of the body. PNPs can increase the

solubility of poorly soluble drugs by attaching solubilizing moieties. The drug having

short half-life can be effectively encapsulated inside polymeric nanoparticles, thus

tremendously increasing the stability and circulating half-life. Drug release from the

polymeric nanoparticles can be controlled by modulating the characteristics of

polymers (Parveen and Sahoo, 2008). PNPs can be used for targeted delivery to tumor

tissue by exploiting the anatomical differences between normal and tumor tissue.

Tumor’s vasculature is large is size, more permeable and leakier as compared to tight

endothelium of healthy tissue. Increased production of vasodilation mediators also

facilitates extravasation. Thus polymeric nanoparticles can reside at tumor side and

increased therapeutic effects (Maeda et al., 2000).

4

Despite of several advantages of polymeric nanoparticles, they also suffer from some

drawbacks. Polymeric nanoparticles are not an ideal system for delivery of protein

and peptides, instability of protein could result during degradation of the polymer.

Polymer can influence the conformational changes of the protein. Burst release of

protein also occur from the polymeric nanoparticles that can lead to unwanted effects

(Mohammadi-Samani and Taghipour, 2015). Polymeric nanoparticle sometimes

results in aggregate formation that can affect the release of drug and overall

physicochemical properties of the drug delivery system (Feng, 2004). Most of the

polymers used for polymeric nanoparticles have hydrophobic surface, that might be

recognized as a foreign body that result in rapid clearance from the body (Hu et al.,

2011).

Lipid-polymer hybrid nanoparticles (LPHNPs) combine the advantages of both

liposomal and polymeric drug delivery system. These types of nanoparticles are

typically consisting of a hydrophobic core and a lipid covering and a hydrophilic

polymer sheath to provide long circulating characteristics. They can incorporate both

hydrophilic and hydrophobic drugs (Zhang and Zhang, 2010). Polymeric

nanoparticles may suffer from the drawback of membrane permeability, combination

of lipid with polymer result in additional biomimetic properties and enhanced

therapeutic efficacy (Rajendiran et al., 2016). Lipids are usually attached to the

polymer layer via ionic interaction or hydrophobic interaction. Ionic interaction

results due to oppositely charged polymer and lipid. LPHNPS have much higher

transfection efficiency as compared to liposomal based and polymer based gene

delivery (Bose et al., 2016). Lipid-polymer hybrid nanoparticles alter the

pharmacokinetic properties of the drug and a suitable vehicle for controlled and

sustained drug delivery. Because of both hydrophilic and hydrophobic portions,

hybrid nanoparticles can deliver multiple agents at the same time. Co-loading of

siRNA and drug is possible, and two drugs can also be delivered at the same time

with excellent loading efficiency. LPHNPs can also co-deliver diagnostic and

therapeutic agents at the same time. Surface modification of hybrid nanoparticles can

also serve as a suitable vehicle for the oral delivery of chemotherapeutic agents

(Zhang et al., 2017). The hybrid nanoparticles usually exhibit core-shell structure;

polymeric core can efficiently deliver drugs with small molecular weight and

diagnostic agents, while lipid core imparts stability, biomimetic and biocompatible

5

properties resulting in enhanced therapeutic efficacy. PEG coating over the lipid layer

further enhance the long circulating properties of these nanoparticles (Wakaskar,

2018). Some studies suggest that LPHNPS have built-in MDR reversal properties,

They have the ability to down regulate the P-gp and increase the toxicity of the

chemotherapeutic agents (Wong et al., 2006).

Chitosan is a naturally occurring polysaccharide and produce from the chitin, which is

naturally occurring structural element of crab and lobsters. Chitosan also occurs in

some fungi. Structure of Chitosan is like cellulose, but it has additional hydroxyl

group, free amino group, due to which it exhibits different functional properties.

Chitosan can respond to different environmental stimuli, such as pH. Chitosan has

great applications in pharmaceutical field due to its intrinsic favorable properties such

as biocompatibility, susceptible to hydrolysis by enzyme and ability to bind with

some organic compounds. Chitosan has stimuli responsive drug delivery properties; it

can be easily protonated in acidic environment and can form complex with negatively

charged polymers, lipids and genes. Chitosan also has mucoadhesive properties and

can lead to significantly enhanced bioavailability. Chitosan has the advantage for

controlled drug delivery, which result in increased efficacy and reduced side effects

(Elgadir et al., 2015). Most of the conventional drug delivery system result in

immediate release of drug that cause fluctuation in the plasma level of drug and

results in unwanted side effect. The drug incorporated in suitable polymer result in

controlled diffusion and slow release. Chitosan is one of suitable polymer for

controlled release formulations (Kumar, 2000). Chitosan has unique cationic

characteristics and gel forming ability, it can form complex with oppositely charged

molecules for the controlled delivery of drug, protein and peptides (Shu and Zhu,

2000). Chitosan has also been investigated for oral controlled drug delivery in

stomach due to its gel forming ability at lower pH and increased gastrointestinal

retention time. Chitosan also has mucoadhesive and antacid properties, which reduce

the drug irritation in the stomach. Chitosan is suitable for both oral and intravenous

controlled drug delivery (Chandy and Sharma, 1992).

Lipids are used in drug delivery system. Most commonly used lipids in drug delivery

are phosphatidylcholine (PC) and phosphatidylethanolamine (PE), phosphatidylserine

(Ps). These are naturally occurring phospholipids and obtained from plants and

animals and are constituent of biological membrane. Lipids used in drug delivery

6

mostly contain phosphatidylcholine and very little amount of PE, because PE can

disrupt bilayer structure under physiological pH (Yingchoncharoen et al., 2016).

Lipids have gain importance for the delivery of drugs having poor water solubility.

Lipids based drug delivery system also enhances the bioavailability of the drugs. The

absorption of drug from the lipid formulation depends on the degree of dispersion and

emulsification of lipid. In general lipid based formulation accelerate the absorption by

increasing dissolution and formation of solubilized phase (Kalepu et al., 2013).

Incorporating the poorly absorbed drug with phosphatidylcholine has been shown to

increase the bioavailability. Intestinal permeability of some drugs have been shown to

increase 20 time by incorporating into phosphatidylcholine containing lipids

(Fagerholm et al., 1998). The lipid used in the formulation is LIPOID S75 that

contain 75% phosphatidylcholine and is suitable for better bioavailability and

controlled drug delivery (Hafner et al., 2009).

Folate receptors were first recognized as a tumor marker when monoclonal antibodies

raised against ovarian tumor were found to recognize the alpha form of folate

receptor. Antibodies that produce against alpha folate receptor found its application as

diagnostic agent in gynecological tumor. After these initial finding the overexpression

of folate receptor was found in more than 90% of ovarian tumors. Because of their

high affinity to folate linked drug they have exploited as an agent for targeted drug

delivery to tumor (Lu and Low, 2003). Folic acid is now used as a targeting agent for

the targeted delivery to the tumor because of its intrinsic small size, penetration

ability, stability, solubility in organic solvents and ability to conjugate with various

therapeutic agents (Cho et al., 1997). Targeting folate receptor is a promising

approach by conjugating folic acid with the polymer that result in significantly

increased uptake of drug by the tumor cells (Saul et al., 2003). Folate membrane

receptors have emerged as a target for drug delivery, in fact, folate metabolism is

primary in replication of DNA and drugs that inhibit folate metabolism can be

targeted. Folate receptors are overly expressed in tumors, which are not in normal

cells. Folate is a vitamin, which is water soluble and carbon donor in the synthesis of

purines, which lead to synthesis of DNA. Folate is required by the normal cells for

multiplication and it enters the cell via anion exchange pathway. Folate is not

overexpressed in the healthy cells, while rapidly dividing tumor cell overexpress the

folate receptor and present as a target for drug delivery (Marchetti et al., 2014).

7

Cisplatin has been used as a first line agent in the treatment of ovarian cancer since

decades. But it exerts side effect on major organs particularly kidney. Several

researches have been made to reduce the side effects and increase the therapeutic

efficacy of the cisplatin by formulating in various nanoparticles and liposomes.

Controlled drug release from the nanoparticles could be an approach to reduce the

side effects associated with the cisplatin (Cheng et al., 2011). Cisplatin inhibit the

tumor growth by inhibiting the DNA mediated functions. Cisplatin inhibit the cell

cycle that result in abnormal mitosis and leads to apoptosis of cells. Cisplatin is

known to cause the inhibition of cell cycle at G2/M phase of cell cycle. Cisplatin form

the bifunctional adduct with the DNA and inhibit the tumor growth (Eastman, 1999).

Here we formulated the novel chitosan lipid hybrid nanoparticles for controlled drug

delivery of cisplatin to reduce the side effect and increase the therapeutic efficacy.

Then chitosan was conjugated with the folic acid and folate targeting was done with

the LPHNPs that added the advantage of lipid layer with folate targeting.

8

1.2. Main objective of study

1. To formulate LPHNPs consisting of chitosan as a polymer and LIPOID S -75

as a lipid by ionic-gelation method and perform physicochemical

characterization.

2. To develop the controlled release formulation of cisplatin loaded in chitosan

LPHNPs.

3. To evaluate the therapeutic efficacy and cell uptake of cisplatin loaded

LPHNPs on ovarian cell line A2780.

4. To evaluate bioavailability, bioavailability studies will be performed in animal

species.

5. To evaluate the in vivo toxicity studies with higher doses of cisplatin and

observe the effect on biochemical parameters and tissue level.

6. To formulate chitosan- folate conjugate for folate targeting and prepare folate

LPHNPS from conjugate and perform physicochemical characterization.

7. To evaluate the effect of folate targeting on ovarian cell line A2780 and

SKOV3 and breast cancer cell lines MCF-7.

8. To perform qualitative cell uptake studies of folate targeted LPHNPs using

fluorescence microscopy and perform quantitative cell uptake of folate

targeted LPHNPS and compare that with untargeted LPHNPS using flow

cytometry

9. To evaluate the cell cycle inhibition mechanism of cisplatin and cell apoptosis

using ovarian cell line.

10. To formulate the three-dimensional (3D) tumor cell culture model to mimic

the in vivo microenvironment of cultured tumor cell

9

11. To study the cell cytotoxicity and cell uptake of folate LPHNPs on 3 D

spheroids to establish their efficacy in in vivo model.

10

Bjs Waltham

Literature Review

CHAPTER 2

11

2.1. Cancer

Cancer is characterized by abnormal cell growth that invades into other body parts

and symptoms include lump, loss of appetite and abnormal bleeding but the

symptoms varies in different types of cancer (Heim and Mitelman, 2015). Cancer cell

differ from normal body cells in some characteristics such as loss of differentiation

and reduced sensitivity to drug and increased invasiveness (Klein, 1987). Cancer

results from the changes in the genome in which oncogene results in increase in their

function and dominance and decrease in function of suppressor gene. Studies reveal

that tumorigenesis is a multistep process that leads to the conversion of normal cell

into malignant cell (Hanahan and Weinberg, 2000). Some studies suggest that

external environmental factors play key role in the development of type of cancer as

compared to genetic factors (Blackadar, 2016).

Cancer results from a series of mutations in genes and these mutations results in

change in cellular function. Chemical compounds also play their role in type of

genetic mutation and cancer (Aizawa et al., 2016). During proliferation the cell

replicates its genome and then divide into daughter cells, the process of proliferation

required energy. In the tumor cell the proliferation is uncontrolled, and cells lost its

check on the process of proliferation. In order to meet the energy demand of

uncontrolled proliferation the cells may adopt different pathway for energy and

metabolism (Garber, 2006). Some studies suggest that alteration in energy metabolism

process may be among root cause of cancer. Warburg proposed that cancer cell use

glycolysis instead of oxidative phosphorylation for ATP production even in the

presence of large amount of oxygen (Warburg, 1956). Increased metabolic demand is

an inherent challenge for the tumor cell and tumor cell modify the metabolism at

cellular level to support the proliferation and adopt the strategies to survive during the

period of metabolic stress (Deberardinis et al., 2008). The major metabolic change in

the proliferating cells is the start of aerobic glycolysis. Glucose is one of primary

nutrient for the proliferating cells. In the proliferating tumors, the pyruvate is

converted into lactate which is then secreted externally and cell recovers NAD+,

which is important to maintain glycolysis and is used for ATP production and

proliferation of tumor cell (Jones and Thompson, 2009).

12

2.2. Ovarian Cancer

Ovarian cancer is one of the leading causes of death. It can be broadly divided into

three different categories, most prevalent one is epithelial cell carcinoma, others are

stromal and germ cell cancer (Agarwal and Kaye, 2003). Ovarian cancer accounts for

0.23 million new cases and 0.15 million deaths annually. The highest rate are

prevalent in the central Europe while around 21000 new cases and 14000 death are

expected to occur in USA annually from ovarian cancer, while in china the rate is low

as compared to population and incidence is 4 in 100,000 peoples (Atlanta, 2015).

The risk of developing ovarian cancer in women in one in 75, while the risk of death

is one in 100. The diagnosis of ovarian cancer is difficult, and is usually diagnosed at

late stage with poor prognosis.

2.3. Classification of ovarian cancer

The exact etiology and cellular origin of ovarian cancer is not well understood in most

cases the tumor seems to be originated from the gynecological tissue and then spread

to the ovary. Broadly ovarian tumors are divided into three types epithelial, stromal

and germ cell origin but more than 90% of ovarian tumors arise from the epithelial

cells (Sankaranarayanan and Ferlay, 2006).

2.3.1. Epithelial tumors

These are most prevalent form of ovarian tumors. These usually originate from the

surface epithelium of the ovary. The epithelial tumor could be benign, atypical

proliferating and malignant. Epithelial ovarian tumors accounts for 61% of the total

ovarian tumors and 90% of the malignant form of ovarian cancer (Scully et al., 1999).

The epithelial cell tumors may include serous, mucinous, endometrial, clear cell

tumor and transitional cell tumor.

Serous tumors originate from the cells that are similar to the cell that lines the internal

fallopian tubes. Benign serous tumors usually do not spread to the other parts and are

characterize by the single chamber that is filled by the colored fluid. Internal linings

of serous tumors are sometimes flat but sometimes show the papillary projections.

Benign serous tumors account for the 2/3rd

of serous tumors and these usually occur in

both ovaries and surgical resection is curative. Borderline serous tumors have more

13

papillary projections. They usually do not invade other body tissues but are

sometimes invasive and accounts for 15% of all serous tumors. Treatment is usually

surgical removal and recurrence could occur and 5 year survival rate is 75%. Mostly

malignant serous tumors contain cystic chambers and have some solid areas. They

also contain papillae projections on the outer surface of tumor (Scully et al., 1998).

Mucinous tumors originate from the cells like endocervical epithelium and intestinal

epithelium. These are usually filled with mucoid tumor and account for 1/4th

of

benign epithelial cell tumors. Endometroid tumors originate from the cells that are

like the endometrium. They may present with the aberrant growth of internal lining of

uterus. Transitional tumors usually arise from the urothelium (internal lining of

urinary bladder). The benign transitional tumors are usually asymptomatic and

clinically non-significant while malignant may contain internal papillary projections

(Kurman, 2013).

2.3.2. Stromal cell tumors

These are believed to originate from the theca cells and granulosa cells. These usually

accounts for 8% of total ovarian tumors. Granulosa cell tumors are rare form of sex

cord tumors and derived from the germinal cells that line the follicles.

Thecoma are solid tumors which are formed by a form of stromal cells that resemble

theca cells. These usually develop in postmenopausal age and are unilateral. Fibromas

are solid tumors that arise from the spindled stromal cells (Chen et al., 2003).

2.3.3. Germ cell tumors

These tumors are derived from the primordial germ cell. These usually account for

6% of malignant ovarian tumors. The prevalence is high in Asia and adolescents.

These include dysgerminomas, yolk sac and embryonal tumors.

Dysgerminomas are solid tumors and are usually unilateral. The incidence of

dysgerminomas is high is early adulthood. These are usually presented with high level

of lactic dehydrogenase in serum. Yolk sac tumors are a form of germ cell tumors

their shape is like primitive yolk sac. These are also called as endometrial sinus

tumors. These are solid and presented with cyst. These are mostly malignant and

invade the surrounding tissues (Chen et al., 2003).

14

2.4. Risk Factors of ovarian cancer

2.4.1. Hormonal

The incessant ovulatory hypothesis suggests that increase in no. of ovulatory cycles

results in higher rate of cell division and increased chances of mutation (Tung et al.,

2005). It relates to the age at first menarche and age at menopause. The higher no of

ovulation cycles results in higher exposure to the gonadotropin, FSH and luteinizing

hormone (Cramer and Welch, 1983).

2.4.2. Pregnancy and infertility

Pregnancies cause stoppage of ovulation cycle and suppression of release of

gonadotropin and thus reduce the chances of ovarian cancer. The studies suggest that

increased number of pregnancy reduce the risk of ovarian tumors by 30-60%. Studies

also showed the increased risk of ovarian tumors in females with first pregnancy

(Tavani et al., 1993).

2.4.3. Lactation

Lactation decreases the secretion of gonadotropin and cause anovulation in initial

months after delivery. Both hypothesis suggest that lactation reduce the risk of

ovarian tumor (Schottenfeld and Fraumeni Jr, 2006).

2.4.4. Use of oral contraceptives

The studies suggest that use of inverse relation of ovarian cancer with use of oral

contraceptives. There is 20% reduction in the risk with 5 year usage of oral

contraceptives (La Vecchia, 2006).

15

2.5 Treatment of ovarian cancer

Treatment of ovarian cancer includes surgery to reduce the tumor size, chemotherapy,

radiations and immunotherapy.

2.5.1. Surgery

The purpose of surgery before chemotherapy is to reduce the tumor size as much as

possible and establish staging and help in diagnosis (Prat and Oncology, 2014).

Recent trend is optimum debulking and omentectomy. Studies from various

retrospective data suggest that maximum removal of tumor during surgery is

associated with the longer survival (Du Bois et al., 2009). Interval debulking is

performed during the process of chemotherapy (Trimbos, 2003).

2.5.2. Chemotherapy

Platinum based regime has been considered as a standard of treatment for ovarian

cancer (Mcguire et al., 1996). Cisplatin is considered as a first line agent for all forms

of ovarian cancer. Studies have shown enhanced efficacy with the addition of

paclitaxel. The response rate is assessed by measuring the serum CA 125 level (Rustin

et al., 2006).

2.6. Cisplatin

Cisplatin was first discovered by Rosenberg as anti-cancer agent (Rosenberg et al.,

1969). In 1971 Cisplatin was approved for clinical trials and in 1978 it got approval

for the treatment of ovarian and testicular cancer (Wong and Giandomenico, 1999).

Metals are important for cellular process, some have redox activity, some are

associated with pathological condition and some have anticancer activity (Frezza et

al., 2010). Cisplatin in one of the most commonly used anti-cancer agent in the

treatment of various malignancies including head and neck, lung, ovarian and

testicular carcinoma (Mattheolabakis et al., 2009). After the discovery of cisplatin

various platinum based drugs were tested but only carboplatin and oxalipatin has

drawn some clinical attention (Desoize and Madoulet, 2002). Cisplatin is also

associated with severe side effects particularly on the renal function. Effective

hydration is necessary to decrease side effects on renal parameters and decrease

dosing (Loehrer and Einhorn, 1984).

16

2.6.1. Chemical structure

Cisplatin is made up of 2 chlorides and 6 hydrogen, 2 Nitrogen and one platinum

compound in the center. The molecular formula of Cisplatin is Cl2H6N2 Pt. The

chemical name of cisplatin is cis-diamminedichloroplatinium, being a cis- isomer

make it more effective (Dhar et al., 2008).

Figure 2.1: Chemical structure of Cisplatin

2.6.2. Mechanism of action

The target of the Cisplatin is DNA, It interacts with DNA and forms adduct, which is

responsible for the anti-cancer activity of the drug (Pinto and Lippard, 1985).

Cisplatin enters the cell by passive diffusion. After entering into the cell, it’s chloride

ion interact with water molecule which form a positively charged specie that can form

nonfunctional adduct with DNA, which further reacts with chloride ion to form bi-

functional adduct (Lepre and Lippard, 1990). The major adduct is bidentate, which

bind to guanine base and unwind the DNA, thus blocking the proliferation of tumor

(Sip et al., 1992). The intermediate adducts like APG and GPG are responsible for 80-

90 % of cytotoxic activity (Kelland, 1993). These adducts stop DNA replication and

resulting in DNA damage of over twenty proteins. These DNA damage proteins

transfer signal to downstream cascade including P53 and P73, which results in

apoptosis (Damia et al., 2001). P73 is a pro-apoptotic protein and that accumulate in

cell treated with cisplatin. Thus induction of p53 pathway and p73 cause apoptosis in

cancer cell (Viktorsson et al., 2003).

