Nanocarriers-encapsulating phytochemicals as potent

Nanocarriers-encapsulating phytochemicals as potent

therapeutics

in cancer therapy

A thesis submitted in fulfilment of the requirements for

the degree of

Doctor of Philosophy

Rasika Radhakrishnan

School of Science

College of Science, Engineering and Health

RMIT University, Australia

Declaration

I certify that except where due acknowledgement has been made, the work

is that of the author alone; the work has not been submitted previously, in

whole or in part, to qualify for any other academic award; the content of the

thesis is the result of work which has been carried out since the official

commencement date of the approved research program; and, any editorial

work, paid or unpaid, carried out by a third party is acknowledged; and ethics

procedures and guidelines have been followed.


Acknowledgement

First and foremost, I would like to thank my supervisors Dr. Ravi Shukla,

RMIT, Dr. Sistla Ramakrishna at IICT, Hyderabad and Prof. Suresh

Bhargava in RMIT for giving me the opportunity to work with them for my

PhD.

I would also like to thank RMIT University and IICT, Hyderabad, India for

allowing me the use of their space and services.

It was an utmost pleasure working with Dr. Ravi. He has taught me virtues

beyond science, which are only going to strengthen me for the time to come.

My experience with him has made me realise the importance of upholding

ethics in a workplace, lessons that I will remember throughout my scientific

career. He has also made me understand the qualities that separate a bad

researcher from good ones. I will be forever indebted to you for this.

Dr. Ramakrishna graciously welcomed me into his group despite me not

being from the field. He has high ideals for living and he translates it into his

work. His hard work, resilience and immense patience are qualities I aspire

to have someday. One of the things I most respect him for is that he does not

compromise when it comes to his ideals. I will be grateful to you Sir.

Prof. Suresh is easily one of the most positive influences in my PhD tenure.

Every discussion with him has always had me feeling optimistic, even when

things are low. Continued faith and belief as a supervisor is one of the

greatest gifts that can be given to a student. He has always encouraged me to

believe in myself and never give up. He has exemplary patience and

understanding to shortcomings. Words cannot explain how grateful I am to

you, Prof. Suresh.

Next, I would like to thank my family, my father, mother and brother, each

supporting me in their own different ways, making me the person I am. They

have always been and will continue to be my pillar of strength and support.

I would like to thank my mentors Dr. Hitesh and Dr. Pooja who initiated me

into the field of pharmaceutics and have been with me throughout most of

my PhD. They have been extremely helpful, critical at times but it was

always constructive. My experience with Dr. Pooja has taught me not to

compromise on work even if things are not going your way. She is a strong

woman and I admire her for it.

I would like to thank Mrs. Nadia Zakhartchouk for her help and support in

the cell culture lab. I would also like to thank Dr. Zeyad for his assistance at

MNRF Facility. I would also like to thank Dr. Madhusudana and Dr. Halley

for helping me during animal studies. Dr. Halley was an immense support,

working with me throughout the study.

I would like to thank my colleagues and friends, Arpita, Ram, Vijay, Sudha,

Naresh, Deepthi, Halley, Suma, Tejashri, Salma, Riyaz and Vatsalya, who

had made working in the lab a fun experience. Special mentions to Arpita

and Sudha with whom I could share my thoughts, even the most abstract

ones. A special mention to Dr. Blake Plowman, who has helped me

immensely during drafting of this thesis.

I would also like to thank the lab assistants at IICT, Hyderabad; Laxman,

Chinna and Satish for their help in lab and the animal house.

I would like to honour and remember the precious beings that sacrificed their

lives during my study.

Last, but in no way least I would like to thank God, without Him I would not

be where I am. His continual presence is what keeps me motivated.


Table of Contents

List of Figures i

List of Tables v

List of Abbreviations vi

Abstract 1

Chapter 1: Introduction

1.1. Cancer 4

1.2. Common causes of cancer 4

1.3. Conventional approaches to combat cancer 4

1.4. Chemotherapy 6

1.5. Problems with current cancer chemotherapy 8

1.6. Nanoparticles used for chemotherapy 10

1.7. Mechanisms for targeting 13

1.8. Rationale 15

1.9. Nanoparticles used in this work 17

1.9.1. Solid lipid nanoparticles 17

1.9.2. Chitosan nanoparticles 22

1.10. Drugs used in this study 25

1.10.1. Epigallocatechin gallate 25

1.10.2. Piperine 27

1.10.3. Mangiferin 27

1.11. Ligands used in this study 28

1.11.1. Bombesin 28

1.11.2. Mannose 29

1.12. Techniques used in this study 30

1.13. Aims and objectives 32

1.14. References 34

Chapter 2: Enhancement in cancer cytotoxicity of EGCG by

encapsulation within solid lipid nanoparticles

2.1. Background 49

2.2. Materials 53

2.3. Methods 53

2.3.1. Aqueous Stability 53

2.3.2. Optimization 54

2.3.3. Preparation of EGCG loaded solid lipid nanoparticles (EGCG-

SLNs)

54

2.3.4. Determination of drug entrapment efficiency 55

2.3.5. Physico-chemical characterization of nanoparticles 55

2.3.5.1. Particle size and surface potential 55

2.3.5.2. FTIR spectroscopy (FTIR) 55

2.3.5.3. Differential scanning calorimetry (DSC) 55

2.3.6. In-vitro drug release 56

2.3.7. In-vitro cytotoxicity studies 56

2.3.8. Colloidal and Serum stability studies 57

2.3.8.1. Colloidal stability of nanoparticles in serum and

physiological conditions

57

2.3.8.2. Electrolyte-induced aggregation 57

2.4. Results 58

2.4.1. Aqueous stability 58

2.4.2. Optimization of different parameters for the blank SLN 60

2.4.3. Influence of formulation parameters 60

2.4.4. Influence of process parameters 61

2.4.5. Encapsulation efficiency of EGCG-SLN 64

2.4.6. Physico-chemical characterization 64

2.4.6.1. FTIR spectroscopy (FTIR) 64

2.4.6.2. Differential scanning calorimetry (DSC) 65

2.4.7. In-vitro drug release studies 67

2.4.8. In-vitro cytotoxicity studies 68

2.4.9. Apoptosis studies 72

2.4.10. Colloidal stability studies 74

2.4.10.1. Stability of NPs in serum and physiological conditions 74

2.4.10.2. Electrolyte induced aggregation 76

2.5. Conclusion 77

2.6. References 78

Chapter 3: Peptide conjugated solid lipid nanoparticles for

breast cancer therapy

3.1. Background 87

3.2. Materials 88

3.3. Methods 89

3.3.1. Preparation of nanoparticles 89

3.3.2. Calculation of Entrapment efficiency 89

3.3.3. Bioconjugation 90

3.3.4. Physicochemical characterization 91

3.3.4.1. Particle size and surface charge 91

3.3.4.2. FTIR analysis 92

3.3.4.3. DSC analysis 92

3.3.5. In-vitro studies 92

3.3.5.1. In-vitro cytotoxicity 92

3.3.5.2. Cellular uptake 93

3.3.5.3. Apoptosis assay 93

3.3.5.4. Migration studies 93

3.3.6. Animal studies 94

3.4. Results 95

3.4.1. Particle size and surface charge 95

3.4.2. FTIR analysis 97

3.4.3. DSC analysis 99

3.4.4. In-vitro studies 99

3.4.4.1. In-vitro cytotoxicity 99

3.4.4.2. Phase contrast microscopy studies 104

3.4.4.3. Cellular uptake studies 107

3.4.4.4. Apoptosis studies 108

3.4.4.5. Migration studies 110

3.4.5. In-vivo studies 111

3.4.6. Conclusions 114

3.4.7. References 116

Chapter 4: Mangiferin and piperine encapsulated

nanoparticles for cancer therapy

4.1. Background 120

A. Mangiferin encapsulated solid lipid nanoparticles for

targeting towards cancer therapy

120

4.2. Materials 122

4.3. Methods 122


4.3.2. Determination of entrapment efficiency 123

4.3.3. Bio-conjugation 123

4.3.4. Calculation of conjugation efficiency 124



4.3.5.2. FTIR Analysis 124


4.4. Results 125







4.4.5.2. FTIR Analysis 130


B. Piperine encapsulated chitosan nanospheres conjugated

with mannose for glycol-targeting towards lectin receptors

overexpressed in cancer

133

4.5. Materials 135

4.6. Methods 135





4.6.5. Physico-chemical characterization 136



4.7. Results 137








4.8. Conclusions 143

Chapter 5: Summary and future perspectives

i

Figure

No. LIST OF FIGURES Page

No.

1.1. Different types of nanoparticles used in chemotherapy 11

1.2. Different mechanisms of targeting 14

1.3. Structure of solid lipid nanoparticle 18

1.4. Structure of chitosan polymer 22

1.5. Preparation of chitosan nanoparticles 23

1.6. Structure of epigallocatechin gallate 26

1.7. Structure of piperine 27

1.8. Structure of mangiferin 27

1.9. Structure of bombesin receptor 29

1.10. Structure of bombesin 29

1.11. Structure of D-mannose 30

2.1. Structure of epigallocatechin gallate (EGCG) 52

2.2. UV/Visible spectra of EGCG (50 µg/mL) at different time points in (a)

distilled water (b) 0.1 N HCl (c) normal saline (0.9 %w/v NaCl) (d)

phosphate buffer saline pH 7.4 (e) phosphate buffer pH 5 (f) phosphate

buffer pH 7 (g) phosphate buffer pH 9

59

2.3. Particle size distribution of a) blank solid lipid nanoparticles and b)

EGCG-loaded solid lipid nanoparticles (EGCG-SLN), measured by

dynamic light scattering

62

2.4. Fourier transform infrared (FTIR) spectra of epigallocatechin gallate

(EGCG), blank solid lipid nanoparticles (blank SLN) and EGCG -

loaded solid lipid nanoparticles (EGCG-SLN)

65

2.5. Differential scanning calorimetric analysis of epigallocatechin gallate

(EGCG) and EGCG loaded solid lipid nanoparticles (EGCG-SLN)

66

2.6. In-vitro drug release studies of EGCG loaded solid lipid nanoparticles

(EGCG-SLN) in sodium acetate buffer pH 5.

67

2.7. Percent cell viability of (a) MDA-MB-231 human breast cancer cells

and (b) DU-145 human prostate cancer cells after 24 h of treatment

with pure epigallocatechin gallate (EGCG), blank solid lipid

nanoparticles (blank SLN) and EGCG-loaded SLN (EGCG-SLN)

69

ii

2.8. Phase contrast microscopy images of (a) MDA-MB-231 human breast

cancer cells and (b) DU-145 human prostate cancer cells after 24 h of

treatment with blank solid lipid nanoparticles (blank SLN), pure

epigallocatechin gallate (EGCG), or EGCG-loaded SLN (EGCG-SLN)

equivalent to 20μg/mL EGCG.

71

2.9. Nuclear fragmentation studies: Florescent microscopic images of (a)

MDA-MB-231 human breast cancer cells and (b) human prostate

cancer cells after 24 h of treatment with blank solid lipid

nanoparticles (blank SLN), pure epigallocatechin gallate (EGCG), or

EGCG-loaded SLN (EGCG-SLN) equivalent to 20 μg/mL EGCG

followed by staining with Hoechst 33342

73

2.10. Change in (a) particle size and (b) zeta potential of EGCG-loaded

solid lipid nanoparticles (EGCG-SLN) with respect to time.

75

2.11. Effect of flocculating agent (sucrose sulphate) on the stability of

EGCG loaded solid lipid nanoparticles

76

3.1. Structure of bombesin 81

3.2. Mechanism of EDC-NHS reaction 90

3.3. Particle size distribution of a) blank solid lipid nanoparticles (Blank-

SLN) b) EGCG-loaded solid lipid nanoparticles (EGCG-SLN) and

Bombesin conjugated solid lipid nanoparticles (EB-SLN), measured

by dynamic light scattering

96

3.4. Fourier transform infrared (FTIR) spectra of epigallocatechin gallate

(EGCG), blank solid lipid nanoparticles (Blank SLN), EGCG -loaded

solid lipid nanoparticles (EGCG-SLN) and Bombesin conjugated

solid lipid nanoparticles (EB-SLN)

98

3.5. Differential scanning calorimetric analysis of epigallocatechin gallate

(EGCG) and EGCG loaded solid lipid nanoparticles (EGCG-SLN)

99

3.6. Percent cell viability of MDA-MB-231 human breast cancer after 24

h of treatment with pure EGCG, EGCG-loaded SLN (EGCG-SLN)

and bombesin-conjugated EGCG-SLN (EB-SLN)

102

iii

3.7. Percent cell viability of DU145 prostate cancer cells after 24 h of

treatment with pure EGCG, EGCG-loaded SLN (EGCG-SLN) and

bombesin-conjugated EGCG-SLN (EB-SLN).

102

3.8. Percent cell viability of A549 prostate cancer cells after 24 h of

treatment with pure EGCG, EGCG-loaded SLN (EGCG-SLN) and

bombesin-conjugated EGCG-SLN (EB-SLN).

103

3.9. Percent cell viability of HEK-293 kidney cells after 24 h of treatment

with pure EGCG, EGCG-loaded SLN (EGCG-SLN) and bombesin-

conjugated EGCG-SLN (EB-SLN).

103

3.10. Percent cell viability of L-132 lung cells after 24 h of treatment with

pure EGCG, EGCG-loaded SLN (EGCG-SLN) and bombesin-

conjugated EGCG-SLN (EB-SLN).

104

3.11. Phase contrast microscopy images of MDA-MB-231 human breast

cancer after 24 h and 48 h of treatment with blank solid lipid

nanoparticles (blank SLN), pure epigallocatechin gallate (EGCG),

EGCG-loaded SLN (EGCG-SLN) and bombesin conjugated SLN

(EB-SLN) equivalent to 20 μg/mL EGCG.

106

3.12. Cellular uptake studies: Florescent microscopic images of MDA-MB-

231 human breast cancer cells after 24 h of treatment with Rhodamine

loaded SLN (R-SLN) and bombesin conjugated SLN (RB-SLN).

108

3.13. Nuclear fragmentation studies: Florescent microscopic images of

MDA-MB-231 human breast cancer cells after 24 h of treatment with

blank solid lipid nanoparticles (blank SLN), pure epigallocatechin

gallate (EGCG), EGCG-loaded SLN (EGCG-SLN) and bombesin

conjugated SLN (EB-SLN) equivalent to 20 μg/mL EGCG followed

by staining with Hoechst 33342.

110

3.14. Scratch assay images for MDA-MB-231 human breast cancer cells

after 24 h of treatment with blank solid lipid nanoparticles (blank

SLN), pure epigallocatechin gallate (EGCG), EGCG-loaded SLN

(EGCG-SLN) and bombesin conjugated SLN (EB-SLN) equivalent to

20 μg/mL EGCG along with control cells for reference

111

3.15. Kaplan-Meier plot for survival following administration of pure

epigallocatechin gallate (EGCG), EGCG-loaded SLN (EGCG-SLN)

113

iv

and bombesin conjugated SLN (EB-SLN) formulations with control

mice for reference

3.16 Change in body weight and tumour volume in mice with the number

of days following administration of pure epigallocatechin gallate

(EGCG), EGCG-loaded SLN (EGCG-SLN) and bombesin conjugated

SLN (EB-SLN) formulations with control mice for reference

114

4.1. Structure of Mangiferin 121

4.2. Calibration curve of Mangiferin 126

4.3. Particle Size distribution for blank formulation (Blank-SLN),

Mangiferin-loaded SLN (MgF-SLN) and bombesin-conjugated BM-

SLN formulations measured by dynamic light scattering

129

4.4. Fourier transform infrared (FTIR) spectra for blank formulation

(Blank-SLN), Mangiferin-loaded SLN (MgF-SLN), Mangiferin and

bombesin-conjugated BM-SLN formulations

131

4.5. In vitro drug release studies of Mangiferin-loaded SLN (MgF-SLN)

in sodium acetate buffer pH 5 and phosphate buffer (pH 7.4)

132

4.6. Structure of Piperine 133

4.7. Calibration curve of piperine 139

4.8. Particle Size distribution for blank formulation (B-NP), piperine-

loaded nanoparticle (P-NP) and mannose-conjugated (MP-NP)

formulations measured by dynamic light scattering

142

4.9. Fourier transform infrared (FTIR) spectra for blank formulation (B-

NP), Piperine-loaded chitosan formulation (P-NP), Piperine and

mannose-conjugated chitosan (MP-NP) formulations

143

v

Table

No. LIST OF TABLES Page

No.

2.1 Optimization of formulation parameters for preparation of solid

lipid nanoparticles by double-emulsification method

63

2.2. Optimization of encapsulation of epigallocatechin gallate (EGCG)

in solid lipid nanoparticles

64

2.3. IC50 values (µg/mL) for epigallocatechin gallate (EGCG) and

EGCG-loaded solid lipid nanoparticles (EGCG-SLN) against

MDA-MB-231 human breast cancer cells and DU-145 human

prostate cancer cells after 24 h of treatment

70

3.1. Particle size and surface charge for blank nanoparticles (Blank-

SLN), EGCG-loaded SLN (EGCG-SLN) and bombesin

conjugated SLN (EB-SLN) determined by DLS

96

3.2. IC50 values (µg/mL) for epigallocatechin gallate (EGCG), EGCG-

loaded solid lipid nanoparticles (EGCG-SLN) and bombesin

conjugated SLN (EB-SLN) against MDA-MB-231, DU-145,

A549, HEK-293 and L-132 cells after 24 h of treatment

101

4.1. Optimization of formulation parameters for mangiferin-loaded

solid lipid nanoparticles prepared by single-emulsification method

126

4.2. Optimization of encapsulation of mangiferin and entrapment

efficiency in Mangiferin-loaded SLN (MgF-SLN)

127

4.3 Particle Size distribution, polydispersity and zeta potential values

for blank formulation (Blank-SLN), mangiferin-loaded SLN

(MgF-SLN) and bombesin-conjugated BM-SLN formulations

determined by dynamic light scattering

129

4.4. Optimization of formulation parameters for piperine loaded-

chitosan nanoparticles (P-NP) prepared by ionic gelation method

138

4.5. Optimization of encapsulation of piperine and entrapment

efficiency in piperine loaded chitosan nanoparticles (P-NP)

139

4.6. Particle Size distribution, polydispersity and zeta potential values

for blank formulation (B-NP), piperine-loaded nanoparticle (P-

NP) and mannose-conjugated (MP-NP) formulations determined

by dynamic light scattering

141

vi

LIST OF ABBREVIATIONS

BAK BCL-2 homologous antagonist killer

BAX BCL-2-associated X protein

BBN Bombesin

BCL-2 B-cell lymphoma 2

BCL-XL B cell lymphoma- extra large

BM-SLN Bombesin conjugated mangiferin SLN

CRD Carbohydrate recognition domain

CS Chitosan

DSC Differential scanning calorimetry

DMEM Dulbecco’s modified eagle medium

DMSO Dimethyl sulfoxide

EB-SLN Bombesin conjugated solid lipid nanoparticle

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

EGCG Epigallocatechin gallate

EGCG-SLN EGCG-loaded solid lipid nanoparticle

EPR Enhanced permeation and retention

FBS Fetal bovine serum

FTIR Fourier transform infrared

GMS Glycerol monostearate

GRP Gastrin-releasing peptide

GRPR Gastrin releasing peptide receptor

KBr Potassium bromide

MAPK Mitogen activated protein kinases

MES 2-(n-morpholino) ethane sulfonic acid

MgF Mangiferin

MgF-SLN Mangiferin-loaded SLNs

MTT

3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium

bromide

NFκB Nuclear factor kappa-B

NMB Neuromedin B

NHS N-hydroxysuccinimide

PA Poly(acrylic) acid

PAMAM Poly(amido amine)

PBS Phosphate buffer saline

PD Mean particle diameter

PDI Polydispersity

PEG Poly (ethylene glycol)

PEI Polyethyleneimine

PGA Poly(glutamic) acid

PLA Polylactide

PLGA Poly(lactic-co-glycolic) acid

vii

PVA Polyvinyl alcohol

RES Reticulo-endothelial system

ROS Reactive oxygen species

RPMI Roswell Park Memorial Institute 1640 medium

SA Stearic acid

SAB Sodium acetate buffer

SD Standard deviation

SFC Supercritical fluid

SLN Solid lipid nanoparticles

TAAs Tumour-associated antigens

VEGF Vascular endothelial growth factor

1

Abstract

Cancer is a disease concerning the abnormal, uncontrolled growth of cells that possess a

potential to invade and injure other parts of the body. Chemotherapy involves

administration of drugs targeted towards the mechanisms responsible for this rapid

division and proliferation. However, most chemotherapy drugs are non-specific, also

causing allied toxicity to normal cells undergoing cell division.

In this work, phytochemicals were used as anti-cancer drugs. These molecules are non-

toxic and biocompatible. However, they also display striking anti-cancer potential, which

was explored in this thesis. In this work, epigallocatechin gallate, piperine and mangiferin

have been explored for their anti-cancer efficacy.

Although phytochemicals are effective agents in cancer chemotherapy, many of them

suffer from disadvantages such as pH instability and limited bioavailability in the body.

To address this problem, nanocarriers were designed to facilitate their increased

bioavailability and stability inside the body. Two nanoparticle matrices were explored in

this work; solid lipid nanoparticles and chitosan nanoparticles. Solid lipid nanoparticles

were used as they are biocompatible, help improve drug stability and show controlled

release. Chitosan nanoparticles are biodegradable, biocompatible and have shown

excellent absorption properties for chemotherapeutic drugs.

Chemotherapy mainly deals with two targeting approaches; active and passive. While

passive targeting relies on the characteristics of the tumour for effective therapy, active

targeting involves attachment of ligands on the nanocarrier for more specific targeting.

Both the targeting approaches have been explored in this work. In addition, two targeting

ligands were used in this work; bombesin and mannose, which are specific for factors

overexpressed in cancer.

2

In summary, this thesis involves the design and fabrication of nanocarriers in order to

increase the anti-cancer efficacy of phytochemicals, utilising different targeting

approaches, matrix components and targeting ligands. The anti-cancer efficacy was

evaluated by in-vitro cytotoxicity studies and in-vivo animal studies. The results validate

the increased anti-cancer efficacy provided by the encapsulation of the drugs within

nanocarrier systems thus offering encouraging, novel possibilities in the field of cancer

therapy.

3

4

1.1. Cancer

Cancer remains one of the world’s most crippling diseases, accounting for about 1 in

every 7 deaths worldwide – more than HIV/AIDS, tuberculosis, and malaria combined[1].

It persists as an unsolved enigma for modern medicine, a cure for which has been sought

since times immemorial. The word 'cancer' is derived from the Greek word for crab

(karkinos), indicating the crab-claw like patterns of veins surrounding the tumour.

Cancerous tissues are characterized by the abnormal growth of cells beyond their usual

boundaries invading adjoining parts of the body and/or spreading to other organs[2].

Lung, prostate, colorectal, stomach and liver cancers are the most common types of

cancer in men, while breast, colorectal, lung, cervix and stomach cancers are most

common among women[3]. Although cancer-related mortality has decreased owing to

better understanding of tumour biology and improved diagnostic devices and treatments,

further advances in this field are necessary to understand the basis of disease progression,

which will in turn lead to better therapy[4].

1.2. Common causes of cancer

Cancer involves the irregular and abnormal growth of cells, perpetuating beyond their

usual lifespan. This diseased condition can be triggered by a number of factors, with the

most well-known being excessive smoking and use of tobacco, exposure to carcinogens,

inadequate diet and physical activity, exposure to radiation, overall genetics, viral and

other infections[3]. However, other factors such as the sex and age of the patient also

influence the development of certain cancers[5].

1.3. Conventional approaches to combat cancer

Current approaches for cancer treatment include surgery, chemotherapy, radiation

therapy, immuno-therapy and gene therapy, as outlined in the following sections:

5

1.3.1. Surgery

Surgery is the preferred method for the excision of solid tumours which are mostly

contained to one area[6]. Excision can be complete or partial (debulking), depending on

the magnitude of expansion of the cancerous tissue, wherein complete removal is

attempted when the tumour is restricted to one area or organ while debulking is carried

out when complete resection might damage an organ or functioning of the body as a

whole[7]. While surgical resection is often a straightforward and plausible line of

treatment, it suffers from the disadvantage of not promising complete removal of

cancerous tissue in cases where metastasis may have taken place, but is yet dormant or

undetected[8]. It also cannot be used for treatment of leukemias.

1.3.2. Radiation therapy

Radiation therapy uses high energy radiations such as gamma rays and X-rays to damage

the DNA in cancerous cells[9]. It is of two types, external beam therapy and internal

radiation therapy. External beam therapy involves the use of a linear accelerator for the

production of X-rays. Internal radiation involves the implantation of a solid or liquid

radioactive source inside the body for the emission of radiations.

1.3.3. Chemotherapy

Chemotherapy involves the administration of an anti-cancer drug (or multiple drugs) as a

part of a regimen for either curative or palliative treatment[10]. It can be used as a

neoadjuvant, wherein chemotherapeutics are administered before radiation therapy or

surgery to reduce the tumour volume or sensitize it for more efficient therapy[8]. It is also

used in tandem with other procedures as an adjuvant in a combinatorial chemotherapeutic

regimen[11].

6

1.3.4. Immunotherapy

Cancer immunotherapy involves stimulating the immune system through the active or

passive targeting against tumour cells[12]. It uses genetically modified cells and viral

particles to stimulate the immune system to destroy cancer cells. Tumour cells express

tumour-associated antigens (TAAs) which can be attacked by the immune system[13].

Active immunotherapy helps the immune system to better detect the TAAs and hence

assist in their removal. In passive immunotherapy, monoclonal antibodies, lymphocytes

and cytokines are used to enhance anti-tumour responses.

1.3.5. Gene therapy

Gene therapy involves the use of genes (in-vitro or in-vivo) to manipulate cancer

cells[14]. However, ineffective delivery of the therapeutic gene to the target cells and

multiple precautions to be taken to ensure the therapeutic gene does not integrate into

unwanted cells, are a few challenges that gene therapy has to circumvent[15].

1.4. Chemotherapy

Chemotherapy is the use of anti-cancer drugs to destroy cancer cells, alone or in

combination with other drugs and sensitizers(As mentioned in Section 1.3)[16]. It mainly

deals with targeting the mechanisms responsible for conferring immortality to cancer

cells. The main types of chemotherapeutic drugs used are as follows[1]:

1.4.1. Alkylating agents

This class includes alkyl group containing synthetic compounds interfering in different

phases of the tumour cell division process, preventing it from further replication. The

alkyl groups react to form irreversible covalent linkages with the various components of

the genetic assembly, such as DNA, RNA or proteins, halting cell division and hence

proliferation[17]. They do suffer from the disadvantage of non-specificity wherein

replicating non-cancerous cells may also be damaged[18]. However, defective DNA

7

repair mechanisms in cancer cells may lead to higher mortality rates in comparison to

normal cells. Cisplatin, oxaliplatin and cyclophosphamide are common examples of

alkylating agents.

1.4.2. Anti-metabolites

Anti-metabolites are modified analogs of purines, pyrimidines, nucleotides and

nucleosides, which are components of the genetic assembly. The similarity of these

analogs to genetic components causes them to be picked up during DNA or RNA strand

synthesis. However irregularity in the strand structure during assimilation of these altered

elements in DNA/RNA strand creates problems in the recoiling and coiling process

during cell division which results in effective cytostatis[19]. Examples include

gemcitabine, 5-fluoro uracil and methotrexate.

1.4.3. Anti-tumour antibiotics

Anti-tumour antibiotics are fermentation products of microbial cultures which are used in

cancer therapeutics. They work by varied mechanisms. Actinomycin and mithromycin

bind to the DNA and inhibit the DNA-dependent RNA synthesis while the anthracyclines

(doxorubicin and daunorubicin) intercalate between base pairs. Bleomycin causes DNA

strand scission whereas mitomycin C carries out DNA alkylation. The common

mechanism of action throughout these agents is their reaction with DNA to interfere in

the DNA replication and division process[20].

1.4.4. Topoisomerase inhibitors

Topoisomerases are enzymes necessary for the supercoiling and relaxation of DNA

strands during the process of cell division. Topoisomerase inhibitors interfere with the

functioning of these enzymes[21,22]. They are classified depending on the enzyme they

inhibit as Topoisomerase I inhibitors, such as irinotecan and topotecan and

Topoisomerase II inhibitors such as etoposide and daunorubicin.

8

1.4.5. Mitotic inhibitors

These agents commonly act by interrupting cell mitosis through the inhibition of

microtubule polymerization which is an indispensable step during cell division[23]. They

are generally derived from plant alkaloids. Common examples include paclitaxel,

docetaxel, vinblastine and vincristine.

1.4.6. Corticosteroids

Corticosteroids are primarily cholesterol-derived hormones of the adrenal cortex. Their

mechanism of action involves multiple signalling processes to induce cell apoptosis in

tumour cells[24]. Common examples include dexamethasone and prednisone.

1.5. Problems with current cancer chemotherapy

Although these enlisted chemotherapeutic drugs have been effective via various

mechanisms against tumour tissue, there are a few drawbacks associated with their use

which are mentioned in the following section.

1.5.1. Non-specificity

Chemotherapy strategies kill cells by targeting important pathways in rapid cell divisions

characteristic of tumourous cells. However, as these therapies lack specificity, they also

affect cellular processes in normal cells undergoing cell division, thus causing allied non-

specific cytotoxicity[25]. Many singular therapies involving one chemotherapeutic drug

therefore have to be co-administered with agents to prevent or alleviate associated non-

specific cytotoxicity[26]. This causes overburdening of the amount of foreign substances

increasing the chances of the reticulo-endothelial system to eliminate the therapeutic

agent[27].

1.5.2. Multi-drug resistance

Development of multi-drug resistance in cancer cells is one of the leading causes for

failure of cancer chemotherapy. Resistance can be effected by numerous mechanisms

9

such as increased drug efflux, activation of DNA repair mechanisms, produce detoxifying

systems and evasion of drug-induced apoptosis[28]. Multi-drug resistance eventually

causes an increase in drug dosages for beneficial therapy as the cells do not take up the

drug in the required amounts. Also, this can increase dose-dependent toxicity at other

sites.

1.5.3. Biological barriers

The human body contains a number of biological and biophysical barriers such as the

blood brain barrier (BBB), cells of the reticulo-endothelial system (RES) and endothelial

cell junctions. The BBB is comprised of tight junctions between epithelial cells, halting

extravasation of chemotherapeutic agents. The BBB also has high specificity in the size

and solubility characteristics of the molecules passing through it. The endothelial cells

and cells of the RES act as immunological barriers by effectively hoarding therapeutic

agents[29]. This causes an inability to deliver therapeutic drugs at the target site and

increased drug dosages.

1.5.4. In-vivo degradation

The metabolic enzymes within the human body, such as proteases, lipases, hydrolases

etc. can rapidly degrade chemotherapy drugs by chemical reactions with their susceptible

reactive groups. This may cause reduction in activity, if the altered functional groups are

important for the therapeutic activity. This causes a lack of significant therapy at the

prescribed dosages and hence, a subsequent increase in drug administration [30].