2.6.3. Pharmacokinetic properties

Cisplatin is administered by intravenous route over one-hour infusion, peak plasma

concentration reaches within no time after administration, the plasma concentration

decreases to 50% after two hours of administration. The clearance of cisplatin from

Pt

Cl

Cl

NH3

NH3

17

the body is triphasic, distribution is achieved immediately as distribution half-life in

thirteen minutes, and elimination half-life in forty-five minutes, while terminal half-

life is almost five days. Major route of excretion is renal as 90% of drug is excreted

via this route. 25% of drug is eliminated within 24 hours of administration (Sturgeon,

2004). Ultra-filterable platinum, which is non-protein bound drug, is responsible for

major pharmacological action of drug. After administration of 110mg/m2 of Cisplatin

the blood plasma level of almost 6 µg/ml reaches immediately after administration

and declines to 2 µg/ml after two hours of administration (Go and Adjei, 1999).

2.6.4. Therapeutic uses

Cisplatin is used in variety of solid tumors including testicular, ovarian and bladder. It

is most commonly used in combination with other chemotherapeutic agents, as the

combination therapy is more effective and has better results.

2.6.4.1. Cisplatin and ovarian cancer

Ovarian cancer is gynecological malignancy with high mortality rate due to late

diagnosis. Most commonly used regime in ovarian cancer is combination of paclitaxel

and cisplatin. Similarly cisplatin in combination with gemcitabine is also used in

ovarian cancer (Agarwal and Kaye, 2003).

2.6.4.2. Cisplatin and lung cancer

At present Lung cancer is a fatal malignancy, Cisplatin is a key drug used in small

cell lung cancer. Non-small cell lung cancer is also treated with cisplatin based

adjuvant therapy (Youlden et al., 2008).

2.6.4.3. Cisplatin and breast cancer

Breast cancer is one of the most prevalent forms of cancer in women throughout the

world. Chemotherapeutic agents are effective in the treatment of breast cancer and

reducing the tumor size before surgery. Cisplatin is important anti-cancer drug used in

the treatment of breast cancer (Dhar et al., 2011).

2.6.4.4. Cisplatin and brain cancer

Brain cancer is one of the most fatal diseases. Cisplatin is used effectively in

childhood brain tumors. It improves the survival rate (Khan et al., 1982).

18

2.6.5. Side effects of cisplatin

Cisplatin interacts with DNA and cause the apoptosis of cell , this interaction with

DNA of cell is major cause of side effects of cisplatin (Yousef et al., 2009). The

major side effects of cisplatin are hepatotoxicity, nephrotoxicity and cardiac toxicity.

2.6.5.1. Hepatotoxicity

Cisplatin induce the oxidative stress resulting in reduced level of glutathione and

major side effects occur due to this , such as toxic effects on liver (Yilmaz et al.,

2004). Transaminase are released in blood circulation immediately after the cellular

damage and are biomarker for the toxicity and cellular damage, increased in level of

liver enzymes is indication of disturbance in liver function (İşeri et al., 2007).

Cisplatin induced hepatotoxicity has been observed to be greater in patients having

overexpression of CYP P450-2E1 enzyme (Caro and Cederbaum, 2004). Side effect

on liver include degeneration of hepatocytes and tissue necrosis (Kart et al., 2010).

The hepatotoxicity induced by cisplatin may be reduced by using Selenium ad vitamin

E (Liao et al., 2008).

2.6.5.2. Nephrotoxicity

The major route of excretion of cisplatin is via urine hence it accumulates in the

proximal tubules of kidney and its concentration is five times greater in proximal

tubules of kidney as compared to serum concentration. The increased level of

accumulation of cisplatin in the kidney contribute to renal damage (Ali and Al

Moundhri, 2006). Cisplatin is excreted via glomerular filtration and tubular secretion

and its accumulation cause side effects on renal function (Yao et al., 2007).

Concentration of cisplatin increase in kidney as compared to blood particularly in

renal parenchymal cell (Ishida et al., 2002).

2.6.5.3. Cardiotoxicity

Cisplatin may cause lipid peroxidation in cardiac tissues and cardiac myocytes may

release creatinine kinase and lactic dehydrogenase. Degeneration and necrosis of

cardiac muscles may occur (Al-Majed et al., 2006).

19

2.7. Novel drug delivery system

Since the start of life on earth, human has been trying to develop drug delivery system

from chewing leaves and plant roots and inhaling smoke of plant for treatment of

disease to the development of tablets and capsules for the delivery of drugs. In the

start of 20th

century efforts have been made to move from traditional and uncontrolled

drug delivery system to controlled drug delivery system. Invention of new drug

molecules having larger molecular weight and poor solubility lead to the development

of capsulated drug delivery system to enable drug to deliver at the site and show its

pharmacological action. Sustained drug delivery systems lead to the decrease in the

frequency of administration and ease to the patients. Controlled drug delivery system

control both rate of release of drug and site of release of drugs for maximum

therapeutic output (Barbe et al., 2004).

The chemotherapy currently being used is nonspecific and exerts serious side effects

to the normal tissues. To overcome this problem various efforts has been made to

achieve site specific drug delivery by using novel technologies. The liposomal drug

delivery system has proved to be very effective for the targeted delivery of

chemotherapeutic agent but is suffers problem of stability and storage (Juliano and

Stamp, 1975). The leakage of liposomes is covered by the PEG coating that enhance

circulation time. DOXIL® a liposomal formulation containing doxorubicin is

approved for cancer treatment in 1995(Lasic and Martin, 1995).

Hydrogels are biodegradable and safe drug delivery systems for targeted delivery to

bone tumors. Hydrogels can be used for thermoresponsive drug delivery as it is liquid

at room temperature and convert to gel at body temperature. Hydrogels can also be

used for pH sensitive drug delivery(Ta et al., 2009). Niosomes are also one of the

novel system for delivery of drugs and they resemble with liposomes except they have

non-ionic surfactant. Different types of surfactant are used which have the ability to

entrap both water loving and hydrophobic drugs. The presence of cholesterol increase

the rigidity of the system and their presence improve the permeability and fluidity

(Rajera et al., 2011).

20

Microspheres have excellent carrier capacity and having small size and suitable for

drug delivery, they have disadvantage of decrease residence time at the site of

absorption. This disadvantage can be overcome by formulating novel bio-adhesive

microspheres. Bio-adhesive microspheres can stick to the mucosal tissues such as in

GI tract, nasal and other surfaces. This offers highly controlled and localized drug

delivery system (Vasir et al., 2003).

Hollow microspheres are used for site specific drug delivery to the GI tract, because

normal drug suffer from the problem of decrease GI transit time, this can be overcome

by formulating floating microspheres, which provide prolong retention in GI tract and

reliable release of drug (Kawashima et al., 1992).

2.8. Nanotechnology

Nanotechnology is a multidisciplinary field paving its role in academia and research.

Due to its emerging role, the US government has established the National

Nanotechnology Institute (NNI) that support the research and commercialization in

the field of nanotechnology (Science and . 2005). Broadly nanotechnology is defined

as, the understanding application of materials in range of 1-100nm. Systematic

reviews suggest that almost 90% of newly discovered molecules have poor

pharmacokinetic properties (Brayden, 2003).

Nanotechnology enables the delivery of therapeutic molecule to the targeted tissue or

organ. Nanotechnology also enable the future ways for delivery of new therapeutic

molecules as ― nanomedicines‖ that helps the targeted and controlled delivery and

reduce the side effect associated with that drug molecule (Science and . 2005). The

emergence of nanotechnology has played a pivotal role in revolution of drug delivery.

The nanotechnology helps to increase the bioavailability of drugs with poor solubility,

passage of drug across tight epithelium barriers such as blood brain barrier, delivery

of large molecular weight drugs and protein at desired site of action. Nanotechnology

enable the co-delivery of drug and imaging agents at the site of action that enable the

visualization of desired site (Liong et al., 2008).

Nanomaterial provides an opportunity to enhance efficacy by modifying size, shape

and surface properties. The shape of nanoparticle affects bio distribution, drug loading

and extravasation at site of action. Squashable nanoparticles have long circulating

21

characteristics because of their ability to avoid from phagocytic cells (Anselmo and

Mitragotri, 2017). Nanomaterials made up of inorganic silica allow higher drug

loading and multistage delivery of therapeutic agent. A combination approach involve

the lipid coating over the porous material that gives higher drug loading and long

circulating characteristics (Ashley et al., 2011).

2.9. Nanotechnology and cancer

Nanotechnology is no stranger to oncology. The liposomes were first ever approved

nanomedicines for the treatment of cancer. Cancer nanotechnology is broad area of

research with application of nanocarrier in drug delivery, imaging and diagnosis.

Nanocarrier are conjugated with the functional groups and used for targeting of

tumors (Ferrari, 2005). Researchers have developed nanocarriers that are covalently

linked with the biological peptide and proteins. These also have diagnostic

applications in cancer such as nanoparticles made up of iron oxide are used as a

contrasting agent for prostate cancer. Personalized nano-oncology enables the

quantification to which extent the drug has reached the desired site of action. The

treatment of transtuzumab, decision is based on the measure of extent of

overexpression of HER2 receptors in individual patients (Evans and Relling, 2004).

Figure 2.2: Areas of nanotechnology applications

The early diagnosis and detection of neoplastic lesion has always remained an elusive

goal. To identify the invading tumor, cell imaging technologies does not provide the

22

enough resolution for early detection. Recent trends in imaging technology require

contrasting agent that is conjugated to a specific targeting agent. Nanoparticle

technologies are helping in early detection by co-delivery of targeting moiety and

contrasting agent that enable the anatomical explanation of lesion (Sullivan and

Ferrari, 2004). Nanocarrier probes loaded with molecular targeting agents provide

information about relative distribution of tumor marker in tumor microenvironment

(Li et al., 2004). Nanoparticles loaded with iron oxide are conjugated with Annexin-V

that detect the phosphatidylserine present in apoptotic cell, which is then used for

MRI identification of jurket T cell (Schellenberger et al., 2002). Nanotechnology has

provided new horizons form the treatment of cancer. The optimum success of

nanoparticle depends on the stability of nanoparticle during circulating in blood

stream and their ability to cross the tight physiological barrier and reach at the

targeted site of action (Grodzinski et al., 2019).

Nanoparticles have therapeutic and diagnostic applications. Magnetic nanoparticles

are well known for diagnostic, drug delivery and thermal properties. Recent

theranostics involve delivery of drugs and diagnostic agents. Biomedical technologies

require methodology to manipulate the living cells that are magnetically responsive

and can be controlled by applying external magnetic field. Cell surface engineering

provide the platform to tailor the magnetic nanoparticles on the surface of cell (Setua

et al., 2018). Recently developed iron oxide based magnetic nanoparticles consist of a

magnetic core coated with the biocompatible material embedded inside is sustained

release drug and a biomarker for diagnostic application (Shevtsov and Multhoff,

2016). Liposomes also serve as a theranostics agent to deliver the drug and diagnostic

agents by encapsulating one agent in the hydrophilic domain of liposomes and other

agent in the hydrophobic chain embedded within the liposomal bilayer. Liposomes

also have the superiority of high biocompatibility and can be conjugated with the

targeted moiety and can be used for passive and active targeting of tumor to serve the

diagnostic and therapeutic purpose at the same time (Yue and Dai, 2018).

23

2.10. Nanoparticles

Nanotechnology is a scientific technique undergoing explosive development and

being used in the delivery of drugs, protein and peptides. Nanotechnology has opened

new therapeutic application for poorly soluble drugs to be delivered orally.

Nanoparticles can easily cross the minute membrane barrier including blood-brain

barrier (Emerich and Thanos, 2003). One of the latest novel drug delivery

technologies is the use of nanoparticles for drug delivery. Polymeric biodegradable

nanoparticles are effective biodegradable delivery system. Various polymers are being

used to achieve the targeted drug delivery. Nanoparticles has the advantage over

liposomes due to better stability and efficient controlled release profile (Soppimath et

al., 2001). Particulate drug delivery system is capable of targeting tumors with

relative better efficacy and safety profile. Nanoparticles are novel technology in the

field of particulate based drug delivery system (Paciotti et al., 2004). Nanoparticles

have the ability to penetrate deep into the tissue and controlled release of drug. They

have advantage over microparticles due to better encapsulation efficiency and

bioavailability (Nagpal et al., 2010).

Nanoparticles have shown large number of advantages over other delivery systems

Nanoparticles have the advantage of their size, which is large enough to avoid

leakage into capillaries and small enough to be captured by macrophages. The

size gap between endothelial cell is 100-600nm, so nanoparticles can reach to

the tumor site for the site specific delivery of drug (Cho et al., 2008).

Polymeric nanoparticles can be tailed to achieve disease-specific delivery and

controlled release of drug. They can be concentrated at the tumor site and may

work as a depot of local delivery for solid tumors by using a suitable carrier.

They can be used for sustained drug delivery by protecting against the

enzymatic degradation (Singh and Lillard, 2009).

Better stability profile of nanoparticles enable oral administration feasible and

sustained release of drug can be achieved after oral administration. Bio

adhesive properties of polymeric nanoparticles enable increased absorption of

drug and better bioavailability. Nanoparticles can be delivered via different

routes from topical to inhalation (Gelperina et al., 2005).

24

Nanoparticles based drug delivery system improves the solubility of

hydrophobic drugs, increase the half-life during circulation by reducing

immune attack, enable stimuli responsive release of drug and decrease

frequency of administration. Moreover more than one drug can be

administered simultaneously this increasing the patient compliance (Zhang et

al., 2008).

Nanoparticles have gain special applications in the delivery of

chemotherapeutic agents. Conventional delivery of anti-cancer drugs also

causes severe harmful effects on normal cells. Nanoparticles have enhanced

permeability and retention effect and may concentrate at the tumor site. Hence

higher intratumoral drug concentration is achieved and less distribution of

drug toward normal tissues sites. This reduces the dose related side effects of

drugs. secondly poorly soluble anti-cancer drugs can also be delivered at the

targeted site easily (Wang et al., 2012).

Nanoparticles have disadvantage of decreased loading capacity that is not greater

than 5% of total weight of nanoparticles. They also suffer from the problem of

burst release after approaching the site of action (Couvreur, 2013).

2.11. Polymeric nanoparticles

Over the decades, polymeric nanoparticles are promising for delivering drugs to the

target site and cover wide range of diseases. The efficiency at the target site of disease

is due to their flexible nature, nano-sized, composition, molecular weight, and stability

of the polymeric drug carrier. The effectiveness of the polymeric drug carrier covers

all the limitation that is the major issues with the conventional dosage form. Polymeric

nanoparticles enhanced the solubility of the drug, leading to enhanced bioavailability

in the systemic circulation and sustained the release of drug at target site. For the

delivery of nanoparticles, various types of natural and synthetic polymers are

employed for drug delivery to the active target site (Naahidi et al., 2013).

25

Figure 2.3: Structure of polymeric nanoparticles

Size range of polymeric nanoparticles ranges from 1nm-1000nm (Soppimath et al.,

2001). Polymeric nanoparticles must have the property of sustained release of

entrapped drug, protection from enzyme degradation, escape from reticuloendothelial

cells and have active and passive targeting ability (Bamrungsap et al., 2012, Steichen

et al., 2013). Polymeric nanoparticles may be branched, spherical or core-shell

structures. These nanoparticles are fabricated by various methods such as solvent

evaporation, solvent diffusion, interfacial polymerization, spontaneous emulsification,

emulsification diffusion, and by use of supercritical carbon dioxide. The biodegradable

and natural polymers are widely used for the fabrication of polymeric nanoparticles to

deliver the drug at the active site. These biodegradable polymers include polyacrylates,

PLGA, polycaprolactone and natural polymers include chitosan, albumin, gelatin,

alginate and collagen (Panyam and Labhasetwar, 2003).

Polymeric nanoparticles deliver the drug to the target site by active and passive

targeting. For targeting, active drug should be able to reach the active site after its

administration with minimum side effects and also kills only diseased tissue

without harming the healthy tissue. The above criteria may be fulfilled by using

passive and active targeting strategies (Bamrungsap et al., 2012). Passive

targeting, exploiting the natural conditions of the target organs of tissues without

involvement of any ligands and its direct attachment of the drug to the target site.

This type of targeting takes the advantage of the unique pathophysiological

characteristics of the tumor/diseased tissue due to its leaky vasculature that

26

enables the nano-drug carrier(s) that leads to the accumulation at the site. The

leaky vasculature allows the migration of molecules having size range up to 400

nm in diameter refers to the enhanced permeability retention (EPR) effect in the

tumor region (Kubik et al., 2005).Active targeting is the attachment of affinity

ligand(s) such as antibodies, biological macromolecules (peptides), vitamins,

aptamers, small molecules such as small interfering RNS (siRNA) and DNA for

co-delivery action. These ligands only bind to specific receptors or epitope on the

cell surface for the delivery of drug to the site of action. The novel nanocarriers

will recognize and then binds to the target cells through the mechanism of ligand-

receptor complex (Sudimack and Lee, 2000).

Polymeric nanoparticles have been considered to be the effective drug delivery system

in the field of therapeutics. This efficacy is due to maximum therapeutic benefit and

minimum side-effects. For this, polymeric nanoparticles have been used as a potential

drug delivery carrier system for loading of drug for sustained/controlled release. The

carrier system used in polymeric nanoparticles may be matrix or core system

depending upon method of preparation. Firstly, the drug used for the carrier system is

dissolved, then loaded, and entrapped to the carrier system (Soppimath et al., 2001).

Besides the promising characteristics of polymeric nanoparticles, their applications

have been limited due to several factors such as control size range and loading of

hydrophilic drugs. The control of size in the polymeric network is quite difficult due to

use of high molecular weight polymers. Mostly drugs for parenteral delivery should be

in size range of less than 400nm because if the size range is more than 400nm than

these will be engulfed by the RES system (Acharya and Sahoo, 2011). Polymeric

nanoparticles have low loading capacity of hydrophilic drugs due to the limited

portioning of more hydrophilic drugs into the lipophilic polymer coat (Yoo and Park,

2001).

2.12. Chitosan

Chitin is highly abundant naturally occurring polysaccharide and is found in the

shells of crabs and other insects. Chitin has poor solubility in water and has poor

biodegradability that limits its vast applications. The degradation product of chitin

are chitosan and chitosan oligosaccharide, which are highly biodegradable and

27

compatible with biological fluids and have vast application in food and

pharmaceutical industry (Du et al., 2009).

Figure 2.4: Chemical structure of chitosan

Chitosan is obtained by N-deacetylation of chitin. It is insoluble in water and is

soluble in acidic solution. It is a moderate cationic polymer and have amino and

carboxylic group in repeated subunits that can be utilized in conjugation chemistry

(Agrawal et al., 2010). Chitosan is believe to act as a scavenger for free radicals

and provide antioxidant effect (Zou et al., 2013). It is readily uptake by the

intestinal cells and biocompatible properties make it a suitable antioxidant. It can

also increase the activity of antioxidant enzymes. It also reduce the lipid

peroxidation (Xia et al., 2011).

Chitosan is also believed to have antimicrobial effects, which also depends on the

molecular weight of chitosan, degree of deacetylation and pH. The Minimum

inhibitory concentration(MIC) of chitosan is found to be 0.1% and also depends

on the type of bacterial growth in culture (No et al., 2002). The investigation of

mechanism of antibacterial effect has shown that amino group of glucosamine

ring of chitosan interact with carboxylic group of bacterial cell wall and form a

complex and inhibit their growth (Kim et al., 2003).

28

The structural integrity, availability in different molecular weight ranges,

conjugation chemistry, and biological compatibility make chitosan an ideal

polymer for drug delivery applications.

2.13. Liposomes

The word liposome is derived from two Greek word ―Lipos‖ and ―Soma‖ which

means ―fat body‖. They can be unilamellar or multilamellar with phospholipid as

a major structural component. They were first discovered by Alec D Bangham in

1965 (Bangham, 1993). The composition of liposomes is similar to the cell

membrane. The major structural component of biological membrane is

phospholipid, which is composed of hydrophilic head and a hydrophobic tail

(Papahadjopoulos et al., 1978).

Liposomes are bilayer membrane structure made up of phospholipids. Liposomes

have attracted a lot of attention due to its high biocompatibility and able to

delivery various therapeutic agents including drugs and genes. Liposomes are first

nanomaterials to be approved for clinical usage in 1995 (Torchilin, 2005). The

phospholipid membrane is a cell like boundary suitable for cellular investigation

and has the ability to mimic the biological system (Hua and Wu, 2013). Since the

liposomes have provided a breakthrough in the field of nanotechnology various

efforts have been done to improve the drug delivery via liposomes by attaching

targeting agents and preparing stimuli responsive liposomes (Lemière et al.,

2015). Some of the recent approaches include increasing the circulating life of

liposomes by attaching suitable amphiphiles. Triggered release approach include

enabling the release of drug in response to pH, temperature or radiation (Bibi et

al., 2012). Liposomes are suitable for gene delivery and offer protection to the

DNA, RNA. They can form complexes with cationic and anionic molecules and

are suitable for targeting the specific site (Alavi et al., 2017).

29

Figure 2.5: Structure of liposomes with PEG coating

Traditional liposomes suffer from the problem of rapid clearance from the body

and have stability issues. It happens because membrane of traditional liposomes is

affected by the proteins circulating in the blood, which cause opsonization and

rapid clearance from the body. In order to compensate these problems, efforts

have been done to modify the surface charge of liposomes and enabling long

circulating characteristics. Stealth liposomes were prepared by modifying the

surface with polyethylene glycol (PEG) that helps liposomes in protection from

circulating protein and increase the circulation life of liposomes (Van Slooten et

al., 2001). Stealth liposomes provide sustained delivery of drug and can also be

used in localized depot formulation. The first example is enhanced efficacy of

cytosine arabinose in treatment of lymphoma and first subcutaneous formulation

of liposomes was to deliver vasopressin as sustained release (Allen et al., 1992).

The surface modification includes attaching the lipid with hydrophilic polymer

such as polyethylene glycol. The covering of PEG prevents the adhesion of

plasma protein to the surface of liposomes that otherwise would have cleared by

mononuclear phagocytic system (MPS). The coating of PEG enables the

liposomes to oppose the uptake by the MPS thus incorporating long circulating

characteristics to the liposomes (Gabizon et al., 1994). Stealth liposomes can be

prepared by various chain lengths of PEG. PEG is biocompatible and have good

30

solubility and in non-ionic in nature. Some studies suggest that size of liposomes

should be less than 300nm in order to have protected stealth properties (Nag and

Awasthi, 2013). Stealth liposomes with PEG coating are also suitable candidate

for active targeting of therapeutic drugs. Active liposomes are used for targeting

folate and transferrin receptors, which are overexpressed in many solid tumors

(Torchilin, 2007). Although PEG is considered as a gold standard for increasing

the circulating life of liposomes and for steric protection most recent include

addition of biodegradable polymer and preparing lipid-polymer conjugating and

incorporating the long circulating characteristics of polymers with lipid (Metselaar

et al., 2003). The clinical trials of stealth liposomes loaded with doxorubicin in

human has produces significant results as compared to doxorubicin drug. The

results indicate that drug remain encapsulated for 7 days after injection. The

presence of metabolites of drug at tumor site indicates the release of drug at tumor

site. There was 10 times greater absorption of drug at tumor site as compared to

control drug solution. These results showed stealth liposomes have achieved

substantial improvement in the treatment of tumor (Daraee et al., 2016).

Due to the stability issues, liposomes are required to be stored at controlled

temperature. At freezing temperature, they lead to crystal formation and breakage

of phospholipid bilayer structure (Perche and Torchilin, 2013). Liposomes with

ultra-small size have less interaction with ligand and receptor. Liposomes with

positive charge have greater electrostatic interaction with anionic charged cell

membrane. Higher ligand density could lead to aggregation of small particles

(Albanese et al., 2012). Liposomes suffer from disadvantages like leakage of

encapsulated drug. Lipid in liposomes may undergo oxidation and hydrolysis that

leads to the unwanted release of therapeutic agent. They have short half-life and

difficulty in solubility of hydrophilic drugs (Daraee et al., 2016).

2.14. Lipid-polymer hybrid nanoparticles (LPHNPs)

Nanotechnology has provided the innovation in the delivery of drug, after many

pre-clinical studies liposomal drug delivery system and polymeric drug delivery

system has proven to be useful. The latest technology is the combining the

beneficial effects of both liposomal and polymeric drug delivery system and

achieving lipid-polymer hybrid nanoparticles. They have the advantage of

31

sustained and targeted delivery. They have better stability as lipid coat provide

diffusional barrier from external stimuli (Kong et al., 2013).

Very few studies have been reported on the development of lipid-chitosan

nanoparticles for the delivery of drugs through oral, topic and parenteral

administration. The reported studies are summarized as:

Nayab,et al (2019) prepared hybrid nanoparticles for controlled delivery of

doxorubicin hydrochloride and doxorubicin base. The prepared nanoparticles

showed enhanced encapsulation and higher internalization and greater cytotoxicity

as compared to doxorubicin solution (Tahir et al., 2019).

Q tan, Weidong Liu et al, (2011) developed the lecithin-chitosan nanoparticle

system for the topical delivery of the drug. They prepared quercitin loaded

nanoparticles and these nanoparticles showed enhanced permeation ability and

greater accumulation in epidermis. Interaction between nanoparticles and skin

surface enhance the skin permeation and nanoparticles are ideal for the topical

delivery of drugs (Tan et al., 2011).

Anita Hafner et al, (2009) prepared the lecithin-chitosan nanoparticles for the

trans-mucosal delivery of drugs. The prepared nanoparticles were of 120 to

340nm. The permeability of melatonin was studies on Caco2 cell lines for in-vitro

evaluation. Different types of lecithin and chitosan ratios were used in the study.

The permeability was better by using Lipoid S45 and Lecithin/Chitosan ratio of

20:1. The results showed that lecithin-chitosan nanoparticles are successful for

trans-mucousal drug delivery and does not induce any damage to plasma

membrane (Hafner et al., 2009).

YS Chhonker et al, (2015) prepared the hybrid nanoparticles for ocular drug

delivery of amphotericin. The Lecithin- Chitosan nanoparticles were prepared by

ionic gelation method with lecithin to chitosan ratio (10:1). The nanoparticles

were of 162 to 230nm size and entrapment efficiency was around 75%. The

prepared nanoparticles were more effective as compared to marketed formulations

and showed enhanced muco-adhesive properties. In-vivo studies showed two fold

enhanced bioavailability and increase in corneal residence time (Chhonker et al.,

2015).

32

Anita Hafner et al, (2011) formulated lecithin-chitosan nanoparticles loaded with

melatonin. The ratio of lecithin-chitosan was 20:1 and have size of 160-328nm.

The prepared nanoparticle suspension was then lyophilized to retain the physical

and chemical stability for longer period of time, which is not possible in nano-

suspension. trehalose was used as excipient for lyophylization of nanoparticles.

The freeze-dried nanoparticles have amorphous nature as indicated by DSC. The

nanoparticles have glass-transition temperature above room temperature and were

capable of retaining their characteristics and appearance for seven months at 4 °C

(Hafner et al., 2011).

Ipek Ozcan et al, (2013) prepared the hybrid lecithin-chitosan nanoparticles for

the topical preparation of betamethasone valerate. The prepared nanoparticle were

compared with the commercially available formulation and results showed that

Lecithin-chitosan nanoparticles showed 1.58 time increase permeation in

epidermis. The prepared formulations also showed skin bleaching and anti-

inflammatory effects (Özcan et al., 2013).

Ana Grenha et al, (2008) formulated the lipid-polymer nanoparticles for the

pulmonary delivery of drugs using mannitol as excipient. These nanoparticles

were then encapsulated in the microspheres. The physicochemical properties of

lipid-polymer nanoparticles were dependent on phospholipid concentration, which

provide controlled release of insulin. They have deep inhalation properties and

release the drug after contacting with the lung surface (Grenha et al., 2008).

Manuel J. Santander-Ortega et al, (2010) prepared the nanoparticles consisting of

lipid core which is surrounded by polymer chitosan for the delivery of DNA and

Hydrophobic drugs. Different formulation were prepared by varying the

concentration of polymer and lipid to measure the effects on physicochemical

properties. The nanoparticles were prepared by solvent-displacement technique.

The prepared nanoparticles have better colloidal stability and inert to

physiological fluid, thus presenting an ideal system for delivery of DNA

(Santander-Ortega et al., 2010).

Marthyna P. Souza et al, (2104) prepared the lipid-chitosan nanoparticles by ionic

gelation method. They have encapsulated the quercitin with excellent loading

capacity of 95%. The possess average size of 168nm and have better stability

33

profile. The loaded quercitin showed much better antioxidant properties as

compared to the normal formulation. Hence Lecithin-chitosan nanoparticles can

be used in preparation of functional food material (Souza et al., 2014).

Renu Chadha et al, (2012) formulated the artsunate loaded lipid-chitosan

nanoparticles. Artesunate has poor bioavailability and low aqueous solubility. The

nanoparticle provided controlled and sustained release of artesunate. The

nanoparticles were prepared by ionic gelation method and prepared particles have

size below 300nm. The drug loading capacity was 90%. The prepared

nanoparticles have shown increased in-vivo anti-malarial activity (Chadha et al.,

2012).

2.15. Types of lipid-polymer hybrid nanoparticles

LPHNPs have the advantage that they provide the sustained release of drug due to

the presence of polymeric core and drug can also be released in a controlled

manner. They also provide enhanced biocompatibility and stability due to the

presence of lipid layer (Chan et al., 2009). The polymer plays its role in release

behavior of drug and structural integrity of LPHNPs. The lipid provide

biomimetic properties and efficacy of system is enhanced as compared to both of

them alone (Zhang et al., 2008). Depends on the nature of lipid and nature of

polymer lipid-polymer HNPs may adopt one of the following arrangements of

lipid and polymer:

1. Polymer inside as a core and lipid as a shell

2. Polymer-caged lipids

3. Lipid and polymer mixed with each other

4. Lipid based then polymer core and lipid coating on the outer side

2.15.1. Lipid-polymer hybrid core-shell structure

In these cases, the polymer used is usually hydrophobic and dissolved in organic

solvent that is embedded inside and serve as a core of the system, while lipid

provide the outer layer and serve as a shell with hydrophobic end of the lipid is

inside and hydrophilic end on the outer surface of lipid-polymer hybrid

nanoparticles. Such type of system is effectively used in drug resistant tumor for

the delivery of therapeutic agent and a moiety to reduce the drug resistance (Zeng

34

et al., 2017). Such type of system is formed by intermixing of lipid and polymer.

The core polymer is surrounded by the layer of cationic or anionic lipids.

Hydrophobic drug can be incorporated inside the polymer core. Efforts have been

done to incorporate ionic drug by interaction of drug molecule with the polymer.

The lipid layer provide stability to the system (Wong et al., 2006).

Figure 2.6: Core- shell lipid-polymer hybrid nanoparticles

2.15.2. Polymer caged lipid hybrid nanoparticles

The type of hybrid nanoparticles has an outer polymer layer to achieve the desire

structure integrity and functionality. It depends on the nature of polymer such as

polyacrylic acid can be coated on the outer surface of lipid. In this cases method of

preparation differ as they involve preparation of liposomes followed by coating of

polymer with functional group that can be linked with different moiety to impart

specific properties such pH responsive behavior and to protect liposomes from

different pH environment (Lee et al., 2007).

35

Figure 2.7: Lipid-polymer cage hybrid nanoparticle

2.15.3. Mixed lipid-polymer hybrid nanoparticles

Such types of hybrid nanoparticles are formed by the interaction of lipid with

polymer. The lipid and polymer are entangled with each other via positive and

negative charge throughout the hybrid system (Gao et al., 2008). Sometimes these

are presented as a vesicle like structure but mostly the polymer and lipid and mixed

and uniformly present through the hybrid system. They possess lipoplex like

structure.

Figure 2.8: Mixed lipid-polymer hybrid system

36

2.15.4. Lipid-polymer-lipid hybrid nanoparticles

They usually consist of a first coat of lipid, which is then followed by the polymeric

core and then a coating of lipid on the outer surface (Shi et al., 2011). The shape is

usually depending on the method of preparation such hybrid nanoparticles are usually

formed by double emulsion method. The outer lipid layer is conjugated with Peg,

which provide enhance circulation time and prevent them to be engulfed by

macrophages system and provide more stability to the system (Pautot et al., 2003).

This system is suitable for co-delivery of different drug and drug and siRNA.

Figure 2.9: Lipid-polymer-lipid hybrid nanoparticles

2.16. Factor affecting hybrid nanoparticles

Lipid-polymer hybrid NP is formulated to a certain particle size via different methods

of preparation. The size and shape of nanoparticles is affected by the lipid to polymer

ratio, pegylation, lipid coat, type of polymer.

37

2.16.1. Lipid to polymer (L/P) ratio

Lipid to polymer ratio is prime factor to control the size and shape of particle. To

obtain the appropriate size of nanoparticles and to provide a proper lipid shell over the

core of polymer the ratio of lipid to polymer is of crucial importance. In case of

PLGA-Lecithin nanoparticles, the optimum ratio of lipid to polymer is 15% (w/w) to

obtain the desire size and shape of hybrid nanoparticles increase in lipid concentration

result in liposomes as a separate formulation (Chan et al., 2009). In a different study

the nanoparticle with size of 65nm were prepared with 10% L/p ratio using PEG coat

and PLA as a polymer and lipid shell. PEG prevents the stearic hindrance in absence

of PEG a higher ratio L/P ratio will be required (Zheng et al., 2010).

2.16.2. Lipid coating

The role of lipid coating is of prime importance. On one hand it act as barrier and

increase the encapsulation efficiency by preventing the leakage of drug one the other

hand it provide the controlled release behavior, which is different form polymeric

nanoparticles where polymer is only controlling factor in release of drug the lipid

coating provide additional controlled behavior (Chan et al., 2009).

2.16.3. Pegylation

The ratio of lipid to PEG is very important to provide colloidal stability. In PLGA

nanoparticle the lipid shell is not sufficient to confer the stability and aggregation of

particles occurs. PEG coating provides the stearic stability to the hybrid nanoparticles

at physiological pH. The optimum size of nanoparticles is obtained with lipid-PEG

ratio of 25%. It is important to note that the ratio of PEG does not affect the

encapsulation and release characteristics (Hadinoto et al., 2013).

2.16.4. Nature of Polymer

Polymer being the prime component of hybrid nanoparticles plays a crucial role in the

size, shape and release behavior of hybrid nanoparticles. The density and charge of

the polymer effect on zeta potential of hybrid nanoparticles and is important for the

stability of the hybrid nanoparticles. High density polymer may have high chances for

aggregation. Anionic polymer have more affinity towards cationic lipid like DOTAP

38

and cationic polymers formed the stable conjugation with anionic lipid (Thevenot et

al., 2008).

2.17. Targeted Drug Delivery

Targeted drug delivery system enables the drug to be delivered at the targeted site for

enhanced therapeutic output. Targeted drug delivery could be achieved by passive or

active targeting.

Figure 2.10: Advantage of targeted drug delivery system

For successful targeting the drug has to reach at its target site and retain and evade at

target side and then release drug (Bae and Park, 2011).

2.17.1. Passive Targeting

Passive targeting is mostly achieved in inflammatory diseases like tumor and

inflammatory tissue. Angiogenesis increase the leakiness of tumor vasculature and

cytokines causes enhanced permeability. Angiogenesis represents as irregular

diameter vessels (Oeffinger and Wheatley, 2004). Increased level of bradykinin cause

vasodilation and leads of extravasation and retention of molecules in tumor

(Matsumura et al., 1988). Most of the solid tumors have vascular pore size in range of

400-800nm, while organization of tumor may differ based on type of tumor (Hobbs et

al., 1998).

39

The size of the nanoparticle should be in this range in order to permeate the leaky

vasculature, in contrast the vasculature of normal cells is impermeable to cells greater

than 4nm (Firth, 2002). The nano size vasculature window provides the opportunity

for drug accumulation at the targeted site and reduces the drug distribution to the

healthy tissue. Recent researched have also shown the role of hyperthermia to further

increase the vascular permeability (Meyer et al., 2001). To ensure passive targeting

nanoparticles must circulate for longer times to have maximum possibilities to pass

through the target site. Nanocarrier usually have short circulating life and are

eliminated by the phagocytic system of the body and pegylation increase the

circulating life (Moghimi et al., 2001).

2.17.1.1. Enhanced permeability and retention (EPR) effect

The main concept of passive tumor targeting is based on the EPR effect, this idea was

first proposed by Dr.Maeda (Maeda et al., 1985). The enhance permeability at the

infection site and tumors is related to different factors including secretion of

bradykinin. Bradykinin cause the increase in vascular permeability, some studies

related to finding the root cause of ascites have revealed that bradykinin is also

responsible for extravasation of ascitic fluid (Maeda et al., 2003). Enhanced

permeability also occur in normal infectious tissue but the retention is not enhanced

because of proper functioning of lymphatic system and inflammation usually ends in

couple of days, which in malignant tissue there is enhanced permeation and retention

occur for longer period because of lack of lymphatic drainage system (Maeda et al.,

2001). So, it was recognized that EPR effect could be utilized for targeted drug

delivery to tumor site. EPR effect involves that increased extravasation of large

molecules like albumin from tumor vessels and their retention in tumor tissue for

longer period, which is not observed in healthy tissue (Matsumura and Maeda, 1986).

It was found that nitric oxide also plays its role in enhanced permeability and some

pro-inflammatory mediators such as VEGF (vascular endothelial growth factor) and

prostaglandins. VEGF plays their role to increase the vascular permeability and in

angiogenesis. The role of nitric oxide in tumor growth is also established

(Papapetropoulos et al., 1997). There is also anatomical difference in vasculature of

tumor and normal tissue. Scanning electron images have revealed that the

endothelium in tumor tissue could be 4.7µm in diameter (Hashizume et al., 2000).

40

The EPR effect can be summarized in following points:

1. Increased in leakage from tumor vessel particularly under high blood pressure

condition as vessels lack smooth muscles.

2. Irregular architecture of vessels and large fenestrations.

3. Irregular blood flow in vessels.

4. Poor lymphatic drainage that leads to accumulation of lipid nanoparticles and

macromolecules in tumor interstitium.

5. Decreased venous return that cause increased retention of macromolecules.

6. Extensive angiogenesis at tumor site.

2.17.2. Active targeting

In addition to the leaky vasculature the tumors also express some receptors or

epitopes that can be used as a targeting agent for active targeting of tumor. Therefore,

nanomedicines can be conjugated with these receptor targeting agents and used for the

active targeting (Farokhzad et al., 2004). Active targeting and enhanced cellular

uptake is important for medicines that are not actively taken up by the cells and

required endocytosis or facilitated diffusion to access the active site of cell (Forssen

and Willis, 1998).

Active targeting also increase the bio distribution of medicine specifically within the

tumor (Drummond et al., 1999). Different techniques are being used to couple the

targeting ligand with the nanocarrier. Mostly employed techniques are covalent and

non-covalent conjugation. Covalent conjugation involve the formation of disulfide

bonds, linkage between two amine group and linking of amine group with carboxylic

and aldehyde group (Nobs et al., 2004). Active targeting via use of nanocarrier is

superior to the drug-ligand conjugate. The use of nanocarrier enables the delivery of

higher drug concentration and delivery of payload at the targeted site. Secondly, drug-

ligand conjugate may modify the activity of the therapeutic molecule. Thirdly,

multiple therapeutic agents can be encapsulating inside the nanocarrier (Sethi et al.,

2003).

Different types of targeting agents are used for active targeting such as antibody

targeting, folate targeting, transferrin targeting, aptamer targeting based on the type of

tumor and conjugation chemistry of nanocarrier.

41

Monoclonal antibodies are target specific reagent used for the therapeutic and

diagnostic purpose. These were first agent used for active targeting based on the

surface antigen present on the tumor (Steinhauser et al., 2006). These antibodies can

be conjugated with the nanocarrier to deliver the payload at the targeted site. It is of

prime importance that the functionality of antibody and drug should be preserved till

the desired site of action. Antibody targeted drug delivery increase the bioavailability

of drug at tumor site and provide controlled release profile with enhanced therapeutic

output (Carter et al., 2016). The first ever antibody to target cancer cell was

developed in 1975 but the potential role of monoclonal antibody in cancer therapy

was established after 20 years (Béduneau et al., 2007). Monoclonal antibodies utilize

different types of receptors for targeting such as HER2 receptors, EGFR receptor, and

transferrin receptors.

Figure 2.11: Antibody targeted drug delivery system

Some types of breast cancer overexpress the human epidermal growth factor 2

receptors on the surface particularly it is overexpressed in 25% of invasive breast

tumors. This serves as a targeting agent for monoclonal antibody such as

transtuzumab. HER2 receptors not only overexpressed but also have extracellular

accessibility and well defined mechanism to internalize the antibody after

conjugation, thus suitable to serve targeting receptors for breast cancer (Wartlick et

al., 2004). Transtuzumab serve as active targeting agent specifically for HER2

42

positive breast cancer and its ability to do so have been proven on various HER2

positive breast cell lines in multiple studies (Ruan et al., 2012).