1.5.5. Poor solubility and stability

Drugs used in cancer treatment are divided into two types based on their solubilities as

hydrophilic and hydrophobic. While hydrophilic drugs are immediately assimilated in the

blood stream if the drug administration route is intravenous, hydrophobic drugs are not

easily soluble and hence require greater therapeutic concentrations to be as effective.

10

Also, certain drugs rapidly deteriorate under certain pH environs[31]. This is a major

challenge as the human body contains many pH conditions for example the physiological

pH of blood is 7.4 while that of a cancer environment is more acidic at around pH 5. This

creates problems in delivering drugs at specific sites.

Nanoparticles have been used as vectors to help protect and deliver anti-cancer drugs by

attempting to circumvent these problems. They are described in greater detail in the

following section.

1.6. Nanoparticles used for chemotherapy

Nanoparticles employed in chemotherapy are typically materials in the size range of 1-

1000 nm, capable of delivering therapeutics in or around the tumour

microenvironment[32]. The small size of nanoparticles allows them to accumulate in

tumour tissues due to the increased permeability and confinement of tumour tissue as a

result of the enhanced permeability and retention (EPR) effect. A size between 10-200

nm is the most preferable size range for anti-cancer drug delivery as particles smaller than

6 nm can be excreted by the kidney, and those larger than 300 nm can be rapidly

recognized and removed by the reticulo-endothelial system (RES)[33]. Therefore,

nanoparticles with this size range are circulated more in the blood after intravenous

administration which provides more opportunity to accumulate in tumour tissues. Over

the years, different types of nanoparticles have been constructed with the view to develop

a fool-proof system for cancer treatment (Refer Fig 1.1.)[34]. Herein, the types of

nanoparticle that have been used for cancer therapy have been discussed.

11

Fig 1.1: Different types of nanoparticles used in chemotherapy (Reproduced with permission from

Masserini et al. 2013)

1.6.1. Polymeric nanoparticles

Polymeric nanoparticles are composed of polymer and co-polymer matrices in the form

of nanospheres and nanovesicles[35]. Nanospheres are matrix systems, wherein the

nanoparticle core is solid and drug molecules may be adsorbed at the sphere surface or

encapsulated within the particle. Nanocapsules are vesicular systems where the entrapped

substances are confined to a cavity consisting of a liquid core (either oil or water)

surrounded by a solid material shell[36]. Commonly used polymeric materials are

poly(lactic-co-glycolic acid) (PLGA), polylactide (PLA), polyethylene glycol (PEG),

polyglutamic acid (PGA), polyethyleneimine (PEI), polyacrylic acid (PA), etc.

1.6.2. Lipid based nanocarriers

Commonly used lipid based nanoparticles include liposomes, nanostructured lipid

carriers and solid lipid nanoparticles. These nanoparticles are favoured more for in-vivo

applications as the lipid matrix is predominantly constructed using physiological lipids

12

which are endogenous to, and hence can be easily degraded within, the cellular milieu

after drug delivery[37]. Commonly used lipid matrix constituents include triglycerides,

fatty acids and phospholipids such as phosphodylcholine and phosphoethanolamine.

1.6.3. Inorganic nanoparticles

Inorganic nanoparticles have demonstrated commendable success in in-vivo and in-vitro

cancer therapies and imaging due to their unique optical-electronic properties. Quantum

dots, carbon nanotubes, gold nanoparticles (spheres, shells, rods and cages), iron oxide

magnetic nanoparticles and ceramic nanoparticles have been employed in tumour

targeting, imaging, photothermal therapy and drug delivery applications[38]. While they

do suffer from several drawbacks such as non-biodegradability and non-biocompatibility,

considerable advances have been made to address their cytotoxicity and give them stealth-

like properties[39].

1.6.4. Carbohydrate-based nanoparticles

Carbohydrate-based nanoparticles have received considerable interest as drug carriers

due to their natural origin and inherent biodegradability and biocompatibility[40].

Commonly used carbohydrates include cyclodextrins, chitosan, amylose, hyaluronic acid

and heparin[41].

1.6.5. Protein-based nanoparticles

Protein-based nanoparticles also display biocompatibility and biodegradability[42].

However, these nanoparticles may show immunogenicity. Abraxane, a human serum

albumin based nanoparticle formulation, has been successfully introduced in the market.

Other proteins explored for nanoparticle synthesis include gelatin, legumin and gliadin.

1.6.6. Dendrimers

Dendrimers are synthetic organic compounds with spherical 3D structural morphology

showing a branched structure. Features like nanoscopic size, narrow polydispersity index,

13

control over molecular structure, the presence of interior cavities and high surface

functionalities makes them attractive candidates for delivery in biological

applications[43]. Commonly used matrix polymers include polypropylene imine,

polyethyleneimine and polyamidoamine (PAMAM). However, they do suffer from

inherent toxicity due to the synthetic nature of the matrix which also makes them more

susceptible to expulsion by the reticulo-endothelial system[44].

1.7. Mechanisms for targeting

Nanoparticle-mediated chemotherapy relies on two types of delivery strategies; active

and passive (Fig. 1.2). Passively-targeted nanoparticle assemblies contain the therapeutic

load but do not have a targeting moiety fabricated in them. This passive targeting relies

on the enhanced permeation and retention (EPR) effect, distinctive of tumour vasculature,

for efficient therapy[45]. The EPR effect accounts for the increased vascular permeability

due to interendothelial gap defects, allowing extravasation of nanoparticles up to 400 nm

in diameter, and also poor lymphatic drainage in the tumour microenvironment aiding the

accumulation of and further release from nanoparticles[46,47].

Passively targeted nanocarriers first reached clinical trials in the mid-1980s, wherein few

liposomes and polymer–protein conjugates based products were marketed in the mid-

1990s[35]. Passive targeting however holds several limitations as the non-specific nature

of the process does not warrant a complete payload delivery at the side of the tumour.

Also certain drugs do not diffuse efficiently inside the tumours and furthermore certain

tumours do not exhibit the EPR effect which makes unmitigated therapy improbable[48].

14

Fig 1.2: Different mechanisms of targeting: Passive tissue targeting achieved by the increased

permeation and retention in tumours. Active cellular targeting (inset) can be achieved by

functionalizing the surface of nanoparticles with ligands that promote cell-specific recognition and

binding. (Reproduced with permission from Peer et al. 2007)

To overcome this limitation, specific molecules can be attached on the surface of the

nanocarrier which are specific to a receptor or protein overexpressed on tumour cells and

can therefore serve as markers for effective ligand binding. These surface modified

nanoparticles will get internalized into the target cells through ligand–receptor

interactions. Targeting ligands such as proteins (antibodies and antibody fragments),

peptides, vitamins, carbohydrates and nucleic acids (aptamers) have been widely explored

for this purpose[49].

However, targeted delivery does have limitations such as the receptor capacity, tumour

volume, cancer cell heterogeneity and differences in overexpression of cancer

markers[50].

15

The density of the surface target receptors plays an important role in determining doses

and dosage regimens. If the density of the targeting receptor is less, any excess targeting

nanocarriers will not have available binding surfaces and thus will be removed[45].

If the targeting agent has a very high affinity for the receptor they can get trapped within

and extravasate in the part of the tumour surrounding the capillaries, and consequently

they do not diffuse throughout the tumour, especially in the case of large solid tumours,

hence recurrence of cancer is more likely, even after sufficient therapeutic dosage

administration[51].

The expression of a particular receptor itself may vary within the same tumour owing to

the high frequency of mutations which are characteristic of cancer cell divisions[52].

Therapies which rely on targeting overexpressed receptors in cancer do not consider the

fact that although these receptors are overexpressed in cancer cells as compared to non-

cancerous cells, the number of non-cancerous cells in the body is far more than the

number of tumorous cells and thus targeting could be considered futile.

With the advantages and disadvantages of the targeting approaches in mind, the work in

this thesis explored both approaches, the rationale for which is explained in the following

section.

1.8. Rationale

Many of the chemotherapy drugs used today target the cellular processes such as

increased cell multiplication which are proven cancer markers as cancerous cells require

a greater amount of energy to account for the increased cell division, metastasis and

angiogenesis[52]. Although these processes are amplified in cancer cells, they are not

exclusive to them alone. Hence, normal cells undergoing mitosis and cell division can

also be affected leading to simultaneous non-specific toxicity and cell death at areas not

intended for action by the drugs[25]. This can in turn lead to several undesirable effects

16

such as hair loss, suppression of bone marrow, multi-drug resistance, neurological

dysfunction and cardiac toxicity[53].

Phytochemicals with potent anti-cancer activity have been used as the drugs of choice in

this thesis. The potential advantage of using phytochemicals is that they have shown

considerable specificity for cancer cells, have a better tolerance within a living system as

they are components of a regular diet and hence will not be harmful in case of non-specific

delivery[54].

Epigallocatechin gallate (EGCG) has shown potent chemopreventive and

chemotherapeutic activity in many cancer cell lines the mechanisms of activity also being

diverse[55–57]. Piperine, one of the most pharmacologically active components of black

pepper, has also shown anti-cancer, anti-inflammatory and anti-oxidant activity[58,59].

Mangiferin from Mangifera indica has also shown a wide range of therapeutic properties

as an anti-diabetic, anti-obesity and anti-cancer agent[60–62].

Two kinds of nanovehicles have been used in this study; solid lipid nanoparticles which

have a matrix made of physiological lipids that are non-toxic. Solid lipid nanoparticles

have a number of advantages including an improvement in drug stability and drug

entrapment, biocompatibility and efficient scale up[63]. Chitosan nanoparticles can also

be easily broken down within the mammalian cell, and thus do not show the accumulation

induced toxicity associated with many other conventional nanoparticle systems such as

the ones with inorganic metals as matrix[64].

Ligands have been employed in two of the projects to confer additional specificity to the

delivery vehicles. One of these ligands is bombesin, which is a tetradecapeptide that is a

natural homolog to the gastrin-releasing peptide receptor which is commonly

overexpressed in many types of cancers such as breast, prostate, ovarian, lung and liver

cancers, and it is therefore an excellent cancer biomarker[65]. The second ligand,

17

mannose, was investigated as a ligand for targeting in cancer as it is a natural homolog to

lectin receptors which are commonly overexpressed in breast, liver, prostate and brain

cancers[66]. A further advantage of ligand conjugation is that it can reduce the toxicity

associated with nanoparticle delivery as the cytotoxic drug will only be delivered to the

cells where it is aimed at and not non-tumorous cells[67].

Prior to investigating their anti-cancer properties, the physico-chemical natures of the

nanoparticle systems were well characterised in order to ensure their suitability for

delivery within living systems. The mean particle size of the nanoparticles was

characterised using dynamic light scattering technique (DLS). To study effective

encapsulation and conjugation differential scanning calorimetry (DSC) and Fourier

transform infrared analysis (FTIR) were used. The in-vitro anti-cancer activity was

checked by assays in cancer cell lines. Uptake and migration assays were carried out to

check the efficiency of the nanoparticle system to be taken up by the cells and suppress

proliferation, respectively.

Thus, different combinations of delivery vehicles (lipid and carbohydrate based),

different approaches for delivery (active and passive) and different targeting ligands

(protein and carbohydrate-based) have been explored in this work.

1.9. Nanoparticles used in this work

1.9.1. Solid lipid nanoparticles (SLNs)

18

Fig 1.3: Structure of an SLN (Reproduced with permission from Khatak et al. 2004.)

Solid lipid nanoparticles (SLNs) are submicron sized colloidal nanocarriers prepared

from physiological lipids which are solid at room temperature[68]. These nanoparticles

have shown an improvement in drug stability and drug entrapment, are biocompatible (as

they are composed of easily biodegradable lipids) and can be efficiently scaled up for

production[69]. The most important advantage of SLNs is that they aid in the controlled,

gradual release of the drug, thus improving the retention time and effectiveness of the

drug while preventing premature degradation[70,71].

SLNs can be prepared as a variety of emulsions. The oil-in-water emulsion (O/W) is

effective to encapsulate hydrophobic drugs that can be dissolved in the inner oil phase

while the water-in-oil emulsion (W/O) emulsion can be used to encapsulate hydrophilic

drugs in the inner aqueous core. In addition, a water-in-oil-in-water (W/O/W) double

emulsion can be used to encapsulate drugs which are sensitive to pH changes. The many

methods for preparation of SLNs are enlisted below:

1.9.1.1. Homogenization

This method of preparation involves the dispersion of the organic phase with the aqueous

phase by the use of strong cavitation forces provided by homogenizers to help in

formation of submicron sized nanoparticles[72]. Homogenisation is of two types:

19

1.9.1.1.1. Hot homogenization

Hot homogenization involves the heating of both the aqueous and organic phases to the

same temperature followed by homogenisation to form a stable emulsion[73]. The

temperature used is higher than the melting point of the lipids used, hence the organic

phase exists as a lipid melt. Also, the higher temperature helps in the formation of smaller

droplets and hence smaller nanoparticle sizes[74].

This method is useful for heat resistant drugs which are not affected by the increase in

temperature during heating. However, care must be taken as the temperature can increase

during homogenisation due to the mechanical stress caused by the cavitation. Hence this

method is not suitable for thermo-labile drugs or those susceptible to structural damage.

1.9.1.1.2. Cold homogenization

This method is employed for encapsulating temperature or shear sensitive drugs where

hot homogenization cannot be used for preparation. The drug is initially dissolved in the

lipid melt to form a uniform organic phase. The melt is then rapidly cooled using liquid

nitrogen to form a solid lipid. Then solid lipid is milled with a mortar to form

microparticles, which are dispersed in an aqueous surfactant solution then into the

aqueous phase followed by homogenization. This method typically gives larger particles

compared to hot homogenization, however the extent of milling of the solid lipid greatly

affects the size of the final nanoparticles.

1.9.1.2. Solvent evaporation and emulsification

In this method, the lipids are initially dissolved in a water-immiscible solvent to form a

continuous organic phase[75]. This organic phase is then homogenized into an aqueous

phase containing surfactants to form a stable emulsion. The formed emulsion is now

subjected to solvent evaporation by stirring, wherein the solvent is removed and the lipids

gradually precipitate into the aqueous phase as nanoparticles. However, if the

20

nanoparticles are to be used for biomedical purposes care must be taken while choosing

the solvents, as there could be additional toxicity induced by the use of toxic solvents[76].

1.9.1.3. Microemulsion

Microemulsions are formed by mixing a hot homogenous lipid phase into a greater

volume of cold aqueous phase[77]. A few factors such as the velocity of addition and the

lipophilicity of the solvents used must be considered for determining particle size. The

faster the distribution in aqueous phase, better the polydispersity of the nanoparticle

suspension. Also, more lipophilic solvents yield bigger particle sizes[78]. This could

again be correlated to the easier distribution in the aqueous phase.

1.9.1.4. Solvent emulsification diffusion technique

This method makes use of a solvent partially miscible with water. Initially, both the water

and the solvent are mixed till the system reaches saturation and are in thermodynamic

equilibrium with each other[79]. Following this, the drugs and the lipids are dissolved in

the water saturated solvent phase. The dissolution is carried out at a higher temperature if

the lipids used have a higher melting temperature. The surfactant phase is made by

saturating the surfactant containing aqueous phase with the solvent. The lipid phase is

now emulsified into the surfactant containing aqueous phase to form a primary emulsion.

After this, more water is added to the system to allow the solvent to diffuse into the

continuous phase and thereby causing the lipid to precipitate in a nanoparticulate form.

The diffused solvent is now eliminated through distillation.

1.9.1.5. Hot melt technique

This method involves melting of the solid lipids to form a hot lipid melt. The drug is

dissolved in lipid melt by vigorous mixing and then emulsified in an aqueous phase that

is heated above the melting temperature of the lipids[80]. The dispersion is then cooled

down to produce nanoparticles.

21

1.9.1.6. Ultrasonication

In this method, the lipid phase is initially melted and the drug is dissolved in this hot

phase[81]. The aqueous phase is then heated to the same temperature and added to the

lipid phase dropwise with ultrasonication. Dispersion occurs due to the strong cavitation

forces supplied by the ultrasonicator. This method does not involve the use of organic

solvents and hence, is preferred for nanoparticle applications due to its minimal toxicity.

1.9.1.7. Solvent injection method

The principle of this method involves lipid precipitation from the dissolved lipid into the

aqueous solution while the lipid is initially dissolved in a water miscible solvent[82]. The

lipid-solvent mixture is then slowly added with an injection needle into the aqueous phase

with or without surfactants. The mixing step is performed while the aqueous phase is

under stirring, to maintain homogeneity and uniform particle size.

1.9.1.8. Supercritical fluid technique

A fluid is called supercritical when its pressure and temperature exceed their critical

value. Beyond the supercritical point, the ability of a fluid to dissolve substances is found

to increase. CO2 has been routinely used as the gas for producing super critical fluid (SFC)

in this method as it is inexpensive, non-inflammable and inert to the drugs. Initially, the

drug is dissolved in the solvent and this drug-solvent mixture is allowed to dissolve in the

SFC. The SFC acts as an anti-solvent, wherein it reduces the solubility of solid in the

solution and therefore the solid precipitates as nanoparticles[83].

1.9.1.9. Double emulsification method

This is a two-step process, wherein in the first step, the drug (hydrophilic) is dissolved in

an aqueous solvent (inner aqueous phase) and then dispersed in a lipid containing an

emulsifier (e.g. lecithin), known as oil phase, to produce a primary emulsion (w/o). A

22

double emulsion (w/o/w) is formed after addition of aqueous solution of the hydrophilic

emulsifier (e.g. Poloxamer, PVA) followed by stirring to evaporate organic solvent[84].

Double emulsification avoids the use of high temperatures to melt the lipid for the

preparation of protein and drug-loaded lipid nanoparticles and there is a scope for surface

modification of the nanoparticles to provide steric stability by using a lipid-PEG

derivative. Steric stabilization significantly improves the stability of these colloidal

systems against pH and electrolyte concentration changes in the gastrointestinal fluids.

1.9.2. Chitosan nanoparticles

Fig 1.4: Structure of chitosan polymer (Reproduced with permission from the Chemistry Glossary)

Chitosan (CS) is a linear copolymer of β-(1–4)-linked D-glucosamine and N-acetyl-D-

glucosamine whose molecular structure comprises a linear backbone linked through

glycosidic bonds[85]. The deacetylated chitosan backbone of glucosamine units has a

high density of basic amine groups which are protonated and thus positively charged in

most physiological fluids[86].

CS is considered hydrophilic, but the percentage of acetylated monomers and their

distribution in the chains has a critical effect on its solubility and conformation in aqueous

media. Because of these molecular features, CS exhibits a pH-dependent behaviour and

interesting biopharmaceutical properties such as muco-adhesiveness and the ability to

open epithelial tight junctions[87].

23

The presence of amino (-NH2) and hydroxyl (-OH) groups in chitosan provides

opportunity for a high degree of chemical modification. Chitosan is an interesting

polymer to prepare nanoparticles, as it is biocompatible, nontoxic, biodegradable and has

shown absorption-enhancing effects[87]. Due to the availability of free amino groups, it

carries a positive charge under physiological conditions and reacts with many negatively

charged surfaces such as the cell membrane. CS is insoluble in water and organic solvents,

however, it is soluble in dilute aqueous acidic solution (pH<6.5), which can convert the

glucosamine units into a soluble form of protonated amine (R–NH3+)[88]. CS is also

known to precipitate in alkaline solutions or with polyanions and form a gel at a lower

pH[89].

Fig 1.5: Preparation of chitosan nanoparticles (Reproduced with permission from Mukhopadhyaya

et al.)

24

Chitosan nanoparticles can be prepared by many methods such as the following:

I. Ionotropic gelation

Ionic cross linking is based on electrostatic interaction to form chitosan nanoparticles

using a negatively charged cross linker such as a triphosphate which can make ionic bonds

with positively charged chitosan molecules, thus forming cross linked polymers. This

method has been widely used to encapsulate proteins and drugs as it avoids the use of

strenuous temperature conditions and organic solvents[90].

II. Covalent conjugation:

Covalent cross linking involves formation of covalent bonds between chitosan chains and

cross linking agents such as glutaraldehydes and polyethylene glycol which helps in the

covalent linking of the chitosan chains[85]. Nanoparticles with a narrow size distribution

have been produced by this method.

III. Precipitation

Precipitation is carried out in two ways. In desolvation, a flocculating agent is added to

an aqueous chitosan solution. The decrease in solubility due to the addition of the

flocculant causes chitosan to precipitate out by the formation of hydrogen bonds. Solvent

emulsification involves the addition of an organic phase containing a drug into the

aqueous chitosan solution. Emulsifiers may be added to reduce the surface tension

between both phases. The interaction of the two phases creates turbulence which results

in of precipitation of chitosan nanoparticles[91].

IV. Radical polymerization

Radical polymerization involves the use of acids and strong conditions such as high

temperatures[92]. Chitosan is dissolved in dilute nitric acid solution and then stirred with

ceric ammonium nitrate, nitric acid and isobutyl cyanoacrylate at 40 °C for 40 minutes in

25

an inert nitrogen environment. This method can be fine-tuned for core and shell properties

by using different monomers.

V. Complex formation

Due to the positive charge on the chitosan chain, it can be used to form ionic complexes

with anionic drugs[93]. Ionic complexes have been formed with retinol encapsulated in

chitosan by this method with a particle size of around 102.6 ± 12.0 nm. Encapsulation of

retinol in chitosan incidentally caused a 1,600-fold increase in its solubility.

VI. Spray drying

This method involves spray drying of chitosan along with another metal oxide that it can

be chelated with. Iron oxide and chitosan nanoparticles have been prepared by this

method[94]. It is a one-step simple method for the preparation of chitosan nanoparticles.

VII. Polyelectrolyte formation

This method involves charge neutralisation of the cationic polymer by use of a negatively

charged anionic component such as a DNA molecule to form a polyelectrolytic

complex[95]. The preparatory method is comparatively mild and requires no

sophisticated instrumentation. A negatively charged anionic solution can be added slowly

to the chitosan dissolved in acetic acid under stirring and slow addition to give chitosan

nanoparticles.

1.10. Drugs used in this study

1.10.1. Epigallocatechin gallate (EGCG)

26

Fig 1.6: Structure of EGCG

IUPAC name: [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl) chroman-3-yl] 3,4,5-

trihydroxybenzoate

Molecular weight: 458.372 g/mol

Solubility: Water, ethanol

Epigallocatechin gallate is one of the most abundant catechins found in green tea. It has

shown anti-diabetic, anti-obesity and anti-cancer potential[96–99]. It has shown to

downregulate mRNA and protein expression of MMP-2 matrix metalloproteinases, which

play an important role in the degradation of the basement membrane, angiogenesis and

metastasis, in MCF-7 breast cancer cell line[100]. It induces prostate cancer cell death by

suppressing androgen receptor acetylation in androgen dependent prostate cancer cell line

LNCaP [101]. EGCG has also shown microRNA targeting potential by upregulating miR-

210 microRNA leading to reduced proliferation and anchorage-independent growth in

H1299 and H460 lung cancer cells[102]. It induces apoptosis in HT-29 colon cancer cells

by upregulation of caspase-3 activity and mitochondrial damage [103]. The structure

consists of three aromatic rings with eight free hydroxyl groups (Fig 1.6). Much of the

beneficial activities of EGCG are linked to its structure. However, the free hydroxyl

groups are susceptible to easy reactions within the biological system by enzymes leading

to an overall loss in activity.

27

1.10.2. Piperine

Fig 1.7: Structure of piperine

IUPAC name: (2E,4E)-5-(1,3-Benzodioxol-5-yl)-1-(1-piperidinyl)-2,4-pentadien-1-on


Solubility: Chloroform, ether, acetic acid

Piperine accounts for 5-9% by weight of the components in black pepper. It has been

known as a prominent anti-oxidant, anti-inflammatory and more recently an anti-cancer

agent[104,105].The anti-cancer effect of piperine may be attributed to the inhibition of

NF-κB, c-Fos, ATF-2 and CREB activities, suppression of angiogenesis by inhibiting Akt

phosphorylation, or blockade of the production of pro-inflammatory cytokines and the

activity of matrix metalloproteinases that are likely to promote tumour growth and

metastasis[104].

1.10.3. Mangiferin

Fig 1.8: Structure of mangiferin

28

IUPAC Name: (1S)-1,5-Anhydro-1-(1,3,6,7-tetrahydroxy-9-oxo-9H-xanthen-2-yl)-D-

glucitol


Solubility: ethanol, DMSO

Mangiferin, obtained from the bark, leaves and pulp of Mangifera indica, is a polyphenol

used in ancient Indian Ayurvedic and Chinese medicine. The structure consists of a C-

glycosidic derivative attached to a xanthenone. It has shown anti-oxidant, anti-diabetic,

anti-allergic, anti-HIV and anti-cancer activities[60].

Mangiferin exhibits its anti-cancer activity by downregulating inflammation, arresting

cell cycle, reducing proliferation/metastasis, promoting apoptosis in malignant cells and

also providing protection against oxidative stress and DNA damage[106]. A study has

shown that mangiferin reduced the tumour volumes similar to that of conventional

anticancer drugs such as Cisplatin[107]. However, the drug shows low bioavailability

which can be addressed by the use of appropriate delivery vehicles[60].

1.11. Ligands used in the study

1.11.1. Bombesin

Bombesin receptors are a type of G-protein coupled receptors and have 4 subtypes- BB1,

BB2, BB3 and BB4. BB1 is known as the neuromedin B (NMB) receptor because of its

affinity towards mammalian ligand NMB. The BB2 receptor with high affinity to

mammalian gastrin releasing peptide (GRP) is called the GRP receptor[65].

Reubi et al. investigated 161 cancer cell lines, out of which 12 prostate cancer cell lines,

41 breast cancer cell lines, 5 gastrinomas and 3 of 9 small cell lung carcinomas expressed

predominantly GRP receptors; 11 of 24 intestinal, 1 of 26 bronchial, and 1 of 1 thymic

carcinoids had preferentially NMB receptors whereas 9 of 26 bronchial carcinoids, 1 large

29

cell neuroendocrine lung carcinoma, and 4 of 9 small cell lung carcinomas had

preferentially BB3 receptors[108].

Fig 1.9: Structure of Bombesin receptor BB2 (Reproduced with permissions from Atlas of Genetics

and Cytogenetics in Oncology and Haematology)

GRPR is a transmembrane glycoprotein with 7 transmembrane domains consisting of 384

amino acids. Bombesin is a tetradecapeptide originally isolated from the toad Bombina

bombina. GRPR has shown to bind bombesin with very high affinity, internalising via

receptor-mediated endocytosis[109].

Fig 1.10: Structure of bombesin

1.11.2. Mannose

C-type lectins are a group of carbohydrate-binding membrane proteins, commonly

functioning as components in cell adhesion, glycoprotein turnover, pathogen recognition

and apoptosis[12]. Most of them contain an extracellular carbohydrate recognition

30

domain (CRD) that helps in the detection of free carbohydrates or in complexed forms

like glycoproteins or glycolipids. Internalization of carbohydrates takes place through

clathrin-mediated endocytosis. C-type lectins are commonly overexpressed in a variety

of cancers such as liver, lung and ovarian cancers. Mannose is an aldohexose sugar (an

epimer of glucose), which is a common ligand to C-type lectin receptors. Hence, mannose

was used as the ligand for lectin targeting. Glyco-targeting is gaining importance as these

targeting ligands are not foreign to the cellular system and have negligible toxicity, thus

preventing expulsion by the RES system[66].

Fig 1.11: Structure of D-mannose (Reproduced with permission from the Medical Biochemistry)

1.12. Techniques used in the study

A. Dynamic light scattering

Dynamic light scattering technique can help measure particle size as small as 1 nm in

diameter. The sample is illuminated with a laser beam. The scattered light is detected by

a photon detector. Analysis of the fluctuation of the scattered light provides the diffusion

coefficient D which can be used to calculate radius of the particle R by the Stokes-Einstein

Equation:

𝐷 =𝑘𝐵T

6πηR

Where kB is the Boltzmann-constant, T is the temperature and η is the viscosity.

The zeta potential indicates the surface charge on a particle, and therefore provides

information about the electrostatic repulsion with adjacent particles and thus its stability.

31

Theoretically, it is the electric potential at the slipping planes between the Stern layer

surrounding the particle and the diffused layer in contact with the dispersion

medium[110]. Thus, the greater the surface charge, the higher the repulsion with similar

particles, and hence the greater its resistance towards aggregation.

In addition to measuring the particle size DLS provides information about the

polydispersity of the sample. Polydispersity measures the heterogeneity or non-

uniformity of a sample between index values of 0 and 1. The more heterogeneous a

sample is, the greater will be the value of the polydispersity index.

B. Fourier transform infrared (FTIR) analysis

FTIR technique helps in obtaining different vibrational frequencies of molecular

interactions within a sample. When a sample is irradiated with infra-red radiation,

absorbed IR radiation excites molecules into a higher vibrational state. Bonds between

different heteroatoms give rise to distinct vibrational frequencies to give an infrared

spectrum which can be used to determine the nature of chemical interactions between

drug molecules, excipients and matrix of nanoparticles. A detector measures the intensity

of transmitted or absorbed light as a function of its wavelength.

The FTIR spectra are represented as plots of intensity versus wavenumber (which is the

reciprocal of the wavelength and is given in units of cm-1), with the intensity plotted as

the percentage of light transmittance or absorbance at each wavenumber.

C. Differential scanning calorimetry (DSC)

DSC is a thermo-analytical technique which evaluates heat changes during phase

transitions and chemical reactions as a function of temperature. In this technique, the

sample and reference are heated to the same temperature in hermetically sealed pans in

an inert environment. The DSC thermogram is a curve of heat flux versus temperature or

32

time. This curve can be used to calculate enthalpies of transitions obtained by integrating

the peak corresponding to a given transition using the given equation:

ΔH = KA

where ΔH is the enthalpy of transition, K is the calorimetric constant, and A is the area

under the curve.

During phase transitions, energy is either absorbed or released. If it is an endothermic

process such as protein denaturation and reduction reactions more heat must be supplied

to the sample for it to remain at the same temperature as the reference. Therefore, the

enthalpy will be positive. In an exothermic reaction, such as crystallization, less heat

needs to be provided to the sample hence the enthalpy change of that of the sample relative

to the reference is negative. DSC was used in the study for the determination of the phase

transition of the drugs post encapsulation inside the nanoparticles.

1.13. Aims and objectives

Aims of this research work:

The main aim of this thesis work was to develop and evaluate various nanoformulations

for the delivery of phytochemicals to enhance their stability and cytotoxicity.

Major Objectives:

➢ To develop different nanocarriers and nano-carrier combinations for

delivery of phytochemicals as anti-cancer drugs.

➢ To improve therapeutic efficacy of phytochemicals by providing a stable

nanocarrier system for delivery

These research aims were achieved by the use of the following methodogies:

➢ To optimize formulation and process variables for the development of

biocompatible nanoparticles.