EGFR receptors are significantly overexpressed in various epithelial tumors.

Cetuximab is a monoclonal antibody that has high affinity for EGFR receptors and

bind competitively to these receptors and used for active targeting of tumors with

overexpression of EGFR receptors (Harding and Burtness, 2005). Different studies

have proved the efficacy of cetuximab conjugated with drug loaded nanoparticles to

specifically target the EGFR positive colorectal cancer cell lines and pancreatic

cancer cell lines (Glazer et al., 2010).

Transferrin receptors is ubiquitously expressed in healthy cells and many fold

overexpressed in malignant cells (Daniels et al., 2012). These are also expressed in

endothelial cells of brain. Loperamide is a drug that does not cross the BBB but if it is

loaded in nanoparticles conjugated with transferrin targeting monoclonal antibody

such as OX26 it can be delivered into brain (Ulbrich et al., 2009).

Transferrin is a glycoprotein that function to transport iron into the cell via transferrin

receptor mediated endocytosis. There is 100 fold high expression of transferrin

receptors in drug resistant malignant tumor, this make transferrin promising agent for

targeted drug delivery (Danhier et al., 2010). Different studies have shown liposomes

conjugated with transferrin loaded with docetaxel efficiently deliver their payload at

neoplastic cell expressing high level of transferrin receptors (Kobayashi et al., 2007).

Aptamers are three-dimensional structure made of single stranded RNA or DNA

nucleotide. They have ability to bind specific target site in biological system. They

have high sensitivity for their target and are specific in their actions (Ni et al., 2011).

Aptamer are composed of nucleotide sequence and can easily be conjugated with

cationic nanoparticles. Nanoparticle loaded with docetaxel and conjugated with A10

RNA has shown higher in-vitro cytotoxicity on cell lines as compared to non-

conjugated (Farokhzad et al., 2006).

43

2.17.3. Folate Targeting

Folate is a vitamin B9 and is present in many nutritional sources including vegetables.

It is required for the normal functions of cells. It acts as a mediator in the transfer of

carbon atom and plays an important role in synthesis of purines and pyrimidines that

leads to DNA and RNA synthesis. It is also involved in the methylation of

phospholipids and proteins. The deficiency of folate could lead to faulty methylation

and abnormal DNA synthesis (Choi and Mason, 2000). Folate receptors are widely

known tumor marker with strong affinity for its substrate folic acid. Folic acid is also

important for synthesis of nucleotide basis. Folate receptors also have strong affinity

for folic acid-drug conjugate and folic acid-polymer conjugate. The mechanism

involve for the internalization is endocytosis (Danhier et al., 2010).

Folate is required in rapid proliferating cells for DNA synthesis and methylation. It is

a vitamin and natural supplement for the body (Locasale, 2013). Dysregulation of

folate metabolism is related to improper embryonic development, heart and brain

defects. The mechanism of transport of folate across the cell membrane is through

reduced folate carrier which assist in transport of dietary folate (Matherly and

Goldman, 2003). The other mechanism involve in the transport of folic acid is

through proton coupled folate transporters. Folate can also be transported via four

different types of folate receptor namely (α, β, γ , δ) having molecular weight in range

of 38 to 45 (Ledermann et al., 2015).

Folate alpha receptors are overexpressed in majority of solid tumors, on contrary in

normal tissues folate alpha receptors are confined to low level in the apical surface

(Toffoli et al., 1997). Folate alpha receptors are particularly overexpressed in tumors

of epithelial origin, ovarian, breast and kidney tumors. The highest concentration

being found in tumors of endometrium, ovary and kidney, while breast, pancreatic

and colorectal tumors also have high expression of folate- α receptors. Folate

receptors are overexpressed in 81% of epithelial ovarian tumors and the extent of

expression in correlated to the grading of tumor. High grade serous tumors have

increased expression of folate receptors. Folate receptors are also used as marker for

tumor aggressiveness and is associated with poor disease survival (Siu et al., 2012).

44

Figure 2.12: Folate receptor internalization mechanism

Folate α receptors are also overexpressed in breast cancer, further studies reveal that

high level of expression is found in ER/PR negative breast cancer patients

(O’shannessy et al., 2012). Physiological hormones such as estrogen, progesterone

have been associated with down regulation of folate alpha receptors and there has

been an inverse relation with expression of folate receptors and estrogen/progesterone

hormone. Triple negative breast cancer that represents 15% of all breast cancer has

been associated with high expression of folate receptors (Anders and Carey, 2009).

Folate cycle is involved in key metabolic reactions for proliferating cells. The folate

taken as a vitamin supplement is reduced and transported inside the cells via folate

carriers. After reaching inside the cells folate play key role in the synthesis of DNA

base pairs purines and pyrimidines (Zwicke et al., 2012).

In the malignant tumors, the folate receptors lose their polarized location and spread

over cell surface of tumor (Kane et al., 1988). Folate receptors are overexpressed in

majority of solid tumors and in 45% of squamous cell tumors and in 40% of lymph

node metastasis. Folate receptors are further divided into alpha and beta forms. The

α-folate receptors are believe to be overexpressed in 40% of tumors , while β-folate

receptors are found in malignant tumors of hematopoietic origin (Low and Kularatne,

2009). Folate receptors have particularly more attraction because these are expressed

at the surface of the receptors. Studies have shown that oral cancer cells incubated

45

with folate conjugated liposomes have shown 31 times more fluorescence compared

to the conventional liposomes (Yang et al., 2014). In non-malignant tissue, the folate

receptors are present on the luminal surface of epithelial cells are inaccessible to

circulation, while in the malignant cells, these cover the surface of the tumor and are

approachable to circulation. Folate receptors also have ability to bind with the folic

acid and can be penetrated inside the tumors via endocytosis. Folic acid can be

conjugated with variety of therapeutic agents to deliver drug in a targeted manner

(Sega and Low, 2008). Recent studies show that folate receptor is present on the

surface of tumor even after the chemotherapy, as it is expressed in a specific manner

at the surface of tumor, thus providing the opportunity to serve as a therapeutic and

diagnostic tool (Salazar and Ratnam, 2007). Currently, different therapeutic agents

utilizing folate targeting are under clinical development. Two therapies that target

folate receptor are under phase-III clinical trial are farletuzumab (Konner et al., 2010)

and vintafolide (Naumann et al., 2013).

Table 2.1: Summary of clinical development of folate targeted ovarian cancer drugs

Trial phase Patient

group

Treatment arm Primary end

point

Result

I Platinum

resistant

ovarian

cancer

Farletizumab Safety of drug

and

tolerability in

patients

Dose can be

increased to

40 mg/m2

III Platinum

resistant

epithelial

tumor of

ovary and

fallopian

tube

Farletizumab+

Paclitaxel

Versus

Farletizumab

Progression

free survival

and overall

survival

No study

results

available

III Platinum

sensitive

ovarian

cancer

Farletizumab in

low dose+

Carboplatin+

Taxane

Vs

High dose

Farletizumab+

carboplatin+taxane

VS

Carboplatin+taxane

Progression

free survival

Some patients

have shown

difference in

progression

free survival

46

I Metastatic

solid tumor

patients

Vintafolide (Low

dose)+ Platinum

drug+ taxane

Vs

Vintafolide (Low

dose)+ Platinum

drug+ taxane

Dose limiting

toxicity

Ongoing

study

I Recurrent

solid tumors

Vintafolide bolus

dose

Vs

Vintafolide IV

infusion over one

hour

Maximum

tolerable dose

2.5 mg as

MTD in both

IV bolus and

IV infusion

group.

Constipation

was dose

limiting

toxicity.

II Platinum

resistant

folate

receptor

positive

ovarian

cancer

Vintafolide To observe

clinical

benefit after 6

cycle of

chemotherapy

without

progression of

disease

5% partial

response.

37% stable

disease

43% disease

control rate

(Morris et al.,

2014).

Farletezumab (A monoclonal antibody) bind selectively to the folate alpha receptors

as a tumor marker and cause immune dependent cytotoxicity. It has high affinity for

the folate alpha receptors and non-selective to healthy cells (Ebel et al., 2007).

Vintafolide is a combination of conjugated folate and vinca alkaloid. This is

selectively taken up by the folate receptors present on the surface of tumors and does

not bind to non-malignant cells, hence providing targeted drug delivery to tumor cells

and avoiding side effect on healthy tissues (Reddy et al., 2007).

Therefore, folate targeting is recent concept and is under clinical trials with different

combinations. Here, we have provided a novel combination of folate conjugation with

lipid covering to provide excellent bioavailability and enhanced therapeutic efficacy.

47

Lipid-Chitosan Hybrid

Nanoparticles for Controlled

Release of Cisplatin

Synthesis and Physicochemical

Characterization

CHAPTER 3

48

3.1. Background

Chemotherapy is an established way of treatment of various forms of cancer. The

important task is to deliver chemotherapeutic agent to the targeted cells and avoiding

side effects on the normal cells (Dayan et al., 2012). Chemotherapy is associated with

various side effects, some studies have shown that 15% of patients suffer from

cognitive function impairment after treatment with conventional chemotherapy,

sometimes called as ―Chemofog‖ (Mehnert et al., 2007). The poor solubility of most

of chemotherapeutic agents and dose related adverse effects instigate the development

of new drug delivery techniques. The use of nanoparticle technology has served as a

tool to avoid the serious side effect on the normal body tissues and delivery the drug

in a controlled manner (Zheng et al., 2015).

The concept of nanocarrier drug delivery was first generated by Sir Paul Ehrlich in

1905 and he termed it as a ―magic bullet‖ which is now termed as ―magic guns‖.

These nanocarrier are effective for enhanced drug loading and site-specific delivery of

drugs (Kumar et al., 2016). The new trend is to use biocompatible, biodegradable

polymers, which have ability to deliver the drugs in controlled and sustained manner

(Tang and Singh, 2009). Two established system of nanocarrier drug delivery system

are polymeric nanoparticles and liposomal drug delivery system (Mieszawska et al.,

2013).

Liposomes are biocompatible and they have enhanced permeability to the lipid based

biological membrane as compared to polymer nanoparticles (Torchilin, 2005).

However they have drawback of poor stability and leakage of drug could occur, they

required storage at controlled temperature (Crist et al., 2001). Liposomes also have

drawback of rapid clearance from the body because of their uptake by phagocytic

cells and pegylation is usually done to enhance their circulation life and to protect

them from phagocytosis (Aad et al., 2012). The methoxy chain in polyethylene glycol

play its role in preventing the activation of defense mechanism of body (Salvador-

Morales et al., 2009).

Polymeric nanoparticles have enhanced encapsulation efficiency and prolong

retention time and suitable vehicle to improve the bioavailability of drugs (Mathew et

al., 2017). They are superior in respect of controlled drug release and targeted

49

delivery of drug. Different kind of targeted moieties can be attached to polymeric

nanoparticles and targeting depends on the size , shape and nature of polymer being

used (Brannon-Peppas and Blanchette, 2012). Polymeric nanoparticles generally have

better cellular internalization, further it depends on the charge of the nanoparticles.

Cationic charge is usually favorable because it can interact with cell membrane and

enhance the internalization of nanoparticles (Chaudhary et al., 2018, Feng, 2004).

Despite of several advantages they have few drawbacks such as not suitable for

delivery of protein and peptides and particle aggregation could occur and release of

drug in acidic environment is aberrant from polymeric nanoparticles (Mohammadi-

Samani and Taghipour, 2015).

Recent technology to overcome the drawbacks of liposomal drug delivery system is to

combine them with polymeric nanoparticles to have structural advantages of the

polymer and to deliver the drug in efficient way at the targeted site of action (Prabhu

et al., 2015). LPHNPs have combined advantage of liposomal and polymeric drug

delivery system. LPHNPs can deliver the drug in controlled and sustained manner

because of polymeric core and enhanced biocompatibility due to liposomal drug

delivery (Chaudhary et al., 2018).

Chitosan is a biodegradable polymer with high degree of acetylation and it is being

used in sponges and sutures for the treatment of wounds. Chitosan undergoes

enzymatic hydrolysis and products are nontoxic and easily excreted form the body. It

is biocompatible and found applications in controlled release of drugs. The rate of

release is also affected by the degree of cross linking and molecular weight of drug

being used. Because of cationic charge it form complexes with negatively charged

lipids via ionic gelation and also suitable for delivery of siRNA and DNA (Stolnik et

al., 1995).

Cisplatin is a potent chemotherapeutic agent that crosslink with DNA and cause

damage to DNA and stop the proliferation of malignant cell but its maximum

therapeutic output is limited due to its severe side effects on renal and hepatic profile

(Rosenberg, 1985). Cisplatin also have less solubility in both hydrohphillic and

hydrophobic environment that limit the development of nanoparticles with excellent

drug loading. Cisplatin has low molecular weight and is rapidly cleared by the body.

50

We have produced chitosan-lipid hybrid nanoparticles for controlled delivery of

cisplatin and enhanced therapeutic output (Khan et al., 2019).

3.2. Materials and Methods

3.2.1. Materials

Low molecular weight chitosan was purchased from Sigma Aldrich. Lipid was kindly

provided by LIPOID®. Zeta Sizer (Malvern, UK). Cisplatin was received as a gift

sample from Pharmedic(PVT) Ltd. Ethanol and acetic acid were purchased from

Thermo Fisher Scientific.

3.2.2 Method of preparation

The nanoparticles were prepared by ionic gelation method as described by F. Sonvico,

et al (Sonvico et al., 2006). Briefly, 5 mg of chitosan was dissolved in 46 ml of acetic

acid solution by overnight stirring and cisplatin was dissolved in that solution. 100mg

of lipid was dissolved in 4ml of ethanolic solution and then ethanolic solution was

added drop wise in the chitosan solution and allowed to stir for 30 min at 10,000 rpm.

The lipid-chitosan hybrid nanoparticles were formed via ionic interaction of chitosan

and lipid. The nanoparticles were prepared with different concentration of lipid and

polymer to further characterize them. The lipid to chitosan ratio was varied from 5:1

to 60:1 in different formulation to optimize the formulation for further

characterization.

51

Figure 3.1: Schematic diagram of lipid-chitosan hybrid nanoparticles

3.2.3. Size and Zeta potential of hybrid nanoparticles

The prepared LPHNPs were analyzed for size, surface charge, polydispersity index

using Zeta Sizer (Malvern, UK). The 50 µl sample was used in a cuvette for the

determination of size and it was diluted with 950 PBS for the measurement of zeta

potential. The readings were performed at 25°C in triplicate.

3.2.4. Determination of drug loading and entrapment efficiency

The entrapment efficiency was determined by the indirect method, by measuring the

amount of unbound drug in the supernatant liquid after the completion of

centrifugation. The drug contents were determined by using UV Spectrophotometer

(Spectrum Scientific). The absorbance of standard and samples were measured at

210nm. The drug was dissolved in PBS at pH 7.4. Suitable dilutions were made from

the supernatant liquid to measure the drug contents. The experiment was performed in

triplicate. The entrapment efficiency and drug loading were determined by the given

formula.

Encapsulation efficiency = amount of entrapped drug / theoretical amount of drug

×100

Percentage drug loading will be calculated by following equation;

Drug loading = Mass of drug in nanoparticles / mass of nanoparticles × 100

52

3.2.5. Morphology of hybrid nanoparticles

Morphology of the cisplatin loaded hybrid nanoparticles was determined by using the

transmission electron microscope (TEM) (Jeol, USA). The prepared nanoparticles

were applied directly on the grid. The excess of sample was removed from the grid

and suitable images were taken at different magnifications.

3.2.6. Fourier transform infrared spectroscopy (FTIR)

The FTIR analysis was performed to evaluate any possible interaction between the

components of the formulation and to ascertain stability within these components by

using Thermo scientific Nicolet iS5 FTIR Spectrometer. Chitosan-lipid hybrid

nanoparticle formulations as well as the individual components lipid, polymer and

cisplatin were analyzed. Graphs were compared among the formulations to detect any

measurable change in the peaks.

3.2.7. Powered X-Ray diffraction analysis (X-RD)

X-RD analysis was performed by using the Xpert Pro Super, PANalytical ((JDX

3532, JEOL, Japan). The sample slides were prepared for the chitosan-polymer hybrid

nanoparticles formulation and the individual components including the lipid, chitosan,

cisplatin and physical mixture. The diffraction was recorded at ambient temperature

with 2θ range of 10-80.

3.2.8. Differential scanning calorimeter (DSC)

DSC analysis was carried out to evaluate the physical form of lipid-chitosan hybrid

nanoparticles. Indium was used for calibration of heat flow and temperature. The

Aluminum pan was used as a reference while other pan was used to analyze the

samples. The samples were heated from 25-450°C at a rate of 11°C/min.

3.2.9. Thermo-gravimetric analysis (TGA)

Thermo gravimetric analysis was performed to measure the change of mass over a

range of temperature. 38 mg of LPHNPs were used and individual components of

LPHNPs such as cisplatin, chitosan, and lipid were put on the gravimetric analyzer

and the temperature was run from 25- 450°C. The loss weight during run time was

recorded for each second.

53

3.2.10. In vitro drug release studies

The drug release study was conducted by using dialysis bag method. The dialysis

membrane of MWCO 10-12 KDa was used. Six different LPHNP formulations were

packed in different dialysis bags and the release was compared with cisplatin solution.

Phosphate buffer saline (pH 7.4) was used as a dissolution medium and temperature

was maintained at 37°C±0.5 with stirring speed at 100 rpm. Samples were withdrawn

at pre-determined time interval and similar amount of fresh medium was added to

maintain the final volume throughout the experiment. Kinetic modeling was used zero

order, first order, higuchi and korsmeyerpeppas models were applied.

54

3.3. Results

3.3.1. Size and surface charge

Size and PDI are important parameters for the nanoparticles, as they effect on the

loading and release of drugs from the formulation. Nanoparticles should have

appropriate size and low polydispersity index (Souza et al., 2014). The particle size of

all 6 formulations varies between 180 nm to 250 nm, which is suitable for passive

targeting of tumor through EPR effect (Cho et al., 2008). The surface charge of 5:1

ratio of lecithin-chitosan ratio to 30:1 ratio remains positive due to electrostatic

interaction of chitosan and lecithin but as the concentration of lipid is increased

further the zeta potential shift from positive to negative value and the formulation of

ratio 60:1 showed poor stability and as the ratio of lecithin is increased further the

formulation showed poor stability (Hafner et al., 2011).

The lecithin to chitosan ratio 60:1 showed that the aggregation of particles occurs

soon after the formation of nano-suspension that occurs due to imbalance of

electrostatic charges and increase in overall negative charge due to higher

concentration of lipid. The ratio of 10:1 and 20:1 showed lower PDI of 0.1 and 0.2

respectively and zeta potential values also showed better stability in these two

formulations. This might be due to proper interaction of negative and positive charges

in appropriate concentration. This already been described in literature (Sonvico et al.,

2006). The particle size and zeta potential of all formulation is shown in Table 3.2.

These results indicate that LPHNPs with lipid to chitosan ratio of 20:1 is most suitable

having particle size of 181nm and low polydispersity index and suitable for further

analysis of morphology of nanoparticles.

55

Table 3.1: Composition of cisplatin loaded lecithin-chitosan hybrid nanoparticles

Formulation

Code Lipid Polymer Drug Ethanol

5:1 50mg 10mg 5mg 2ml

10:1 50mg 5mg 5mg 2ml

20:1 50mg 2.5mg 5mg 2ml

30:1 75mg 2.5mg 5mg 3ml

40:1 100mg 2.5mg 5mg 4ml

60:1 200mg 3.35mg 5mg 8ml

Table 3.2: Effect of lipid to polymer ratio on particle size and surface charge

Formulat

ion Code Lipid Polymer Drug

Particle

Size

(nm) PDI

Surface

charge

(mV)

5:1 50mg 10mg 5mg 213±1.5 0.3 37.1±1.1

10:1 50mg 5mg 5mg 218±2.1 0.2 30.5±1.2

20:1 50mg 2.5mg 5mg 181±0.8 0.2 21.1±0.5

30:1 75mg 2.5mg 5mg 200±1.1 0.3 20.5±0.9

40:1 100mg 2.5mg 5mg 189±1.6 0.4 -19.8±1.2

60:1 200mg 3.65mg 5mg 245±2.1 0.5 -9.8±1.4

56

3.3.2. Morphology of nanoparticles

Morphology of the lecithin-chitosan hybrid nanoparticles was studies by using TEM.

The images showed the spherical shaped nanoparticles of around 200nm with the core

shell morphology of the lipid and the polymer present inside the hybrid particles. The

size of the particle was in accordance with the size determined by DLS. The lipid and

chitosan are entangled with each other through ionic interactions. The core of polymer

and lipid shell is entangled with each other to form a spherical lipoplex like structure.