33

➢ To characterize nanoformulations using different analytical techniques such

as UV-Visible spectroscopy, dynamic light scattering, Fourier transform infrared

and differential scanning calorimetry techniques.

➢ Bioconjugation of a targeting ligand on the surface of drug loaded

nanoparticles to prepare targeted nanoparticles.

➢ To study the release patterns of encapsulated phytochemicals from

nanoparticle systems.

➢ Evaluation of in vitro cytotoxicity of targeted and non-targeted, drug-

encapsulated nanoparticles in comparison to pure drug.

➢ To study the cellular uptake of nanoparticles by using fluorescent dye-

loaded nanoparticles.

➢ To study the in-vivo efficacy of targeted formulations by using suitable

animal model

➢ To study the in-vivo efficacy of targeted formulations by survival studies

and changes in tumour volume and body weight.

34

1.14. References

[1] American Cancer Society, Cancer Facts & Figures, 2016.

doi:10.3322/caac.20121.

[2] WHO | Cancer, (n.d.). http://www.who.int/mediacentre/factsheets/fs297/en/

(accessed December 29, 2015).

[3] WHO | Cancer, WHO. (2017). http://www.who.int/cancer/en/ (accessed March 25,

2017).

[4] A. Urruticoechea, R. Alemany, J. Balart, A. Villanueva, F. Viñals, G. Capellá,

Recent Advances in Cancer Therapy : An Overview, (2010) 3–10.

[5] Worldwide cancer incidence statistics | Cancer Research UK, (n.d.).

http://www.cancerresearchuk.org/content/worldwide-cancer-incidence-statistics

(accessed January 4, 2016).

[6] Surgery for Cancer- National Cancer Institute, (n.d.).

https://www.cancer.gov/about-cancer/treatment/types/surgery (accessed August 5,

2017).

[7] R. Rami-Porta, C. Wittekind, P. Goldstraw, et International Association for the

Study of Lung Cancer (IASLC) Staging Committee, K. Tsuboshima, N. Tsubota, et al.,

Complete resection in lung cancer surgery: proposed definition., Lung Cancer. 49 (2005)

25–33. doi:10.1016/j.lungcan.2005.01.001.

[8] M.D. Walker, Chemotherapy: adjuvant to surgery and radiation therapy., Semin.

Oncol. 2 (1975) 69–72. http://www.ncbi.nlm.nih.gov/pubmed/137527 (accessed June 13,

2017).

[9] M. Ichel, B. Olla, D. Ionisio, G. Onzalez, P. Adraig, W. Arde, et al., Improved

survival with radiotherapy and goserelin in locally advanced prostate cancer improved

survival in patients with locally advanced prostate cancer treated with radiotherapy and

35

goserelin,337(n.d.).http://www.nejm.org.ezproxy.lib.rmit.edu.au/doi/pdf/10.1056/NEJM

199707313370502 (accessed June 13, 2017).

[10] How Chemotherapy Drugs Work,

(n.d.).https://www.cancer.org/treatment/treatments-and-side-effects/treatment-

types/chemotherapy/how-chemotherapy-drugs-work.html (accessed March 25, 2017).

[11] W. Yu, I. Whang, A. Averbach, D. Chang, P.H. Sugarbaker, Morbidity and

mortality of early postoperative intraperitoneal chemotherapy as adjuvant therapy for

gastric cancer, Am. J. Surg. 64 (1998) 1104–1108.

[12] K. Drickamer, M.E. Taylor, Recent insights into structures and functions of C-type

lectins in the immune system, Curr. Opin. Struct. Biol. 34 (2015) 26–34.

doi:10.1016/j.sbi.2015.06.003.

[13] R.A.B. Wood, A.R. Moossa, The prospective evaluation of tumour-associated

antigens for the early diagnosis of pancreatic cancer, Br. J. Surg. 64 (1977) 718–720.

doi:10.1002/bjs.1800641009.

[14] R. Mulligan, The basic science of gene therapy, Science (80-. ). 260 (1993) 926–

932. doi:10.1126/science.8493530.

[15] D. Cross, J.K. Burmester, Gene therapy for cancer treatment: past, present and

future., Clin. Med. Res. 4 (2006) 218–27. doi:10.3121/CMR.4.3.218.

[16] Chemotherapy- Cancer Council Australia, (n.d.). http://www.cancer.org.au/about-

cancer/treatment/chemotherapy.html (accessed March 25, 2017).

[17] G.P. Wheeler, Studies Related to the Mechanisms of Action of Cytotoxic

Alkylating Agents: A Review, Cancer Res. 22 (1962).

http://cancerres.aacrjournals.org/content/22/6/651.short (accessed June 13, 2017).

[18] R. Saffhill, G. Margison, P. Oconnor, Mechanisms of carcinogenesis induced by

alkylating agents, Biochim. Biophys. Acta - Rev. Cancer. 823 (1985) 111–145.

36

doi:10.1016/0304-419X(85)90009-5.

[19] G.J. Peters, C.L. van der Wilt, C.J.A. van Moorsel, J.R. Kroep, A.M. Bergman, S.P.

Ackland, Basis for effective combination cancer chemotherapy with antimetabolites,

Pharmacol. Ther. 87 (2000) 227–253. doi:10.1016/S0163-7258(00)00086-3.

[20] G.P. Sartiano, W.E. Lynch, W.D. Bullington, Mechanism of action of the

anthracycline anti-tumor antibiotics, doxorubicin, daunomycin and rubidazone:

Preferential inhibition of DNA polymerase .ALPHA.., J. Antibiot. (Tokyo). 32 (1979)

1038–1045. doi:10.7164/antibiotics.32.1038.

[21] Y. Pommier, P. Pourquier, Y. Fan, D. Strumberg, Mechanism of action of

eukaryotic DNA topoisomerase I and drugs targeted to the enzyme, Biochim. Biophys.

Acta - Gene Struct. Expr. 1400 (1998) 83–106. doi:10.1016/S0167-4781(98)00129-8.

[22] D.A. Burden, N. Osheroff, Mechanism of action of eukaryotic topoisomerase II and

drugs targeted to the enzyme, Biochim. Biophys. Acta - Gene Struct. Expr. 1400 (1998)

139–154. doi:10.1016/S0167-4781(98)00132-8.

[23] N. Jiang, X. Wang, Y. Yang, W. Dai, Advances in Mitotic Inhibitors for Cancer

Treatment, Mini-Reviews Med. Chem. 6 (2006) 885–895.

doi:10.2174/138955706777934955.

[24] S. Watanabe, E. Bruera, Corticosteroids as adjuvant analgesics, J. Pain Symptom

Manage. 9 (1994) 442–445. doi:10.1016/0885-3924(94)90200-3.

[25] J.-J. Monsuez, J.-C. Charniot, N. Vignat, J.-Y. Artigou, Cardiac side-effects of

cancer chemotherapy., Int. J. Cardiol. 144 (2010) 3–15. doi:10.1016/j.ijcard.2010.03.003.

[26] V. Pannu, P. Karna, H.K. Sajja, D. Shukla, R. Aneja, Synergistic antimicrotubule

therapy for prostate cancer, Biochem. Pharmacol. 81 (2011) 478–487.

doi:10.1016/j.bcp.2010.11.006.

[27] L.M. Kaminskas, B.J. Boyd, Nanosized Drug Delivery Vectors and the

37

Reticuloendothelial System, in: Springer, Dordrecht, 2011: pp. 155–178.

doi:10.1007/978-94-007-1248-5_6.

[28] J.-P. Gillet, M.M. Gottesman, Mechanisms of Multidrug Resistance in Cancer, in:

Methods Mol. Biol., 2010: pp. 47–76. doi:10.1007/978-1-60761-416-6_4.

[29] M. Ferrari, Frontiers in cancer nanomedicine: directing mass transport through

biological barriers, Trends Biotechnol. 28 (2010) 181–188.

doi:10.1016/j.tibtech.2009.12.007.

[30] Q.Y. Zhu, A. Zhang, D. Tsang, Y. Huang, Z.-Y. Chen, Stability of Green Tea

Catechins, J. Agric. Food Chem. 45 (1997) 4624–4628. doi:10.1021/jf9706080.

[31] H. Singh, R. Bhandari, I.P. Kaur, Encapsulation of Rifampicin in a solid lipid

nanoparticulate system to limit its degradation and interaction with Isoniazid at acidic pH,

Int. J. Pharm. 446 (2013) 106–111. doi:10.1016/j.ijpharm.2013.02.012.

[32] I. Brigger, C. Dubernet, P. Couvreur, Nanoparticles in cancer therapy and

diagnosis, Adv. Drug Deliv. Rev. 54 (2002) 631–651. doi:10.1016/S0169-

409X(02)00044-3.

[33] M. Longmire, P.L. Choyke, H. Kobayashi, Clearance properties of nano-sized

particles and molecules as imaging agents: considerations and caveats, Nanomedicine. 3

(2008) 703–717. doi:10.2217/17435889.3.5.703.

[34] M. Ferrari, Cancer nanotechnology: opportunities and challenges, Nat. Rev.

Cancer. 5 (2005) 161–171. doi:10.1038/nrc1566.

[35] C. Errico, A. Dessy, A. Piras, F. Chiellini, Polymeric Nanoparticles for Targeted

Delivery of Bioactive Agents and Drugs, in: Polym.

Biomater., CRC Press, 2013: pp. 593–616. doi:doi:10.1201/b13757-18.

[36] F. Masood, Polymeric nanoparticles for targeted drug delivery system for cancer

therapy, Mater. Sci. Eng. C. 60 (2016) 569–578. doi:10.1016/j.msec.2015.11.067.

38

[37] K. Manjunath, J.S. Reddy, V. Venkateswarlu, Solid lipid nanoparticles as drug

delivery systems, Methods Find. Exp. Clin. Pharmacol. 27 (2005) 127.

doi:10.1358/mf.2005.27.2.876286.

[38] T.A. Pecorelli, M.M. Dibrell, Z. Li, C.R. Thomas, J.I. Zink, Multifunctional

inorganic nanoparticles for imaging, targeting, and drug delivery, in: S. Achilefu, R.

Raghavachari (Eds.), 2010: p. 75760K. doi:10.1117/12.841168.

[39] K.G. Neoh, E.T. Kang, Functionalization of inorganic nanoparticles with polymers

for stealth biomedical applications, Polym. Chem. 2 (2011) 747–759.

doi:10.1039/C0PY00266F.

[40] M. Marradi, I. Garc?a, S. Penad?s, Carbohydrate-Based Nanoparticles for Potential

Applications in Medicine, in: 2011: pp. 141–173. doi:10.1016/B978-0-12-416020-

0.00004-8.

[41] C.A. Peptu, L. Ochiuz, L. Alupei, C. Peptu, M. Popa, Carbohydrate based

nanoparticles for drug delivery across biological barriers., J. Biomed. Nanotechnol. 10

(2014) 2107–48. http://www.ncbi.nlm.nih.gov/pubmed/25992451 (accessed March 25,

2017).

[42] A. MaHam, Z. Tang, H. Wu, J. Wang, Y. Lin, Protein-Based Nanomedicine

Platforms for Drug Delivery, Small. 5 (2009) 1706–1721. doi:10.1002/smll.200801602.

[43] P. Kesharwani, K. Jain, N.K. Jain, Dendrimer as nanocarrier for drug delivery,

Prog. Polym. Sci. 39 (2014) 268–307. doi:10.1016/j.progpolymsci.2013.07.005.

[44] L.M. Kaminskas, B.J. Boyd, Nanosized Drug Delivery Vectors and the

Reticuloendothelial System, in: A. Prokop (Ed.), Intracell. Deliv. Fundam. Appl.,

Springer Netherlands, Dordrecht, 2011: pp. 155–178. doi:10.1007/978-94-007-1248-5_6.

[45] E. Ruoslahti, S.N. Bhatia, M.J. Sailor, Targeting of drugs and nanoparticles to

tumors, J. Cell Biol. 188 (2010). http://jcb.rupress.org/content/188/6/759 (accessed June

39

13, 2017).

[46] M. Gaumet, A. Vargas, R. Gurny, F. Delie, Nanoparticles for drug delivery: The

need for precision in reporting particle size parameters, Eur. J. Pharm. Biopharm. 69

(2008) 1–9. doi:10.1016/j.ejpb.2007.08.001.

[47] H. Maeda, The enhanced permeability and retention (EPR) effect in tumor

vasculature: the key role of tumor-selective macromolecular drug targeting, Adv. Enzyme

Regul. 41 (2001) 189–207. doi:10.1016/S0065-2571(00)00013-3.

[48] R.K. Jain, Barriers to Drug Delivery in Solid Tumors, Sci. Am. 271 (1994) 58–65.

doi:10.1038/scientificamerican0794-58.

[49] L. Brannon-Peppas, J.O. Blanchette, Nanoparticle and targeted systems for cancer

therapy, Adv. Drug Deliv. Rev. 64 (2012) 206–212. doi:10.1016/j.addr.2012.09.033.

[50] K.S. Shohdy, A.S. Alfaar, Nanoparticles targeting mechanisms in cancer therapy:

current limitations and emerging solutions, Ther. Deliv. 4 (2013) 1197–1209.

doi:10.4155/tde.13.75.

[51] Y. Fukumori, H. Ichikawa, Nanoparticles for cancer therapy and diagnosis, Adv.

Powder Technol. 17 (2006) 1–28. doi:10.1163/156855206775123494.

[52] L.A. Loeb, K.R. Loeb, J.P. Anderson, Multiple mutations and cancer., Proc. Natl.

Acad. Sci. U. S. A. 100 (2003) 776–81. doi:10.1073/pnas.0334858100.

[53] A. Coates, S. Abraham, S.B. Kaye, T. Sowerbutts, C. Frewin, R.M. Fox, et al., On

the receiving end - patient perception of the side-effects of cancer chemotherapy, Eur. J.

Cancer Clin. Oncol. 19 (1983) 203–8. doi:10.1016/0277-5379(83)90418-2.

[54] H. Sakagami, Apoptosis-inducing activity and tumor-specificity of antitumor

agents against oral squamous cell carcinoma, Jpn. Dent. Sci. Rev. 46 (2010) 173–187.

doi:10.1016/j.jdsr.2010.01.004.

[55] C. Braicu, C.D. Gherman, A. Irimie, I. Berindan-Neagoe, Epigallocatechin-3-

40

Gallate (EGCG) Inhibits Cell Proliferation and Migratory Behaviour of Triple Negative

Breast Cancer Cells, J. Nanosci. Nanotechnol. 13 (2013) 632–637.

doi:10.1166/jnn.2013.6882.

[56] Y.D. Jung, M.S. Kim, B.A. Shin, K.O. Chay, B.W. Ahn, W. Liu, et al., EGCG, a

major component of green tea, inhibits tumour growth by inhibiting VEGF induction in

human colon carcinoma cells., Br. J. Cancer. 84 (2001) 844–50.

doi:10.1054/bjoc.2000.1691.

[57] Y. Xu, C.-T. Ho, S.G. Amin, C. Han, F.-L. Chung, Inhibition of Tobacco-specific

Nitrosamine-induced Lung Tumorigenesis in A/J Mice by Green Tea and Its Major

Polyphenol as Antioxidants, Cancer Res. 52 (1992) 3875–3879.

http://cancerres.aacrjournals.org/content/52/14/3875.short (accessed December 30,

2015).

[58] L. Lai, Q. Fu, Y. Liu, K. Jiang, Q. Guo, Q. Chen, et al., Piperine suppresses tumor

growth and metastasis in vitro and in vivo in a 4T1 murine breast cancer model, Acta

Pharmacol. Sin. 33 (2012) 523–530. doi:10.1038/aps.2011.209.

[59] C.R. Pradeep, G. Kuttan, Effect of piperine on the inhibition of lung metastasis

induced B16F-10 melanoma cells in mice, Clin. Exp. Metastasis. 19 (2002) 703–708.

doi:10.1023/A:1021398601388.

[60] P. Rajendran, T. Rengarajan, N. Nandakumar, H. Divya, I. Nishigaki, Mangiferin

in cancer chemoprevention and treatment: pharmacokinetics and molecular targets, J.

Recept. Signal Transduct. 35 (2015) 76–84. doi:10.3109/10799893.2014.931431.

[61] H. Li, J. Huang, B. Yang, T. Xiang, X. Yin, W. Peng, et al., Mangiferin exerts

antitumor activity in breast cancer cells by regulating matrix metalloproteinases,

epithelial to mesenchymal transition, and β-catenin signaling pathway, Toxicol. Appl.

Pharmacol. 272 (2013) 180–190. doi:10.1016/j.taap.2013.05.011.

41

[62] R.K. Khurana, R. Kaur, S. Lohan, K.K. Singh, B. Singh, Mangiferin: a promising

anticancer bioactive, Pharm. Pat. Anal. 5 (2016) 169–181. doi:10.4155/ppa-2016-0003.

[63] A. zur Mühlen, C. Schwarz, W. Mehnert, Solid lipid nanoparticles (SLN) for

controlled drug delivery – Drug release and release mechanism, Eur. J. Pharm. Biopharm.

45 (1998) 149–155. doi:10.1016/S0939-6411(97)00150-1.

[64] S.J. Soenen, P. Rivera-Gil, J.-M. Montenegro, W.J. Parak, S.C. De Smedt, K.

Braeckmans, Cellular toxicity of inorganic nanoparticles: Common aspects and

guidelines for improved nanotoxicity evaluation, Nano Today. 6 (2011) 446–465.

doi:10.1016/j.nantod.2011.08.001.

[65] J. Zhou, J. Chen, M. Mokotoff, R. Zhong, L.D. Shultz, E.D. Ball,

Bombesin/Gastrin-Releasing Peptide Receptor: A Potential Target for Antibody-

Mediated Therapy of Small Cell Lung Cancer, (n.d.).

http://clincancerres.aacrjournals.org/content/clincanres/9/13/4953.full.pdf (accessed

March 25, 2017).

[66] H. Ghazarian, B. Idoni, S.B. Oppenheimer, A glycobiology review: Carbohydrates,

lectins and implications in cancer therapeutics, Acta Histochem. 113 (2011) 236–247.

doi:10.1016/j.acthis.2010.02.004.

[67] M. Srinivasarao, C. V. Galliford, P.S. Low, Principles in the design of ligand-

targeted cancer therapeutics and imaging agents, Nat. Rev. Drug Discov. 14 (2015) 1–17.

doi:10.1038/nrd4519.

[68] W. Mehnert, Solid lipid nanoparticles Production, characterization and

applications, Adv. Drug Deliv. Rev. 47 (2001) 165–196. doi:10.1016/S0169-

409X(01)00105-3.

[69] S. Weber, A. Zimmer, J. Pardeike, Solid Lipid Nanoparticles (SLN) and

Nanostructured Lipid Carriers (NLC) for pulmonary application: a review of the state of

42

the art., Eur. J. Pharm. Biopharm. 86 (2014) 7–22. doi:10.1016/j.ejpb.2013.08.013.

[70] C. Qi, Y. Chen, Q.-Z. Jing, X.-G. Wang, Preparation and characterization of

catalase-loaded solid lipid nanoparticles protecting enzyme against proteolysis., Int. J.

Mol. Sci. 12 (2011) 4282–93. doi:10.3390/ijms12074282.

[71] C. Schwarz, W. Mehnert, J.S. Lucks, R.H. Müller, Solid lipid nanoparticles (SLN)

for controlled drug delivery. I. Production, characterization and sterilization, J. Control.

Release. 30 (1994) 83–96. doi:10.1016/0168-3659(94)90047-7.

[72] A.C. Silva, E. González-Mira, M.L. García, M.A. Egea, J. Fonseca, R. Silva, et al.,

Preparation, characterization and biocompatibility studies on risperidone-loaded solid

lipid nanoparticles (SLN): High pressure homogenization versus ultrasound, Colloids

Surfaces B Biointerfaces. 86 (2011) 158–165. doi:10.1016/j.colsurfb.2011.03.035.

[73] R. Abdullah, N.A. Alhaj, S. Ibrahim, A. Bustamam, Tamoxifen Drug Loading Solid

Lipid Nanoparticles Prepared by Hot High Pressure Homogenization Techniques, Am. J.

Pharmacol. Toxicol. 3 (2008) 219–224.

http://thescipub.com/PDF/ajptsp.2008.219.224.pdf (accessed August 5, 2017).

[74] K. Manjunath, V. Venkateswarlu, Pharmacokinetics, tissue distribution and

bioavailability of clozapine solid lipid nanoparticles after intravenous and intraduodenal

administration, J. Control. Release. 107 (2005) 215–228.

doi:10.1016/j.jconrel.2005.06.006.

[75] D. Liu, S. Jiang, H. Shen, S. Qin, J. Liu, Q. Zhang, et al., Diclofenac sodium-loaded

solid lipid nanoparticles prepared by emulsion/solvent evaporation method, J.

Nanoparticle Res. 13 (2011) 2375–2386. doi:10.1007/s11051-010-9998-y.

[76] A. Sharma, S. V Madhunapantula, G.P. Robertson, Toxicological considerations

when creating nanoparticle-based drugs and drug delivery systems, Expert Opin. Drug

Metab. Toxicol. 8 (2012) 47–69. doi:10.1517/17425255.2012.637916.

43

[77] Z. Rahman, A.S. Zidan, M.A. Khan, Non-destructive methods of characterization

of risperidone solid lipid nanoparticles, Eur. J. Pharm. Biopharm. 76 (2010) 127–137.

doi:10.1016/j.ejpb.2010.05.003.

[78] S. Mukherjee, S. Ray, R.S. Thakur, Solid lipid nanoparticles: a modern formulation

approach in drug delivery system., Indian J. Pharm. Sci. 71 (2009) 349–358.

doi:10.4103/0250-474X.57282.

[79] M. Trotta, F. Debernardi, O. Caputo, Preparation of solid lipid nanoparticles by a

solvent emulsification?diffusion technique, Int. J. Pharm. 257 (2003) 153–160.

doi:10.1016/S0378-5173(03)00135-2.

[80] V. Venkateswarlu, K. Manjunath, Preparation, characterization and in vitro release

kinetics of clozapine solid lipid nanoparticles, J. Control. Release. 95 (2004) 627–638.

doi:10.1016/j.jconrel.2004.01.005.

[81] G. Suresh, K. Manjunath, V. Venkateswarlu, V. Satyanarayana, Preparation,

characterization, and in vitro and in vivo evaluation of lovastatin solid lipid

nanoparticles., AAPS PharmSciTech. 8 (2007) 24. doi:10.1208/pt0801024.

[82] M.A. Schubert, C.C. Müller-Goymann, Solvent injection as a new approach for

manufacturing lipid nanoparticles – evaluation of the method and process parameters,

Eur. J. Pharm. Biopharm. 55 (2003) 125–131. doi:10.1016/S0939-6411(02)00130-3.

[83] P. CHATTOPADHYAY, B. SHEKUNOV, D. YIM, D. CIPOLLA, B. BOYD, S.

FARR, Production of solid lipid nanoparticle suspensions using supercritical fluid

extraction of emulsions (SFEE) for pulmonary delivery using the AERx system?, Adv.

Drug Deliv. Rev. 59 (2007) 444–453. doi:10.1016/j.addr.2007.04.010.

[84] M. Gallarate, M. Trotta, L. Battaglia, D. Chirio, Preparation of solid lipid

nanoparticles from W/O/W emulsions: Preliminary studies on insulin encapsulation, J.

Microencapsul. 26 (2009) 394–402. doi:10.1080/02652040802390156.

44

[85] J.H. Park, G. Saravanakumar, K. Kim, I.C. Kwon, Targeted delivery of low

molecular drugs using chitosan and its derivatives, Adv. Drug Deliv. Rev. 62 (2010) 28–

41. doi:10.1016/j.addr.2009.10.003.

[86] K.A. Janes, M.P. Fresneau, A. Marazuela, A. Fabra, M.J. Alonso, Chitosan

nanoparticles as delivery systems for doxorubicin, J. Control. Release. 73 (2001) 255–

267. doi:10.1016/S0168-3659(01)00294-2.

[87] M. Garcia-Fuentes, M.J. Alonso, Chitosan-based drug nanocarriers: Where do we

stand?, J. Control. Release. 161 (2012) 496–504. doi:10.1016/j.jconrel.2012.03.017.

[88] S. Kunjachan, S. Jose, T. Lammers, Understanding the mechanism of ionic gelation

for synthesis of chitosan nanoparticles using qualitative techniques, Asian J. Pharm. Free

Full Text Artic. from Asian J Pharm. 4 (2014).

http://www.brnsspublicationhub.org/ajp/index.php/ajp/article/view/220 (accessed June

13, 2017).

[89] K.Y. Lee, W.S. Ha, W.H. Park, Blood compatibility and biodegradability of

partially N-acylated chitosan derivatives, Biomaterials. 16 (1995) 1211–1216.

doi:10.1016/0142-9612(95)98126-Y.

[90] W. Fan, W. Yan, Z. Xu, H. Ni, Formation mechanism of monodisperse, low

molecular weight chitosan nanoparticles by ionic gelation technique, Colloids Surfaces B

Biointerfaces. 90 (2012) 21–27. doi:10.1016/j.colsurfb.2011.09.042.

[91] C. Yuwei, W. Jianlong, Preparation and characterization of magnetic chitosan

nanoparticles and its application for Cu(II) removal, Chem. Eng. J. 168 (2011) 286–292.

doi:10.1016/j.cej.2011.01.006.

[92] F. Atyabi, S. Saremi, S.P. Akhlaghi, R. Dinarvand, S.N. Ostad, Thiolated chitosan

nanoparticles for enhancing oral absorption of docetaxel: preparation, in vitro and ex vivo

evaluation, Int. J. Nanomedicine. (2011) 119. doi:10.2147/IJN.S15500.

45

[93] published by Dove Press, NSA-91785-factors-determining-the-stability--size-

distribution--and-ce, (n.d.). doi:10.2147/NSA.S91785.

[94] Z. Zeng, Recent advances of chitosan nanoparticles as drug carriers, Int. J.

Nanomedicine. (2011) 765. doi:10.2147/IJN.S17296.

[95] A. Grenha, Chitosan nanoparticles: a survey of preparation methods, J. Drug

Target. 20 (2012) 291–300. doi:10.3109/1061186X.2011.654121.

[96] S. Wolfram, D. Raederstorff, M. Preller, Y. Wang, S.R. Teixeira, C. Riegger, et al.,

Epigallocatechin Gallate Supplementation Alleviates Diabetes in Rodents, J. Nutr. 136

(2006) 2512–2518. http://jn.nutrition.org/content/136/10/2512.short (accessed December

30, 2015).

[97] M.-S. Lee, C.-T. Kim, Y. Kim, Green tea (-)-epigallocatechin-3-gallate reduces

body weight with regulation of multiple genes expression in adipose tissue of diet-

induced obese mice., Ann. Nutr. Metab. 54 (2009) 151–7. doi:10.1159/000214834.

[98] E. Tedeschi, H. Suzuki, M. Menegazzi, Antiinflammatory Action of EGCG, the

Main Component of Green Tea, through STAT-1 Inhibition, Ann. N. Y. Acad. Sci. 973

(2002) 435–437. doi:10.1111/j.1749-6632.2002.tb04678.x.

[99] Z.P. Chen, J.B. Schell, C.-T. Ho, K.Y. Chen, Green tea epigallocatechin gallate

shows a pronounced growth inhibitory effect on cancerous cells but not on their normal

counterparts, Cancer Lett. 129 (1998) 173–179. doi:10.1016/S0304-3835(98)00108-6.

[100] T. Sen, S. Moulik, A. Dutta, P.R. Choudhury, A. Banerji, S. Das, et al.,

Multifunctional effect of epigallocatechin-3-gallate (EGCG) in downregulation of

gelatinase-A (MMP-2) in human breast cancer cell line MCF-7., Life Sci. 84 (2009) 194–

204. doi:10.1016/j.lfs.2008.11.018.

[101] Y.-H. Lee, J. Kwak, H.-K. Choi, K.-C. Choi, S. Kim, J. Lee, et al., EGCG

suppresses prostate cancer cell growth modulating acetylation of androgen receptor by

46

anti-histone acetyltransferase activity, Int. J. Mol. Med. 30 (2012) 69–74.

http://www.spandidos-publications.com/ijmm/30/1/69/abstract (accessed December 30,

2015).

[102] H. Wang, S. Bian, C.S. Yang, Green tea polyphenol EGCG suppresses lung cancer

cell growth through upregulating miR-210 expression caused by stabilizing HIF-1alpha,

Carcinogenesis. 32 (2011) 1881–1889. doi:10.1093/carcin/bgr218.

[103] C. Chen, G. Shen, V. Hebbar, R. Hu, E.D. Owuor, A.-N.T. Kong, Epigallocatechin-

3-gallate-induced stress signals in HT-29 human colon adenocarcinoma cells.,

Carcinogenesis. 24 (2003) 1369–78. doi:10.1093/carcin/bgg091.

[104] D. Ouyang, L. Zeng, H. Pan, L. Xu, Y. Wang, K. Liu, et al., Piperine inhibits the

proliferation of human prostate cancer cells via induction of cell cycle arrest and

autophagy, Food Chem. Toxicol. 60 (2013) 424–430. doi:10.1016/j.fct.2013.08.007.

[105] J.S. Bang, D.H. Oh, H.M. Choi, B.-J. Sur, S.-J. Lim, J.Y. Kim, et al., Anti-

inflammatory and antiarthritic effects of piperine in human interleukin 1β-stimulated

fibroblast-like synoviocytes and in rat arthritis models, Arthritis Res. Ther. 11 (2009)

R49. doi:10.1186/ar2662.

[106] F. Gold-Smith, A. Fernandez, K. Bishop, Mangiferin and Cancer: Mechanisms of

Action, Nutrients. 8 (2016) 396. doi:10.3390/nu8070396.

[107] African Networks on Ethnomedicines., African journal of traditional,

complementary, and alternative medicines : AJTCAM, [African Networks on

Ethnomedicines], 2004. http://www.bioline.org.br/request?tc11025 (accessed August 6,

2017).

[108] J.C. Reubi, S. Wenger, J. Schmuckli-Maurer, J.-C. Schaer, M. Gugger, Bombesin

Receptor Subtypes in Human Cancers, Clin. Cancer Res. 8 (2002).

http://clincancerres.aacrjournals.org/content/8/4/1139.article-info (accessed June 13,

47

2017).

[109] A.A. Begum, P.M. Moyle, I. Toth, Investigation of bombesin peptide as a targeting

ligand for the gastrin releasing peptide (GRP) receptor, Bioorg. Med. Chem. 24 (2016)

5834–5841. doi:10.1016/j.bmc.2016.09.039.

[110] 2 Origin of charge at interfaces : Structure of the electrical double layer, (n.d.).