The images of hybrid nanoparticles are shown in figure 3.2.

Figure 3.2: Transmission electron microscopy images of lipid-chitosan hybrid

nanoparticles

3.3.3. Entrapment efficiency and Drug loading

The entrapment of drug in lipid-chitosan hybrid nanoparticles was measured by

indirect method i.e. by calculating the amount of un-entrapped drug in the

nanoparticles. Lipid-polymer nanoparticles with ratio of 20:1 showed the highest

encapsulation efficiency (89.54%), while 60:1 showed the lowest encapsulation

efficiency of the nanoparticles. The low entrapment in 60:1 Formulation is attributed

to the greater concentration of lipid as compared to chitosan and hence imbalance of

positive and negative charges and weaker electrostatic interaction (Chuah et al.,

2009). The formulation 30:1 showed the highest drug loading that may be due to

presence of greater number of particles with multilamellar structure (Gerelli et al.,

57

2008). The hybrid nanoparticles demonstrate significantly increased encapsulation

and drug loading as compared to polymeric nanoparticles, owing to the presence of

lipid layer (Zhang and Zhang, 2010). The effect of lipid to polymer ratio of

entrapment and drug loading is shown in Table 3.3.

Table 3.3: Effect of Lipid to Polymer ratio on entrapment efficiency and Drug

Loading

Formulation

Code Lipid Polymer Drug

Entrapment

Efficiency

Drug

Loading

5:1 50mg 10mg 5mg 83.18±1.2 1.69±0.6

10:1 50mg 5mg 5mg 83.81±2.3 1.86±0.4

20:1 50mg 2.5mg 5mg 89.58±1.1 2.07±0.2

30:1 75mg 2.5mg 5mg 89.44±2.5 2.11±0.5

40:1 100mg 2.5mg 5mg 73.87±2.1 1.75±0.3

60:1 200mg 3.65mg 5mg 73.17±1.9 2.15±0.8

3.3.4. FTIR spectroscopy

The lipid-polymer hybrid nanoparticle formulation was analyzed by FTIR to check

the compatibility of excipients used in the formulation (Figure 3.3). The FTIR of pure

drug cisplatin, chitosan, lipid and nanoparticle formulations were carried out to check

the compatibility among structural components. The FTIR spectra of cisplatin showed

a typical band at 3275.4 cm−1

stretch of –NH3 group (Neault and Tajmir-Riahi, 1998).

The FTIR spectra of chitosan exhibited a typical N-H stretch at 3285.62 cm−1

and a C-

O-C band stretch at 1025 cm−1

(Pawlak and Mucha, 2003). The spectra of lipid

showed an O-H stretch at 2922.25 cm−1

and C=O stretch at 1735.24 cm−1

and a

asymmetric C-O-C band stretch at 1060.06 cm−1

(Whittinghill et al., 2000).The FTIR

spectra of the lipid-chitosan formulation at 3273.62 cm−1

predicted the presence of

cisplatin and chitosan in the formulation and exhibited an extended peak due to

presence of N-H group in both cisplatin and chitosan. The formulation peak at

1637.26 cm−1

and 1044.99 cm−1

indicated the presence of lipid in the formulation.

The N-H of chitosan shifted from 3285.62 cm−1

to 3273.62 cm−1

in the formulation and

58

the C=O stretch shifted from 1735.24 cm−1

to 1637.26 cm−1

in the formulation and the

C-O-C band shifted from 1060.06 cm−1

to 1044.99 cm−1

. The absence of any new

peak in the formulation indicated that there was no chemical change during

entrapment of drug in the lipid-polymer hybrid nanoparticles and that the different

structural components are compatible with each other.

Figure 3.3: FTIR Spectra of individual components and lipid-polymer hybrid

nanoparticle formulation

3.3.5. XRD Studies

The promising sharp peaks of cisplatin were identified at 15,28, 46 and 70 2θvalues,

which indicated that cisplatin is of crystalline nature (Jayasuriya and Darr, 2013).

The broader peak between 15 and 25 depicted the amorphous nature of lipid (2 θ

scattering) (Lavanya et al., 2016). The characteristic peak of chitosan at 20 (2 θ value)

illustrated its crystalline nature (Pang and Zhitomirsky, 2005). Similarly, the sharp

peaks at 15 and 28 appeared in the physical mixture also portrayed crystalline nature

of cisplatin, However, reduction in intensity and diffused sharp peaks of cisplatin and

chitosan were observed in the lipid-chitosan formulation, illustrating encapsulation of

cisplatin the hybrid nanoparticles. These results were also in good agreement with the

59

findings of DSC and FTIR that confirmed the absence of any significant interaction

among all formulations components.

Figure 3.4: X-ray diffraction analysis of components and LPHNPs

3.3.6. Differential Scanning Calorimetry (DSC)

The DSC analysis was performed to check the crystalline/amorphous nature of drug

and components in lipid-chitosan hybrid nanoparticles. The cisplatin loaded LPHNPs

exhibit a typical endothermic peak at 102°C, this is due to the evaporation of water

associated with the drug. The cisplatin pure drug showed a dehydration peak at

around 100°C due to water associated with it and then an endothermic peak at 270°C,

which related to the melting point of the drug (Dixit et al., 2015). However there is no

sharp endothermic peak of cisplatin in the nanoparticle formulation, which showed

that the drug has loose its crystalline behavior in the formulation, as also reported in

previous studies (Dixit et al., 2015). The curve of chitosan typically showed the broad

endothermic peak in temperature range of 70-150°C, this corresponds to the loss of

water of crystallization and melting point of the chitosan (Cervera et al., 2011). The

thermal decomposition of chitosan starts at 300°, which is an exothermic process and

its peak is seen at around 320°C (Drebushchak et al., 2006). This behavior of chitosan

is also seen in the formulation with endothermic peak starting at 70°C and broader

endothermic peak in the formulation is due to presence of chitosan and lipid and

cisplatin converted to amorphous form. Therefore, no physical interaction between the

components was seen.

60

Figure 3.5: Differential Scanning calorimetry graph of LPHNPs and components

3.3.7. Thermogravimetric analysis (TGA)

TGA was performed to measure the percentage of weight loss over time at certain

range of temperature. TGA of cisplatin, chitosan and lipid and formulation was

61

performed. The results showed that the weight loss of cisplatin occur at 270 °C, which

corresponds to the melting point of drug (Prodana et al., 2014). Chitosan showed

slight weight loss at 70°C and then at around 290°C there is gradual weight loss

(Javaid et al., 2018). Weight loss in lipid mixture also starts at 250°C. The melting

points of all three components differ from each other and in formulation there is

combined peak of weight loss in the same range, which also confirms absence of any

physical interaction between components and thermal stability of the formulation.

Figure 3.6: Thermo gravimetric analysis of cisplatin loaded lipid-chitosan hybrid

nanoparticles

63

3.3.8. In vitro Dissolution Studies

Drug release studies were performed in phosphate buffer saline pH 7.4 at 37°C and

100rpm using the dialysis membrane method (Jeong et al., 2008). None of the

formulation showed burst release, studies on release pattern of polymer-lipid hybrid

system also showed absence of immediate burst release and hence drug can be

released in controlled manner (Kim et al., 2008).

The delayed release in 40:1 and 60:1 formulation might be due to excessive lipid layer

over the polymer, which retard diffusion of drug (Kim et al., 2008). After that there is

a sustained release over 24 hours from the lipid-polymer complex (Mandal et al.,

2016). Kinetic modeling was applied to the cisplatin release data including zero order,

first order, higuchi and korsmeyerpeppas and the results showed that best fit model

for the formulation is korsmeyerpeppas model which is usually followed in lipid-

polymer hybrid system (Tran et al., 2015). The controlled release of drug is attributed

to lipid layer and minor contribution of polymer and change is concentration of lipid

effect the rate of release of drug from formulations (Chan et al., 2009). The value of n

suggest that the drug follow super case II transport mechanism in most of the

formulation, the release mechanism if different in 40:1 and 60:1 due to higher

concentration of lipid and imbalance of positive and negative charges (Sonawane et

al., 2016). The drug release pattern of different formulation is shown in Figure 3.7

and kinetic modeling is shown in Table 3.4.

64

Figure 3.7: In vitro drug release profile of LPHNPs with various ratios of lipid to

polymer.

65

Table 3.5: Kinetic Modeling of Drug Release profile of Cisplatin loaded Lecithin-

Chitosan Hybrid Nanoparticles

Formulation Zero Order

R2

First

Order

R2

Higuchi

Model

R2

Korsmeyer

Pepas

Model

R2

n

5:1 0.6626

0.7324

0.5855

0.8930

1.750

10:1 0.6845

0.8700

0.8333

0.8457

1.591

20:1 0.7054

0.8401

0.7536

0.9069

1.067

30:1 0.7538

0.7686

0.6899

0.8846

1.187

40:1 0.1375

0.8731

0.5040

0.9478

0.350

60:1 -0.0727

0.8412

0.3954

0.9686

0.322

Drug 0.6643 0.7248

0.6600

0.9887

0.253

66

3.5. Discussion

Lipid polymer hybrid nanoparticles have the advantage of both polymeric and

liposomal drug delivery systems and have the ability to entrap both hydrophilic and

hydrophobic drugs. The chitosan-lipid hybrid nanoparticles have been formulated for

controlled delivery of cisplatin and to achieve maximum therapeutic benefit. The

formed LPHNPs were evaluated by both physicochemical tests.

Physicochemical test include size, entrapment, morphology, compatibility among the

excipients and thermal stability and in vitro release profile. Size and polydispersity

index have effects on the loading and release of drug from the nanoparticle

formulation (Souza et al., 2014). LPHNPs were formulated with various ratio of

chitosan to lipid, at a ratio of 20:1 they showed mono-dispersity and a better stability

profile because of an interaction of negative and positive charges in appropriate

concentration (Sonvico et al., 2006). The comparative low entrapment in the 60:1

formulation is attributed to the greater concentration of lipid as compared to chitosan

and hence an imbalance of positive and negative charge and a weaker electrostatic

interaction (Chuah et al., 2009). The 30:1 formulation showed the highest drug

loading. This may be due to the presence of a multiple layered structure and strong

interaction of positive and negative charges (Gerelli et al., 2008).

The images exhibited a core shell morphology with some lipid covering which

prevented diffusion of drug and water penetration into the system the shell of

chitosan-lipid provides long circulating characteristics (Mandal et al., 2013). The

absence of any new peak in the formulation indicated that there was no chemical

change during entrapment of drug in the lipid-polymer hybrid nanoparticles and that

the different structural components are compatible with each other. The release

pattern of the polymer-lipid hybrid system also showed an absence of immediate burst

release and suggested that drug can be released in a controlled manner.

The lipid-polymer hybrid nanoparticles with drug distributed inside the polymer

showed a controlled release profile. The release of drug from the polymer matrix

depends on diffusion. Further lipid layering prolongs the drug release (Li et al., 2008).

The value of release component (n) suggests that the drug follows a super case II

transport mechanism in most of the formulations. The release mechanism is different

67

in 40:1 and 60:1 due to a higher concentration of lipid and imbalance of positive and

negative charges (Sonawane et al., 2016). The results indicated that best fit model for

the formulation is a korsmeyerpeppas model which is usually followed in lipid-

polymer hybrid systems (Tran et al., 2015).

Cisplatin loaded lipid-chitosan hybrid nanoparticles were successfully fabricated. The

best formulation with a suitable combination of lipid and chitosan showed mono-

dispersity, small size and a controlled release profile. Further release of drug was

affected by an increase in the lipid concentration. The rate of release of drug was

attributable to both polymer and lipid. The release of drug is controlled by the

polymer matrix and further by a lipid layer that prevents leakage of drugs. There was

an absence of burst release of cisplatin due to its entrapment in the inner polymer

layer and outer lipid covering.

68

3.6. Conclusion

The physicochemical tests concluded that cisplatin loaded lipid-polymer hybrid

nanoparticles with ratio of lipid to chitosan (20:1) have core shell morphology with

high drug encapsulation efficiency and loading capacity. The components are

compatible with each other and cisplatin is converted from crystalline to amorphous

form in LPHNPs. The LPHNPs showed excellent thermal stability and a controlled

drug release profile. The lipid-chitosan hybrid nanoparticles with ratio of (20:1) will

be used for further biological characterization.

69

Lipid-Chitosan Hybrid

Nanoparticles for Controlled

Release of Cisplatin

Biological characterization

CHPATER 4

70

4.1. Background

In previous studies, we have prepared cisplatin loaded folate-chitosan hybrid

nanoparticles and were characterize for size, potential, shape, drug release and

thermal studies. The optimized formulation is now required to be tested on the ovarian

cell lines to establish the therapeutic efficacy of prepared hybrid nanoparticles and

biocompatibility of blank hybrid nanoparticles.

Cell culture is the growth of cells in artificial environment and is recognized for

studying the physiology of cells and effect of drug on particular cell lines. Every cell

type grow in specific medium that contain all necessary nutrient required for the

growth of that cell lines (Antoni et al., 2015). Cells grow as a monolayer attach to the

surface of flask and some cell lines grow in the suspension form and growth of

particular cell line depends on its cycle of exponential growth. Cell lines are used for

variety of studies to establish the efficacy of drug and to check the uptake of

nanoparticle system inside the specific cell line (Ravi et al., 2015).

The lipid-chitosan hybrid nanoparticles are hybrid system made up of chitosan as a

polymer, which is biocompatible and have structural integrity. The lipid is component

of biological membrane and enhances the biocompatible properties. Previous studies

of lipid-polymer nanoparticles show the biocompatible nature of nanoparticles

(Tezgel et al., 2018).

Ovarian cell line A2780 is extensively used to evaluate the efficacy of drug delivery

system. A2780 are of epithelial origin and grow as a monolayer. Cisplatin is first line

agent in the treatment of ovarian cancer. Cisplatin loaded lipid-chitosan hybrid

nanoparticles were evaluated by using A2780 cell lines (Wojewódzka et al., 2019).

OECD has issued guidelines for the conduct of acute oral toxicity studies to establish

the safety of polymer and lipids being used in large quantity. These studies enable the

evaluation of target organ and clinical symptoms and any hematological changes

observed during the high dose of drug delivery system (Jonsson et al., 2013).

71

4.2. Material and Methods

4.2.1. Material

The Cell titer blue for cytotoxicity studies was purchased from Promega®. A2780

ovarian cell lines were purchased from Sigma Aldrich. RPMI media for growth of

A2780 cell lines and fetal bovine serum and antibacterial and antimycotic

solutions were purchased from Cell Gro (VA, USA). Rabbits and Rats were

obtained from animal house of The Islamia University of Bahawalpur.

4.2.2. Cell viability

Cell cytotoxicity studies were carried out using A2780 ovarian cancer cell lines.

Cells (5000) were seeded in each well of 96-well plate. After overnight

incubation, cells were treated with blank LPHNPs, cisplatin-loaded LPHNPs and

cisplatin solution at a cisplatin concentration range of 1.25 to 50 µg/ml to check

the effect at different concentrations (Wang et al., 2014). Formulations were

washed out after 4 hours treatment and replaced with fresh RMPI media. The

effect on cytotoxicity was observed at 20 and 44 hours after treatment using a Cell

TiterBlue®

assay following manufacturer instructions.

4.2.3. Fluorescence microscopy

Microscopic slides were carefully placed in each well of 12 well plates and cells

(100,000) were seeded a 12-well plate. After overnight incubation cells were

treated with the cisplatin loaded LPHNP formulation labeled with Rh-PE and a

control blank formulation for 4hours. After four hours, cells were washed with

phosphate buffer saline (pH 7.4) and fixed with same solution containing 4%

paraformaldehyde (PFA) for 30 min at room temperature. Cells were then washed

with PBS (pH 7.4) three times and stained with 10 µg/ml Hoechst 33342 in PBS

pH 7.4 for 15 min. Cells were washed again with PBS, pH 7.4, and mounted on

microscope slides with mounting buffer for analysis by keyence fluorescence

microscope.

4.2.4. Cellular uptake

The uptake of cisplatin loaded LPHNP was evaluated using flow cytometry

(Beckton Dickinson FACS Calibur™, NJ, USA). Cells (500,000) were seeded in

72

each well of 6-well plate. After 24-hour incubation, cells were treated with Rh-

123 containing formulations (0.5mol %) for 4h. After completion of incubation

period cells were detached using trypsin and washed with PBS, pH 7.4, three

times and centrifuged at 2500 rpm for 5 min and then re-suspended in 300µl of

PBS. This solution was analyzed by flow cytometry and a total of 10,000 gated

live cell events were collected.

4.2.5. In vivo toxicity

Acute toxicity studies were conducted following approval from the research ethics

committee of the faculty of pharmacy and alternative medicines Islamia

University Bahawalpur. Female white Albino rats were selected for acute toxicity

studies according to guidelines of Organization of Economic Cooperation and

development (Oecd, 2000). Rats were divided into three groups. Female rats are

more sensitive hence selected for the toxicity studies. One group served as a

control and another was treated with lipid-polymer hybrid nanoparticles as a blank

formulation at the dose of 2000 mg/kg to check the toxicity of the components.

The third group was treated with cisplatin loaded lecithin-chitosan hybrid

nanoparticles at a dose of 30mg/kg. The signs of toxicity were evaluated after 14

days of treatment.

4.2.6. In vivo pharmacokinetic

Twelve healthy rabbits were selected for the pharmacokinetic studies (average

weight, 2.4 ± 0.4 kg). Rabbits were divided into two groups. One group was

administered 4mg/kg of the cisplatin drug solution as a reference while the other

group received 4mg/kg of the cisplatin loaded lecithin-chitosan hybrid

nanoparticles. Rabbits were kept on fasting overnight before the start of the study.

Blood samples were collected at defined time intervals over a period of 24 hours

and centrifuged immediately to separate plasma. Plasma was stored at -20°C. The

samples were treated to separate protein and 20 µl samples were used to determine

drug concentration using HPLC (Huo et al., 2005). The mixture of acetonitrile:

Water: Methanol (30:30:40) was used as a mobile phase and detector was adjusted

at 210nm for flow rate was 1.5ml/min

73

4.3. Results

4.3.1. Cell viability studies

The cytotoxicity studies were carried out using cell titer blue by measuring

fluorescence from cells using a plate reader. The reading from plate reader indicated

that blank chitosan-lipid hybrid nanoparticles have no cytotoxic effect on the cells and

are safe. The readings were taken after 24 hours and 48 hours of incubation period.

The results after 24 hours period showed that there is not much significant difference

in the cytotoxic effect of cisplatin drug solution and cisplatin loaded lipid-chitosan

hybrid nanoparticles. This is due to slow release of drug from the LPHNPs. The

cisplatin solution showed more cytotoxic effect only at one concentration of

12.5µg/ml as compared to cisplatin LPHNPs and at remaining all concentration range

there was not any significant difference in the cytotoxic effect of cisplatin loaded

LPHNPs on A2780 ovarian cell lines. This is due to easy access of cisplatin solution,

while in LPHNPs the drug in embedded inside lipid and polymer layers.

The reading from second 96 well plate was measured after 48 hours and the results

showed that the cisplatin loaded lipid-chitosan hybrid nanoparticles have significantly

higher cytotoxic effect as compared to cisplatin solution particularly at concentration

of 6.2µg/ml, while at concentration of 3.1µg/ml and 1.6 µg/ml there is also significant

higher toxicity in cisplatin LPHNPs as compared to cisplatin. The significant higher

cytotoxic effect after 48 hours showed that cisplatin loaded LPHNPs release the drug

in a controlled manner and significant higher effect are shown after 48 hours of

incubation period.

74

Figure 4.1: Cytotoxicity studies of cisplatin LPHNPs compared with cisplatin solution

and blank LPHNPs on A2780 cell lines after 24 hours of treatment

75

Figure 4.2: Cytotoxicity studies of cisplatin LPHNPs compared with cisplatin solution

and blank LPHNPs on A2780 cell lines after 24 hours of treatment

76

4.3.2. Cellular uptake Studies

The cell uptake studies were carried out to measure the quantitative uptake of

nanoparticles by the A2780 ovarian cell lines. The lipid-chitosan hybrid nanoparticles

loaded with rhodamine-123 were prepared and cells were treated with same, while

one well of 6-well plate was kept as a control. The results were measure using flow

cytometry, which indicate that there is 8-times more uptake of nanoparticle loaded

with fluorescence dye Rh-123 as compared to the control. The results were measured

in triplicate for each sample.