48

Enhancement of cancer cytotoxicity of

EGCG by encapsulation within solid

lipid nanoparticles

49

2.1. Background

Tea, obtained from the cured leaves of the tea shrub Camellia sinensis, has been known

as a health promoting beverage since ancient times; its medicinal properties discovered

as early as the 5th century AD[1]. Most of the health benefits associated with drinking

tea including its anti-obesity and anti-oxidant effects have been attributed to its

polyphenolic constituents, especially its catechins including epigallocatechin gallate

(EGCG), (-)-epigallocatechin, (-)-epicatechin gallate and (-)- epicatechin [2–4]. EGCG is

one of the most abundant and active constituents of green tea with extensive therapeutic

properties[5,6]. Research on EGCG has shown that it exhibits a wide array of therapeutic

potencies including anti-inflammatory, anti-diabetic, anti-obesity and anti-cancer

effects[5,7–10]. EGCG has been found effective not only as a chemopreventive but also

as a potent chemotherapeutic agent[11–13]. It has been documented to possess anti-

cancer activity in many cancer cell lines by inhibiting tumorigenesis, proliferation and

angiogenesis[14–16]. Many pathways have been elucidated to explain the anti-cancer

activity of EGCG as described below:

a. Telomerase activity inhibition

Telomeres are long repeating, highly-conserved, end-nucleotide sequences in eukaryotic

cells that help cells to divide without losing any of the crucial genes in the process[17].

During cell replication, the enzyme DNA polymerase uses each strand of the DNA as a

template to synthesize two daughter strands[18].

However, the enzyme is not able to completely replicate the entire strand, and hence there

is a shortening of the end of the strand following each division. Telomeres help in

preserving the important genetic sequences by getting shortened themselves in the

process[17]. Telomerase is an RNA-dependent DNA polymerase (reverse transcriptase)

that helps replenish the units of the telomeres. In most somatic cells, there is no telomerase

50

so the cells cease replicating following a certain number of divisions[19]. Expression of

telomerases is upregulated in a number of cancers thus leading to the maintenance and

the continuous replication of genetic material[20]. EGCG has shown to inhibit telomerase

activity in a number of cancers, thus leading to cell division cessation[21].

b. Gene silencing

DNA hypermethylation is the process of the addition of methyl groups to the DNA strand

by the enzyme DNA methyltransferase. When the DNA subunits at the promoter region

of a gene are hypermethylated, there is no binding of the transcription factors at this

portion leading to gene silencing[22]. Cancer cells are found to induce hypermethylation

at the regions of the DNA that contain pro-apoptotic genes in order to maintain continuous

cell multiplication[23]. EGCG is found to directly inhibit DNA methyltransferase and

also causes reactivation of methylation-silenced genes leading to increased apoptosis

among cancer cell populations [24].

c. Angiogenesis

Angiogenesis is a process of neovascularization wherein new blood vessels are formed

from existing ones. Cancer cells upregulate angiogenesis in order to compensate for the

greater blood supply and nutrients required for the highly escalated rate of cell

division[25]. The process includes disruption of the basement membrane to allow

migration of angiogenic factors which help recruit new vessels. Vascular endothelial

growth factor (VEGF) is one such potent angiogenic factor upregulated in angiogenesis

[26].

EGCG has been reported to inhibit VEGF expression by downregulating expression of

hypoxia-inducible factor-1α which is a VEGF stimulator. Also, the hypoxia and oxidative

stress in cancer cells leads to production of oxygen radicals, which activate transcription

factors such as nuclear factor kappa-B(NFκB), that also cause overexpression of VEGF.

51

EGCG is also found to downregulate expression of NFκB, thus indirectly reducing

VEGF[27].

d. Metastasis

Metastasis is the movement of cancer cells from the primary area of tumour development

to other parts of the body through blood vessels or the lymphatic system. It involves the

degradation of the basement membrane which causes disruption of the membrane wall,

helping dissemination of cancer cells to cause tumour formations elsewhere[28]. EGCG

has been found to inhibit matrix metalloproteinases 2, 3 and 9 which are important in the

degradation of the basement membrane, thus preventing the spread of tumour to other

areas[29].

e. Apoptosis

Apoptosis is a process of programmed cell death that occurs through two pathways-

intrinsic and extrinsic. The extrinsic pathway functions by the activation of death

receptors by binding which triggers a cascade to allow the release of caspases that bring

out events in apoptosis. The intrinsic pathway involves pro-apoptotic proteins such as

BCL-2-associated X protein (BAX) and BCL-2 homologous antagonist killer (BAK) that

triggers activation of caspases. This pathway is regulated by anti-apoptotic proteins such

as B-cell lymphoma 2 (BCL-2) and B cell lymphoma extra-large (BCL-XL), which

antagonize with BAX and BAK. Cancer cells have shown to supress apoptotic pathways

to maintain immortality[30]. EGCG has been shown to promote the expression of pro-

apoptotic proteins BAX and BAK while also consequently inhibiting the expression of

BCL-2 and BCL-XL in many cancer types[31,32].

f. Anti-proliferative activity

Mitogen activated protein kinases (MAPK) are enzymes involved in important steps for

cell survival, proliferation and development of cancer cells[33]. EGCG has shown to

52

inhibit MAPK pathway in many cancer cell types. It has also been shown to inhibit

insulin-like growth factor-I receptor activity which helps to activate the MAPK pathway

and plays an important role in proliferation[34].

Fig 2.1: Structure of epigallocatechin gallate

EGCG’s diverse therapeutic properties are attributed to its polyphenolic structure that

allows electron delocalization on the benzene ring helping quench free radicals[35]. It has

a trihydroxy group substitution on one ring and an esterified gallate group attached to

another. However, it has been found to be unstable in neutral and alkaline conditions;

undergoing rapid degradation involving the deprotonation of the hydroxyl groups[36].

Also, the hydroxyl groups are susceptible to various biotransformation reactions within

the living system such as glucuronidation, methylation and sulphonidation leading to a

loss in biological activity[37]. This instability has also been cited as one of the reasons

for the poor bioavailability of EGCG and other catechins within the biological

system[38]. In this study, the stability of EGCG in solutions of different pH was initially

investigated to determine the pH range where EGCG is stable at room temperature. Then,

EGCG was encapsulated in solid lipid nanoparticles (SLNs) for enhancing the stability

and anti-cancer activity of EGCG. SLN were prepared from biocompatible lipids which

are solid at room and body temperature[39]. They provide multiple advantages over other

conventional nanocarriers such as lipid nanoemulsions and polymeric nanoparticles.

These advantages include an improvement in drug stability, drug entrapment and

53

biocompatibility, as they are composed of easily biodegradable excipients, and offers

efficient scaling up for production[40,41]. Moreover, SLN control the release of a pH-

sensitive drug such as EGCG and therefore, prevent the premature degradation of

encapsulated drug in the biological system[42,43]. Post encapsulation of EGCG in SLN,

the anti-cancer activity of pure EGCG and EGCG-loaded SLN (EGCG-SLN) was

checked by cellular proliferation assays in MDA-MB-231 human breast cancer and DU-

145 human prostate cancer cell lines.

2.2. Materials

Glycerol mono-stearate (GMS) was purchased from Alfa Aesar (Johnson Matthey

Chemicals, Hyderabad, India). EGCG, tristearin (TS), tripalmitin (TP), stearic acid (SA),

Pluronic F68 (P F68), polyvinyl alcohol (PVA), Roswell Park Memorial Institute

(RPMI)-1640 medium, fetal bovine serum (FBS), trypsin–EDTA, phosphate buffered

saline (Ca2+, Mg2+ free), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

(MTT), dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich (St. Louis, MO,

USA). Soya lecithin was purchased from HiMedia (Mumbai, India). MDA-MB-231

human breast cancer cells and DU-145 human prostate cancer cells were obtained from

American Type Culture Collection (ATCC, Manassas, USA). Dichloromethane was

obtained from Merck chemicals (Mumbai, India). Tween180 was purchased from sd Fine

Chemicals Limited (Hyderabad, India).

2.3. Methods

2.3.1. Aqueous Stability

The stability of EGCG was determined in different aqueous solutions; 0.1 N HCl (pH

1.5), phosphate buffer saline pH 7.4 (PBS), normal saline and phosphate buffer solutions

of different pH (5, 7.4 and 9). A stock solution of EGCG was prepared (1 mg/mL) and

dilutions were made in the study medium to give a final drug concentration of 50 µg/mL.

54

UV-Visible spectroscopy was used to evaluate changes in the absorption characteristics

of EGCG throughout the study at λmax of EGCG at 274.5 nm. Samples were scanned in

the range of 200-800 nm and spectra were obtained in the various solutions at different

time points of 0, 4, 8, 24, 48 and 96 h using a Jasco-UV650 spectrophotometer.

2.3.2. Optimization

The most important aspect of nanoparticle preparation for biomedical applications is the

regulation of nanoparticle sizes and particle surface charges for obtaining optimum

dimensions as the cell endothelia and tumour microenvironments have stringent

limitations. The synthesis of nanoparticles was optimized with respect to different process

and formulation parameters such as the type of lipid, lipid to co-lipid ratio, surfactant used

and its concentration, amount of co-emulsifier, inner aqueous phase volume and

sonication time.

2.3.3. Preparation of EGCG loaded solid lipid nanoparticles (EGCG-SLNs)

EGCG-SLNs were prepared by emulsion-solvent evaporation method as reported in the

literature[43]. Briefly, 80 mg GMS, 20 mg SA and 20 mg soya lecithin were dissolved

in dichloromethane and sonicated for 3 min. EGCG was dissolved in 500 µL distilled

water and 2 % w/v Pluronic F68 solution was then added to the lipid mixture to form a

water-in-oil (w/o) primary emulsion. The mixture was sonicated for 5 min and then

transferred into 1 % w/v Pluronic F68 solution to form a double emulsion. The emulsion

was stirred for 2 h to evaporate the organic solvent. The nanoparticle suspension was

centrifuged and washed thrice with distilled water to remove any free drug adhering to

the nanoparticle surface. Blank nanoparticles (Blank-SLNs) were prepared in a similar

manner, excluding the drug.

2.3.4. Determination of drug entrapment efficiency

The drug entrapment efficiency (EE) was calculated by the indirect method. The

55

supernatant obtained after centrifugation of the nanoparticle suspension was used for

estimation of the free drug content by UV/Visible spectroscopy at the absorption maxima

of EGCG at 274.5 nm. The percent entrapment efficiency was calculated by the following

formula:

% 𝐸𝐸 =𝑇𝑜𝑡𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓𝐸𝐺𝐶𝐺 𝑎𝑑𝑑𝑒𝑑 − 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓𝐸𝐺𝐶𝐺 𝑖𝑛 𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡

𝑇𝑜𝑡𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐸𝐺𝐶𝐺 𝑎𝑑𝑑𝑒𝑑×100

2.3.5. Physico-chemical characterization of nanoparticles

2.3.5.1. Particle size and surface potential

The mean particle diameter (PD), polydispersity index (PDI) and surface potential of

Blank-SLN and EGCG-SLN were measured by photon correlation spectroscopy using a

Zetasizer Nano-ZS (Malvern Instrument Ltd., Malvern, UK). Samples were diluted

appropriately and were analysed at 25 °C with a backscattering angle of 173°.

2.3.5.2. FTIR spectroscopy (FTIR)

FTIR analysis of EGCG-SLN and Blank-SLNs was carried out by potassium bromide

(KBr) pellet method, using an IR spectrophotometer (Perkin Elmer, USA). Pellets were

prepared by adding approximately 3 mg of the sample to 100 mg of KBr. Samples were

scanned in the range of 400–4000 cm−1.

2.3.5.3. Differential scanning calorimetry (DSC)

DSC analysis of EGCG-SLN and EGCG were carried out on a DSC1 instrument (Mettler

Toledo, Switzerland). The instrument was calibrated using pure indium. Approximately

5 mg of sample was heated in a hermetically sealed flat-bottom aluminium pan and

scanned from 50 °C to 240 °C at a heat flow rate of 10 °C/min, under a nitrogen

environment. Empty aluminium pans were used as standards.

2.3.6. In-vitro drug release

In-vitro drug release studies were performed by dialysis method in sodium acetate buffer

56

pH 5 (SAB). EGCG-SLN, equivalent to 2 mg of EGCG, was placed in dialysis tubing

and kept in a beaker containing 100 mL of SAB and maintained at 37 °C. At

predetermined time interval, a definite volume was withdrawn and replaced with same

volume of fresh media. The EGCG concentration was determined by measuring the

absorbance at 274.5 nm using a UV/Visible spectrophotometer.

2.3.7. In-vitro cytotoxicity studies

In-vitro cytotoxicity of native EGCG and EGCG-SLN against MDA-MB-231 human

breast cancer cells and DU-145 human prostate cancer cells was evaluated by MTT assay.

MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells were

cultured in RPMI-1640 and DMEM medium, respectively, supplemented with 10% FBS,

100 mg mL-1 streptomycin, 100 U mL-1 penicillin, and at 37 °C in a 5% CO2 incubator.

Cells (5 × 103 cells per well) were seeded in a 96-well plate and incubated with different

concentrations of test formulations. After 48 h, the medium was replaced with serum-free

medium containing 0.5 mg mL-1 of MTT reagent and further incubated for 4 h. Then the

medium was removed and formazan crystals generated by live cells were dissolved by

adding 150 µL of DMSO in each well. The absorbance was measured using a microplate

reader at a wavelength of 570 nm. The relative cell viability (%) was determined by

comparing the absorbance with control wells. Data are presented as average ± SD (n =

4).

Cell apoptosis was detected by assessment of nuclear morphology staining with Hoechst

33342. MDA MB-231 and DU145 cell lines were seeded in a 12-well culture plate at a

density of 1 x 105 cells per well, cultured at 37 ºC, 5% CO2 incubator to obtain confluency.

The cells were treated with EGCG and EGCG-SLN formulation at IC-50 concentration

of drug-loaded formulations and incubated for 24 hrs. After incubation, the cells were

washed with ice cold PBS for twice, fixed with 4% paraformaldehyde in PBS (pH 7.4)

57

for 15 min at room temperature, stained with 5 µg/mL Hoechst 33342 in PBS for 15 min

and washed twice with ice cold PBS. The cells were then examined using fluorescence

microscopy.

2.3.8. Colloidal and serum stability studies

2.3.8.1. Colloidal stability of nanoparticles in serum and physiological

conditions

Colloidal stability of EGCG-SLNs with time was tested in PBS, plasma and distilled

water. A stock solution (1 mg/mL) of nanoparticle suspension was prepared in distilled

water. Aliquots of the stock solution were then taken and dispersed in the respective

medium. The samples were stored at room temperature and the particle size,

polydispersity and zeta potential were measured at different time intervals using a

Zetasizer Nano-ZS.

2.3.8.2. Electrolyte-induced aggregation

The effect of electrolytes on the solid lipid nanoparticle formulation was studied by

electrolyte-induced aggregation technique as reported in literature[44]. Sucrose sulphate

solutions were prepared in varying concentrations to study the behaviour of EGCG-SLNs

in the presence of a flocculating agent. Briefly, 16.7 g of sucrose was dissolved in 100

mL double distilled water to give 16.7 % sucrose solution. Sodium sulphate was added to

this native solution to make a 2 M sucrose sulphate solution. Serial dilutions were made

from this stock solution to prepare final concentrations ranging from 0.1 to 2 M.

Nanoparticle suspensions equivalent to 1 mg/mL were added to the serial dilutions to

make a final volume of 3 mL and immediately analysed for particle size, zeta potential,

polydispersity and UV-Visible absorbance.

2.4. Results and discussion

2.4.1. Aqueous stability

58

The stability of EGCG in solutions of different pH was evaluated with UV/Visible

spectroscopy at 274.5 nm which is the absorption maxima for EGCG (Fig.2.2). The

absorption characteristics for EGCG in 0.1 N HCl (pH 1.5), PBS (pH 7.4), normal saline

(0.9 %w/v) and phosphate buffer solutions with three different pH values 5, 7 and 9 were

recorded. This study was carried out to evaluate the behaviour of free EGCG when it is

released in solutions simulating the different environment it encounters when released in

the blood stream. Spectra were recorded in the wavelength range of 200-800 nm up to 96

h to compare the changes in the drug absorption characteristics with changes in the study

environment. EGCG was found to be most stable at highly acidic pH 1.5 in 0.1 N HCl,

showing practically no degradation up to 96 h. In distilled water, the peak intensity for

EGCG is found to change after 4 h but the absorption characteristics remained same till

96 h. At 96 h, the peak is found to disintegrate, which could be due to the formation of

degradation products. In 0.9 % w/v normal saline, the peak distorted at around 4 h, but

complete peak disintegration occurred at 24 h. In PBS, the peak absorption showed a

slight change after 4 h but after 24 h complete peak disintegration was observed. At pH

5, the absorption characteristics remain unchanged up to 24 h after which distortion is

seen at 48 h. At pH 7, peak changes were observed at 8 h but peak distortion occurred at

24 h. EGCG was most unstable at alkaline pH 9. At this pH, changes in the absorption

characteristics are observed immediately after addition and complete disintegration of the

peak was observed after 4 h. These results suggested that EGCG is highly stable at acidic

pH while its stability decreases as the pH tends towards neutral and shows very less or no

stability at highly alkaline conditions.

59

Fig 2.2: UV/Visible spectra of EGCG (50 µg/mL) at different time points in (a) distilled

water (b) 0.1 N HCl (c) normal saline (0.9 % w/v NaCl) (d) phosphate buffer saline pH 7.4

(e) phosphate buffer pH 5 (f) phosphate buffer pH 7 (g) phosphate buffer pH 9

60

2.4.2. Optimization of different parameters for the blank SLN

Blank nanoparticles without EGCG encapsulation were prepared by double

emulsification method followed by solvent evaporation. This method shows high

potential for encapsulation of hydrophilic drugs alike. Stearic acid served as charge

modifier as the free carboxylic acid groups of stearic acid renders generation of

electronegative nanoparticles which in-turn can be harnessed for other applications

including ligand conjugation. Formulation and process parameters such as type of lipid,

lipid to co-lipid ratio, type of surfactant and concentration, amount of co-surfactant,

aqueous phase volume, sonication and stirring time were sequentially optimized

2.4.3. Influence of formulation parameters

Lipid crystallinity, lipophilicity, loading capacity, melting point and purity are important

factors to be considered when choosing a lipid for a nanoparticle formulation[45,46].

Tristearin (TS), tripalmitin (TP) and glycerol mono-stearate (GMS) were used for

optimization of particle diameter and surface potential (Table 2.1). The observed particle

size for SLN prepared with TS, TP and GMS were 131.1 ± 2 nm, 133.8 ± 2.7 nm and

121.4 ± 2.6 nm respectively. SLN prepared with GMS not only showed the least particle

size, but also had the least polydispersity. The co-lipid used in SLN preparation was

stearic acid which also acted as a charge modifier. Greater surface potential correlates

with greater stability of nanoparticles as there is a corresponding increase in the

electrostatic repulsive forces with an increase in the charge on each particle[47]. Stearic

acid content was varied between 30%, 20% and 10%w/w of total lipid content. Stearic

acid at 20% w/w of lipid showed a particle diameter of 125.7 ± 2.7 nm and good surface

potential values with least polydispersity. Surfactants stabilize the nanoparticles in the

colloidal state and prevent agglomeration during growth and storage.

The choice of stabilizers is important not only to control particle size and stabilization of

61

dispersions but also to control crystallization and polymorphic transitions[48]. SLN made

with polyvinyl alcohol, Tween® 80 and Pluronic F68 showed a particle diameter of 175

± 3.9 nm, 153.6 ± 3.4 nm and 127.5 ± 2.4 nm, respectively. SLN prepared with Pluronic

F68 showed the smallest particle size compared to the other surfactants. The

concentration of surfactant was varied from 1 to 3% Pluronic F68. SLN prepared with

2% Pluronic solution showed greater uniformity with lesser particle size and hence was

chosen as the surfactant utilized in preparation. Excess lipid molecules employed in the

production form small, stray, unilamellar vesicles which exhibit limited mobility and

therefore they are unable to immediately cover the newly created crystalline interfaces

during lipid recrystallization, leading to aggregation, wherein co-emulsifiers help in

providing additional stability during recrystallization events[49]. The co-emulsifier

employed was soya lecithin (30% phosphatidylcholine) as it has been found to be efficient

in the solvent-evaporation method for preparation of SLN[40]. It was optimized between

10 and 30 mg of which 20 mg soya lecithin showed a higher surface potential with lesser

particle diameter. The inner aqueous phase volume was optimized for particle size.

Increase in the aqueous phase was observed to cause a corresponding increase in the total

particle size. A volume of 400 μL of inner aqueous phase increased the particle size by

around 30 nm i.e. 153 nm compared to that obtained with 200 μL of inner aqueous phase,

which was 115.7 nm.

2.4.4. Influence of process parameters

Sonication helps in the emulsifying process, causing further size reduction of nanoparticle

formulations[50]. Sonication time was optimized for total sonication times of the two

cycles. The optimum sonication time was found to be 8 min for emulsion preparation as

it showed the optimum particle diameter and good surface potential of 118 ± 2.01nm and

-32.2 ± 1.5mV respectively. Solvents are used to dissolve the lipids in the oil phase in

62

emulsion preparation. Dichloromethane was found optimal for our study. However,

solvents must be removed in the final preparation to eliminate any additional solvent-

induced toxicity. In solvent evaporation, the emulsion can stir for adequate amount of

time to allow for effective removal of solvents by evaporation. The particle size was found

to increase with longer stirring times. This may be due to the aggregation of particles post

solvent-evaporation. A stirring time of 2 h was found to be optimum for preparation.

Fig 2.3: Particle size distribution of a) blank solid lipid nanoparticles and b) EGCG-loaded solid

lipid nanoparticles (EGCG-SLN), measured by dynamic light scattering.

63

Table 2.1: Optimization of formulation parameters for the preparation of solid lipid nanoparticles

by the double-emulsification method

Code Parameter Particle

diameter

(nm)

PDI ZP (mV)

F1

Type of lipid

GMS* 121.4 ± 2.6 0.131 ± 0.07 -28.3 ± 3.4

F2 Tripalmitin 133.8 ± 2.7 0.289 ± 0.17 -29.4 ± 0.9

F3 Tristearin 131.1 ± 2.0 0.284 ± 0.2 -23.8 ± 3.04

F4 Lipid: co-lipid

ratio

70:30 225.3 ± 2.4 0.452 ± 0.22 -33.8 ± 2.9

F1 (GMS:SA) 80:20* 125.7 ± 2.7 0.128 ± 0.06 -30.4 ± 1.7

F5 90:10 134.2 ± 1.7 0.234 ± 0.12 -32.5 ± 1.8

F6 Surfactant used PVA 175 ± 3.9 0.411 ± 0.24 -4.23±2.01

F7 Tween 80 153.6 ± 3.4 0.345 ± 0.26 -21.8 ± 3.4

F1 Poloxamer F68* 127.5 ± 2.4 0.237 ± 0.15 -30.4 ± 2.4

F8 Concentration of

surfactant (%)

1 133.7 ± 3.7 0.154 ± 0.01 -29.7 ± 2.8

F1 2* 122.3 ± 3.4 0.157 ± 0.12 -24.5 ± 1.6

F9 3 132.6 ± 2.6 0.264 ± 0.21 -31.1 ± 2.9

F10 Amount 10 133.4 ± 2.3 0.128 ± 0.1 -30.9 ± 1.5

F11 of co-emulsifier 15 127.1 ± 1.0 0.313 ± 0.22 -28.6 ± 3.4

F1 (Soya lecithin) 20* 124.5 ± 1.6 0.294 ± 0.26 -35.1 ± 3.2

F12 mg 30 130.4 ± 3.8 0.314 ± 0.21 -27.4 ± 2.3

F1 Inner aqueous

phase volume

(µL)

200* 115.7 ± 2.2 0.131 ± 0.1 -32.3 ± 1.6

F13 300 135.2 ± 2.3 0.186 ± 0.11 -35.1 ± 2.4

F14 400 153 ± 2.3 0.161 ± 0.14 -32.8 ± 1.8

*denotes the optimized formulation

GMS: Glyceryl mono-stearate; SA: Stearic acid; PVA: Polyvinyl alcohol

64

2.4.5. Encapsulation efficiency of EGCG-SLN

After optimising the nanoparticles for process and formulation parameters, they were

further optimised for the amount of drug loaded in the nanoparticles. EGCG was

dissolved in the inner aqueous phase of the double emulsion and the rest of the procedure

for preparation remained the same as that of the blank nanoparticles. EGCG-SLNs were

prepared by keeping the lipid concentration constant at 5% w/v and varying the

concentration of EGCG between 3% w/w and 5% w/w with respect to the lipid (Table

2.2). The results showed that an increase in the concentration of the lipid led to an increase

in the drug loading and hence the encapsulation efficiency. SLN with 5% w/w of drug

showed an encapsulation efficiency of 67.2% and particle size of 157 ± 2.5 nm, hence

was chosen as the optimum for future preparations (Fig. 2.3). Higher loading of drug was

not possible due to super-saturation of the drug in the limited inner aqueous volume.

Table 2.2: Optimization of encapsulation of epigallocatechin gallate (EGCG) in solid lipid

nanoparticles

EGCG to lipid

ratio (%w/w)

Particle size

(nm)

Polydispersity

index (PDI)

Zeta potential

(mV)

% EE

3 112.5 ± 3.1 0.14 ± 0.12 -30.1 ± 3.2 89.5 ± 4.7

4 128.2 ± 4.3 0.132 ± 1.21 -31.5 ± 3.7 75.8 ± 2.3

5 157.4 ± 2.5 0.268 ± 0.14 -37.2 ± 2.5 67.2 ± 4.5

PDI: Polydispersity index; ZP: Zeta potential; EE: Encapsulation efficiency

2.4.6. Physico-chemical characterization

2.4.6.1. FTIR analysis

Infrared spectroscopy was used to determine the compatibility of drug with the

components of the nanoparticles (Fig 2.4)[51]. The FTIR spectrum of EGCG shows a

characteristic peak at 3356 cm−1 for the O-H group attached to the aromatic ring, a strong

peak at 1691 cm−1 for the C=O group that links the trihydroxybenzoate group and

65

chroman group, and a peak at 1543 cm−1 for the C-C stretch in aromatic ring. Blank SLN

shows a broad peak at 3347 cm−1 for the O-H stretching in glycerol mono-stearate which

is one of the bulk components of the lipid core. It also shows a strong peak at 2917 cm−1

for the O-H stretch and at 1737 cm−1 for the C-O stretch in carboxylic group in stearic

acid. It also shows a peak at 1456 cm−1 for the C-H bending in alkanes. EGCG-SLN also

shows peaks at 3358 cm−1 for the O-H stretching in GMS, peaks at 2917 cm−1 for the O-

H stretch and 1731 cm−1 for the C-O stretch in stearic acid and at 1468 cm−1 for the C-H

bend in alkanes. Thus, EGCG post-encapsulation in SLN did not interact chemically and

hence, is compatible with the excipients.

Fig 2.4: Fourier transform infrared (FTIR) spectra of epigallocatechin gallate (EGCG), blank solid

lipid nanoparticles (Blank SLN) and EGCG -loaded solid lipid nanoparticles (EGCG-SLN)

2.4.6.2. DSC analysis

The physical state of EGCG as a free drug and encapsulated as EGCG-SLN was

determined by DSC analysis (Fig. 2.5). DSC analysis for Blank SLN shows a sharp peak

66

at 74 °C for melting point of stearic acid in blank formulation. The DSC thermogram of

pure EGCG showed an endothermic peak at 224°C which corresponds to its melting

point. This peak was not observed in EGCG-SLN suggesting the conversion of the

crystalline form of drug into an amorphous, disordered crystalline or solid solution

state[52]

It indicates that the drug was solubilized inside the lipid matrix of EGCG-SLN. The peak

for GMS appears at 62.5 °C which is close to its melting point of 59 °C. This peak,

however, showed a broad shouldering. This can be attributed to the fact that lipids behave

differently in bulk, native form and in the form of SLN formulation[53,54]. The shift in

melting point can be explained by the fact that the small particle size of SLN results in

high surface energy[44,55]. Also, lattice defects are created during preparation of SLN

which leads to decrease in the crystallinity of the lipids being used[47]. These less ordered

crystalline or amorphous solids require less energy to melt than pure crystalline

substances and show a broad and less intense peak[56].

Fig 2.5: Differential scanning calorimetric analysis of epigallocatechin gallate (EGCG) and EGCG


50 100 150 200 250

He

at

Flo

w (

mW

)

EGCG-SLN

Temperature (oC)

EGCG

67

2.4.7. In-vitro drug release studies

In-vitro release of EGCG from EGCG-SLN was studied in SAB at pH 5 to determine the

pattern of release of drug from the nanoparticles. Fig. 2.6 shows the drug release pattern

of EGCG-SLN. About 10% of encapsulated drug was released in the first hour increasing

to 83.9% after 12 h and complete drug release (>90%) after 24 h. The sustained release

of the EGCG from EGCG-SLN could be attributed to release of the drug by diffusion or

erosion of the lipid nanocarriers[57,58]. Fangueiro et al. (2014) suggested that

hydrophilic drugs are released from lipid nanoparticles by erosion or metabolization of

the constituent lipids making up the nanoparticle shell[58]. Interestingly, because of the

pH-dependent stability of EGCG (unstable at neutral pH while stable at acidic pH),

EGCG-SLN could be a potential formulation for the delivery of EGCG to cancer cells in-

vivo. The drug released during its transport through the blood stream will degrade and

only the drug reaching to the cancer cells through endocytosis of the nanoparticles will

be effective. The system thus shows promise in reducing toxicity of EGCG in normal

cells.

Fig 2.6: In vitro drug release studies of EGCG loaded solid lipid nanoparticles (EGCG-SLN) in

sodium acetate buffer pH 5 (Mean ± SD, n=3)

68

2.4.8. In-vitro cytotoxicity studies

The in-vitro anti-cancer efficacy of pure EGCG, blank SLN and EGCG-SLN was

investigated by anti-proliferation assay against two cancer cells i.e. MDA-MB 231 and

DU-145 cells. Fig. 2.7 shows the % cell viability of MDA-MB 231 cells (Fig. 2.7a) and

DU-145 cells (Fig. 2.7b) after incubation with blank SLN, EGCG and EGCG-SLN in a

concentration range of 10–100 μg/mL for 24 hours. In the case of Blank-SLN, cell

viability in both cancer cell lines was more than 90% at all tested concentrations which

indicated its high biocompatibility and non-toxicity. After treatment with EGCG, cell

viability in both cancer cells (MDA-MB-231 and DU-145) does not decrease

substantially with an increase in EGCG concentration. However, EGCG-SLN showed a

rapid decline in the viability of cancer cells. The cell viability of MDA-MB-231 was

77.07% after treatment with 10 μg/mL of EGCG which progressively decreased to

42.96% viability at the maximum concentration.