Figure 4.3: Cellular uptake of LPHNPs by A2780 cell lines

77

4.3.3. Cellular association

The cell association of the nanoparticles was also observed by staining with the

fluorescent dye and observing under Keyence fluorescence microscope. Hoechst is

used for staining nucleic acid and it produces blue color after interaction with DNA of

the cell. Rhodamine PE produces the red color fluorescence in the cells. It can be

predictive from the images that nanoparticle showed internalization in A2780 ovarian

cancer cells lipid present in the LPHNP may facilitate the internalization by

interacting with the lipid layer at the surface of cell membrane (Guo et al., 2015). The

cells exhibit association with LPHNPs and produce fluorescence.

78

Figure 4.4: Uptake of chitosan-lipid hybrid nanoparticles loaded with Rh-PE by

A2780 cells

79

4.3.4. Toxicological studies in rats

In vivo toxicity studies were conducted in rats (Co-Operation and Development,

2002). The study was conducted in accordance with the protocol of animal ethics

committee of The Islamia University of Bahawalpur. The weight of rats was noted at

the start of the study period, after 7 days of administration of cisplatin and at the end

of the 14 days study period. There was no significant difference in the weight of the

rats in that time period (Kim et al., 2008).

Table 4.1: Body weight and food, water intake of rats of different groups

Parameters Control Group Blank

Formulation

Drug Loaded

Formulation

Body weight (Kg)

Pre-treatment 1.40±1.21 2.20±1.8 2.10±1.6

Day 7 1.40±1.32 2.14±1.9 1.98±1.6

Day 14 1.34±1.40 1.70±1.2 1.60±1.2

Water In-take

(ml)

Pre-treatment 180 ± 1.57 201 ± 2.03 220 ± 2.03

Day 7 194 ± 2.01 210 ± 1.98 230 ± 1.98

Day 14 201 ± 1.95 215 ± 2.15 198± 2.15

Food Intake (gm.)

Pre-treatment 65.2 ± 1.50 72.30 ± 1.50 74.30 ± 1.50

Day 7 68.3 ± 1.43 71.20 ± 1.93 69.20 ± 1.93

Day 14 63.4 ± 1.24 68.50 ± 1.82 72.50 ± 1.82

Change in

Behavior

Nil Nil Nil

Death Nil Nil Nil

80

4.3.4.1. Biochemical and blood analysis

Biochemical blood analysis was carried out before and after treatment. Liver function

parameters and renal function parameters were also observed (Table 4.2). The blood

was collected after the completion of study periods in vials containing EDTA as an

anticoagulant for the complete blood count measurement while for renal function test

the blood was collected in other vial. After collection of blood samples, the samples

were tested in a laboratory to check the level of different hematological parameters.

The hematological study was carried out to evaluate the effect of lipid-polymer hybrid

system and to evaluate the cellular response to nanomaterial (Medina et al., 2007).

The effect of blank LPHNPS and cisplatin loaded LPHNPs was measured on blood

parameters and liver function and renal function parameter to check the compatibility

of the drug delivery system and to measure whether blank LPHNPs effect any

parameters or not. The results confirmed the safety profile of blank LPHNPs, which is

in accordance with out in vitro results on A2780 cell lines.

The effect of cisplatin loaded hybrid nanoparticles was also observed at a dose of

30mg/kg cisplatin. The dose of cisplatin was kept high to measure any noticeable

changes at higher doses. There was no substantial difference in hematological

parameters. However, slight change in the liver alkaline phosphatase level was

observed. These data are in accordance with previous studies (Dhar et al., 2011).

81

Table 4.2: Comparative hematological parameters in rats

Parameters Control Drug free LPHNP Cisplatin-LPHNP

Hemoglobin (g/dl) 14.2±0.3 13.7±0.20 12.9±0.31

Neutrophils (%) 24±3.0 23 ± 2.0 28±2.1

Lymphocytes (%) 58 ± 4.1 66 ± 1.0 61 ± 3.5

Monocytes (%) 5 ± 1 7 ± 2 8 ± 2

Eosinophils (%) 2 ± 1 2 ± 1 2 ± 1

Total leukocytes

(count/mm3)

5800 ± 400 4600 ± 200 2600 ± 300*

Platelet

(count/mm3)

410,000 ± 2000 464000 ± 3000 448000 ± 3000

82

Table 4.3: Comparative liver and renal function parameters in rats

Liver function test

Parameters

Control

Drug free LPHNP

Cisplatin-LPHNP

Bilirubin (IU/L) 0.4 ± 0.2 0.4 ± 0.1 0.3 ± 0.1

ALT(IU/L) 50 ± 2 66 ± 3 71 ± 2*

Alkaline Phosphatase

(IU/L)

78 ± 7 125 ± 10 310 ± 30*

Renal Function

Urea (mg/dl) 55 ± 3 60 ± 4 64 ± 6

Creatinine (mg/dl) 0.7 ± 0.1 0.7 ± 0.2 0.8 ± 0.2

All results indicate mean ± SD, n=6. *P < 0.05 (compared to control).

4.3.4.2. Histopathological examination

For histopathological examination rats were sacrificed after 14 days of treatment, and

the vital organs were separated including heart, lungs, stomach, liver and kidneys and

preserved in 10% formalin solution. Slides were prepared from these organs and

observed under optical microscopy for changes at the cellular level (Figure 4.5).

There were no changes in vital organs of blank LPHNPs group. However, rats

administered with the cisplatin containing formulation showed a slight change in

hepatic cells but, there were no signs of disruption or deformation of the other vital

organs. This is in accordance with previous studies with cisplatin (Mattheolabakis et

al., 2009).

83

Figure 4.5: Representative histopathological images of rat vital organs (A) Control

(B) Blank LPHNPs (C) Cisplatin loaded LPHNPs

84

4.3.5. In vivo pharmacokinetics

To determine the pharmacokinetic behavior of drug inside biological system, in vivo

studies were carried out on rabbits. The rabbits were divided into two groups and

were injected with same amount of drug as a cisplatin solution and cisplatin loaded

lipid-chitosan hybrid nanoparticles. The plasma samples were run in HPLC using

acetonitrile: Water: Methanol (30:30:40) as a mobile phase. The concentration versus

time relationship graph was drawn to evaluate the pharmacokinetic parameters

(Figure 4.6).

These studies suggest that combination of polymer and phospholipid can strongly

influence the pharmacokinetic parameters and could serve as a vehicle for controlled

drug delivery (Feng et al., 2011). The time to reach maximum concentration was

1±0.05 hours in the cisplatin solution group and 6 ±0.15 hours in the cisplatin loaded

lipid-chitosan hybrid formulation group. Maximum serum concentration was

observed at 1.01µg/ml compared to 4.07 µg/ml in the drug solution group. A lower

peak concentration is considered effective for prolonged exposure and reduced side

effects of chemotherapeutic drugs (Cheng et al., 2015). There was no burst release of

drug that is useful in case of anticancer drugs because immediate exposure to high

concentration of chemotherapeutic drug cause harm to normal body cells.

The half-life of the cisplatin loaded lipid-chitosan formulation was 14.0±0.4 hours

which was much greater than 1.25±0.04 hours for the drug solution group, which

indicates a prolonged release of drug in controlled manner. Mean residence time

(MRT) was 20.8±0.3 compared to 6.0±0.5 in cisplatin drug solution, indicating that

the lipid-polymer hybrid formulation provides a controlled release of cisplatin. A

similar increase in MRT was observed for a cisplatin formulation by Nakano, et

al(Nakano et al., 1997). The AUC with the lipid-chitosan formulation was 9.83±0.3

µg h/ml as compared to drug solution at 18.4±0.5 µg h/ml. Similarly, the lipid-

polymer formulation group showed a 4.6 fold increase in volume of distribution of

cisplatin as compared to the cisplatin solution Kai, et al also showed a 4.2 fold

increase in volume of distribution of cisplatin (Kai et al., 2015). Overall the

pharmacokinetic parameters of cisplatin were vastly improved with the lipid-polymer

hybrid nanoparticle system.

85

Figure 4.6: Concentration versus time profile curve of cisplatin LPHNPs and cisplatin

solution (Mean± SD n=6)

86

4.4. Discussion

Nanotechnology is progressing leaps and bounds in present era. Nanotechnology has

wide range of applications in the material sciences and drug delivery it deals with the

synthesis of materials at molecular level and delivery of drug at cellular level (Pal et

al., 2011). The lipid polymer hybrid nanoparticles are novel drug delivery system

with advantages of both polymeric and liposomal drug delivery system. Polymer

provided the structural advantages while out lipid confers the stability and serve as a

barrier and enhance the biocompatibility of the system. It is suitable for the controlled

delivery of drug (Wakaskar, 2018).

Therapeutic efficacy of cisplatin loaded LPHNP was evaluated on A2780 ovarian cell

lines. The LPHNPs cause more than 70% of cell death. The cytotoxic effect of

cisplatin is time and concentration dependent. Optimum activity after 48hours is in

agreement with the previous studies (Zhang et al., 2017). It is also important that after

48 hours, the cisplatin loaded lipid-polymer hybrid nanoparticles showed a 20-30%

increased cytotoxicity at same concentration as compared to 24hours and up to 50%

of cell death at the lowest concentration. These results are in agreement with the

controlled release formulation of cisplatin observed by Readon, et al (Reardon et al.,

2017). The controlled release of formulation is also evident from the in vitro

dissolution and in vivo pharmacokinetic studies.

Cell uptake studies showed significant increase uptake of the cisplatin loaded LPHNP

formulation and cellular internalization was observed using fluorescence microscope.

Cisplatin is rapidly excreted due its low molecular weight and long retention in the

blood is important to retain efficacy for longer period. The efforts have been made to

increase the retention and ensure controlled delivery of cisplatin. In vivo

pharmacokinetic studies suggest that combination of polymer and phospholipid can

strongly influence the pharmacokinetic properties and could serve as a vehicle for

controlled delivery (Feng et al., 2011). The cisplatin encapsulated in LPHNPs showed

controlled release profile and can be used as an effective system for delivery of

cisplatin.

Cell viability studies confirmed the cytotoxic effect on the A2780 ovarian cancer cell

line over a 48hours period. Cell uptake studies showed increased cellular uptake of

87

LPHNPs. In vivo pharmacokinetic studies in rabbits showed a controlled release

behavior. Toxicity studies in rats provided safety profile of the LPHNPs. Based on the

characterization and in vitro release profile, the lipid-chitosan hybrid nanoparticles

can provide controlled delivery of cisplatin and act as a useful platform for the

potential delivery of cisplatin to tumors. Further studies in tumor animal models

should be undertaken to examine the effectiveness of treatment of tumors with

LPHNPs.

88

4.5. Conclusion

It was concluded from that chapter that cisplatin loaded lipid-chitosan hybrid

nanoparticles have better cytotoxic effects on ovarian cell lines as compared to

cisplatin drug solution after 48 hours of treatment. The cell uptake studies showed 8-

fold increase in uptake as compared to control. Fluorescence microscope images

prove the cellular association. Toxicity studies prove the safety profile of lipid-

chitosan hybrid nanoparticles. In vivo pharmacokinetic studies prove the controlled

release of cisplatin. This cisplatin loaded lipid-chitosan hybrid nanoparticles can be

effectively used for the controlled release of cisplatin to the tumor cell with enhanced

efficacy.

89

Bjs Waltham

Folate Targeted Chitosan-Lipid

Hybrid Nanoparticles for

Enhanced Therapeutic Efficacy

Synthesis and Physicochemical

Characterization

CHAPTER 5

90

5.1. Background

Chemotherapy is considered as a front line treatment for cancer; however, it is

associated with injurious effects to normal body cells. The side effects associated with

the chemotherapy are major point of concern for the patients as well as for clinician.

Patients are put on additional drug to counteract the side effects of chemotherapy

which sometimes fails to resolve the issue and only add pain to the patients. Most of

the patients undergoing chemotherapy experience with nausea, vomiting,

gastrointestinal disturbances and peripheral neuropathy. It is time to deliver

chemotherapeutic agents to the targeted tumor cell and avoid the harmful effects on

normal body cells (Nurgali et al., 2018).

Nanotechnology is emerging hope to fight with plethora of diseases like cancer. It is

effectively used to deliver the drug and diagnostic agents. The drugs and diagnostic

agents are encapsulated inside the nanoparticles and release mechanism depends on

the nature of polymer used or on the external stimuli. The size of nanoparticles

enable the targeted delivery at tumor site (Kim et al., 2010). Nanoparticles also enable

the drug to be delivered at targeted site by conjugation with active targeting agents

and using the advantage of enhanced permeation and retention (Gu et al., 2007).

Polymeric nanoparticles have structural superiority and stability during shelf life and

ability to delivery drugs in a controlled release manner. They can be used for active

targeting. PEG coating further enhance their long circulating life and immune-

compatibility (Owens Iii and Peppas, 2006). Polymeric nanoparticles generally have

better cellular internalization, further it depends on the charge of the nanoparticles.

Cationic charge is usually favorable because it can interact with cell membrane and

enhance the internalization of nanoparticles (Chaudhary et al., 2018, Feng, 2004).

Despite of several advantages they have few drawbacks such as not suitable for

delivery of protein and peptides and particle aggregation could occur and release of

drug in acidic environment is aberrant from polymeric nanoparticles (Mohammadi-

Samani and Taghipour, 2015).

Liposomal drug delivery system has the advantage of superior biocompatibility as the

phospholipid mimic the biological membrane. Liposomes have short circulating life

and are easily cleared by reticuloendothelial system of body and pegylation is usually

done to enhance their circulating life. However liposomes have disadvantage of

91

structural integrity and leakage of drug could occur during storage (Maurer et al.,

2001).

To overcome the drawbacks of liposomal and polymeric drug delivery system a new

class of lipid-polymer hybrid nanoparticles has been introduced. They have the

structural superiority of polymers and enhanced biocompatibility of liposomes. They

provide controlled release profile with enhanced encapsulation efficiency. They also

have immune-compatibility and serve as a suitable controlled drug delivery system

(Salvador-Morales et al., 2009).

Folate receptors were first recognized as a tumor marker when monoclonal antibodies

raised against an ovarian tumor were found to recognize the alpha form of folate

receptor. Antibodies that produce against alpha folate receptor found its application as

diagnostic agent in gynecological tumor. After these initial finding the overexpression

of folate receptor was found in more than 90% of ovarian tumors. Because of their

high affinity to folate linked drug they have exploited as an agent for targeted drug

delivery to tumor (Lu and Low, 2003). Folic acid is now used as a targeting agent for

the targeted delivery to the tumor because of its intrinsic small size, penetration

ability, stability, solubility in organic solvents and ability to conjugate with various

therapeutic agents (Cho et al., 1997). Targeting folate receptor is a promising

approach by conjugating folic acid with the polymer that result in significant

increased uptake of drug by the tumor cells (Saul et al., 2003). Folate membrane

receptors have emerged as a target for drug delivery, in fact, folate metabolism is

primary in replication of DNA and drugs that inhibit folate metabolism can be

targeted. Folate receptors are overly expressed in tumors, which are not in normal

cells. Folate is a vitamin, which is water soluble and carbon donor in the synthesis of

purines, which lead to synthesis of DNA. Folate is required by the normal cells for

cell division it enter the cell via anion exchange pathway. Folate is not overexpressed

in the healthy cells, while rapidly dividing tumor cell overexpress the folate receptor

and present as a target for drug delivery (Marchetti et al., 2014).

Cisplatin has been used as a first line agent in the treatment of ovarian cancer since

decades. But it exerts side effect on major organs particularly kidney. Several

researches have been made to reduce the side effects and increase the therapeutic

efficacy of the cisplatin by formulating in various nanoparticles and liposomes.

92

Controlled drug release from the nanoparticles could be an approach to reduce the

side effects associated with the cisplatin (Cheng et al., 2011). Cisplatin inhibit the

tumor growth by inhibiting the DNA mediated functions. Cisplatin inhibit the cell

cycle that result in abnormal mitosis and leads to apoptosis of cells. Cisplatin is

known to cause the inhibition of cell cycle at G2/M phase of cell cycle. Cisplatin form

the bi-functional adduct with the DNA and inhibit the tumor growth (Eastman, 1999).

Here we formulated the folate targeted lipid-chitosan hybrid nanoparticles for active

targeting of tumor cell. This ensures site specific delivery of cisplatin and reduces the

side effects on the nearby health cells with enhanced cell penetration and efficacy.

93


5.2.1. Materials

Low molecular weight chitosan, folic acid and 1-ethyl-3-(3- dimethylaminopropyl)

carbodiimide hydrochloride (EDC) were purchased from Sigma Aldrich (USA). Lipid

was kindly provided by LIPOID® (Germany). Cisplatin was received as a gift sample

from Pharmedic (PVT) Ltd. (Pakistan). Ethanol and acetic acid were purchased from

Thermo Fisher Scientific (USA). Transmission electron microscope (JEOL, USA)

was used for imaging.

5.2.2. Preparation of folate-chitosan conjugate

Folic acid was conjugated with chitosan using carbidoiimide chemistry as described

by wang et al, (Wang et al., 2015). Briefly, chitosan was dissolved in 20 ml of acetic

acid solution by overnight stirring. Folic acid and 1-ethyl-3-(3- dimethylaminopropyl)

carbodiimide hydrochloride (EDC) were dissolved in anhydrous dimethyl sulfoxide.

After that the mixture of folic acid and EDC was added drop wise into the chitosan

solution and allowed to stir for 16 hours under dark. The pH of the mixture was

adjusted to 9 by using 1M sodium hydroxide solution.

The mixture was then centrifuged at 2000rpm for 10 minutes and the collected

precipitates were dialyzed against phosphate buffer saline using dialysis bag with

MWCO 10k for 3 days and then the same mixture was again dialyzed for 4 days

against deionized water to remove the unconjugated folic acid and to purify the

conjugated. Then the folic acid-chitosan conjugate was lyophilized overnight to

obtain the folate-chitosan conjugate as yellow colored sticky powder.

94

+

Figure 5.1: Schematic chemistry of folate-chitosan conjugate

EDC Dialysis

95

5.2.3. Preparation of folate-chitosan conjugated lipid hybrid nanoparticles

The folate-chitosan conjugate was then used for the preparation of hybrid

nanoparticles. The folate-chitosan conjugate was dissolved in the acetic acid solution

and lipid was dissolved in ethanol. The ethanolic solution of lipid was then added

drop wise to the acetic acid solution at varying ratio of lipid at 1000 rpm. The hybrid

nanoparticles were then formed by interaction of positively charged chitosan in folate-

chitosan conjugated and negatively charged lipid. The prepared nanoparticles were

then centrifuged and dried by lyophilization for further characterization.

Figure 5.2: Schematic diagram of folate targeted lipid-chitosan hybrid nanoparticles.

96

+

Figure 5.3: Schematic chemistry of formation of folate lipid-chitosan hybrid

nanoparticles

97

Figure 5.4: Mechanism of internalization of folate targeted lipid-chitosan hybrid

nanoparticles via endocytosis.

Folate targeted lipid-chitosan hybrid nanoparticles internalize the cell via

overexpressed folate receptors on the tumor cell and show targeted action via active

targeting.

98

5.2.4. Purity of folate-chitosan conjugate

The purity of folate chitosan conjugate was confirmed by thin layer chromatography.

The silica gel coated on aluminum foil plates were used as a stationary phase, while

methanol was used as a mobile phase. Two drops were placed on the stationary phase.

One spot of free folic acid solution and other of folate chitosan conjugate and mobile

phase was ran through the stationary phase till completion of time to check the

presence of free folic acid in the conjugate.

5.2.5. Nuclear magnetic resonance

Nuclear magnetic resonance was performed to confirm the conjugation of folic acid

and chitosan (500 MHZ Bruker). 1H-NMR was performed using Bruker nuclear

magnetic resonance spectroscopy. Folic acid was dissolved in deuterium dimethyl

sulfoxide solution and chitosan was dissolved in deuterium acetic acid solution and

folate-chitosan conjugate was also dissolved in deuterium acetic acid solution and

prepared solution were filled in NMR tubes and NMR was performed using standard

shim for DMSO and water.

5.2.6. Size and zeta potential of hybrid nanoparticles

The prepared folate LPHNPs were analyzed for size, surface charge, polydispersity

index using Zeta Sizer (Malvern, UK). The 50 µl sample was used in a cuvette for the

determination of size and it was diluted with 950 PBS for the measurement of zeta

potential. The readings were performed at 25°C in triplicate.

5.2.7. Determination of drug loading and entrapment efficiency

The entrapment efficiency was determined by the indirect method, by measuring the

amount of unbound drug in the supernatant liquid after the completion of

centrifugation. The drug contents were determined by using HPLC method. The

mixture of acetonitrile: water: Methanol (30:30:40) was used as a mobile phase and

detector was set at 210nm to detect cisplatin and flow rate was 1.5ml/min. The

absorbance of standard and samples were measured at 210nm. The drug was

dissolved in PBS at pH 7.4. Suitable dilutions were made from the supernatant liquid

to measure the drug contents. The experiment was performed in triplicate. The

entrapment efficiency and drug loading were determined by the given formula.

99

Encapsulation efficiency = amount of entrapped drug / theoretical amount of drug ×

100

Percentage drug loading will be calculated by following equation;

Drug loading = Mass of drug in nanoparticles / mass of nanoparticles × 100

5.2.8. Morphology of hybrid nanoparticles

Morphology of the cisplatin loaded folate LPHNPs was determined by using the

Transmission electron microscope (TEM) (Jeol, USA). The prepared nanoparticles

were applied directly on the grid. The excess of sample was removed from the grid

and suitable images were taken at different magnifications.

5.2.9. In vitro drug release studies

The drug release study was carried out by using dialysis bag method. The dialysis

membrane of MWCO 10-12 KDa was used. Folate LPHNP formulations were packed

in different dialysis bags and the release was compared with cisplatin solution.

Phosphate buffer saline (pH 7.4) was used as a dissolution medium and temperature

was maintained at 37°C±0.5 with stirring speed at 100 rpm. Samples were withdrawn

at pre-determined time interval and similar amount of fresh medium was added to

maintain the final volume throughout the experiment. The samples were run in HPLC

to determine the drug content. The mixture of acetonitrile: water: Methanol (30:30:40)

was used as a mobile phase and detector was set at 210nm to detect cisplatin and flow

rate was 1.6ml/min.

100

5.3. Results

5.3.1 Purity of the conjugate

Purity of the conjugate was determined by this layer chromatography. The free folic

acid and folate-chitosan conjugate were run together on TLC plate and methanol was

used as a mobile phase. After completion of run time the plate was observed under

UV light. The folic acid spot on right side of plate showed that free folic acid

throughout the run area, while no free folic acid was observed in folate-chitosan

conjugate on left side of plate. No free folic acid in region of folate-chitosan spot

confirms the proper conjugation of folate-chitosan and absence of free folic acid.

Figure 5.5: Thin layer chromatography of folic acid and folate-chitosan conjugate

101

5.3.2. Nuclear magnetic resonance spectroscopy

The conjugation of folic acid with chitosan was confirmed by 1H-NMR. The

individual spectra of folic acid was taken using deuterium dimethyl sulfoxide as a

solvent and spectra of chitosan and folate-chitosan conjugate were taken using

deuterium acetic acid as a solvent.

The spectra of chitosan showed typical peaks of glucosamine ring of chitosan at 3.4 to

3.7ppm (these are linked to carbon atom in chitosan ring C1 to C4) and folic acid

showed the spectra at 6.6 to 8.6ppm that correspond to the aromatic ring of folic acid.

The spectra of folic acid-chitosan conjugate clearly depicted peaks at 3.4 to 3.78 ppm

that confirmed the presence of chitosan in the conjugate and peaks at 6.8, 7.8, 8.5ppm

confirmed that presence of folic acid and presence of characteristic peaks of both folic

acid and chitosan in spectra of conjugate confirmed the successful conjugation of folic

acid with chitosan. The characteristic spectra of folic acid, chitosan and folate-

chitosan conjugate is shown in figure 5.6.

102

Figure 5.6: 1H-NMR spectra of folate-chitosan conjugate, chitosan and folic acid

103

5.3.3. Size and zeta potential of hybrid nanoparticles

The size and zeta potential were measured of six different formulations with varying

ratio of folate-chitosan conjugate to lipid by using zetasizer. The hybrid nanoparticles

were prepared from 5:1 ratio to 30:1 of lipid to folate-chitosan conjugate. The hybrid

nanoparticles showed good colloidal stability with zeta potentials values in +20 to

+39.

The size of prepared hybrid nanoparticles with lipid to folate-chitosan ratio of 5:1 to

20:1 was in range of 200nm to 300nm while the hybrid nanoparticles with lipid to

chitosan ratios greater than 20:1 showed large particle size and greater polydispersity

index. This showed that increasing the amount of lipid increase the negative charge

too much, while there is not enough cationic charge available to folate-chitosan

conjugate to interact with negatively charged lipid. Hence the particles formed have

greater particle size and showed polydispersity with particles of different size in same

formulation. Further the hybrid nanoparticles with lipid to folate-chitosan ratio of 30:1

were quite unstable and aggregation of particles occurs soon after the formation of

nano-suspension. The hybrid nanoparticles with lipid to folate-chitosan ratio of 15:1

showed low PDI and particle size in range of 210nm that is suitable for passive and

active targeting.

Table 5.6: Composition of cisplatin loaded lecithin-chitosan hybrid nanoparticles

Formulation

Code

Folate-

chitosan

conjugate

(mg)

Lipid

(mg) Drug Ethanol

5:1 0.312 1.56 500 µg 63µl

10:1 0.156 1.56 500 µg 63µl

15:1 0.156 2.34 500 µg 94µl

20:1 0.156 3.12 500 µg 125µl

25:1 0.156 3.91 500 µg 157µl

30:1 0.156 4.68 500 µg 186µl

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Table 5.7: Effect of lipid to polymer ratio on particle size and surface charge

Formulat

ion Code

Folate-

chitosan

(mg)

Lipid

(mg) Drug

Particle

Size

(nm) PDI

Surface

charge

(mV)

5:1 0.312 1.56 500 µg 296± 1.36 0.3 40.1±1.2

10:1 0.156 1.56 500 µg 312± 1.12 0.2 37.2±1.1

15:1 0.156 2.34 500 µg 210± 0.61 0.2 30.5±0.7

20:1 0.156 3.12 500 µg 260± 0.91 0.3 37.6±0.9

25:1 0.156 3.91 500 µg 375± 2.12 0.4 27.6±1.4

30:1 0.156 4.68 500 µg 525± 2.85 0.5 21.5±1.8

5.3.4. Drug entrapment and loading efficiency

The entrapment of drug was calculated using indirect method by measuring the

amount of un-entrapped drug. The standard sample was prepared and run in HPLC to

draw a calibration curve. The prepared formulations were centrifuged, and

supernatant was run into HPLC using mobile phase and the amount of un-entrapped

drug was calculated and results showed the all formulations with varying ratios of

lipid to folate-chitosan conjugate have good encapsulation efficiency of more than

80% drug entrapped inside the hybrid nanoparticles and nanoparticles with ratio of

15:1 showed highest encapsulation efficiency of more than 75% and good drug

loading.

The hybrid nanoparticles with lipid to folate-chitosan ratio of 15:1 were selected for

further characterization based on particle size, PDI, zeta potential and encapsulation

efficiency.

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Table 5.8. Effect of Lipid to polymer ratio on entrapment efficiency and drug loading

Formulation

Code

Folate-

chitosan

(mg)

Lipid

(mg) Drug

Entrapment

Efficiency (%)

Drug

Loading (%)

5:1 0.312 1.56 500 µg 84.7±2.1 1.5±0.7

10:1 0.156 1.56 500 µg 83.5±1.9 2.1±0.6

15:1 0.156 2.34 500 µg 90.6±0.8 3.0±0.3

20:1 0.156 3.12 500 µg 86.5±1.2 2.8±0.5

25:1 0.156 3.91 500 µg 78.5±1.4 1.6±0.4

30:1 0.156 4.68 500 µg 76.3±1.5 1.4±0.5

5.3.5. Morphology of folate LPHNPs

The morphology of folate targeted lipid-polymer hybrid nanoparticles was checked by

using transmission electron microscopy. The TEM images showed the lipoplex like

structure that is formed by ionic interaction of positively charged folate-chitosan

conjugate and negatively charged lipid and covering of folate on the outer side of the

particles. The images showed that the outer surface of particles is black, which is due

to coating of folate on the outer surface. The size of nanoparticles was in range of

200nm which is in accordance with the size determined by differential light scattering

method. The shape of hybrid nanoparticles is shown in figure 5.7.

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Figure 5.7: Transmission electron microscopy image of folate targeted lipid-chitosan

hybrid nanoparticles

5.3.6. In vitro drug release profile

Drug release studies were conducted using dialysis bag method. The hybrid

nanoparticles with lipid to folate-chitosan ratio of 15:1 were placed in a dialysis bag

of MWCO value 12k and cisplatin drug solution was placed in other bag and both

were placed in phosphate buffer saline pH 7.4 as a dissolution medium. The

temperature of the medium was kept at 37°C and stirring speed of 100rpm. The

samples were taken after defined time intervals. In cisplatin drug solution group, more

than 90% drug was released in 24 hours while the folate lipid-chitosan hybrid

nanoparticles showed sustained release behavior and only 60% of drug was released

after 24 hours. In FLPHNPs formulation group the drug release reach to 90% after 48

hours. The sustained released from the FLPHNPs group is due to entrapment of drug

inside the polymer and a lipid layer on the outer side to prevent the leakage of drug

and ensure sustained release behavior of formulation.

107

Figure 5.8: In vitro release profile of folate lipid-chitosan hybrid nanoparticles and

cisplatin drug solution

108

5.4. Discussion

Folic acid has attracted a lot of attention as a targeting agent due to its high

internalization. It is water soluble compound and has low molecular weight and it is

biocompatible and has the advantage of its conjugation chemistry that enables it to be

conjugated with variety of polymers including chitosan. We have conjugated folic

acid with chitosan using carbidoiimide chemistry (Shia et al., 2009). Folate receptors

are overexpressed in tumor cell and belong to high affinity protein family these are

mainly overexpressed in tumors of epithelial origin (Vllasaliu et al., 2013).

The nuclear magnetic resonance spectroscopy was performed to confirm the

conjugation of folic acid with chitosan. The conjugation of folic acid with chitosan

occurs via carbidoiimide chemistry. First EDC reacts with folic acid and activate the

carboxylic group of folic acid which is then conjugated with amine group of chitosan.

FA-CS conjugate showed the typical spectral peaks at 3.8 3.9 and 4.2ppm that

correspond to the chitosan group and spectral peaks at 6.8 7.7ppm confirmed the

presence of folic acid. The same spectral peaks of FA-CS were also observed in

previous studies (Yang et al., 2014). The coupling of folic acid with chitosan leads to

slight vibration in the peaks as compared to pure folic acid and chitosan (Li et al.,

2011).

The size of prepared nanoparticles and zeta potential are of critical importance in

defining the characteristics of the nanoparticles. The charge on the nanoparticles will

affect the stability in the suspension. The value of surface charge of all prepared

formulation is above +20 that showed better stability of the nanoparticles. The

spherical shape of the prepared nanoparticles is evident from the transmission electron

microscope images. The similar findings of morphology and surface charge have been

observed with folate-chitosan nanoparticles in previous studies (Wang et al., 2015).

In vitro release studies showed sustained release of cisplatin over a period of 48 hours

that is due to entrapment of drug inside the polymer core and lipid layer serve as a

barrier and prevent the leakage of drug from the hybrid nanoparticles. Lipid layer

provides a diffusional barrier and drug is released in a sustained manner and results in

increased therapeutic benefits. Immediate release of large amount of

chemotherapeutic agent could cause harmful effects on normal body tissues, thus,

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sustained release pattern may results in decreased side effects and provision of

therapeutic dose over a prolong time period (Kim et al., 2008).

5.5. Conclusion

Folic acid has been successfully conjugated with chitosan and hybrid nanoparticles

have been prepared by ionic gelation method using anionic lipid by ionic interaction

of positively charged folate-chitosan conjugate and negatively charged lipid. The

prepared hybrid nanoparticles have suitable size and PDI and cationic charge with

excellent stability in the suspension form. The morphology of hybrid nanoparticles

was confirmed by transmission electron microscopy and images showed spherical

shaped nanoparticles. The entrapment efficiency was more than 75% and drug release

studies provide sustained release for 48 hours. The prepared folate conjugated lipid-

chitosan hybrid nanoparticles could be used for sustained delivery of cisplatin further

cell lines studies needed to be carried out to establish the therapeutic efficacy of


110

Bjs Waltham

Folate Targeted Chitosan-

Lipid Hybrid Nanoparticles

for Enhanced Therapeutic

Efficacy

Biological characterization

CHAPTER 6

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6.1. Background

The hybrid nanoparticle drug delivery system provides enhanced therapeutic efficacy

and biocompatibility. The biocompatible properties of nanoparticle drug delivery

system are usually assessed by cell culture. Cell cultures are suitable to measure the

efficacy of the drug delivery system. Cell culture has established pivotal role in

measuring the therapeutic efficacy and biocompatibility of drug delivery system since

the development of first ever HeLa cell lines. Cells either attached to the surface of

flask as a monolayer or remain in the suspension form. The doubling time of cells

vary depending on cell type and cell cycle (Ravi et al., 2015).

In recent era, 3D cell cultures have gain importance that mimic the in vivo

environment. They are more realistic and closer to in vivo environment to establish

the efficacy of drug delivery system. To obtain 3D shape that is closer to in vivo

environment different type of scaffold are used and most common type is agarose gel

(Vinci et al., 2012). The major advantage of using 3D culture spheroids over 2D

culture is to minimize the gap between in vitro cell culture and physiology of cell. In

traditional 2D cell culture system most of important parameters such as cell to cell

interaction and cell to matrix interaction cannot be studied. The 3D cell culture

provides more clear mechanism of tumorigenesis and in vivo conditions

(Muthuswamy, 2011). 3D cell cultures are also extensively used for drug screening

and to check the pharmacological effects at initial stage and exclude the toxic and

relatively ineffective substance and it also reduce the unethical animal studies and

thereby the cost associated with the controversial animal studies (Pampaloni et al.,

2009).

3D spheroids made up of cancer cell lines have three layers the upper proliferating

layer and central necrotic layer and inner quiescent layer that mimic the environment

of solid tumors. 3D spheroids have most of features of in vivo solid tumors including

hypoxic environment and cellular interaction (Lee et al., 2007). 3D spheroids have

many peculiar features of in vivo tumors particularly of at initial stage of tumor

growth. They also have necrotic area and proliferative index. The drug resistance in

spheroids is also found to be similar to the in vivo animal studies. These are also

suitable to study the pattern of cell proliferation and cell cycle mechanism. The

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microenvironment of tumor plays key role in 3D spheroids and are useful to mimic

tumor’s microenvironment (Mazzoleni et al., 2009).

Here, we studies the biocompatibility of blank folate LPHNPs and cytotoxicity of

cisplatin loaded folate LPHNPs, LPHNPs on ovarian cell lines A2780 and SKOV3

and also on MCF-7 breast cell lines and prepared 3D spheroids using breast cell lines

to mimic the in vivo environment and studies the efficacy of cisplatin loaded lipid

chitosan hybrid nanoparticles against 3D spheroids. Cell uptake sstudies were also

carried out on 3D spheroids model to check the folate mediated penetration and

uptake of folate LPHNPs.

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6.2.1. Materials

Cell Titer Blue and Cell titer Glo (CTG) were purchased from Promega (WI, USA).

The ovarian cell lines SKOV3 and A2780 were purchased from Sigma Aldrich, breast

cancer cell line MCF-7 was purchased from Sigma Aldrich. Roswell Park Memorial

Institute medium (RPMI), DMEM media, fetal bovine serum (FBS), and penicillin-

streptomycin solution were obtained from Cell Gro (VA, USA). Hoechst 33342 was

purchased from Molecular Probes Inc. (Eugene, OR). Paraformaldehyde was from

Electron Microscopy Sciences (Hatfield, PA, USA). Trypan blue solution was

obtained from Hyclone (Logan, UT, USA). Rdodamine-123 and rhodamine-PE were

purchased from Thermo Fischer Scientific (USA).

6.2.2. Cell viability studies

Cell viability studies were conducted on A2780 and SKOV3 ovarian cell lines and

MCF-7 breast cell lines. Cells (5000) were seeded in each well of two 96 well plates.

After overnight incubation the cells were treated with folate LPHNPs, LPHNPs and

cisplatin solution and blank folate LPHNPs to check the cytotoxic effect on cell lines.

The reading was taken after 24 and 48 hours of treatment on different plates using

plate reader. Cell titer blue was used to measure the cell viability. It is based on the

ability of live cells to convert resazurin, which is a redox dye into fluorescent dye

resorufin. The dead cell does not have the metabolic activity and cannot convert the

redox dye and only living cells are counted using plate reader.


Cells (100,000) were seeded in a 6 well plate using SKOV3 cell lines. After 24 hours

of incubation periods the cells were treated with folate LPHNPs loaded with cisplatin

and rhodamine-123 that produce green fluorescence and other group of folate

LPHNPs was loaded with cisplatin and rhodamine-PE that produce red fluorescence.

The third group of folate LPHNPs was loaded with both fluorescence dyes

rhodamine-123 and rhodamine-PE. Similar lipid-chitosan hybrid nanoparticles were

also loaded with fluorescent dyes. After four hours, cells were washed with PBS (pH

7.4) and fixed with same solution containing 4% paraformaldehyde (PFA). After

114

fixation cells washed with again using PBS (pH 7.4) three times and stained with 10

µg/ml Hoechst for 15 min. Cells were washed again with PBS, pH 7.4, and mounted

on microscope slides with mounting buffer for analysis by keyence fluorescence

microscope.

6.2.4. Cellular uptake

Flow cytometry was used for quantitative determination of cell uptake. Cells

(400,000) were seeded overnight in each well of 6-well plate. After overnight

incubation period, cells were treated with Rh-123 containing formulation (0.1 mol %)

for 4hours. After four hours of treatment cells were detached using trypsin and

washed with PBS, pH 7.4, three times and centrifuged at 2000 rpm for 5 min and the

re-suspended in 300µl of PBS. The re-suspended cell suspension was then used for

determination of cell uptake by flow cytometry using fluorescence signals.

6.2.5. Cell apoptosis

Cell apoptosis studies were performed using annexin-V FITC apoptosis detection kit.

Cells (100,000) were seeded in each well of 6 well plate and after overnight

incubation period cells were treated with cisplatin loaded folate LPHNPs, LPHNPs

and cisplatin drug solution to compare the cell apoptosis. The cells were collected

after 24 and 48 hours of treatment using trypsin and then centrifuged and washed

using PBS and cell were incubated with propidium iodide (PI) for 15 minutes and

kept on ice and then cell apoptosis was measured using flow cytometry.

6.2.6. Cell cycle

Cell cycle studies were conducted to evaluate the inhibition mechanism. The cells

(700,000) were seeded in each well of 6 well plate. After overnight incubation they

were treated with cisplatin loaded folate LPHNPs, LPHNPs and cisplatin solution.

After 24 and 48 hours of treatment periods the cells were collected and fixed using

10% ethanol. After fixation the cells were centrifuged and washed with PBS and

incubated with propidium iodide for 30 minutes and then reading was taken using

flow cytometry.

115

6.2.7. Preparation of 3 D spheroids

The spheroids were prepared in 96 well plated. 96 well plates were coated with 1.5%

agarose and kept at 4°C. Cells (10,000) were then seeded in each well of 96 well plate

already coated with agarose. The cells were then centrifuged at 15000rpm at 15°C for

20 minutes and then kept in the incubator at 25°C and 5% CO2.The shape of spheroids

was monitored using microscope. The plates were kept in incubator for about 7 days

until they attain a proper 3D spheroids shape.

6.2.8. Cell viability towards 3D spheroids

Cell viability studies were performed on spheroids using 96 well plates. Cells

(10,000) were then seeded in each well of 96 well plate already coated with agarose.

The cells were then centrifuged at 15000rpm at 15°C for 20 minutes and then kept in

the incubator at 25°C and 5% CO2. After 7 days of incubation the spheroids were

ready for treatment. They were treated with cisplatin loaded folate LPHNPs, LPHNPs

and cisplatin solution. After 24 hours and 48 hours of treatment the cells were treated

with cell titer Glo (Promega) using standard protocol and luminescence was measured

using plate reader.

6.2.9. Cell uptake studies toward 3 D spheroids

Cell uptake studies were performed on spheroids using 96 well plate. Cells (10,000)

were then seeded in each well of 96 well plate already coated with agarose. The cells

were then centrifuged at 15000rpm at 15°C for 20 minutes and then kept in the

incubator at 25°C and 5% CO2. After 7 days of incubation the spheroids were ready

for treatment. They were treated with cisplatin loaded folate LPHNPs, LPHNPs and

cisplatin solution. After 24 hours and 48 hours of treatment the spheroids were

collected in eppendorf tubes and washed with PBS and uptake was measured using

flow cytometry.