Fig 2.7a

0

20

40

60

80

100

10 20 40 80 100

%

Cell

via

bilit

y

Concentration (μg/mL)

MDA-MB-231 Blank SLN

EGCG

EGCG-SLN

69

Fig 2.7b

Fig 2.7: Percent cell viability of (a) MDA-MB-231 human breast cancer cells and (b) DU-145

human prostate cancer cells after 24 h of treatment with pure epigallocatechin gallate (EGCG),

blank solid lipid nanoparticles (blank SLN) and EGCG-loaded SLN (EGCG-SLN)

In EGCG-SLN, in contrast, cell viability was found to be 46.65% and 3.12% at 10 μg/mL

and 40 μg/mL of EGCG-SLN, respectively. In a concentration beyond 40 μg/mL, there

was no viability seen in MDA-MB-231. A similar pattern was observed in DU-145,

wherein EGCG displays no change in cell viability up to 40 μg/mL beyond which there

was reduction in cell viability with increasing EGCG concentration, finally giving a cell

viability percentage of 60.41% at the highest concentration (100 μg/mL). EGCG-SLN

showed a remarkable decrease in cell viability of DU-145 as well. About 86.27% of DU-

145 cells were viable in the minimum test concentration (10 μg/mL) and 16.9% viable

cells present at the 100 μg/mL. Observed IC50 values for EGCG and EGCG-SLN was

78.9 ± 4.3 μg/mL and 9.7 ± 0.6 μg/mL against MDA MB-231 and 126.74 ± 3.4 μg/mL

and 33.23 ± 0.7 μg/mL against DU-145 (Table 2.3). EGCG-SLN was 8.1 times more

cytotoxic than EGCG against MDAMB-231 and about 3.8 times more cytotoxic against

0

20

40

60

80

100

120

10 20 40 80 100

% C

ell

via

bilit

y

Concentration (µg/mL)

DU-145Blank SLN

EGCG

EGCG-SLN

70

DU-145. This increased cytotoxicity of EGCG-SLN in cancerous cells can be harnessed

in future in-vivo applications as tumours are often characterized by a defective, leaky

vascular architecture because of the poorly regulated nature of tumour angiogenesis and

the interstitial fluid within a tumour is usually inadequately drained by a poorly formed

lymphatic system. All of these factors contribute towards enhanced extravasation and

selective retention of sub-micron particles within tumour tissues[59]. This phenomenon

is called the enhanced permeation and retention (EPR) effect which is can be extensively

used by nanoparticle systems like EGCG-SLN for passive targeting in cancer therapy.

Table 2.3: IC50 values (µg/mL) for epigallocatechin gallate (EGCG) and EGCG-loaded solid lipid

nanoparticles (EGCG-SLN) against MDA-MB-231 human breast cancer cells and DU-145 human

prostate cancer cells after 24 h of treatment

Formulation

MDA-MB-231 DU-145

EGCG 78.9 ± 4.3 126.7 ± 3.4

EGCG-SLN 9.7 ± 0.6 33.2 ± 1.5

71

Fig 2.8a

Fig 2.8b

Fig 2.8: Phase contrast microscopy images of (a) MDA-MB-231 human breast cancer cells and (b)

DU-145 human prostate cancer cells after 24 h of treatment with blank solid lipid nanoparticles

(blank SLN), pure epigallocatechin gallate (EGCG), or EGCG-loaded SLN (EGCG-SLN)

equivalent to 20 μg/mL EGCG.

72

Fig. 2.8 shows phase contrast images of control cells, cells treated with pure EGCG, blank

SLN or EGCG-SLN after 24 h incubation. Fig. 2.8a and 2.8b show MDA-MB 231 and

DU-145 cells treated with the above formulations, respectively. The cell morphology in

MDA-MB 231 and DU-145 control cells is characterized by elongated epithelial cells. A

change in morphology from elongated to spherical and cell shrinkage was visible in both

the cell lines treated with EGCG and EGCG-SLN. However, a greater amount of cell

shrinkage was visible in cells treated with EGCG-SLN suggesting greater cytotoxicity in

the EGCG-SLN formulation compared to pure EGCG in agreement with the results in

Fig. 2.7.

2.4.9. Apoptosis studies

Apoptosis of EGCG-SLN and EGCG in MDA-MB-231 and DU-145 cells was studied

using the nucleus staining dye Hoechst 33342. The cells were treated with EGCG and

EGCG-SLN for 24 h and then stained with Hoechst 33342 (Fig. 2.9). Nuclear

fragmentation and apoptotic bodies were observed at a magnification of 10x with a scale

of 100 µm. Untreated or control cells exhibited spherical and intact nuclei. Cells treated

with EGCG-SLN showed more apoptotic bodies (32.7% in MDA-MB-231 and 24.9% in

DU-145) and nuclear shrinkage compared to the EGCG-treated cells (14.5% in MDA-

MB-231 and 11.8% in DU-145) which are consistent with the results from the anti-

proliferation assay (Section 2.4.7.), thus proving that encapsulation in SLN facilitated

greater anti-cancer efficiency and hence enhanced apoptosis of EGCG.

73

Fig 2.9a

Fig 2.9b

Fig 2.9: Nuclear fragmentation studies: Florescent microscopic images of (a) MDA-MB-231 human

breast cancer cells and (b) DU-145 human prostate cancer cells after 24 h of treatment with blank

solid lipid nanoparticles (blank SLN), pure epigallocatechin gallate (EGCG), or EGCG-loaded SLN

(EGCG-SLN) equivalent to 20 μg/mL EGCG followed by staining with Hoechst 33342

74

2.4.10. Colloidal stability studies

2.4.10.1. Stability of NPs in serum and physiological conditions

The colloidal stability of the nanoparticles was determined in distilled water and PBS and

expressed as a change in the particle size and zeta potential with time (Fig. 2.10). After

intravenous administration, the size and charge of the nanoparticles are mainly

determined by the adsorbed serum components[60]. Hence, a similar study was also

carried out in rat serum. Particle size in distilled water remains constant with the size of

112 nm at 0 h and 118.5 nm at 4 h. As expected, an initial increase in size (189.1 nm) in

EGCG-SLN is observed in serum owing to the adsorption of serum proteins, subsequently

falling to 134.3 nm at 2 h, following gradual protein desorption[61].

Particle size increases to 154.3 nm at the end of 8 h and 207 nm at 24 h. Particle size in

PBS increases from 112 nm to 247.15 nm at 2 h, after which it stays constant. Zeta

potential values for EGCG-SLN in distilled water remain constant with negative

potentials of −30.3, −28.4, −32.3, −28.9, −31.3 and −29.5 mV at 0, 1, 2, 4, 8 and 24 h

respectively. In PBS, the surface potential is −16.2, −19.2, −12.3, −10.7, −16.2 and −17.4

mV at 0, 1, 2, 4, 8 and 24 h, respectively. The reduced negative potential of the

nanoparticle suspension may be attributed to electrostatic interaction of opposite ions

present in PBS. Surface potential in serum was −7.01, −7.39, −4.43, −7.64, −6.99 and

−7.17 mV at 0, 1, 2, 4, 8 and 24 h, respectively. The reduced negative potential of EGCG-

SLN after exposing to serum may be due to the cumulative surface charges caused by the

adsorption of serum proteins and other serum components[62].

75

Fig 2.10a

Fig 2.10b

Fig 2.10: Change in (a) particle size and (b) zeta potential of EGCG-loaded solid lipid nanoparticles

(EGCG-SLN) with respect to time.

0

50

100

150

200

250

300

0 4 8 12 16 20 24

Pa

rtic

le d

iam

ete

r (n

m)

Time (h)

Distilled water PBS Serum

-40

-30

-20

-10

0

0 4 8 12 16 20 24

Ze

ta p

ote

nti

al

(mV

)

Time (h)

Distilled water PBS Serum

76

2.4.10.2. Electrolyte induced aggregation

Electrolyte-induced aggregation method is used to determine the resistance of

nanoparticles against flocculating agents[63]. EGCG-SLN were prepared by using

Poloxamer 188 as a surfactant. Coating with surfactants provides a hydrophilic surface to

the nanocarriers vehicle reducing the binding of opsonins and cells of the reticulo-

endothelial system[64]. EGCG-SLN show a high negative zeta potential which helps to

stabilize nanoparticles in the dispersion by electrostatic repulsion (Fig. 11). The addition

of electrolytes caused disturbances in this steric barrier resulting in particle aggregation.

EGCG-SLN showed a steady increase in absorbance with an increase in sucrose sulphate

concentration. Particle absorbance is constant until a concentration of 1.2 M after which

an abrupt increase in absorbance was observed at concentrations of 1.4 M and higher.

This suggests that EGCG-SLN are stable through electrostatic repulsion up to a

concentration of 1.2 M sucrose sulphate after which excess electrolyte causes disruptions

in the electrical double layer leading to increased flocculation.

Fig 2.11: Effect of flocculating agent (sucrose sulphate) on the stability of EGCG loaded solid lipid

nanoparticles

0

0.05

0.1

0.15

0.2

0.25

0 0.4 0.8 1.2 1.6 2 2.4

Ab

so

rba

nc

e

Sucrose sulphate concentration (M)

77

2.5. Conclusions

In summary, EGCG loaded solid lipid nanoparticles were formulated to improve the

stability and anti-cancer activity of EGCG. SLNs were prepared by a double-

emulsification method and optimized to provide nanoparticles with a particles size less

than 200 nm and high drug encapsulation efficiency. EGCG-SLN showed significantly

increased stability of encapsulated EGCG which was unstable at physiological conditions

and showed complete degradation in pure form. In-vitro cytotoxicity studies showed that

EGCG-SLN caused an 8.1fold increase in cytotoxicity of EGCG against MDA-MB-231

cells and 3.8 times increase against DU-145 cancer cells. Further, in colloidal stability

studies, EGCG-SLN showed stability in both serum and PBS and high resistance to

electrolyte-induced aggregation. Therefore, EGCG-SLN can be considered a promising

system for delivering EGCG as a potential chemotherapeutic agent.

78

2.6. References

[1] H. Lu, J. Zhang, Y. Yang, X. Yang, B. Xu, W. Yang, et al., Earliest tea as evidence

for one branch of the Silk Road across the Tibetan Plateau, Sci. Rep. 6 (2016) 18955.

doi:10.1038/srep18955.

[2] C. Cabrera, R. Artacho, R. Giménez, Beneficial Effects of Green Tea—A Review,

J. Am. Coll. Nutr. 25 (2006) 79–99. doi:10.1080/07315724.2006.10719518.

[3] C.J. Dufresne, E.R. Farnworth, A review of latest research findings on the health

promotion properties of tea, J. Nutr. Biochem. 12 (2001) 404–421. doi:10.1016/S0955-

2863(01)00155-3.

[4] H. Mukhtar, N. Ahmad, Tea polyphenols: Prevention of cancer and optimizing

health, Am. J. Clin. Nutr. 71 (2000) 1698–1702.

http://ajcn.nutrition.org/content/71/6/1698s.full.pdf (accessed October 1, 2015).

[5] G.-J. Du, Z. Zhang, X.-D. Wen, C. Yu, T. Calway, C.-S. Yuan, et al.,

Epigallocatechin Gallate (EGCG) is the most effective cancer chemopreventive

polyphenol in green tea., Nutrients. 4 (2012) 1679–91. doi:10.3390/nu4111679.

[6] N. Khan, H. Mukhtar, Tea polyphenols for health promotion., Life Sci. 81 (2007)

519–33. doi:10.1016/j.lfs.2007.06.011.

[7] M.E. Cavet, K.L. Harrington, T.R. Vollmer, K.W. Ward, J.-Z. Zhang, Anti-

inflammatory and anti-oxidative effects of the green tea polyphenol epigallocatechin

gallate in human corneal epithelial cells., Mol. Vis. 17 (2011) 533–42.

/pmc/articles/PMC3044696/?report=abstract (accessed December 30, 2015).

[8] S. Klaus, S. Pültz, C. Thöne-Reineke, S. Wolfram, Epigallocatechin gallate

attenuates diet-induced obesity in mice by decreasing energy absorption and increasing

fat oxidation., Int. J. Obes. (Lond). 29 (2005) 615–23. doi:10.1038/sj.ijo.0802926.

[9] M.-S. Lee, C.-T. Kim, Y. Kim, Green tea (-)-epigallocatechin-3-gallate reduces

79

body weight with regulation of multiple genes expression in adipose tissue of diet-

induced obese mice., Ann. Nutr. Metab. 54 (2009) 151–7. doi:10.1159/000214834.

[10] E. Tedeschi, h. suzuki, m. menegazzi, Antiinflammatory Action of EGCG, the

Main Component of Green Tea, through STAT-1 Inhibition, Ann. N. Y. Acad. Sci. 973

(2002) 435–437. doi:10.1111/j.1749-6632.2002.tb04678.x.

[11] A.M. El-Mowafy, M.M. Al-Gayyar, H.A. Salem, M.E. El-Mesery, M.M.

Darweish, Novel chemotherapeutic and renal protective effects for the green tea (EGCG):

role of oxidative stress and inflammatory-cytokine signaling., Phytomedicine. 17 (2010)

1067–75. doi:10.1016/j.phymed.2010.08.004.

[12] M. Shimizu, Y. Shirakami, H. Moriwaki, Targeting receptor tyrosine kinases for

chemoprevention by green tea catechin, EGCG., Int. J. Mol. Sci. 9 (2008) 1034–49.

doi:10.3390/ijms9061034.

[13] H. Tachibana, Molecular basis for cancer chemoprevention by green tea polyphenol

EGCG., Forum Nutr. 61 (2009) 156–69. doi:10.1159/000212748.

[14] C. Braicu, C.D. Gherman, A. Irimie, I. Berindan-Neagoe, Epigallocatechin-3-

Gallate (EGCG) Inhibits Cell Proliferation and Migratory Behaviour of Triple Negative

Breast Cancer Cells, J. Nanosci. Nanotechnol. 13 (2013) 632–637.

doi:10.1166/jnn.2013.6882.

[15] Y.D. Jung, L.M. Ellis, Inhibition of tumour invasion and angiogenesis by

epigallocatechin gallate (EGCG), a major component of green tea, Int. J. Exp. Pathol. 82

(2002) 309–316. doi:10.1046/j.1365-2613.2001.00205.x.

[16] Y. Xu, C.-T. Ho, S.G. Amin, C. Han, F.-L. Chung, Inhibition of Tobacco-specific

Nitrosamine-induced Lung Tumorigenesis in A/J Mice by Green Tea and Its Major

Polyphenol as Antioxidants, Cancer Res. 52 (1992) 3875–3879.

http://cancerres.aacrjournals.org/content/52/14/3875.short (accessed December 30,

80

2015).

[17] D.E. Shippen, Telomeres and telomerases, Curr. Opin. Genet. Dev. 3 (1993) 759–

763. http://ac.els-cdn.com.ezproxy.lib.rmit.edu.au/S0959437X05800954/1-s2.0-

S0959437X05800954-main.pdf?_tid=06936214-5c05-11e7-857f-

00000aab0f6b&acdnat=1498656411_c66add9d63a10db92be00e1f5db484ec (accessed

June 28, 2017).

[18] L.S. Beese, J.R. Kiefer, C. Mao, J.C. Braman, Visualizing DNA replication in a

catalytically active Bacillus DNA polymerase crystal, Nature. 391 (1998) 304–307.

doi:10.1038/34693.

[19] K. Collins, J.R. Mitchell, Telomerase in the human organism, Oncogene. 21 (2002)

564–579. doi:10.1038/sj.onc.1205083.

[20] N.W. Kim, M.A. Piatyszek, K.R. Prowse, C.B. Harley, M.D. West, P.L.C. Ho, et

al., Specific Association of Human Telomerase Activity with Immortal Cells and Cancer,

Science (80-. ). 266 (n.d.) 2011–2015. doi:10.2307/2885288.

[21] J.B. Berletch, C. Liu, W.K. Love, L.G. Andrews, S.K. Katiyar, T.O. Tollefsbol,

Epigenetic and genetic mechanisms contribute to telomerase inhibition by EGCG, J. Cell.

Biochem. 103 (2008) 509–519. doi:10.1002/jcb.21417.

[22] J.G. Herman, S.B. Baylin, Gene Silencing in Cancer in Association with Promoter

Hypermethylation, N. Engl. J. Med. 349 (2003) 2042–2054.

doi:10.1056/NEJMra023075.

[23] S.B. Baylin, DNA methylation and gene silencing in cancer, Nat. Clin. Pract.

Oncol. 2 (2005) S4–S11. doi:10.1038/ncponc0354.

[24] M.Z. Fang, Y. Wang, N. Ai, Z. Hou, Y. Sun, H. Lu, et al., Tea Polyphenol (−)-

Epigallocatechin-3-Gallate Inhibits DNA Methyltransferase and Reactivates

Methylation-Silenced Genes in Cancer Cell Lines, Cancer Res. 63 (2003).

81


[25] J. Folkman, Role of angiogenesis in tumor growth and metastasis, Semin. Oncol.

29 (2002) 15–18. doi:10.1016/S0093-7754(02)70065-1.

[26] N. Ferrara, VEGF and the quest for tumour angiogenesis factors, Nat. Rev. Cancer.

2 (2002) 795–803. doi:10.1038/nrc909.




doi:10.1054/bjoc.2000.1691.

[28] I.J. Fidler, Origin and biology of cancer metastasis, Cytometry. 10 (1989) 673–680.

doi:10.1002/cyto.990100602.

[29] S. Garbisa, L. Sartor, S. Biggin, B. Salvato, R. Benelli, A. Albini., Tumor

gelatinases and invasion inhibited by the green tea flavanol epigallocatechin-3-gallate,

Cancer. 91 (2001) 822–832. doi:10.1002/1097-0142(20010215)91:4<822::AID-

CNCR1070>3.0.CO;2-G.

[30] J. Koff, S. Ramachandiran, L. Bernal-Mizrachi, A Time to Kill: Targeting

Apoptosis in Cancer, Int. J. Mol. Sci. 16 (2015) 2942–2955. doi:10.3390/ijms16022942.

[31] J. Sonoda, R. Ikeda, Y. Baba, K. Narumi, A. Kawachi, E. Tomishige, et al.,

Experimental and therapeutic medicine., [Spandidos Pub.], 2014. https://www.spandidos-

publications.com/10.3892/etm.2014.1719 (accessed June 28, 2017).

[32] M. Leone, D. Zhai, S. Sareth, S. Kitada, J.C. Reed, M. Pellecchia, Cancer

Prevention by Tea Polyphenols Is Linked to Their Direct Inhibition of Antiapoptotic Bcl-

2-Family Proteins, Cancer Res. 63 (2003).


[33] J.S. Sebolt-Leopold, R. Herrera, Targeting the mitogen-activated protein kinase

82

cascade to treat cancer, Nat. Rev. Cancer. 4 (2004) 937–947. doi:10.1038/nrc1503.

[34] M. Li, Z. He, S. Ermakova, D. Zheng, F. Tang, Y.-Y. Cho, et al., Direct Inhibition

of Insulin-Like Growth Factor-I Receptor Kinase Activity by (−)−Epigallocatechin-3-

Gallate Regulates Cell Transformation, Cancer Epidemiol. Prev. Biomarkers. 16 (2007).

http://cebp.aacrjournals.org/content/16/3/598.short (accessed June 28, 2017).

[35] J.D. Lambert, R.J. Elias, The antioxidant and pro-oxidant activities of green tea

polyphenols: a role in cancer prevention., Arch. Biochem. Biophys. 501 (2010) 65–72.

doi:10.1016/j.abb.2010.06.013.

[36] Q.Y. Zhu, A. Zhang, D. Tsang, Y. Huang, Z.-Y. Chen, Stability of Green Tea

Catechins, J. Agric. Food Chem. 45 (1997) 4624–4628. doi:10.1021/jf9706080.

[37] S. Sang, J.D. Lambert, C.-T. Ho, C.S. Yang, The chemistry and biotransformation

of tea constituents., Pharmacol. Res. 64 (2011) 87–99. doi:10.1016/j.phrs.2011.02.007.

[38] L. Zhang, Y. Zheng, M.S.S. Chow, Z. Zuo, Investigation of intestinal absorption

and disposition of green tea catechins by Caco-2 monolayer model., Int. J. Pharm. 287

(2004) 1–12. doi:10.1016/j.ijpharm.2004.08.020.

[39] W. Mehnert, Solid lipid nanoparticles Production, characterization and

applications, Adv. Drug Deliv. Rev. 47 (2001) 165–196. doi:10.1016/S0169-

409X(01)00105-3.

[40] D. Pooja, H. Kulhari, L. Tunki, S. Chinde, M. Kuncha, P. Grover, et al.,

Nanomedicines for targeted delivery of etoposide to non-small cell lung cancer using

transferrin functionalized nanoparticles, RSC Adv. 5 (2015) 49122–49131.

doi:10.1039/C5RA03316K.

[41] S. Weber, A. Zimmer, J. Pardeike, Solid Lipid Nanoparticles (SLN) and

Nanostructured Lipid Carriers (NLC) for pulmonary application: a review of the state of

the art., Eur. J. Pharm. Biopharm. 86 (2014) 7–22. doi:10.1016/j.ejpb.2013.08.013.

83

[42] C. Qi, Y. Chen, Q.-Z. Jing, X.-G. Wang, Preparation and characterization of

catalase-loaded solid lipid nanoparticles protecting enzyme against proteolysis., Int. J.

Mol. Sci. 12 (2011) 4282–93. doi:10.3390/ijms12074282.

[43] C. Schwarz, W. Mehnert, J.S. Lucks, R.H. Müller, Solid lipid nanoparticles (SLN)

for controlled drug delivery. I. Production, characterization and sterilization, J. Control.

Release. 30 (1994) 83–96. doi:10.1016/0168-3659(94)90047-7.

[44] K. Vivek, H. Reddy, R.S.R. Murthy, Investigations of the effect of the lipid matrix

on drug entrapment, in vitro release, and physical stability of olanzapine-loaded solid

lipid nanoparticles., AAPS PharmSciTech. (2007). doi:10.1208/pt0804083.

[45] A. Radomska-Soukharev, Stability of lipid excipients in solid lipid nanoparticles.,

Adv. Drug Deliv. Rev. 59 (2007) 411–8. doi:10.1016/j.addr.2007.04.004.

[46] D. Pooja, L. Tunki, H. Kulhari, B.B. Reddy, R. Sistla, Optimization of solid lipid

nanoparticles prepared by a single emulsification-solvent evaporation method., Data Br.

6 (2016) 15–9. doi:10.1016/j.dib.2015.11.038.

[47] C. Freitas, R.H. Müller, Correlation between long-term stability of solid lipid

nanoparticles (SLNTM) and crystallinity of the lipid phase, Eur. J. Pharm. Biopharm. 47

(1999) 125–132. doi:10.1016/S0939-6411(98)00074-5.

[48] T. Helgason, T.S. Awad, K. Kristbergsson, D.J. McClements, J. Weiss, Effect of

surfactant surface coverage on formation of solid lipid nanoparticles (SLN), J. Colloid

Interface Sci. 334 (2009) 75–81. doi:10.1016/j.jcis.2009.03.012.

[49] H. Bunjes, M.H.J. Koch, K. Westesen, Influence of emulsifiers on the

crystallization of solid lipid nanoparticles., J. Pharm. Sci. 92 (2003) 1509–20.

doi:10.1002/jps.10413.

[50] F. Franco, L.A. Pérez-Maqueda, J.L. Pérez-Rodríguez, The effect of ultrasound on

the particle size and structural disorder of a well-ordered kaolinite, J. Colloid Interface

84

Sci. 274 (2004) 107–117. doi:10.1016/j.jcis.2003.12.003.

[51] S. Thomas, D. Rouxel, D. Ponnamma, Spectroscopy of polymer nanocomposites,

n.d.

[52] H. Gleiter, Nanostructured materials: basic concepts and microstructure, Acta

Mater. 48 (2000) 1–29. doi:10.1016/S1359-6454(99)00285-2.

[53] S. Das, W.K. Ng, R.B.H. Tan, Are nanostructured lipid carriers (NLCs) better than

solid lipid nanoparticles (SLNs): development, characterizations and comparative

evaluations of clotrimazole-loaded SLNs and NLCs?, Eur. J. Pharm. Sci. 47 (2012) 139–

51. doi:10.1016/j.ejps.2012.05.010.

[54] A. Kovacevic, S. Savic, G. Vuleta, R.H. Müller, C.M. Keck, Polyhydroxy

surfactants for the formulation of lipid nanoparticles (SLN and NLC): effects on size,

physical stability and particle matrix structure., Int. J. Pharm. 406 (2011) 163–72.

doi:10.1016/j.ijpharm.2010.12.036.




[56] A.P. Nayak, W. Tiyaboonchai, S. Patankar, B. Madhusudhan, E.B. Souto,

Curcuminoids-loaded lipid nanoparticles: novel approach towards malaria treatment.,

Colloids Surf. B. Biointerfaces. 81 (2010) 263–73. doi:10.1016/j.colsurfb.2010.07.020.

[57] A. zur Mühlen, C. Schwarz, W. Mehnert, Solid lipid nanoparticles (SLN) for

controlled drug delivery – Drug release and release mechanism, Eur. J. Pharm. Biopharm.

45 (1998) 149–155. doi:10.1016/S0939-6411(97)00150-1.

[58] J.F. Fangueiro, T. Andreani, L. Fernandes, M.L. Garcia, M.A. Egea, A.M. Silva, et

al., Physicochemical characterization of epigallocatechin gallate lipid nanoparticles

(EGCG-LNs) for ocular instillation., Colloids Surf. B. Biointerfaces. 123 (2014) 452–60.

85

doi:10.1016/j.colsurfb.2014.09.042.

[59] K. Greish, Enhanced permeability and retention (EPR) effect for anticancer

nanomedicine drug targeting., Methods Mol. Biol. 624 (2010) 25–37. doi:10.1007/978-

1-60761-609-2_3.

[60] C. Lourenco, Steric stabilization of nanoparticles: Size and surface properties, Int.

J. Pharm. 138 (1996) 1–12. doi:10.1016/0378-5173(96)04486-9.

[61] T. Verrecchia, P. Huve, D. Bazile, M. Veillard, G. Spenlehauer, P. Couvreur,

Adsorption/desorption of human serum albumin at the surface of poly(lactic acid)

nanoparticles prepared by a solvent evaporation process, J Biomed Mater Res. 27 (1993)

1019–1028.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Cita

tion&list_uids=8408114.

[62] R. Vinluan, J. Zheng, Serum protein adsorption and excretion pathways of metal

nanoparticles, Nanomedicine. 10 (2015) 2781–2794. doi:10.2217/nnm.15.97.



lipid nanoparticles., AAPS PharmSciTech. 8 (2007) E83. doi:10.1208/pt0804083.

[64] V.S. Shenoy, R.P. Gude, R.S.R. Murthy, Paclitaxel-loaded glyceryl palmitostearate

nanoparticles: in vitro release and cytotoxic activity., J. Drug Target. 17 (2009) 304–10.

doi:10.1080/10611860902737938.

86

Peptide conjugated solid lipid

nanoparticles for breast cancer therapy

87

3.1. Background

In 2012, an estimated 14.1 million new cases of cancer occurred worldwide. Four of the

most common cancers occurring worldwide are lung, female breast, bowel and prostate

cancers, together constituting around 40% of all the cancer cases[1]. Breast cancer is the

most frequent cancer in women, with 1.67 million cases diagnosed in 2012, accounting

for one in four of all cancer cases in the world for women[2].

Although many different chemotherapy drugs and drug regimens have been explored for

the treatment of cancer, the disadvantages of non-specificity with cytotoxic drugs have

to be circumvented to reduce non-specific organ damage[3]. The dissipation of the drug

in other areas also results in the concomitant increase in drug dosages, as the intended

organ may not get the total therapeutic dose required[4].

Active targeting involves the attachment of ligands specific to factors overexpressed in

cancer[5] to the nanoparticle. This leads to more accurate delivery as only cancer cells

will receive the cytotoxic drug. Active targeting is therefore being explored in order to

overcome the significant shortcomings associated with traditional chemotherapy. Gastrin

releasing peptide receptor (GRPR) has been observed to be overexpressed in a variety of

cancer types such as prostate, breast, cervical, glioblastomas, small cell lung cancer,

gastric, pancreatic, and colon cancers[6]. It was therefore used as a cancer marker in this

work. GRPR is a glycoprotein receptor (Chapter 1; Fig 1.9) with 7 transmembrane

domains consisting of 384 amino acids[7].

Fig 3.1: Structure of Bombesin

88

Bombesin (BBN) was the targeting ligand used in this study towards GRPR targeting.

BBN is a 14 amino acid peptide with very high affinity to the gastrin releasing peptide

receptor[8]. It was originally isolated from the skin of the toad Bombina bombina.

Bombesin is the amphibian analog to GRPR’s natural ligand mammalian gastrin releasing

peptide (GRP). GRP is a 27 amino acid peptide which shares a homologous C-terminal

seven amino acid long sequence Trp-Ala-Val-Gly-His-Leu-MetNH2 with bombesin[9].

This sequence has been reported to be important for receptor binding and ligand

internalisation via clathrin-mediated endocytosis[9]. Therefore, BBN is taken up by

GRPR with the same mechanism as in its interaction with gastrin-releasing peptide.

In this study, bombesin was conjugated to the EGCG-encapsulated SLN system that was

optimised in Chapter 2 (Please refer to Section 2.3.2.). BBN-conjugated solid lipid

nanoparticles were then explored for their anti-cancer efficacy, examining whether the

addition of a targeting ligand enhances the anti-cancer potential. In-vivo studies on

C57BL6 mice were performed to evaluate changes in tumour volumes and survivability

in comparison to control animals.

3.2. Materials

Glycerol mono-stearate (GMS) was purchased from Alfa Aesar (Johnson Matthey

Chemicals, Hyderabad, India). EGCG, bombesin, Pluronic F68 (P F68), Roswell Park

Memorial Institute (RPMI)-1640 medium, fetal bovine serum (FBS), trypsin–EDTA,

phosphate buffered saline (Ca2+, Mg2+ free), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma

Aldrich (St. Louis, MO, USA). Soya lecithin was purchased from HiMedia (Mumbai,

India). MDA-MB-231 human breast cancer cells, DU-145 (prostate cancer), A549(lung

cancer), HEK-293 (embryonic kidney) and L-132 lung cell lines were obtained from

89

American Type Culture Collection (ATCC, Manassas, USA). Dichloromethane was

obtained from Merck Chemicals (Mumbai, India). Tween 80 was purchased from sd Fine

Chemicals Limited (Hyderabad, India).

3.3. Methods

3.3.1. Preparation of nanoparticles

Solid lipid nanoparticles were prepared by the solvent emulsification-evaporation method

as explained in Chapter 2 (Section.2.3.3.). Briefly, 80 mg glycerol mono-stearate, 20 mg

stearic acid and 20 mg soya lecithin were dissolved in 2 mL of dichloromethane. The

inner aqueous phase containing dissolved EGCG was added to this organic phase

followed by 3 mL of 2% Pluronic F68 solution under sonication at 65% amplitude to

form a primary water-in-oil (w/o) emulsion. This emulsion was added to 7 mL of 2%

Pluronic solution to give a water-in-oil-in-water (w/o/w) emulsion. The nanoparticle

suspension was allowed to stir for 2 hours for evaporation of the solvent and further

centrifuged to precipitate out nanoparticle pellets. The supernatant was used for

evaluation of entrapment efficiency.