6.2.10. Fluorescence microscopic images of 3D spheroids

Cells (10,000) were then seeded in each well of 96 well plate already coated with

agarose. The cells were then centrifuged at 15000rpm at 15°C for 20 minutes and then

kept in the incubator at 25°C and 5% CO2. After 7 days of incubation the spheroids

were ready for treatment. They were treated with cisplatin loaded folate LPHNPs,

116

LPHNPs and cisplatin solution. After 24 hours and 48 hours of treatment the

spheroids were collected in eppendorf tubes and washed with PBS and then fixed

containing 4% paraformaldehyde (PFA). After fixation cells washed with again using

PBS (pH 7.4) three times and stained with 10 µg/ml Hoechst for 15 min. Cells were

washed again with PBS, pH 7.4, and placed in 85mm tubes and images of spheroids

were taken using fluorescence microscope and depth of penetration of checked by

using Z-stacking of fluorescence microscope.

117

6.3. Results

6.3.1. Cytotoxicity studies

Cytotoxicity studies were carried out on ovarian cancer cell lines and breast cancer

cell lines. A2780 and SKOV3 were used as ovarian cell lines and MCF-7 was used as

breast cancer cell lines.

Two 96 well plates were seeded with A2780 cell line and cells were treated with

blank folate LPHNPs and cisplatin loaded folate LPHNPS and LPHNPs and cisplatin

solution. Cell viability was measured using cell titer blue after 24 hours for one plate

and after 48 hours for other plate using a plate reader. The results showed that blank

folate LPHNPs does not have any cytotoxic effect and are safe drug delivery system.

The results showed that after 24 hours of incubation folate LPHNPs have more

significant cytotoxicity as compared to cisplatin drug solution and LPHNPs at

concentration 12.5µg/ml and 6.25µg/ml while there in no significant difference at

other concentration. After 48 hours of treatment folate LPHNPs have more significant

cytotoxic effect compared to LPHNPS and cisplatin drug solution at concentration of

(25,12.5,6.25,3.12 µg/ml). This is due to folate receptors mediated endocytosis of

folate LPHNPs and enhanced uptake of folate LPHNPs that result in enhanced

cytotoxic effect.

Two 96 well plates were seeded with SKOV3 cell line and cells were treated with

blank folate LPHNPs and cisplatin loaded folate LPHNPS and LPHNPs. Cell viability

was measured using cell titer blue after 24 hours for one plate and after 48 hours for

other plate using a plate reader. The results showed that blank folate LPHNPs does

not have any cytotoxic effect and are safe drug delivery system. The results showed

that after 24 hours of incubation there is not much significant difference in cytotoxic

effect of folate LPHNPS and LPHNPS. After 48 hours of treatment folate LPHNPs

have more significant cytotoxic effect compared to LPHNPS and cisplatin drug

solution at concentration of (50, 25, 12.5, 6.25, 3.12 and 1.6µg/ml). This is due to

folate receptors mediated endocytosis of folate LPHNPs and enhanced uptake of

folate LPHNPs that result in enhanced cytotoxic effect. There is no significant

difference after 24 hours while much enhanced significant cytotoxic effect after 48

hours was due to sustained release of cisplatin from folate LPHNPs.

118

Two 96 well plates were seeded with MCF-7 cell line and cells were treated with

blank folate LPHNPs and cisplatin loaded folate LPHNPS and LPHNPs. Cell viability

was measured using cell titer blue after 24 hours for one plate and after 48 hours for

other plate using a plate reader. The results showed that blank folate LPHNPs does

not have any cytotoxic effect and are safe drug delivery system. The results showed

that after 24 hours of incubation folate LPHNPs have significant more cytotoxic effect

as compared to cisplatin drug solution at concentration of 12.5µg/ml and 3.1µg/ml but

there is not much significant difference in cytotoxic effect of folate LPHNPS and

LPHNPS. After 48 hours of treatment folate LPHNPs have more significant cytotoxic

effect compared to LPHNPS and cisplatin drug solution at concentration of (12.5 and

3.1µg/ml). This is due to folate receptors mediated endocytosis of folate LPHNPs and

enhanced uptake of folate LPHNPs that result in enhanced cytotoxic effect.

119

Figure 6.1: Cell viability study on A2780 cell lines after 24 hours of incubation

comparison of blank folate LPHNPS, folate LPHNPS and LPHNPs

120

Figure 6.2: Cell viability study on A2780 cell lines after 48 hours of incubation


121

Figure 6.3: Cell viability study on SKOV3 cell lines after 24 hours of incubation


122

Figure 6.4: Cell viability study on SKOV3 cell lines after 48 hours of incubation


123

Figure 6.5: Cell viability study on MCF-7 cell lines after 24 hours of incubation


124

Figure 6.6: Cell viability study on MCF-7 cell lines after 48 hours of incubation


125

6.3.2. Cell uptake studies

Cell uptake studies were performed using flow cytometry to measure the quantitative

difference of cell uptake between folate LPHNPs and LPHNPs using rhodamine-123

as a fluorescent dye.

The results showed 12-fold more uptake of LPHNPs loaded with rhodamine-123

compared to control and folate LPHNPs have 24 fold increase in uptake as compared

to control and 12 fold increased in uptake as compared to LPHNPs. The statistical

analysis showed that there is significant difference of uptake of LPHNPs as compared

to control and folate LPHNPs also showed significantly higher uptake as compared to

LPHNPs that is due to folate receptor mediated endocytosis and enhanced uptake by

the cells.

Figure 6.7: Cell uptake studies using flow cytometry. Comparison of LPHNPs and

Folate LPHNPs loaded with rhodamie-123

126


Qualitative uptake of hybrid nanoparticles by SKOV3 cells was checked by using

fluorescence microscopy. Folate targeted lipid-chitosan hybrid nanoparticles were

loaded with fluorescent dyes rhodamine-123 and rhodamine-PE. Rhodamine-123

stains the cell green color and rhodamine-PE stains red color. Cells were already

stained by HOECST a blue color dye that stains the nucleic acid during the process of

fixation of cells.

The results showed that SKOV3 cell actively uptake the folate targeted lipid-chitosan

hybrid nanoparticles and showed green and red fluorescence. The uptake of folate

LPHNPs is due to folate receptors mediated endocytosis.

Figure 6.8: Fluorescence microscopy images of SKOV3 cells treated with folate

LPHNPs and folate LPHNPs loaded with Rh-123 and Rh-PE

127

6.3.4. Cell cycle studies

Cell cycle studies were performed using propidium iodide and fixation of cells using

70% ethanol. The cell population was recorded using flow cytometry after 24 and 48

hours of treatment. The results showed that after 24 hours of treatment control group

have 65% of population in G0/G1 phase and 15 % of population is S phase and

remaining population is G2/M phase of cell cycle. After 24 hours of treatment the

population of cells in drug treated group has shifted from G0/G1phase to S phase and

there is significant more shift in population of folate LPHNPs as compared to

LPHNPs and cisplatin solution group. After 48 hours of treatment the cell population

has shifted to G2/M phase and there is significant more population in folate LPHNPs

as compared to cisplatin drug solution of LPHNPs. The results showed that folate

LPHNPs more significantly halt the cell cycle as compared to cisplatin loaded

LPHNPs and cisplatin drug solution.

Figure 6.9: Cell cycle studies after 24 hours of treatment

128

Figure 6.10: Cell cycle studies after 48 hours of treatment

6.3.5. Cell apoptosis studies

Cell apoptosis studies were carried out using propidium iodide and annexin-V to

detect the early and late apoptosis of the cells. The results were compared between

cisplatin drug solution, LPHNPs and folate LPHNPs. The results showed that after 24

hours of treatment there is significant shift of cells toward early and late apoptosis as

compared to control group and folate LPHNPs have significantly higher apoptosis as

compared to LPHNPs and cisplatin drug solution. After 24 hours of treatment

cisplatin solution has 10% of cells in early apoptosis and LPHNPs have 15% of cells

while folate LPHNPs have 26% of cells at early apoptosis and 39% of cells at late

apoptosis as compared to only 18% in late apoptosis of LPHNPs.

After 48 hours of treatment there is an increase in late apoptosis and decrease in early

apoptosis of the cells. Folate LPHNPs showed significantly increased in late apoptosis

as compared to LPHNPs and cisplatin drug solution but cisplatin solution showed

increase in early apoptosis as compared to folate LPHNPs. Late apoptosis of cells is

considered as of critical importance as cells in early apoptosis might have chances to

revert. The result showed significant increase in late apoptosis by folate LPHNPs

where more than 60% cells are in late apoptosis as compared to 40% in cisplatin drug

solution group.

129

Figure 6.11: Cell apoptosis after 24 and 48 hours of treatment comparison of cisplatin

solution, LPHNPs and folate LPHNPs

130

Figure 6.12: Control group cell apoptosis showing early and late apoptosis

131

Figure 6.13: Cell apoptosis of cisplatin drug solution showing early and late

apoptosis

132

Figure 6.14: Cell apoptosis of lipid- chitosan hybrid nanoparticle loaded with

cisplatin

133

Figure 6.15: Cell apoptosis of folate targeted lipid-chitosan hybrid nanoparticles

134

6.3.6. Cell Cytotoxicity toward cancer cell in 3D spheroids

Cell cytotoxicity studies were conducted on 3D spheroids model that mimic the in

vivo environment to evaluate the in vivo efficacy of folate targeted LPHNPs and

compare the cytotoxicity on 3D spheroids with LPHNPs. The spheroids were grown

for 10 days and treated with cisplatin loaded folate LPHNPs, LPHNPs and cisplatin

drug solution and CTG protocol was used to calculate the cell viability that measure

the luminescence by the cell in 3D spheroids.

The results showed that after 24 hours of treatment folate LPHNPs have more

significant cytotoxic effect against tumor cell line as compared to cisplatin drug

solution at concentration of (50, 12.5, 3.1µg/ml) and more cytotoxic effect as

compared to LPHNPs at concentration of 6.25 µg/ml.

After 48 hours of treatment folate LPHNPs have more significant cytotoxicity as

compared to cisplatin drug solution at concentration of (25, 12.5, 6.2 µg/ml) and more

significant cytotoxic effect as compared to LPHNPs at concentration of (25, 12.5, 6.2,

3.1 µg/ml). This showed that folate targeted lipid-chitosan hybrid nanoparticles are

more effective as compared to LPHNPs and cisplatin drug solution and exert greater

cytotoxic effects.

135


comparison of folate LPHNPs, LPHNPs and cisplatin drug solution

136


comparison of folate LPHNPs, LPHNPs and cisplatin drug solution

137

6.3.7. Cell uptake studies toward cancer cell in 3D spheroids

Cell uptake studies were conducted in 3D spheroids as in vivo model. Spheroids were

treated with folate LPHNPs and LPHNPs and the uptake was measured after 4 hours

of treatment using flow cytometry. The results showed that LPHNPs are have 2-fold

more uptake in 3D spheroids as compared to control and folate LPHNPs have double

uptake as compared to LPHNPs and 4 times more uptake as compared to control. This

enhanced uptake by spheroids is due to folate mediated endocytosis of folate LPHNPs

that results in enhanced uptake by the cell and results in enhanced efficacy.

Figure 6.18: Cell uptake studies using flow cytometry comparison of folate LPHNPs,

LPHNPs

138

6.3.8. Fluorescence microscope images of cell uptake towards 3D spheroids

Cell uptake by 3D spheroids was also measured qualitatively by taking images of

spheroids using flow cytometry. Rhodamine-123 is a fluorescent dye and was loaded

inside the nanoparticles and used to measure the uptake. It gives green color after

uptake inside the cells.

The results showed that folate targeted lipid-chitosan hybrid nanoparticles produce

more fluorescence as compared to LPHNPs and there is more bright green color as

compared to LPHNPs. The images of spheroids were also taken using z-stacking to

confirm that weather the cells in the lower layer of spheroids have uptake the folate

LPHNPs loaded with rhodamine and results showed that there is greater uptake as

compared to LPHNPs.

Figure 6.19: Cell uptake towards 3D spheroids using fluorescence microscope

comparison of folate LPHNPs with LPHNPs

139

Figure 6.20: Z-stack images of 3D spheroids control group

140

Figure 6.21: Z-stack image of lipid-chitosan hybrid nanoparticle group of 3D

spheroids

141

Figure 6.22: Z-stack image of folate targeted lipid-chitosan hybrid nanoparticle

group of 3D spheroids

142

6.4. Discussion

The lipid-chitosan hybrid nanoparticles are made of lipid that are component of

biological membrane and polymer chitosan that is biocompatible and obtained from

the chitin. The folate is conjugate with chitosan which is a vitamin and required by the

body. The studies on different ovarian and breast cell lines proved the

biocompatibility of blank LPHNPs and folate conjugated lipid-chitosan hybrid

nanoparticles. The results prove that there is not any cytotoxic effect of blank folate

LPHNPs on the cell lines and cell count is almost similar to the control group. Several

previous studies also support the biocompatibility of folate-chitosan nanoparticles (Jin

et al., 2016).

The cell viability studies on ovarian cell lines showed that folate LPHNPs have more

cytotoxic effect on A2780 cell lines as compared to LPHNPs and cisplatin drug

solution that is due to enhanced internalization of folate LPHNPs and exhibit greater

cytotoxic effect. The greater cytotoxic effect was observed after 48 hours of treatment

because of controlled release of drug from folate LPHNPs. Previous studies of folate

targeting also showed that folate targeted nanoparticles showed more cytotoxic effect

as compared to without folate conjugation (Mansouri et al., 2006).

The cell uptake studies were conducted to measure the internalization of

nanoparticles. Qualitative uptake was measured using fluorescence microscope and

quantitative uptake was measured fluorescence microscopy. The results showed folate

targeted nanoparticles showed 2 times more cell uptake as compared to untargeted

hybrid nanoparticles. The enhanced uptake was folate targeted LPHNPs as compared

to untargeted LPHNPs is due to folate mediated internalization and uptake via folate

receptors. The enhanced cell uptake of folate targeted nanoparticles is also established

in previous studies (Xu et al., 2013).

The cell cycle studies were conducted using cisplatin loaded folate LPHNPs and

cisplatin loaded LPHNPs and cisplatin solution. The results showed that after 24

hours of treatment there is regular reduction in G0/G1phase and increase in S phase

population of cells and after 48 hours of treatment there is significant reduction in

G0/G1phase and increase in G2/M phase and folate LPHNPs have significant more

increase in G2/M phase population as compared to without folate. Previous studies on

143

folate targeted cisplatin nanoparticles also report increase in G2/M phase population

of cell (Wang et al., 2013).

The studies on 3D spheroids were conducted to mimic the in vivo environment. The

cytotoxicity studies on 3D spheroids showed that folate targeted LPHNPs have more

cytotoxic effect as compared to without folate targeted. There is also enhanced cell

uptake of folate LPHNPs due to folate mediated endocytosis both qualitative and

quantitative uptake studies established the internalization of folate LPHNPs.

144

6.5. Conclusion

It was concluded from the biological characterization that folate lipid-chitosan hybrid

nanoparticles have enhanced cytotoxic effect as compared to LPHNPs on A2780

ovarian cell lines after 24 and 48 hours of treatment. Similar studies were conducted

on SKOV3 ovarian cell lines and enhanced efficacy of folate LPHNPs was

established. MCF-7 cell lines were used to check the cytotoxic effects against breast

cancer cell lines the results also showed greater cytotoxic effects as compared to

LPHNPs and cisplatin drug solution. The cell uptake studies showed greater uptake of

folate LPHNPs as compared to without folate targeting. The enhanced effect on cell

uptake is due to folate receptors mediated endocytosis. The cytotoxicity studies on 3D

spheroids established that folate LPHNPs could showed enhanced cytotoxic effect in

in vivo environment. The results showed that folate targeted lipid-chitosan hybrid

nanoparticles could serve as a system for active targeting of tumor cells.

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Conclusions and

Recommendations

CHAPTER 7

146

7. Conclusions and recommendations

LPHNPs are drug delivery system for controlled drug delivery. In this dissertation

we used chitosan and lipid for the preparation of hybrid nanoparticles. The

LPHNPs were then loaded with cisplatin for controlled drug delivery to the tumor

site. The chitosan was then conjugated with folic acid for active targeting of

tumor. The efficacy of cisplatin loaded hybrid nanoparticles was then evaluated by

using different ovarian cell lines.

The following conclusion were made from these studies

1. Cisplatin loaded lipid-chitosan hybrid nanoparticles were successfully fabricated

using different lipid-to chitosan ratios. The lipids to chitosan ratios of (10:1 to

60:1) were used and 6 different formulations were prepared. The size, surface

charge, entrapment efficiency and drug loading was measured and it was

concluded that LPHNPs with ratio of 20:1 have excellent size with low PDI and

good entrapment efficiency and drug loading and these were used for further

analysis.

2. Cisplatin loaded LPHNPs were evaluated for the thermal stability using

differential scanning calorimetry and thermogravimetric analysis and they showed

excellent thermal stability and drug encapsulation within the system. The prepared

hybrid nanoparticles were further evaluated by using FTIR to check the

compatibility among the formulation excipients and it was concluded that

components are compatible as there are no new peaks.

3. The drug release studies were performed and it was concluded that LPHNPs

showed controlled release of drug due to entrapment of drug inside polymer core

and lipid provide diffusional barrier and drug is release in a controlled manner.

The kinetic modeling was applied, and it was concluded that cisplatin loaded

LPHNPs follow korsmeyerpeppas model and super case II transport mechanism.

4. The therapeutic efficacy of cisplatin loaded LPHNPs was evaluated in vitro using

A2780 ovarian cell lines. It was concluded from the results that cisplatin loaded

LPHNPs have more cytotoxic effect as compared to cisplatin drug solution and

uptake studies confirmed the cellular association and internalization of LPHNPs.

5. The cisplatin loaded LPHNPs system was further evaluated on rats to confirm the

safety profile of LPHNPs system. Toxicological studies on rats were performed

147

and evaluated for biochemical evaluation and histopathological evaluation using

higher doses of LPHNPs and cisplatin. The results showed safety profile of

LPHNPs and there are not any significant changes in hematological parameters

and histopathological evaluation. The pharmacokinetic studies were performed on

rabbits to check the in vivo profile of cisplatin and confirmed higher mean

residence time of LPHNPs compared to cisplatin solution group.

6. The chitosan was then successfully conjugated with folic acid for active targeting.

The TLC analysis confirms the purity of conjugate and nuclear magnetic

resonance spectroscopy confirmed the conjugation of folic acid with chitosan.

7. The folate LPHNPs were successfully fabricated by the ionic gelation method.

The folate LPHNPs containing lipid to folate-chitosan ratio of (15:1) showed

better size and stability with good entrapment efficiency and drug loading.

8. The morphology confirmed the spherical shaped nanoparticles with folate

covering on the outer surface. The drug release studies showed that sustained

release of cisplatin over the periods of 48 hours compared to cisplatin solution.

9. The therapeutic efficacy of folate LPHNPs was evaluated on ovarian cell lines and

breast cell lines. A2780 and SKOV3 were used as ovarian cell lines and it was

concluded from the results that after 48 hours of treatment folate LPHNPs have

more significant cytotoxic effect against ovarian cell lines compared to LPHNPs

and cisplatin drug solution. The studies on MCF-7 breast cell lines also showed

the significantly enhanced cytotoxic effect of folate LPHNPs as compared to

LPHNPs and cisplatin solution.

10. To evaluate the in vivo efficacy of folate LPHNPs we prepared 3D spheroids that

mimic the in vivo environment and performed the cell cytotoxicity and cell uptake

studies on 3D spheroids. The results of 3D spheroids further establish the

enhanced cytotoxic effect of folate LPHNPs as compared to LPHNPs without

folate targeting and cisplatin solution. It was concluded the folate targeted

LPHNPs are more effective to provide targeted and controlled drug delivery at

tumor site.

148

7.1 . Future prospects

This study provides the promising results for the fabrication and optimization of

lipid-chitosan hybrid nanoparticles for the delivery of chemotherapeutic agents.

This study also provides a new drug delivery system for folate targeting. Folate-

chitosan conjugate was used to prepared nanoparticles using anionic lipid which is

new system of folate targeting having advantage of lipid over traditional folate

targeting.

1. Different synthetic and natural polymers can be evaluated with combination of

cationic and anionic lipids to prepared LPHNPs for controlled delivery of

different chemotherapeutic agents.

2. These lipid-chitosan hybrid nanoparticles can be evaluated for co-delivery of

siRNA and drug to combat with drug resistance and provide optimum therapeutic

effectiveness.

3. LPHNPs can be evaluated for the sustained and controlled delivery of different

antidiabetic, antihypertensive and other drugs.

4. Folate targeted LPHNPs can also be used for co-delivery of siRNA and

chemotherapeutic agents because of their cationic charge and ability to interact

with negatively charged siRNA and DNA.

5. 3D spheroids establish the estimated in vivo efficacy of folate LPHNPs further

studies on animal tumor models and pre-clinical studies can be conducted to

establish folate LPHNPs as an effective drug delivery system compared to

traditional folate targeting and moving towards FDA approval.

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150

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