3.3.2. Calculation of entrapment efficiency

The drug entrapment efficiency (EE) was calculated by the indirect method as previously

mentioned in Chapter 2 (Section 2.3.3.). Briefly, the supernatant after centrifugation of

the nanoparticle suspension was used for estimation of the free drug content by

UV/Visible spectroscopy at the absorption maxima of EGCG at 274.5 nm. The percent

entrapment efficiency was calculated by the following formula:

% 𝐸𝐸 =𝑇𝑜𝑡𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐸𝐺𝐶𝐺 𝑎𝑑𝑑𝑒𝑑 − 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐸𝐺𝐶𝐺 𝑖𝑛 𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡

𝑇𝑜𝑡𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐸𝐺𝐶𝐺 𝑎𝑑𝑑𝑒𝑑 ×100

90

3.3.3. Bioconjugation

Conjugation, in this work, involves the formation of an amide bond between the peptide

ligand and the lipid excipients of the nanoparticle matrix. Conjugation of bombesin to the

drug loaded nanoparticles was carried out by the EDC-NHS method as described

below[10].

1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) is a cross linking

agent used to couple carboxylic acids to primary amines. EDC reacts with a carboxyl

group of the molecule to form an unstable amine-reactive O-acylisourea intermediate.

This could also react with a primary amine to give a stable amide bond, but the instability

of the O-acylisourea intermediate does not allow efficient coupling. If this intermediate

does not encounter an amine, it will hydrolyse and regenerate the carboxyl group.

Addition of sulpho-NHS helps to improve the stability of the intermediate to allow

reaction with the primary amine[11] Sulpho NHS (N-hydroxysulfo succinimide) reacts

with the acylisourea intermediate to form a more stable amine reactive NHS ester, which

can now react with the primary amine leading to the formation of a stable amide bond[11].

Fig 3.2: Mechanism of the EDC-NHS reaction (Reproduced with permission from Pierce et al.)

91

Briefly, the nanoparticle suspension was incubated with 15 mg N-hydroxysuccinimide

(NHS) and 20 mg N-(3-dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride

(EDC). The mixture was allowed to stir for one hour in an inert nitrogen atmosphere.

Bombesin (0.25 mg) was added to this and further allowed to stir for 4 hours. The reaction

was carried out in the presence of MES buffer (pH 6.2). Conjugation of bombesin to the

lipid nanoparticles has been found to be optimum around pH 6.2 based on previous studies

[12].

The nanoparticle dispersion was collected and centrifuged at 10,000 rpm for 10 min and

washed twice with distilled water. The supernatant was collected to estimate the

conjugation efficiency using a standard Bradford protein assay. The amount of bombesin

in the supernatant gives the amount of peptide not conjugated to the nanoparticles. A

calibration curve in the range of 0.5–10 g/mL was prepared using a standard BSA stock

solution (2 mg/mL). The supernatant was diluted appropriately and the absorbance was

measured spectrophotometrically at 595 nm to determine the concentration of bombesin.


3.3.4.1. Particle size and surface charge

The particle size and surface charge was determined by a Malvern Zetasizer using the

protocol mentioned in Chapter 2 (Section 2.3.5.1.). Briefly, the mean particle size and

zeta potential of blank nanoparticles (Blank SLN), EGCG-loaded SLNs (EGCG-SLN)

and bombesin conjugated SLNs (EB-SLN) were measured by photon correlation

spectroscopy using a Malvern Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern,

UK). Samples were diluted 10 times with distilled water and analyzed at 25°C with a

backscattering angle of 173°.

92

3.3.4.2. FTIR Analysis

FTIR analysis of blank nanoparticles (Blank SLN), EGCG-loaded SLNs (EGCG-SLN)

and bombesin conjugated SLNs (EB-SLN) was carried by potassium bromide (KBr)

pellet method following the same protocol in Chapter 2 (Section 2.3.5.2.). Samples were

scanned in the range of 400–4000 cm−1.

3.3.4.3. DSC Analysis

DSC analysis of EGCG-SLN and EGCG were carried out on a DSC1 instrument (Mettler

Toledo, Switzerland) using the protocol mentioned previously in Chapter 2 (Section

2.3.5.3.)

3.3.5. In-vitro studies

3.3.5.1. In-vitro cytotoxicity

MDA-MB-231 (breast cancer), DU-145 (prostate cancer), A549(lung cancer), HEK-293

(embryonic kidney) and L-132 lung cell lines were grown in DMEM and RMI medium

supplemented with 10% fetal bovine serum, 100 µg/mL penicillin, 200 µg/mL

streptomycin and 2 mM L-glutamine. The cultures were maintained in a humidified

atmosphere with 5% CO2. Dilutions were made with sterile PBS to get the required

concentration. Formulations were filtered with a 0.22 µm sterile filter before adding to

the wells containing cells.

The cytotoxicity of formulations was determined by MTT assay based on mitochondrial

reduction of yellow MTT tetrazolium dye to a highly coloured blue formazan product

which shows absorption at 570nm[13]. 1 × 104 cells (counted by the Trypan blue

exclusion dye method) in 96 well plates were incubated with EGCG-SLN, EB-SLN and

pure EGCG with a series of concentrations for 48 h at 37 ºC in DMEM with 10% FBS

medium. Then the above media was replaced with 90 µL of fresh serum free media and

10 µL of MTT reagent (5 mg/mL) and the plates were incubated at 37 °C for 4 h. After

93

removing the media, 200 µL of DMSO was added and incubated at 37 °C for a further

10 min. The absorbance at 570 nm was measured using spectrophotometer (Spectramax

Plus, Molecular Devices, USA).

3.3.5.2. Cellular uptake

Rhodamine-B loaded nanoparticles (R-SLN) were prepared and conjugated with BBN

(RB-SLN) for comparative cellular uptake studies. For quantitative studies, 1 × 105 cells

per well were seeded in 12-well plates and allowed to adhere for 24 h. Cells were

incubated with R-SLN and RB-SLN formulations for time intervals of 12 and 24 h. After

removal of the culture media, the cells were washed twice with cold PBS and observed

under a fluorescence microscope.

3.3.5.3. Apoptosis assay

Cell apoptosis was detected by assessment of nuclear morphology staining with Hoechst

33342[14]. MDA-MB-231 cells were seeded in a 12-well culture plate at a density of 1 x

105 cells per well and cultured at 37 ºC in a 5% CO2 incubator to obtain confluency. The

cells were treated with EGCG, EB-SLN and EGCG-SLN formulation at a concentration

of 20 μg/mL of EGCG and incubated for 24 hrs. After incubation the cells were washed

with ice cold PBS twice, fixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 min at

room temperature, stained with 5 µg/mL Hoechst 33342 in PBS for 15 min and washed

twice with ice cold PBS. The cells were then examined under fluorescence microscopy.

3.3.5.4. Migration assay

MDA-MB-231 (5 × 105 cells/mL) were seeded in petri dishes and allowed to grow to

80% confluence. Wounds were created with sterile 250 μL pipette tips. Cells were

incubated with EGCG, EGCG-SLN or EB-SLN, equivalent to 20 μg/mL EGCG. The

zone of wound healing was observed at 0 and 24 h using a bright field microscope. The

94

percentage of wound closure was determined by measuring the wound area using Image

J analysis software.

3.3.6. Animal studies

The animal experimental protocol for this study was approved by the Institutional Animal

Ethics Committee of the CSIR-Indian Institute of Chemical Technology, Hyderabad

(approval no. IICT/PHARM/SRK/FEB/2013/8). All the studies performed on the animals

were in accordance with the guidelines of the Committee for the Purpose of Control and

Supervision of Experiments on Animals (CPCSEA). Male C57/BL6 mice (6–8 weeks)

weighing between 20–25 g were used for the survival studies. Animals were kept in

polypropylene cages under standard laboratory conditions (12:12 h light/ dark cycle) at

24 °C. The animals were housed five per cage and had free access to food and water. The

mouse melanoma cancer cells B16F10 were grown and maintained in RPMI medium,

supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin. Mice were

injected with B16F10 cells (3 × 105 /mouse in 100 µL PBS) subcutaneously into the right

flank via a 30G needle. The control group (PBS), EGCG group (1 mg EGCG/mouse),

EGCG-SLN group (1 mg EGCG equivalent of EGCG-SLN/mouse) and EB-SLN (1 mg

EGCG equivalent of EB-SLN/mouse) were tested in tumour-bearing mice. PBS, EGCG,

EGCG-SLN and EB-SLN were administrated independently via intra-peritoneal injection

every third day from day 14 after tumour implantation. The tumour size and body mass

were measured regularly following the inoculation of tumour cells. Callipers were used

to assess tumour growth by measuring two bisecting diameters in each tumour. The

following equation was used to calculate the tumour volume:

95

Tumour volume (mm3) =(width × length2)

2

Scheme 3.1: Diagrammatic representation of the animal study methodology

3.4. Results

3.4.1. Particle size and surface charge

Particle size and charge are important dimensions when constructing nanovehicles for in-

vivo applications. Nanoparticles within the size range of 6-200 nm show better tissue

penetration and hence more efficient cargo delivery[15]. Surface charge of a particle

determines the stability of the nanoparticles in the biological system. The greater the

magnitude of the surface charge (i.e. strongly positive or strongly negative) the greater

will be the repulsion with similar nanoparticles thus preventing aggregation and helping

to sustain the nanoparticulate dimensions.

Tumour cell Inoculation

•B16F10 mouse melanoma cells (3 x 105/100 µL) injected in each mouse.

•Tumour develops in 12-14 days

Formulation testing

Formulations administered

intraperitoneally at a concentration of 50 mg EGCG/kg body weight

every three days

Tumour regression

and Survival Studies

Tumour volume will be evaluated at periodic

time intervals.

Animals will be kept aside from each group for survival and dosage

will be continued till natural death

96

Fig 3.3: Particle size distribution of a) blank solid lipid nanoparticles (Blank-SLN) b) EGCG-

loaded solid lipid nanoparticles (EGCG-SLN) and Bombesin conjugated solid lipid nanoparticles

(EB-SLN), measured by dynamic light scattering

Table 3.1: Particle size and surface charge for blank nanoparticles (Blank-SLN), EGCG-loaded

SLN (EGCG-SLN) and bombesin conjugated SLN (EB-SLN) determined by dynamic light

scattering

Formulation Particle diameter

(nm)

Polydispersity index Zeta potential (mV)

Blank-SLN 127.5 ± 2.4 0.227 ± 0.12 -30.4 ± 3.2

EGCG-SLN 157.4 ± 2.4 0.182 ± 0.21 -37.5 ± 2.5

EB-SLN 163.4 ± 3.2 0.341 ± 0.15 -25.2 ± 2.8

(c) EB SLN

97

Blank nanoparticles showed a diameter of around 127.5 ± 2.4 nm with a PD index of

0.227 ± 0.12 and a zeta potential of -30.4 ± 3.2 mV. The negative charges on the

nanoparticles could be attributed to the carboxylate groups of the excipient lipids in the

nanoparticle matrix. EGCG-SLN shows a particle size of around 157.4 ± 2.4 nm, a PD

index of 0.182 ± 0.21 and a surface charge of -37.5 ± 2.5 mV. Bombesin conjugated EB-

SLN shows a particle size of 163.4 ± 3.2 nm with a PD index of 0.341 ± 0.15 and a zeta

potential of -25.2 ± 2.8 mV. The surface potential has reduced from a more negative -

37.5 mV in EGCG-SLN to -25.2 mV in EB-SLN. This reduction in negative potential

could be attributed to the masking of the negatively charged groups on the nanoparticle

matrix. As more peptides get conjugated to the nanoparticle shell, more of the negative

carboxylate groups of the charged lipids are neutralized by the formation of peptide

bonds, thus reducing the total negative potential on the particle.

3.4.2. FTIR analysis

Infrared spectroscopy was used to determine the compatibility of the drug with the

nanoparticle excipients. It also indicates the formation of a chemical bond between the

lipids in the nanoparticle and bombesin (Fig 3.4). The FTIR spectrum of EGCG shows

a characteristic peak at 3356.74 cm−1 for the O-H group attached to the aromatic ring, a

peak at 1691.30 cm−1 for the C-O group that links the trihydroxybenzoate group and

chroman group and a peak at 1543.88 cm−1 for the C-C stretch in the aromatic ring. The

blank SLN shows a broad peak at 3347 cm−1 for O-H stretching in glycerol mono-stearate

which is one of the bulk components of the lipid core. It also shows a strong peak at 2917

cm−1 for the O-H stretch and a peak at 1737.88 cm−1 for the C-O stretch in the carboxylic

group of stearic acid. It also shows a peak at 1456.05 cm−1 for C-H bending in alkanes.

EGCG-SLN also shows peaks at 3358.08 cm−1 for O-H stretching in GMS, peaks at

2917.17 cm−1 for the OH stretch and 1731.24 cm−1 for the C-O stretch in stearic acid and

98

at 1468.99 cm−1 for the C-H bend in alkanes. Thus, EGCG post-encapsulation in SLN

did not interact chemically and hence, is compatible with the excipients. EB-SLN does

not show the presence of peaks at 1730 cm−1 for carboxylic acid groups, suggesting the

replacement of the carboxyl groups due to the formation of amide bonds with the amine

group of bombesin. Furthermore, a prominent peak at 1640 cm−1 characteristic of amide

C-N interaction is visible in the spectrum for EB-SLN. These changes point to the

formation of amide bonds between the constituent lipids of the nanoparticle matrix and

the peptide ligand.

Fig 3.4: Fourier transform infrared (FTIR) spectra of epigallocatechin gallate (EGCG), blank solid

lipid nanoparticles (BlankSLN), EGCG-loaded solid lipid nanoparticles (EGCG-SLN) and

bombesin conjugated solid lipid nanoparticles (EB-SLN)

99

3.4.3. DSC analysis

The physical state of EGCG as a free drug and encapsulated as EGCG-SLN was

determined to evaluate whether EGCG was infact encapsulated within the SLNs (Fig.

3.5). DSC analysis for EGCG-SLN shows a sharp peak at 62.4°C for the melting point

of glycerol mono stearate in the EGCG-loaded formulation. The DSC thermogram of

pure EGCG showed an endothermic peak at 224.4 °C which corresponds to its melting

point. This peak was not observed in EGCG-SLN suggesting the conversion of the

crystalline form of the drug into an amorphous, disordered crystalline state or a solid

solution state[16]. It indicates that the entire drug was completely solubilized inside the

lipid matrix of EGCG-SLN.

Fig. 3.5: Differential scanning calorimetric analysis of epigallocatechin gallate (EGCG) and EGCG


3.4.4. In-vitro studies

3.4.4.1. In-vitro cytotoxicity

100

The in-vitro cytotoxicity of pure EGCG, EGCG-SLN and EB-SLN against MDA-MB-

231, A549, HEK-293 and L132 cell lines was evaluated by MTT assay. The cell viability

of EGCG, EGCG-SLN and EB-SLN at a concentration range of 5-100 µg/mL was

determined in MDA-MB 231 cells. The observed IC50 values for EGCG, EGCG-SLN and

EB-SLN were 78.92 ± 4.3 µg/mL, 9.71 ± 0.6 µg/mL and 5.57 ± 3.2 µg/mL, respectively

(Fig 3.6). The cell viability for EGCG was 42.94% at the highest tested concentration of

100 µg/mL. For the EGCG-SLN formulation the cell viability was found to be 3.02% at

100 µg/mL. Bombesin-conjugated EB-SLN showed a cell viability of 51.77% at the

lowest concentration of 5 µg/mL, reducing drastically to 1.67% at a concentration of 40

µg/mL and showing no cell viability at higher concentrations. The results indicate that the

pure EGCG has low cytotoxicity in MDA-MB-231. However, its activity increased more

than 8 times after encapsulation in SLNs and more than 14 times post conjugation with

bombesin. For DU-145 cell line, the cell viability for maximum concentration of

100µg/mL of EGCG was 60.4%, that for EGCG-SLN was 16.9% whereas for EB-SLN it

was 7.7%. The observed IC50 values for EGCG, EGCG-SLN and EB-SLN were 126.7 ±

3.4 µg/mL, 33.2 ± 1.5 µg/mL and 19.2 ± 2.4 µg/mL respectively. For A549 cell line, the

cell viability for maximum concentration of 100µg/mL of EGCG was 85.1%, that for

EGCG-SLN was 20.4% whereas for EB-SLN it was 14.5%. The observed IC50 values for

EGCG, EGCG-SLN and EB-SLN were224.5 ± 3.9 µg/mL, 27.6 ± 4.5µg/mL and 24.8 ±

3.8 µg/mL respectively. Of the three tested cancer cell lines, EB-SLN showed greater

cytotoxicity in comparison with EGCG-SLN and EGCG in MDA-MB 231 cell line and

hence this cell line was chosen for further studies.

In addition, cytotoxicity evaluation was also carried out in two non-cancer cell lines HEK-

293 embryonic kidney and L132 lung cell lines. An interesting finding in this study was

that EB-SLN was least toxic to these cells in comparison to EGCG-SLN and EGCG. The

101

observed IC50 values in HEK-293 for EGCG, EGCG-SLN and EB-SLN were19.6 ± 4.7

µg/mL, 153.9 ± 2.5 µg/mL and 163.7 ± 4.1 µg/mL respectively. In L-132 cell line it was

13.2 ± 2.3 µg/mL, 182.02 ± 3.4 µg/mL and 252.5 ± 3.7 µg/mL for EGCG, EGCG-SLN

and EB-SLN respectively. These results point to selective specificity of formulation

towards cancer cells as opposed to non-cancerous cells.

Table 3.2: IC50 values (µg/mL) for epigallocatechin gallate (EGCG), EGCG-loaded solid lipid

nanoparticles (EGCG-SLN) and bombesin conjugated SLN (EB-SLN) against MDA-MB-231, DU-

145, A549, HEK-293 and L-132 cells after 24 h of treatment

Formulation

MDA-MB-231 DU-145 A549 HEK-293 L132

EGCG 78.9 ± 4.3 126.7 ± 3.4 224.5 ± 3.9 19.6 ± 4.7 13.2 ± 2.3

EGCG-SLN 9.7 ± 0.6 33.2 ± 1.5 27.6 ± 4.5 153.9 ± 2.5 182.02 ± 3.4

EB-SLN 5.5 ± 3.2 19.2 ± 2.4 24.8 ± 3.8 163.7 ± 4.1 252.5 ± 3.7

102

Fig 3.6: Percent cell viability of MDA-MB-231 human breast cancer after 24 h of treatment with

pure EGCG, EGCG-loaded SLN (EGCG-SLN) and bombesin-conjugated EGCG-SLN (EB-SLN).

Fig 3.7: Percent cell viability of DU145 prostate cancer cells after 24 h of treatment with

pure EGCG, EGCG-loaded SLN (EGCG-SLN) and bombesin-conjugated EGCG-SLN (EB-SLN).

0

20

40

60

80

100

5 10 20 40 80 100

% V

iab

ilit

y


EGCG

EGCG-SLN

EB-SLN

0

20

40

60

80

100

120

10 20 40 80 100

% V

iab

ilit

y


DU-145

EGCG

EGCG-SLN

EB-SLN

103

Fig 3.8: Percent cell viability of A549 prostate cancer cells after 24 h of treatment with pure EGCG,

EGCG-loaded SLN (EGCG-SLN) and bombesin-conjugated EGCG-SLN (EB-SLN).

Fig 3.9: Percent cell viability of HEK-293 kidney cells after 24 h of treatment with pure EGCG,

EGCG-loaded SLN (EGCG-SLN) and bombesin-conjugated EGCG-SLN (EB-SLN).

0

20

40

60

80

100

120

10 20 40 80 100

% V

iab

ilit

y


A549EGCG

EGCG-SLN

EB-SLN

0

20

40

60

80

100

5 10 20 40 80 100

% V

iab

ilit

y


HEK EGCG

EGCG-SLN

EB-SLN

104

Fig 3.10: Percent cell viability of L-132 lung cells after 24 h of treatment with pure EGCG, EGCG-

loaded SLN (EGCG-SLN) and bombesin-conjugated EGCG-SLN (EB-SLN).

3.4.4.2. Phase contrast microscopy studies

Phase contrast microscopy helps to microscopically observe changes to cell morphology.

Fig 3.11 shows MDA-MB-231 cells treated with blank SLNs (Blank-SLN), EGCG, drug

loaded EGCG-SLN and EB-SLN at 24 hours and 48 hours post treatment with the

respective formulations. Control cells were used as negative controls in the experiment.

When cells are under stress and undergoing apoptosis, a few distinct morphological

changes can be seen. MDA-MB-231 cells are characteristically elongated and tapered.

During cell death, the cell becomes spherical and apoptotic bodies can be seen with the

help of a nuclear staining dye.

Blank SLNs do not show any observable cytotoxicity, as is evident by the elongated

healthy cells at 24 and 48 hours (Fig 3.11). The EGCG treated samples show apoptotic

cells, demonstrated by the rounding of the cells when compared to the control cells at both

time points. EGCG-SLN shows more spherical cells compared to EGCG which suggests

greater toxicity. This could be attributed to the increased stability of the drug within the

0

20

40

60

80

100

120

5 10 20 40 80 100

% v

iab

ilit

y

Concentration (μg/ml)

L132EGCG

EGCG-SLN

EB-SLN

105

SLNs and also the sustained release due to encapsulation. EB-SLN shows more apoptotic

cells, with the number of dead or dying cells increasing at 48 hours compared to 24 hours.

This could again be attributed to the sustained release of the drug within SLNs and more

importantly greater uptake due to targeting of bombesin to GRPR receptors. This

enhanced uptake would automatically lead to increased concentrations of drug within the

cell and hence, greater cytotoxicity, in agreement with the observations in Fig 3.6.

106

Fig 3.11: Phase contrast microscopy images of MDA-MB-231 human breast cancer cells after 24 h

and 48 h of treatment with B) pure epigallocatechin gallate (EGCG), C) blank solid lipid

nanoparticles (blank SLN), D) EGCG-loaded SLN (EGCG-SLN) and E) bombesin conjugated SLN

(EB-SLN) equivalent to 20 μg/mL EGCG and A. control cells for reference.

A

B

C

D

E

24 h 48 h

107

3.4.4.3. Cellular uptake studies

Uptake of nanoparticles by cancer cells was performed by loading a fluorescent dye in

the lipid nanoparticle and observing fluorescence through microscopic detection. In this

work Rhodamine B was chosen as the dye due to its hydrophilic nature, as the dye must

be loaded in the same phase as that of the drug. Uptake was studied in the MDA-MB-231

breast cancer cell line. Rhodamine-B loaded SLNs (RB-SLNs) were prepared in the same

method as EGCG-loaded SLNs (Chapter 2, Section 2.3.3.) but with the drug replaced with

Rhodamine-B dye. They were further conjugated with bombesin to give bombesin

conjugated Rhodamine-B nanoparticles (RB-SLNs). An important advantage of targeting

lies in the specificity it provides to the carrier vehicle. This increases the amount of drug

delivered at the target site due to increased uptake via different mechanisms such as

receptor mediated endocytosis; whereas passive targeting merely relies on EPR effect

which does lead to sufficient uptake of nanoparticles but does not guarantee maximum

payload delivery. The use of fluorescence to study uptake is a more visual way to

determine differences in uptake. Fluorescence images were taken at two time points of 12

and 24 hours (Fig 3.12). At 12 hours, visibly more intense red fluorescence is seen in cells

treated with bombesin-conjugated nanoparticles compared with the unconjugated

nanoparticles. At 24 hours, this difference is more pronounced with not only an increase

in the fluorescence in the cells treated with RB-SLN, but there is also visible

intensification of fluorescence in individual cells. These findings point to continual and

increased uptake of nanoparticles in the cancerous cells conjugated with the targeting

ligand bombesin as opposed to the unconjugated formulation.

108

Fig 3.12: Cellular uptake studies: Fluorescent microscopic images of MDA-MB-231 human

breast cancer cells after 24 h of treatment with Rhodamine-loaded SLN (R-SLN) and

bombesin conjugated SLN (RB-SLN)

3.4.4.4. Apoptosis studies

Apoptosis is a process of programmed cell death that occurs after a cell is no longer

needed in the body. It occurs by two pathways, namely extrinsic and intrinsic pathways

(Refer to Section 2.1.e.). In cancer cells, the factors affecting these two pathways are

downregulated causing the cells to continue to proliferate uncontrollably. EGCG has

shown the ability to induce apoptosis, as explained in Chapter 2. Nuclear condensation is

an important process during apoptosis that forms the principle of the nucleus-staining

apoptosis assay. Hoeschst 33342 was the dye used in this study. Hoeschst 33342 is a cell-

permeable, blue fluorescence dye. When this dye binds to DNA it emits blue fluorescence

at 495 nm[14].

To study apoptosis caused by the different formulations, MDA-MB-231 cells were

incubated with blank-SLN, pure EGCG, EGCG-SLN and EB-SLN for 24 hours followed

R-SLN RB-SLN

12 Hrs

24 Hrs

109

by staining with Hoeschst 33342. Fluorescence images in Fig. 3.13 clearly showed the

difference in nuclear staining. Hoeschst 33342 stains all nuclei, hence blue fluorescence

is visible throughout. However, the intensity of fluorescence is more in cells undergoing

apoptosis, owing to nuclear condensation. Control cells and cells treated with blank-SLN

show no intense fluorescence. However, EGCG does show several intense fluorescence

specks. These specks increase in the EGCG-SLN, however the highest intensity is visible

in the EB-SLN formulation treated cells. An explanation for this is that the cells show an

increased uptake of these nanoparticles in comparison to the other samples due to the

presence of the targeting ligand bombesin. This resulted in the successful delivery of

increased concentrations of the cytotoxic EGCG inside the cells, thus causing a greater

extent of apoptosis.

110

Fig. 3.13: Nuclear fragmentation studies: Fluorescent microscopic images of MDA-MB-231 human

breast cancer cells after 24 h of treatment with blank solid lipid nanoparticles (blank SLN), pure

epigallocatechin gallate (EGCG), EGCG-loaded SLN (EGCG-SLN) and bombesin conjugated SLN

(EB-SLN) equivalent to 20 μg/mL EGCG followed by staining with Hoechst 33342.

3.4.4.5. Migration studies

Angiogenesis is the process by which a tumour develops new blood vessels from existing

ones in order to maintain continual growth and nutrition. Therefore, inhibition of

angiogenesis aids in preventing the growth and metastasis of tumours, by essentially

cutting off its nutrition. EGCG has shown anti-angiogenic effects in a number of cell lines

as mentioned in Chapter 2(Section 2.1). However, this effect was found to be increased

when it was encapsulated as EGCG-SLN and further conjugated with bombesin (Fig

3.14). Wound closure is found to more in control cells as compared to EGCG. Wound

EGCG-SLN

111

closure is almost negligible in EGCG-SLN and EB-SLN. Enhanced activity in EGCG-

SLN and EB-SLN could be attributed to the increased stability of the drug within the

solid lipid nanoparticles and hence greater activity. Sustained release of EGCG from the

nanoparticles also aids in continued anti-migratory effect of the drug leading to better

wound closure in EGCG- loaded nanoparticles than pure drug.

Fig 3.14: Scratch assay images for MDA-MB-231 human breast cancer cells after 24 h of treatment

with blank solid lipid nanoparticles (blank SLN), pure epigallocatechin gallate (EGCG), EGCG-

loaded SLN (EGCG-SLN) and and bombesin conjugated SLN (EB-SLN) equivalent to 20 μg/mL

EGCG, along with control cells for reference

3.4.5. In-vivo studies

The in-vivo anticancer efficacy of a therapeutic agent is assessed in terms of prolonging

the quality of life and increasing the survival time. The anti-tumour efficacy of EGCG,

EGCG-SLN or EB-SLN was evaluated on tumour bearing mice upon administration of

multiple doses equivalent to 50 mg/kg EGCG. In-vivo anti-tumour efficacy was evaluated

in a syngeneic C57/BL6 mouse model.

B16F10 cancer cell line was used to develop melanoma in this study. Gastrin-releasing

peptide receptors are overexpressed in many cancers. B16F10 mouse melanoma cell line

0 hr

24 hr

112

shows expression of GRPR receptors and therefore was used for checking the efficacy of

the bombesin conjugated formulation[17]. Mice were first injected subcutaneously with

3 × 105 B16F10 cells on the flank to grow melanoma in- situ. Forty tumour bearing

C57/BL6 mice were randomly divided into 4 groups (n=10) and intraperitoneally

administered EGCG, EGCG-SLN or EB-SLN at a dose equivalent to 50 mg/kg body

weight of EGCG every third day. The control group was administered an equivalent

volume of saline every third day.

The body weight, tumour volume and overall survival of the mice were recorded. The

mean survival time was determined using a Kaplan-Meier survival plot (Fig 3.15). A

mean survival of 41, 48 and 55 days were observed for EGCG, EGCG-SLN and EB-SLN

treated mice, respectively, compared to control mice (37 days) which were administered

with normal saline. There was a significant extension in the life span of mice treated with

EB-SLN.

The changes in body weight and tumour volume of the animals were also recorded during

the whole experiment. The change in body weight is an indicator of systemic toxicity

caused by the formulation[18]. There was rapid loss of body weight in the saline treated

control group in comparison to EGCG formulation treated animals (Fig 3.16). The control

group receiving only saline recorded a loss of 7.01% in total body weight in 30 days

whereas both EGCG-SLN groups showed increase by 1.58% and EB-SLN, 1.06% loss of

body weight, respectively.

The change in tumour volume was also observed and results revealed that tumour shrank

in the mice receiving EGCG formulation. The percent increase in tumour volume in mice

treated with EGCG-SLN and EB-SLN was 3.78% and 0.78% respectively as compared

to mice receiving pure EGCG and control which were 5.45% and 7.97% respectively.

113

Fig 3.15: Kaplan-Meier Plot for survival following administration of pure epigallocatechin

gallate (EGCG), EGCG-loaded SLN (EGCG-SLN) and bombesin conjugated SLN (EB-SLN)

formulations, with control mice for reference

20

21

22

23

24

25

26

1 7 14 21 30

Bo

dy w

eig

ht

(g)

No. of days

Control

EGCG

EGCG-SLN

EBSLN

114

Fig 3.16: Change in body weight and tumour volume in mice with the number of days

following administration of pure epigallocatechin gallate (EGCG), EGCG-loaded SLN

(EGCG-SLN) and bombesin conjugated SLN (EB-SLN) formulations with control mice for

reference

3.4.6. Conclusions

Bombesin-conjugated nanoparticles were prepared by the double-emulsification solvent

evaporation method followed by conjugation with EDC-NHS. FTIR analysis validated

the formation of the amide bond between the carboxylic C-O group of the lipid with the

free amino group of the peptide. IC50 values indicate that the pure drug EGCG has low

cytotoxicity in MDA-MB-231, but its activity increased more than 8 times after

encapsulation in SLNs and more than 14 times post conjugation with bombesin.

Fluorescence uptake images show more uptake of RB-SLN compared to non-targeted R-

SLN at all time points. Migration studies show substantial inhibition by the targeted EB-

SLN formulation. Animal studies indicate that administration of EB-SLN led to greater

survival rate in C57/BL6 mice in comparison to other formulations. These findings

5000

5500

6000

6500

7000

7500

8000

8500

1 7 14 21 30

Tu

mo

ur

vo

lum

e (

mm

3)

No. of days

Control

EGCG

EGCG-SLN

EBSLN

115

suggest the merits of attachment of a targeting ligand to the EGCG-encapsulated SLN

system in Chapter 2, further corroborating the potential of EGCG-encapsulated systems

in cancer therapeutics.

116

3.4.7. References

[1] Worldwide cancer statistics | Cancer Research UK, (n.d.).

http://www.cancerresearchuk.org/health-professional/cancer-statistics/worldwide-

cancer#heading-Zero (accessed July 16, 2017).

[2] GLOBOCAN Cancer Fact Sheets: Breast cancer, (n.d.).

http://globocan.iarc.fr/old/FactSheets/cancers/breast-new.asp (accessed July 16, 2017).



[4] A. Coates, S. Abraham, S.B. Kaye, T. Sowerbutts, C. Frewin, R.M. Fox, et al., On

the receiving end - patient perception of the side-effects of cancer chemotherapy, Eur. J.

Cancer Clin. Oncol. 19 (1983) 203–8. doi:10.1016/0277-5379(83)90418-2.

[5] K.S. Shohdy, A.S. Alfaar, Nanoparticles targeting mechanisms in cancer therapy:

current limitations and emerging solutions, Ther. Deliv. 4 (2013) 1197–1209.

doi:10.4155/tde.13.75.

[6] G. Halmos, J.L. Wittliff, A. V. Schally, Characterization of Bombesin/Gastrin-

releasing Peptide Receptors in Human Breast Cancer and Their Relationship to Steroid

Receptor Expression, Cancer Res. 55 (1995).

http://cancerres.aacrjournals.org/content/55/2/280.short (accessed July 16, 2017).

[7] R. Markwalder, J.C. Reubi, Gastrin-releasing Peptide Receptors in the Human

Prostate: Relation to Neoplastic Transformation, CANCER Res. 59 (1999) 1152–1159.

http://cancerres.aacrjournals.org/content/canres/59/5/1152.full.pdf (accessed July 16,

2017).




117


March 25, 2017).

[9] A.A. Begum, P.M. Moyle, I. Toth, Investigation of bombesin peptide as a targeting

ligand for the gastrin releasing peptide (GRP) receptor, Bioorg. Med. Chem. 24 (2016)

5834–5841. doi:10.1016/j.bmc.2016.09.039.

[10] H. Xin, X. Jiang, J. Gu, X. Sha, L. Chen, K. Law, et al., Angiopep-conjugated

poly(ethylene glycol)-co-poly(ε-caprolactone) nanoparticles as dual-targeting drug

delivery system for brain glioma., Biomaterials. 32 (2011) 4293–305.

doi:10.1016/j.biomaterials.2011.02.044.

[11] D. Bartczak, A.G. Kanaras, Preparation of peptide-functionalized gold

nanoparticles using one pot EDC/Sulfo-NHS coupling, Langmuir. 27 (2011) 10119–

10123. doi:10.1021/la2022177.

[12] H. Kulhari, D.P. Kulhari, M.K. Singh, R. Sistla, Colloidal stability and

physicochemical characterization of bombesin conjugated biodegradable nanoparticles,

Colloids Surfaces A Physicochem. Eng. Asp. 443 (2014) 459–466.

doi:10.1016/j.colsurfa.2013.12.011.

[13] J.M. Edmondson, L.S. Armstrong, A.O. Martinez, A rapid and simple MTT-based

spectrophotometric assay for determining drug sensitivity in monolayer cultures, J.

Tissue Cult. Methods. 11 (1988) 15–17. doi:10.1007/BF01404408.

[14] S. Allen, J. Sotos, M.J. Sylte, C.J. Czuprynski, Use of Hoechst 33342 staining to

detect apoptotic changes in bovine mononuclear phagocytes infected with

Mycobacterium avium subsp. paratuberculosis., Clin. Diagn. Lab. Immunol. 8 (2001)

460–464. doi:10.1128/CDLI.8.2.460-464.2001.

[15] M. Gaumet, A. Vargas, R. Gurny, F. Delie, Nanoparticles for drug delivery: The

need for precision in reporting particle size parameters, Eur. J. Pharm. Biopharm. 69

118

(2008) 1–9. doi:10.1016/j.ejpb.2007.08.001.



lipid nanoparticles., AAPS PharmSciTech. 8 (2007) E83. doi:10.1208/pt0804083.

[17] J. Fang, Y. Lu, K. Ouyang, G. Wu, H. Zhang, Y. Liu, et al., Specific antibodies

elicited by a novel DNA vaccine targeting gastrin-releasing peptide inhibit murine

melanoma growth in vivo, Clin. Vaccine Immunol. 16 (2009) 1033–1039.

doi:10.1128/CVI.00046-09.

[18] C. Criscitiello, O. Metzger-Filho, K.S. Saini, G. de Castro Jr, M. Diaz, A. La

Gerche, et al., Targeted therapies in breast cancer: are heart and vessels also being

targeted?, Breast Cancer Res. 14 (2012) 209. doi:10.1186/bcr3142.

119

Mangiferin and piperine encapsulated

nanoparticles for cancer therapy

120

4.1. Background

Phytochemicals are plant derived chemical compounds which have been used in ancient

medicine in many parts of the world, with the first literature on their beneficial effects

dating back to 2800 BC[1]. Phytochemicals consist of a wide class of chemical

compounds which includes alkaloids, glycosides, flavonoids, volatile oils, tannins and

resins[2]. They have been known to help in the treatment and prevention of many diseases

such as cardiovascular disease, cancer, osteoporosis and hypercholesterolemia[3].

In this chapter, two phytochemicals have been used to explore anti-cancer efficacy

namely mangiferin and piperine. These formulations were chosen in order to investigate

the use of SLNs with a targeted chemotherapeutic system.

A. Mangiferin encapsulated solid lipid nanoparticles for targeting towards

cancer

Mangiferin (2-b-D-glucopyranosyl-1, 3, 6, 7-tetrahydroxyxanthone) is a C-glucoside

xanthone found in many families such as Anacardiaceae, Bignoniaceae, Moraceae,

Thymelaceae, Aphloiaceae, Malvaceae and Gentianaceae[4]. It is found in higher

concentrations in the root, bark and leaves of Mangifera indica (mango) plant.

Chemically it contains an aromatic ring attached to the C–C bond of a glucose moiety

(Fig. 4.1.), making the molecule polar and water soluble[5].

Its many beneficial medicinal properties such as anti-oxidant, anti-diabetic, analgesic,

anti-inflammatory, anti-hypertensive, immune-modulatory, neuro-protective, anti-viral,

hypo-lipidemic and anti-cancer activities have been reviewed extensively[6–9].

121

Fig 4.1: Structure of mangiferin

Mangiferin has been extensively researched for its anti-cancer properties, exerting anti-

cancer effects by a variety of mechanisms such as anti-inflammatory, anti-proliferative,

apoptotic and immune-modulatory effects[10–12] outlined as follows:

a. Anti-inflammation

Inflammatory processes have been regarded as important contributors in the development

of cancer. Factors such as NFκB (Nuclear factor kappa-B) and interleukins are

overexpressed which are critical mediators of the immune responses that control the

transcription of inflammatory cytokines[13]. Mangiferin downregulates the production of

NFκB by blocking NF-κB and MAPK signalling pathways[14].

b. Proliferation/ metastasis

In normal cells, the rate of cell division and cell death is strictly regulated to prevent

anomalies. In cancer cells, however, the factors responsible for cell death are down-

regulated, causing cells to replicate uncontrollably. Loss of cell adhesion causes cancer

cells to escape from the site of origin and migrate to other sites causing secondary

cancers[15]. Mangiferin has been found to cause induction of miRNA-15b which is a

factor whose upregulation causes cell cycle arrest in cancer cells in the G0/G1 phase,

while simultaneously downregulating matrix metalloproteinase MMP-9, which is an

important factor in the degradation of the basement membrane and metastasis[16].

c. Apoptotic effect

122

Apoptosis is a process of controlled cell death which occurs by two pathways; an extrinsic

pathway which involves activation of death receptors or the intrinsic pathway which

involves the increased permeability of the mitochondria[17]. Mangiferin was found to

activate the intrinsic apoptotic pathway by upregulating caspase-9 cleavage and

activation, which is an initiator caspase that cleaves and activates the executioner caspases

(caspases 3 and 7), leading to apoptosis[18].

d. Immunomodulatory effects

Cancer cells avoid detection by the body’s immune system by the expression of proteins

that dampen the immune response. Mangiferin was found to increase the levels of

immunoglobulins IgM and IgG in animals with benzo(a)pyrene-induced lung

carcinogenesis[19].

In this work, mangiferin was encapsulated in solid lipid nanoparticles to study its anti-

cancer efficacy. The encapsulation was carried out by the single emulsion-solvent

evaporation method. Stearic acid was used as a charge modifier along with glycerol

mono-stearate as the matrix components. Bombesin was conjugated to this system as the

ligand and the release characteristics of the system were studied.

4.2. Materials

Glycerol mono-stearate, stearic acid, Poloxamer F68, mangiferin, chloroform, 1-ethyl-3-

(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and N-

[tris(hydroxymethyl)methyl]-2-aminoethane sulfonic acid (MES) were used as received.

4.3. Methods


SLNs were prepared by the single solvent-emulsification and evaporation method. The

oil in water emulsion of mangiferin was made by dissolving 5 mg of MgF in 2 mL

chloroform and it was then emulsified slowly in 10 mL of 1.5% Tween 20 surfactant

123

solution. The emulsion was sonicated for 15 mins at 50% amplitude using a probe

sonicator. It was stirred for 3 hours to allow for solvent evaporation and mangiferin-

loaded SLNs (MgF-SLNs) were then pelleted by centrifugation at 15000 rpm for 30

minutes and stored for further work. The supernatant was used for determining the

encapsulation efficiency.

4.3.2. Determination of entrapment efficiency

The entrapment efficiency of solid lipid nanoparticles to encapsulate mangiferin was

calculated by the indirect method. The supernatant collected after centrifugation of MgF-

SLNs was used to determine the concentration of free mangiferin by UV-visible

spectroscopy. A calibration curve of mangiferin was constructed and used for the

calculation of unknown concentrations.


MgF-SLNs were conjugated with bombesin using EDC-NHS reaction. Briefly, the

nanoparticle suspension was incubated with 15 mg N-hydroxysuccinimide (NHS) and 20

mg N-(3-dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride (EDC). The

mixture was allowed to stir for an hour for the activation of the carboxylic acid groups of

stearic acid in the SLN matrix. Bombesin (0.25 mg) was then added to this and further

stirred for 4 hours to allow conjugation to take place. The reaction was carried out in the

presence of MES buffer (pH 6.2) as the conjugation of bombesin to lipid nanoparticles

has been found to be optimum around pH 6.2 based on previous studies[20].

The bombesin–conjugated nanoparticles (MB-SLNs) were now centrifuged at 10,000

rpm for 10 minutes to remove unconjugated peptide molecules. The supernatant was

stored for calculation of the unconjugated peptide.

4.3.4. Calculation of conjugation efficiency

124

Unconjugated peptide was calculated by the Bradford assay method for determination of

protein. Briefly, a calibration curve for bombesin was plotted and the unknown

concentration was calculated by spectrophotometric detection at 595nm.



Nanoparticles were optimized for particle size and surface charge. The mean particle sizes

and zeta potentials of blank nanoparticles (Blank-SLN), mangiferin-loaded SLNs (MgF-

SLNs) and bombesin conjugated nanoparticles (MB-SLNs) were measured using a

Malvern Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, UK). Samples were

diluted 10 times with double distilled water and analyzed at 25 ºC with a backscattering

angle of 173º. Surface potentials for the formulations were also determined with the

Zetasizer..


FTIR analysis of Blank-SLN, MgF-SLN and MB-SLN was carried by using an IR

spectrophotometer (Perkin Elmer, USA). Pellets were prepared by adding approximately

3 mg of the sample to 100 mg of KBr. Samples were scanned in the range of 400–4000

cm−1.


In-vitro drug release studies were performed by dialysis in sodium acetate buffer pH 5

(SAB) and phosphate buffer pH 7.4. MgF-SLNs, equivalent to 2 mg of mangiferin, were

placed in dialysis tubing and kept in a beaker containing 100 mL of SAB and PBS and

maintained at 37 ºC. At predetermined time intervals, 1 mL of buffer was withdrawn and

replaced with same volume of fresh media to maintain sink conditions. The mangiferin

concentration was determined by measuring the absorbance at 342 nm, which is the

absorption maxima for mangiferin, using a UV/Visible spectrophotometer.

125

4.4. Results


Solid lipid nanoparticles were prepared by the emulsification-solvent evaporation method

to form an oil-in-water emulsion. Mangiferin was dissolved in chloroform which was the

inner organic phase. Tween 20 solution was used as the outer aqueous phase.

Emulsification was carried out using a probe sonicator for 15 minutes. The formulation

was optimized for particle diameter and charge for the surfactant used and its

concentration. The lipids used and their concentrations were optimized earlier in Chapter

2 (Section 2.4.2) Surfactants used for optimization included polyvinyl alcohol (PVA),

Tween 20 and Poloxamer F68. Tween 20 gave a particle size of 100.2 ± 3.1 nm, while

PVA and Poloxamer F68 gave a particle size of 132.5 ± 2.2 nm and 125.4 ± 3.6 nm,

respectively. The polydispersity of the Tween 20 formulation (F2) was 0.185 ± 0.2, while

that for PVA and Poloxamer 188 were 0.342 ± 0.2 and 0.379 ± 0.3, respectively. The

particle size and polydispersity of F2 were considerably lower than that of the others and

hence it was chosen to carry out further optimization. The concentration of Tween 20 in

the aqueous phase was optimized between 1, 1.5 and 2% in aqueous solution. The particle

size decreases with an increase in surfactant concentration with 1%, 1.5% and 2% Tween

20 giving particle diameters of 117.7 ± 5.4 nm, 100.2 ± 3.1 nm and 96.5 ± 4.7 nm,

respectively. This could be attributed to the fact that an increase in surfactant

concentration increases particle repulsion hence giving better size and polydispersity

measurements. However, the surface potential is found to decrease at 2% Tween 20

concentration which could lead to a reduction in stability. Thus, 1.5% Tween 20

formulation was chosen as the final formulation.

126

Table 4.1: Optimization of formulation parameters for Mangiferin-loaded solid lipid nanoparticles

prepared by the single-emulsification method


Formulation F2 chosen as the final formulation for optimization of drug loading and

entrapment efficiency. Initially a calibration curve of mangiferin was plotted with UV-

Visible spectroscopy at the λmax of 315 nm. Three concentrations of mangiferin were

evaluated for entrapment efficiency, which were 1, 3 and 5 mg of mangiferin.

Fig 4.2: Calibration curve of Mangiferin

y = 0.0078x + 0.0028R² = 0.9833

0

0.05

0.1

0.15

0.2

0.25

0 5 10 15 20 25 30

Ab

sorb

an

ce (

A.U

.)


Mangiferin Calibration curve

Formulation

name

Parameter Type Particle size

(nm)

Polydispersity

index

Zeta potential

(mV)

F1 Surfactant PVA 132.5 ± 2.2 0.342 ± 0.2 -25.7 ± 2.6

F2 used Tween 20 100.2 ± 3.1 0.185 ± 0.2 -24.9 ± 2.5

F3 Poloxamer

F68

125.4 ± 3.6 0.379 ± 0.3 -21.6 ± 3.4

F4 Concentration 1 117.7± 5.4 0.237 ± 0.3 -30.8 ± 2.3

F2 Of 1.5 100.2 ± 3.1 0.185 ± 0.2 -24.9 ± 2.5

F5 Surfactant

(%)

2 96.5 ± 4.7 0.126 ± 0.3 -17.7 ± 3.4

127

Table 4.2: Optimization of encapsulation of mangiferin and entrapment efficiency in mangiferin-

loaded SLN (MgF-SLN)

Particle sizes for 1, 3 and 5 mg mangiferin loaded formulations were found to be 89.52 ±

3.7 nm, 95.1 ± 4.1 nm and 100.2 ± 3.1 nm, respectively. Particle sizes were found to

increase with an increase in the amount of drug, which could be attributed to an increase

in the amount of drug assimilation between the crystallized lipid matrix therefore causing

an increase on hydrodynamic diameter. Entrapment efficiency for 1, 3 and 5 mg were

found to be 85.6 ± 3.7%, 81.4 ± 2.3% and 75.31 ± 2.3% for the F6, F7 and F2

formulations, respectively (Table 4.2). However, the F2 (5 mg) formulation was chosen

as even with a lesser entrapment efficiecy the total amount of drug loaded will be more

in this case compared to the others (3.765 mg).


Bioconjugation of bombesin to MgF-SLN was carried out by the EDC-NHS method as

explained in Chapter 3 (Refer Section 3.3.3.). EDC-NHS reaction helps to activate the

carboxylic groups in the charge modifier lipid (stearic acid) in order to help easier

formation of the amide bond between the carboxylate ion and the amine group of

bombesin. The centrifuged bombesin-conjugated nanoparticle (MB-SLN) formulation

was evaluated for particle size and surface potential. The zeta potential increased from -

24.9 ± 2.5 mV of MgF-SLN formulation to -16.3 ± 2.4 mV in MB-SLN. This could be

Formulatio

n name


(nm)

Polydispersity

index

Zeta

potential

(mV)

Entrapmen

t efficiency

(%)

F6 Amount 1 89.52 ± 3.7 0.131 ± 0.2 -24.4 ± 2.1 85.6 ± 3.7

F7 Of drug 3 95.1± 4.2 0.264 ± 0.4 -25.2 ± 2.3 81.4 ± 2.3

F2 (mg) 5 100.2 ± 3.1 0.185 ± 0.2 -24.9 ± 2.5 75.31 ± 2.3

128

attributed to masking of the negative carboxylate groups due to bond formation and

therefore a decrease in the total negative potential on the particle surface.


The conjugation efficiency of bombesin (BBN) was calculated by the indirect method.

The amount of unconjugated Bombesin in the supernatant was quantified using the

Bradford assay for protein determination[21]. The conjugation efficiency for 250 µg BBN

was found to be 82.34 ± 2.53%.



The particle size and surface charge of (Blank-SLN), MgF-SLN and MB-SLN were

observed by Malvern Zetasizer Nano ZS. The particle size of Blank-SLN, MgF-SLN and

MB-SLN were 96.57 ± 2.8 nm, 100.2 ± 3.1 nm and 121.4 ± 4.2 nm, respectively (Table

4.3). The increase in nanoparticle diameter from Blank SLN to MgF-SLN could be

attributed to the diffusion of the drug between the crystalline lipid matrix therefore

leading to an increase in size[22]. The zeta potential for Blank SLN and MgF-SLN was -

26.9 ± 3.1 mV and -24.9 ± 2.5 mV, respectively. There was not much change in surface

potential suggesting that mangiferin was not adsorbed on the surface and was

encapsulated. The particle size of the MB-SLN increases to 121.4 ± 4.2 nm which could

be due to attachment of the ligand on the particle surface. Also, the zeta potential tends

towards more positive value of -16.3 ± 2.4 mV in MB-SLN. This could be attributed to

masking of the negative carboxylate groups by bond formation with bombesin molecules

on the surface.

129

Table 4.3: Particle size distribution, polydispersity and zeta potential values for blank formulation

(Blank-SLN), mangiferin-loaded SLN (MgF-SLN) and bombesin-conjugated MB-SLN formulations

determined by dynamic light scattering

Fig 4.3: Particle size distribution for the blank formulation (Blank-SLN), mangiferin-loaded SLN

(MgF-SLN) and bombesin-conjugated MB-SLN formulations measured by dynamic light scattering

Formulation

name

Particle size

(nm)

Polydispersity

index

Zeta

potential

(mV)

Blank-SLN 96.57 ± 2.8 0.270 ± 0.1 -26.9 ± 3.1

MgF-SLN 100.2 ± 3.1 0.185 ± 0.2 -24.9 ± 2.5

MB-SLN 121.4 ± 4.2 0.324 ± 0.1 -16.3 ± 2.4

Blank

SLN

MgF-SLN

MB-SLN

130


FTIR spectrum of mangiferin shows a peak at 3367 cm-1 indicating presence of secondary

-OH bond (alcoholic OH), a peak at 2938 cm-1 for C-H anti-symmetric stretching and

peaks at 2830 cm-1 and 1649 cm-1 indicating the presence of the C-H and C-O symmetric

stretching. The blank SLN shows a broad peak at 3308 cm-1 for the O-H stretching in

glycerol mono-stearate which is one of the bulk components of the lipid core. It also

shows a peak at 2917 cm-1 for the O-H stretch and at 1730 cm-1 for the C=O stretch in the

carboxylic group in stearic acid. It also shows a peak at 1456 cm-1 strong for the C-H

bending in alkanes. Drug loaded MgF-SLN also shows peaks at 3307 cm-1 for the O-H

stretching in GMS, peaks at 2917 cm-1 for the O-H stretch and 1730 cm-1 for the C=O

stretch in stearic acid. The spectrum for mangiferin was different from the blank-SLN

and MgF-SLN. The spectra for drug loaded MgF-SLN and blank-SLN were similar which

shows that there were no unpleasant interactions between the drug and the lipid

nanoparticle and it was encapsulated efficiently.

Conjugated-SLN (MB-SLN) shows a broad peak at 3390 cm-1 for secondary alcoholic

OH groups from stearic acid. The peak at 2917 cm-1 for the O-H stretching is also visible.

However, peaks at 1645 cm-1 and 1707 cm-1 for amide linkage, which were not visible in

the blank or MgF-SLN are prominent providing evidence for amide bond formation.

131

Fig 4.4 Fourier transform infrared (FTIR) spectra of for blank formulation (Blank-SLN),

mangiferin-loaded SLN (MgF-SLN), mangiferin and bombesin-conjugated MB-SLN formulations


The drug release of Mangiferin was evaluated in phosphate buffer saline (PBS-pH 7.4)

and sodium acetate buffer (SAB-pH 5). MgF-SLN were placed in a dialysis membrane

and allowed to stir in solutions of a fixed volume (100 mL) of PBS and SAB buffers.

Aliquots of 1 mL were collected at regular intervals and the amount of mangiferin

released was quantified by UV-Vis spectroscopy. 4 mg mangiferin was loaded in both

formulations.

132

Fig 4.5: In- vitro drug release studies of mangiferin-loaded SLN (MgF-SLN) in sodium acetate

buffer pH 5 and phosphate buffer (pH 7.4)

Release data in PBS shows controlled release with 4.3 % released in the first 15 minutes,

16.8 % released at the end of 6 hours. In SAB, 4.4% was released in 15 minutes and 17.1

% in 6 hours. At the end of 24 hours, around 77.5% of the drug is released while at 48

hours, 94% of the drug was released. In SAB, 89.8% is released in the first 24 hours,

while 96.77% is released at the end of 48 hours. The results show that there is gradual

release in both pH conditions. Also, release is greater in SAB while compared to PBS,

suggesting that there is greater release in an acidic environment such as the one found in

cancer cells as compared to physiological blood pH. These findings provide promise that

this system will be appropriate in physiological conditions such as those encountered in

tumour tissue.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 10 20 30 40 50

Co

nce

ntr

ati

on

(µg

)

Time (Hours)

PBS

SAB

133

B. Piperine encapsulated chitosan nanospheres conjugated with mannose for

glycotargeting towards lectin receptors overexpressed in cancer

Pepper has been used as a spice in southeast Asian and European cuisines and medicine

for a long time. Piperine ((2E,4E)-5-(2H-1,3-Benzodioxol-5-yl)-1-(piperidin-1-yl) penta-

2,4-dien-1-one) is one of the major constituents imparting pungency to pepper. It exhibits

diverse pharmacological activities like anti-oxidant, anti-tumor, anti-inflammatory,

immuno-modulatory, anti-apoptotic and anti-metastatic activities[23].

Fig 4.6: Structure of piperine

a. Anti-oxidant activity

Inflammation leads to the production of free radicals and generates inflammatory

response factors like Interleukin-6 which help in increasing tumour development.

Piperine was found to decrease the amount of superoxide ions (O2–) and hydroxyl radicals

(OH•) which are known to injure surrounding tissues and organs in rats fed with a high

fat diet[24]. It also inhibited the expression of IL-6 and matrix metalloproteinase 13 in rat

arthritis models[25] which are important factors helping in inflammation.

b. Immuno-modulatory effects

Mouse splenocytes were used to evaluate the in-vitro immune-modulation of piperine for

cytokine production, macrophage activation and lymphocyte proliferation. Piperine

134

treated mouse splenocytes demonstrated an increase in the secretion of Th-1 cytokines

(IFN-γ and IL-2), increased macrophage activation and proliferation of T and B cells[26].

c. Apoptotic effect

Apoptosis is often downregulated in cancer cells to maintain continued proliferation. The

apoptotic effect of piperine was studied in prostate cancer cell lines wherein treatment

with piperine induced apoptosis by the activation of caspase-3 and by the cleavage of

PARP-1 proteins in different prostate cancer cells like PC-3, DU-145 and LNCaP prostate

cancer cells[27].

d. Anti-metastatic effect

Metastasis is the development of secondary tumours at places other than the site of origin.

Cells treated with piperine showed a reduction in the expression of cyclin B1 which is

important for cell cycle progression in the 4T1 breast cancer cell line. Piperine

significantly decreased the mRNA expression of MMP-9 and MMP-13 which play an

important role in basement membrane degradation. 4T1 cells are shown to metastasize to

organs like the lung 10th day following inoculation of 1×105 tumour cells. The extent of

metastasis in lungs was found to be less in piperine-treated mice as compared to control

mice as determined by colonogenic assay[28].

Piperine has been extensively used as a bioavailability enhancer for other anti-cancer

drugs, but there is not much research on the anti-cancer activities of piperine alone[29].

In this chapter, piperine was encapsulated in chitosan nanospheres by ionic gelation

method. Mannose was conjugated onto the positively charged amine groups of chitosan

using EDC-NHS reaction. The release and surface characteristics of this system were

studied and propose its potential for further anti-cancer work.

4.5. Materials

135

Chitosan, Tween 20, piperine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC),

N-hydroxysuccinimide (NHS), N-[tris(hydroxymethyl)methyl]-2-aminoethane sulfonic

acid (MES) and sodium tripolyphosphate (TPP) were used as received.

4.6. Methods


Piperine encapsulated chitosan nanospheres were made by ionic gelation method as

explained in the first chapter 1(Section B,I). Briefly, chitosan solution was prepared by

adding chitosan to a 1% acetic acid solution (pH 4.8) to yield a solution with a

concentration of 1 mg/mL. The solution was allowed to stir overnight. 10 mL of this

solution was taken in a beaker. Piperine (5 mg) dissolved in 1 mL acetone was added very

slowly dropwise, while stirring. This solution was ultrasonicated with a probe sonicator

at 65% amplitude for 2 minutes. Sodium tripolyphosphate (1 mg/mL) was added

dropwise to this solution with continuous stirring, and the end point was indicated by a

slight turbidity. The above solution was allowed to stir for 3 hours for the evaporation of

the organic solvent and the chitosan encapsulated nanoparticles (P-NP) were collected.

4.6.2. Determination of the entrapment efficiency

The entrapment efficiency was calculated by the indirect method. The supernatant was

collected after centrifugation of the chitosan nanoparticles. The calibration curve of

piperine at known concentrations was constructed using UV-Visible spectroscopy at its

absorption maxima of 342 nm. The amount of unloaded piperine was calculated using the

curve.


P-NPs were conjugated with mannose using a protocol mentioned in the literature[30,31].

Briefly, 10 mg of mannose was allowed to undergo a ring opening reaction by stirring in

SAB buffer (pH 5) for 1-2 hours. Mannose solution was added to P-NPs and incubated

136

with 15 mg N-hydroxysuccinimide (NHS) and 20 mg N-(3-dimethylaminopropyl)-N-

ethyl carbodiimide hydrochloride (EDC). The mixture was stirred for 4 hours in an inert

nitrogen environment to allow conjugation to take place.

Mannose–conjugated chitosan nanoparticles (MP-NPs) were then centrifuged at 10,000

rpm for 10 minutes to remove unconjugated mannose molecules. The supernatant was

stored for calculation of the unconjugated peptide.


Unconjugated mannose was calculated by the phenol-sulphuric acid mannose detection

method[32]. Briefly, a 5% w/v phenol solution was prepared freshly. 50 µL of unknown

sample along with known concentrations of mannose solution were added into 96 well

plate. 150 µL of concentrated sulphuric acid was pipetted into each well and mixed gently,

followed by the addition of 30 µL of the phenol solution. The plate was covered with

aluminium foil and allowed to sit for 10 minutes at room temperature. The absorbance

was now read at 490 nm.



Nanoparticles were optimized for particle size and surface charge. The mean particle size

and zeta potential of blank nanoparticles (B-NP), piperine loaded nanoparticles (P-NPs)

and mannose-conjugated nanoparticles (MP-NPs0 were measured by photon correlation

spectroscopy using a Malvern Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern,

UK). Samples were diluted 10 times with double distilled water and analyzed at 25 °C

with a backscattering angle of 173º.


FTIR analysis of B-NPs, P-NPs and MP-NPs was carried by using an IR

spectrophotometer (Perkin Elmer, USA). Pellets were prepared by adding approximately

137

3 mg of the sample to 100 mg of KBr. Samples were scanned in an inert atmosphere in

the range of 400–4000 cm−1.

4.7. Results


Piperine-encapsulated chitosan nanoparticles were prepared by ionic gelation method as

explained in Chapter 1. The end of nanoparticle formation was noted as slight turbidity.

The formulation was optimized for sodium tripolyphosphate (TPP) added which was in

the range of 1-3 mg. An amount of 2.5 mg TPP gave a particle size of 163.7 ± 3.4 nm

and zeta potential of 23.4 ± 2.4 mV (Table 4.4). An increase in the amount of TPP was

found to cause a decrease in the hydrodynamic size of the nanoparticles. This can be

hypothesized to the increase in the compactness of the nanoparticle by the increased

degree of cross-linking. A further increase in the amount of TPP (3 mg) did cause a

reduction in particle size. However, the surface potential of the particles could not record

a stable value. Hence, formulation F4 was carried forward for further optimization. The

formulation was also optimized for total volume of chitosan solution. Volumes of 8, 10

and 12 mL gave a particle size of 157.9 ± 2.8 nm, 163.7 ± 3.7 nm and 341.1 ± 4.3 nm,

respectively. Although particle size was lower in formulation F7 (8 mL), the zeta potential

could not be recorded which suggested instability in charges. Formulation F4 (10 mL),

showed adequate particle size and surface potential values and hence, was used for further

studies. This formulation was also optimized for process parameters such as sonication

amplitudes and stirring times but the results were constant. Therefore, the same

parameters as the followed protocol were used for further formulations.

Table 4.4: Optimization of formulation parameters for the piperine loaded chitosan nanoparticles

prepared by the ionic gelation method

138

Form

ulatio

n

Parameter Value Particle size

(nm)

Polydispersity

index

Zeta

potential

(mV)

F1 Amount of 1 467.4 ± 5.4 0.614 ± 0.2 NR

F2 TPP 1.5 412.5 ± 3.2 0.468 ± 0.3 NR

F3 (mg) 2 257.4 ± 2.1 0.428 ± 0.4 27.1± 1.4

F4

2.5 163.7 ± 3.4 0.345 ± 0.1 23.4±2.4

F5

3 121.3 ± 3.8 0.254 ± 0.3 NR

F6 Total Volume 12 341.1 ± 4.3 0.475 ± 0.2 25.8 ± 3.5

F4 of Chitosan 10 163.7 ± 3.7 0.345 ± 0.1 23.4±2.4

F7 (mL) 8 157.9 ± 2.8 0.513 ± 0.2 NR

*NR- not recorded


Formulation F4 was used to determine appropriate drug loading concentrations and

examine the entrapment efficiency. A calibration curve of known concentrations of

piperine was constructed using UV-Visible spectroscopy at the λmax of 342 nm. The

solubility of piperine is low in water and hence, a 1:1 water-ethanol mixture was used to

make the standard solutions and also the reference. The amount of piperine encapsulated

was optimized between 1, 3 and 5 mg as beyond this amount, piperine reached its

solubility limit. Particle sizes were found to increase with an increase in the amount of

the drug, which could again be attributed to the increased drug dispersion in the lipid

matrix therefore causing an increase in particle size. The particle sizes for 1, 3 and 5 mg

were found to be 153.27 ± 2.8 nm, 163.7 ± 3.7 nm and 234.21 ± 5.7 nm, respectively. An

abrupt increase of particle size at 5 mg piperine (Formulation F9) could be attributed to

the reducing solubility therefore causing disturbances in the process of nanoparticle

polymerization. Also, the surface potential for this formulation could not be recorded

again possibly pointing to an irregularly distributed lipid surface. The F8 formulation (1

mg) did show lesser particle size and a good entrapment efficiency of 78.2 ± 2.7%.

139

However, the F4 (3 mg) formulation was chosen as even with a lesser entrapment

efficiency (75.3 ± 4.3%), more drug (~ 2.25 mg)will be loaded in the formulation.

Fig 4.7: Calibration curve of piperine

Table 4.5: Optimization of encapsulation of piperine and entrapment efficiency in piperine loaded

chitosan nanoparticles (P-NP)


Bioconjugation with mannose was carried out as mentioned in the protocol (Section

3.3.3.). Ring opening of mannose was carried out by using acidic SAB buffer at pH 5 to

allow ligand conjugation. The conjugation occurs by formation of a Schiff’s base (–

N=CH–) between the aldehyde group of the mannose and the primary amine groups of

the chitosan. Schiff’s base could get reduced to the secondary amine (–NH–CH2–) and

y = 0.1585x + 0.0253R² = 0.9988

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20

Ab

sorb

an

ce

Concentration (µg)

Formulation

name


(nm)

Polydispersit

y

index

Zeta

potential

(mV)

Entrapment

efficiency

(%)

F8 Amount 1 153.27 ± 2.8 0.203 ± 0.2 -24.4±1.13 78.2 ± 2.7

F4 of Drug 3 163.7± 3.7 0.345± 0.1 23.4±2.4 75.3 ± 4.3

F9 (mg) 5 234.21 ± 5.7 0.664 ± 0.3 NR 62.4 ± 3.4

140

remain in equilibrium with the Schiff’s base[31]. The mannose conjugated chitosan

nanoparticles were washed thrice by centrifugation with MilliQ water to remove unbound

mannose. The pellet was stored for further studies.


The conjugation efficiency of the mannose to the MgF-SLNs was evaluated by the

phenol-sulphuric acid assay for the detection of sugars. The method works on the

principle of reaction of the aulphonated phenol with the sugars in a solution to give rise

to a yellow orange coloured furfural compound with an absorption maxima at around 490

nm[32]. The supernatant collected post centrifugation of the MP-NPs was used to

determine the unconjugated mannose. The unknown mannose in the supernatant was

determined by using the standard mannose calibration curve. The conjugation efficiency

after using 10 mg of mannose was found to be 92.34 ± 3.31%.



The particle size and surface charge of blank nanoparticles (B-NP), piperine-loaded P-

NP and conjugated MP-NP nanoparticles were observed by a Malvern Zetasizer Nano

ZS. The particle size of B-NP, P-NP and MP-NP were 152.9 ± 2.8 nm, 163.7 ± 3.7 nm

and 196.4 ± 4.2 nm, respectively (Table 4.6 and Fig. 4.8). There is an increase of about

10 nm in particle size from B-NP to P-NP which could be attributed to the packing of

piperine molecules between the chitosan polymer matrix therefore leading to increase in

size. The zeta potential for B-NP and P-NP were 24.1 ± 2.3 and 23.4 ± 2.4 mV,

respectively, with the positive potentials due to the amine groups present in chitosan. This

also suggests that there was no change in surface properties suggesting that piperine was

encapsulated and not adsorbed onto the particle surface. The particle size of MP-NP is

196.4 ± 4.2 nm, which is more compared to P-NP (163.7± 3.7). This could be due to the

141

attachment of mannose to the surface and hence an increase in the overall particle

diameter. Also, the zeta potential of MP-NP is found to reduce to 16.3 ± 3.2 mV from a

more positive 23.4 ± 2.4 mV in P-NP. This could be due to the attachment of mannose to

the nanoparticle surface, engaging the positive amino groups in chitosan. The negatively

charged alcohol groups (OH-) on the ring-opened mannose could also contribute

additional negative charges to bring down the zeta potential to a less positive value.

Table 4.6: Particle size distribution, polydispersity and zeta potential values for the blank

formulation (B-NP), piperine-loaded nanoparticles (P-NP) and mannose-conjugated (MP-NP)

formulations determined by dynamic light scattering

Formulation

name

Particle size

(nm)

Polydispersity

index

Zeta

potential

(mV)

B-NP 152.9 ± 2.8 0.269 ± 0.2 24.1 ± 2.3

P-NP 163.7± 3.7 0.196 ± 0.4 23.4 ± 2.4

MP-NP 196.4 ± 4.2 0.345± 0.1 16.3 ± 3.2

142

Fig 4.8: Particle size distribution for blank formulation (B-NP), piperine-loaded nanoparticle (P-

NP) and mannose-conjugated (MP-NP) formulations measured by dynamic light scattering

4.7.5.2. FTIR Analysis

FTIR analysis was carried out for B-NP, P-NP, MP-NP and pure piperine (Fig 4.9).

Characteristic peaks of chitosan for O-H stretching frequency between 3200-3400 cm-1

were observed in both B-NP (3424 cm-1) and P-NP (3388 cm-1). Peaks for N-H bending

(amine) appear at 1570 cm-1 and 1415 cm-1 for B-NP. P-NP also shows N-H bending

peaks at 1567 cm-1 and 1384 cm-1. The similarity in spectra between B-NP and P-NP

suggests that no unfavourable interactions took place between piperine and the chitosan

matrix. Piperine shows its characteristics peaks at 2925 cm-1 for C–H stretching vibration

and 1638 cm-1 for C-C stretching vibration. MP-NP shows broad and intense O–H stretch

B-NP P-NP

MP-NP

143

and C–O stretch peaks of mannose around 3462 cm-1 and 1094 cm-1, respectively. The

N–H deformation of secondary amine at 1417 cm-1 confirmed the Schiff’s base formation,

and the amide linkage formed between the aldehyde of mannose and the amine

termination of chitosan was also observed at 1640 cm-1. These results suggest successful

conjugation between the mannose molecules and chitosan nanoparticle surfaces.

Fig 4.9: Fourier transform infrared (FTIR) spectra for blank formulation (B-NP), piperine-loaded

chitosan formulation (P-NP), piperine and mannose-conjugated chitosan (MP-NP) formulations

4.8. Conclusions

In summary, solid lipid nanoparticles encapsulating mangiferin showed good size and

surface potential for application in cancer theray. Conjugation for bombesin was carried

out to rovide sppecificity towards cancers overexpressing GRPR recetors. FTIR studies

144

showed formation of amide bond linkages between SLNs and bombesin. Thus, this

system could be a strong candidate for cancer drug delivery. Mannose conjugated

chitosan nanoparticles encapsulating piperine and mangiferin-encapsulated SLNs are

both promising candidates for futher anti-cancer studies based on their physicochemical

properties. These two systems can be used to study in-vitro and in-vivo efficacy in future

studies.

145

4.9. References

[1] H. Wang, T. Oo Khor, L. Shu, Z.-Y. Su, F. Fuentes, J.-H. Lee, et al., Plants vs.

Cancer: A Review on Natural Phytochemicals in Preventing and Treating Cancers and

Their Druggability, Anticancer. Agents Med. Chem. 12 (2012) 1281–1305.

doi:10.2174/187152012803833026.

[2] N.M. Meybodi, A.M. Mortazavian, A.B. Monfared, F.A. Meybodi, Phytochemicals

in Cancer Prevention : A Review of the Evidence, 10 (2017). doi:10.17795/ijcp-

7219.Review.

[3] D.M. Tham, C.D. Gardner, W.L. Haskell, Potential Health Benefits of Dietary

Phytoestrogens: A Review of the Clinical, Epidemiological, and Mechanistic Evidence1,

Http://Dx.Doi.Org/10.1210/Jcem.83.7.4752. 83 (2011) 2223–2235.

doi:10.1210/jc.83.7.2223.

[4] A. Matkowski, P. Kus, E. Goralska, D. Wozniak, Mangiferin – a Bioactive

Xanthonoid, not only from Mango and not just Antioxidant, Mini Rev. Med. Chem. 13

(2013) 439–455. doi:10.2174/138955713804999838.

[5] R.H. Mirza, N. Chi, Y. Chi, Therapeutic Potential of the Natural Product

Mangiferin in Metabolic Syndrome, J. Nutr. Ther. 2 (2013) 74–79.

[6] R.K. Khurana, R. Kaur, S. Lohan, K.K. Singh, B. Singh, Mangiferin: a promising

anticancer bioactive, Pharm. Pat. Anal. 5 (2016) 169–181. doi:10.4155/ppa-2016-0003.

[7] S. Saha, P. Sadhukhan, P.C. Sil, Mangiferin: A xanthonoid with multipotent anti-

inflammatory potential, BioFactors. 42 (2016) 459–474. doi:10.1002/biof.1292.

[8] L. He, X. Peng, J. Zhu, X. Chen, H. Liu, C. Tang, et al., Mangiferin attenuate sepsis-

induced acute kidney injury via antioxidant and anti-inflammatory effects., Am. J.

Nephrol. 40 (2014) 441–50. doi:10.1159/000369220.

[9] P. Rajendran, T. Rengarajan, N. Nandakumar, H. Divya, I. Nishigaki, Mangiferin

146

in cancer chemoprevention and treatment: pharmacokinetics and molecular targets, J.

Recept. Signal Transduct. 35 (2015) 76–84. doi:10.3109/10799893.2014.931431.

[10] H. Li, J. Huang, B. Yang, T. Xiang, X. Yin, W. Peng, et al., Mangiferin exerts

antitumor activity in breast cancer cells by regulating matrix metalloproteinases,

epithelial to mesenchymal transition, and β-catenin signaling pathway, Toxicol. Appl.

Pharmacol. 272 (2013) 180–190. doi:10.1016/j.taap.2013.05.011.

[11] J. Das, J. Ghosh, A. Roy, P.C. Sil, Mangiferin exerts hepatoprotective activity

against D-galactosamine induced acute toxicity and oxidative/nitrosative stress via Nrf2–

NFκB pathways, Toxicol. Appl. Pharmacol. 260 (2012) 35–47.

doi:10.1016/j.taap.2012.01.015.

[12] D. Du Plessis-Stoman, J. Du Preez, M. Van de Venter, Combination treatment with

oxaliplatin and mangiferin causes increased apoptosis and downregulation of nfκb in

cancer cell lines, African J. Tradit. Complement. Altern. Med. 8 (2011).

doi:10.4314/ajtcam.v8i2.63206.

[13] X. Dolcet, D. Llobet, J. Pallares, X. Matias-Guiu, NF-kB in development and

progression of human cancer, Virchows Arch. 446 (2005) 475–482. doi:10.1007/s00428-

005-1264-9.

[14] W. Dou, J. Zhang, G. Ren, L. Ding, A. Sun, C. Deng, et al., Mangiferin attenuates

the symptoms of dextran sulfate sodium-induced colitis in mice via NF-κB and MAPK

signaling inactivation, Int. Immunopharmacol. 23 (2014) 170–178.

doi:10.1016/j.intimp.2014.08.025.

[15] I.J. Fidler, Origin and biology of cancer metastasis, Cytometry. 10 (1989) 673–680.

doi:10.1002/cyto.990100602.

[16] J. Xiao, L. Liu, Z. Zhong, C. Xiao, J. Zhang, Mangiferin regulates proliferation and

apoptosis in glioma cells by induction of microRNA-15b and inhibition of MMP-9

147

expression, Oncol. Rep. 33 (2015) 2815–2820. doi:10.3892/or.2015.3919.

[17] S. Elmore, Apoptosis: A Review of Programmed Cell Death, Toxicol. Pathol. 35

(2007) 495–516. doi:10.1080/01926230701320337.

[18] M. Cuccioloni, L. Bonfili, M. Mozzicafreddo, V. Cecarini, S. Scuri, M. Cocchioni,

et al., Mangiferin blocks proliferation and induces apoptosis of breast cancer cells via

suppression of the mevalonate pathway and by proteasome inhibition, Food Funct. 7

(2016) 4299–4309. doi:10.1039/C6FO01037G.

[19] P. Rajendran, T. Jayakumar, I. Nishigaki, G. Ekambaram, Y. Nishigaki, J.

Vetriselvi, et al., Immunomodulatory Effect of Mangiferin in Experimental Animals with

Benzo(a)Pyrene-induced Lung Carcinogenesis., Int. J. Biomed. Sci. 9 (2013) 68–74.

http://www.ncbi.nlm.nih.gov/pubmed/23847456 (accessed August 15, 2017).

[20] H. Kulhari, D.P. Kulhari, M.K. Singh, R. Sistla, Colloidal stability and

physicochemical characterization of bombesin conjugated biodegradable nanoparticles,

Colloids Surfaces A Physicochem. Eng. Asp. 443 (2014) 459–466.

doi:10.1016/j.colsurfa.2013.12.011.

[21] J.M. Walker, Handbook Edited by, 2002. doi:10.1385/1592591698.

[22] H. Patel, D.R. Panchal, U. Patel, T. Brahmbhatt, M. Suthar, Matrix Type Drug

Delivery System : A Review, J. Pharm. Sci. Biosci. Res. 1 (2011) 143–151.

[23] Z.A. Damanhouri, A. Ahmad, A Review on Therapeutic Potential of Piper nigrum

L. (Black Pepper): The King of Spices, Med. Aromat. Plants. 3 (2014) 1–6.

doi:10.4172/2167-0412.1000161.

[24] R.S. Vijayakumar, D. Surya, N. Nalini, Antioxidant efficacy of black pepper (

Piper nigrum L.) and piperine in rats with high fat diet induced oxidative stress, Redox

Rep. 9 (2004) 105–110. doi:10.1179/135100004225004742.

[25] J.S. Bang, D.H. Oh, H.M. Choi, B.-J. Sur, S.-J. Lim, J.Y. Kim, et al., Anti-

148

inflammatory and antiarthritic effects of piperine in human interleukin 1β-stimulated

fibroblast-like synoviocytes and in rat arthritis models, Arthritis Res. Ther. 11 (2009)

R49. doi:10.1186/ar2662.

[26] E.. Sunila, G. Kuttan, Immunomodulatory and antitumor activity of Piper longum

Linn. and piperine, J. Ethnopharmacol. 90 (2004) 339–346.

doi:10.1016/j.jep.2003.10.016.

[27] A. Samykutty, A.V. Shetty, G. Dakshinamoorthy, M.M. Bartik, G.L. Johnson, B.

Webb, et al., Piperine, a Bioactive Component of Pepper Spice Exerts Therapeutic Effects

on Androgen Dependent and Androgen Independent Prostate Cancer Cells, PLoS One. 8

(2013) e65889. doi:10.1371/journal.pone.0065889.

[28] L. Lai, Q. Fu, Y. Liu, K. Jiang, Q. Guo, Q. Chen, et al., Piperine suppresses tumor

growth and metastasis in vitro and in vivo in a 4T1 murine breast cancer model, Acta

Pharmacol. Sin. 33 (2012) 523–530. doi:10.1038/aps.2011.209.

[29] C.R. Pradeep, G. Kuttan, Effect of piperine on the inhibition of lung metastasis

induced B16F-10 melanoma cells in mice, Clin. Exp. Metastasis. 19 (2002) 703–708.

doi:10.1023/A:1021398601388.

[30] A. Jain, A. Agarwal, S. Majumder, N. Lariya, A. Khaya, H. Agrawal, et al.,

Mannosylated solid lipid nanoparticles as vectors for site-specific delivery of an anti-

cancer drug, J. Control. Release. 148 (2010) 359–367. doi:10.1016/j.jconrel.2010.09.003.

[31] N. Nimje, A. Agarwal, G.K. Saraogi, N. Lariya, G. Rai, H. Agrawal, et al.,

Mannosylated nanoparticulate carriers of rifabutin for alveolar targeting, J. Drug Target.

17 (2009) 777–787. doi:10.3109/10611860903115308.

[32] P. Rao, T.N. Pattabiraman, Further studies on the mechanism of phenol-sulfuric

acid reaction with furaldehyde derivatives, Anal. Biochem. 189 (1990) 178–181.

doi:10.1016/0003-2697(90)90103-G.

149

Summary and future perspectives

150

Cancer remains one of the world’s most debilitating diseases with 8.8 million deaths

worldwide in 2015, with the number of new cases expected to rise by 70% in the next

two decades[1].

Drug delivery in cancer mainly uses two types of approaches; passive and active

targeting[2]. Passive targeting relies on the distinct properties of tumour tissue (such as

enhanced permeation and retention effect) for delivery of the therapeutic payload.

Increased interendothelial gaps in tumour blood vessels causes increased penetration of

nanoparticles up to a size of 400 nm, with defective lymphatic drainage leading to

improved retention and, therefore, more delivery of cytotoxic drugs at tumour sites[3].

The rationale of this thesis is the preparation of nanoparticle assemblies for cancer therapy

with reduced non-specific toxicity. Many conventional model drugs for cancers lead to

non-specific associated toxicities at other sites, creating undesirable side effects[4].

Phytochemicals were used as cancer therapeutics in this thesis, which are plant-derived,

naturally occurring compounds. Epigallocatechin gallate (EGCG), mangiferin and

piperine are derived from green tea, mango and pepper, respectively. All of these

compounds have shown anti-cancer potential in-vitro[5–7]. These compounds have been

part of the regular human diet for centuries, and hence have mechanisms in place for their

effective metabolism, thus leading to reduced injury to non-targeted sites.

Two types of nanoparticle vehicles have been explored in this thesis viz. solid lipid

nanoparticles and chitosan nanoparticles. Solid lipid nanoparticles are made of a lipid

matrix which is solid at room temperature. The solid core gives added protection to the

encapsulated drugs. Also, these lipids can be broken down within the cells by metabolic

enzymes such as lipases. This helps to circumvent accumulation-induced toxicity which

is one of the major drawbacks associated with nanoparticle delivery[8]. Solid lipid

nanoparticles have also been known to show controlled release of the drugs from the

151

matrix and thus, improved bioavailability and sustained efficacy[9]. Chitosan

nanoparticles have a matrix of covalently cross-linked chitosan molecules. Chitosan is a

naturally occurring polysaccharide extracted from the shells of crabs. The cross linked

polymer also helps in effective encapsulation and increased bioavailability of the

encapsulated drugs within the biological system[10]. These nanoparticles can also be

easily degraded within the biological system and thus do not lead to accumulation[11].

Both the targeting approaches (active and passive) were explored in this study. Passive

targeting was used in Chapter 2 wherein EGCG was encapsulated in SLNs and studied

the effect of encapsulation on the anti-cancer efficacy. Other targeting ligands have been

used in the following chapters, including bombesin which is a tetra-decapeptide isolated

from the toad Bombina bombina. Bombesin is an amphibian analogue of the mammalian

gastrin releasing peptide which is a natural ligand to gastrin releasing peptide receptors,

overexpressed in a variety of cancers and hence used as a tumour marker[12]. The

mechanism of nanoparticle uptake through GRPR binding is via receptor mediated

endocytosis. Mannose was used as a ligand to target lectin receptors which are commonly

overexpressed in lung, liver, breast and brain tumours. Uptake of nanoparticles in this

case is also via receptor mediated or clathrin-mediated endocytosis.

EGCG is the most abundant catechin found in green tea. It has been explored in recent

times for its anti-cancer activity[13]. The structure of EGCG however causes it to be

easily degraded within the cell[14]. It has 8 free hydroxyl groups which can be easily

acted upon by cellular enzymes, causing it to be glucuronidated, sulphonated or amidated

therefore causing a loss in activity[14].

The stability of EGCG with different pH conditions using buffer solutions was initially

studied. EGCG was found to be most stable in acidic pH with stability increasing as the

solution becomes increasingly acidic. However, stability was found to reduce as we move

152

towards the neutral pH and further is found to degrade almost instantly in basic solutions.

Therefore, to combat stability issues EGCG was encapsulated in solid lipid nanoparticles.

Furthermore, the physico-chemical parameters such as particle size and surface charge of

the ECG-encapsulated SLNs were studied. Surface functionalities was studied using

FTIR spectroscopy to study any changes in the SLN surface post encapsulation. Spectra

showed encapsulation of EGCG as opposed to adsorption. Also, DSC studies were carried

out to further test whether EGCG was encapsulated within SLNs. The crystalline melting

peak of EGCG was not seen in the EGCG-encapsulated in SLNs, which therefore

confirms that EGCG was not present in its bulk state. Drug release studies were

performed to determine the release characteristics of EGCG from the SLNs. The results

indicated sustained release in acidic pH which could demonstrate favourable release in

acidic tumour tissue. Colloidal stability of the nanoparticles was studied in different

physiological mediums such as PBS, DW and serum to check the behaviour of the

nanoparticles in biological mimicking environments, wherein EGCG-SLN showed

stability in both serum and PBS and high resistance to electrolyte-induced aggregation.

Finally, in-vitro cytotoxicity was employed to assess the change in anti-cancer efficacy

post encapsulation. EGCG-SLN showed an 8-fold increase in cytotoxicity in the breast

cancer cell line and a 4-fold increase in cytotoxicity in the prostate cancer cell line as

opposed to pure EGCG. It also showed enhanced apoptosis as opposed to the free drug.

EGCG-SLN also showed more apoptosis in both cell lines as opposed to pure EGCG.

Thus, we can conclude that the increase in anti-cancer efficacy was obtained owing to the

encapsulation of EGCG within SLNs.

Bombesin-targeted solid lipid nanoparticles were prepared by the double-emulsification

solvent evaporation method as shown in Chapter1 followed by conjugation with the

peptide by EDC-NHS reaction. Bombesin was used as a ligand to target the gastrin-

153

releasing peptide receptor which is overexpressed in breast cancer[15]. FTIR analysis

validated the formation of an amide bond between the carboxylic COO- of the lipid with

the free amino group of the peptide. In-vitro studies in breast cancer cell line MDA-MB-

231 as it shows increased expression of GRP receptors were carried out. IC50 values

indicate that the pure drug EGCG has low cytotoxicity in MDA-MB 231, but its activity

increased more than 8 times after encapsulation in SLNs and more than 14 times post

conjugation with bombesin. Fluorescence uptake images show more uptake in RB-SLN

compared to non-targeted R-SLN at all time points. Migration studies show substantial

inhibition by the targeted EB-SLN formulation. Survival studies on C57BL6 mice

showed greater survival in mice administered with the targeted formulation compared to

the non-targeted ones, which was in turn more than mice administered with only EGCG.

Reductions in tumour volumes in mice administered with targeted formulations were also

observed. This study shows the potential of bombesin conjugated formulation in efficient

breast cancer therapy.

Mangiferin and piperine are two phytochemicals which have been used in ancient

medicine for their medicinal properties including their anti-cancer activities. Although

their mechanisms of action have been widely explored and documented, the number of

formulations that have been tried with these phytochemicals is comparatively low. In this

work, two novel systems for the delivery of these phytochemicals have been explored.

Mangiferin was encapsulated in solid lipid nanoparticles by a single-emulsification

method and further conjugated with bombesin, which is an efficient ligand for targeting

that was explored in Chapter 3. In this work, the formulation was optimized for particle

size and surface potential and checked for its physicochemical characteristics. Fourier

transform infrared analysis showed successful conjugation between the stearic acid

carboxylic group in SLNs and amine group in bombesin. For piperine, a chitosan

154

formulation was tested to optimise a nano-formulation with a carbohydrate matrix.

Mannose was conjugated onto this nanoparticle for targeting towards lectin receptors.

Zetasizer measurements showed size dimensions of <160 nm and surface charge values

of approximately 24mV. FTIR showed successful conjugation and there were no

incompatibilities between piperine and the excipients used for the nanoparticle system.

Both systems show good promise for further studies representing exciting candidates for

further in-vitro and in-vivo studies.

Future prospective in this work:

EGCG-SLN formulation showed 8 times more cytotoxicity than EGCG in breast

cancer cell lines MDA-MB 231 and 4 times more than EGCG in DU-145 prostate cancer

cell lines. In-vivo studies such as biodistribution and pharmacokinetics can be carried out

to further study the efficacy of encapsulated system compared to free EGCG.

A surprising observation was the differential cytotoxicities of the EGCG

formulations in cancerous and non-cancerous cell lines. EGCG showed more cytotoxity

in non-cancerous cell lines as compared to cancerous cell lines. These findings suggest

differential specificity of the conjugated system which could be a superior advantage if

sufficiently validated. More work remains to be done in this respect. Work on in-vivo

studies with this system such as pharmacokinetic studies and tissue distribution can be

carried out to determine in-vivo efficacy.

Mannose conjugated chitosan nanoparticles encapsulating piperine and mangiferin-

encapsulated SLNs conjugated with bombesin are both promising candidates for futher

anti-cancer studies based on their physicochemical properties. These two systems can be

used to study in-vitro cancer efficacy by cytotoxicity, uptake and apoptosis assays. In-

vivo studies such as pharmacookinetics can be carried out to determine distribution of

these nanoparticle system in-vivo.

155

Key knowledge additions provided by this thesis:

➢ EGCG-loaded SLNs helped improve the stability of a labile polyphenol such as

EGCG which has pH dependent stability issues. The increase in in-vitro efficacy could

be attributed to the additional stability imparted by the lipid core and also the slow release

from the SLN.

➢ EGCG-loaded SLNs conjugated with bombesin showed greater efficacy in-vitro as

well as in-vivo when compared to non-targeted nanoparticles. In-vitro studies have shown

specificity to cancerous cell lines in comparison to non-cancerous cells. Survival studies

showed greater survival of mice injected with targeted formulations.

➢ Piperine and mangiferin remain to be explored as potent chemotherapeutics in a

targeted system. SLN formulation with mangiferin and chitosan formulation with

piperine were optimized for size and charge parameters. The conjugation reactions

with bombesin and mannose were validated by FTIR analysis. Thus, two novel

nanoparticle systems have been optimized for particle size, surface conjugation and

surface potential in this thesis.

Publications from this thesis:

1. Published article:

Radhakrishnan, Rasika, et al. "Encapsulation of biophenolic phytochemical EGCG within

lipid nanoparticles enhances its stability and cytotoxicity against cancer." Chemistry and

physics of lipids 198 (2016): 51-60.

2. Article under preparation:

Bombesin-conjugated solid lipid nanoparticles for targeted breast cancer therapy- Work

represented in Chapter 3.

156

References

[1] WHO | Cancer, WHO. (2017). http://www.who.int/cancer/en/ (accessed March 25,

2017).

[2] L. Brannon-Peppas, J.O. Blanchette, Nanoparticle and targeted systems for cancer

therapy, Adv. Drug Deliv. Rev. 64 (2012) 206–212. doi:10.1016/j.addr.2012.09.033.

[3] Y. Fukumori, H. Ichikawa, Nanoparticles for cancer therapy and diagnosis, Adv.

Powder Technol. 17 (2006) 1–28. doi:10.1163/156855206775123494.



[5] T. Sen, S. Moulik, A. Dutta, P.R. Choudhury, A. Banerji, S. Das, et al.,

Multifunctional effect of epigallocatechin-3-gallate (EGCG) in downregulation of

gelatinase-A (MMP-2) in human breast cancer cell line MCF-7., Life Sci. 84 (2009) 194–

204. doi:10.1016/j.lfs.2008.11.018.

[6] D. Ouyang, L. Zeng, H. Pan, L. Xu, Y. Wang, K. Liu, et al., Piperine inhibits the

proliferation of human prostate cancer cells via induction of cell cycle arrest and

autophagy, Food Chem. Toxicol. 60 (2013) 424–430. doi:10.1016/j.fct.2013.08.007.

[7] M. Cuccioloni, L. Bonfili, M. Mozzicafreddo, V. Cecarini, S. Scuri, M. Cocchioni,

et al., Mangiferin blocks proliferation and induces apoptosis of breast cancer cells via

suppression of the mevalonate pathway and by proteasome inhibition, Food Funct. 7

(2016) 4299–4309. doi:10.1039/C6FO01037G.

[8] Solid Lipid Nanoparticles ( SLN ) and Nanostructured Lipid Carriers ( NLC ):

Occlusive Effect and Penetration Enhancement Ability, (2015) 62–72.

file:///C:/Users/Rasika/Downloads/JCDSA_2015052814334704.pdf (accessed October

5, 2015).

[9] K. Manjunath, J.S. Reddy, V. Venkateswarlu, Solid lipid nanoparticles as drug

157

delivery systems, Methods Find. Exp. Clin. Pharmacol. 27 (2005) 127.

doi:10.1358/mf.2005.27.2.876286.

[10] K.Y. Lee, W.S. Ha, W.H. Park, Blood compatibility and biodegradability of

partially N-acylated chitosan derivatives, Biomaterials. 16 (1995) 1211–1216.

doi:10.1016/0142-9612(95)98126-Y.




[12] R. Markwalder, J.C. Reubi, Gastrin-releasing Peptide Receptors in the Human

Prostate: Relation to Neoplastic Transformation, CANCER Res. 59 (1999) 1152–1159.

http://cancerres.aacrjournals.org/content/canres/59/5/1152.full.pdf (accessed July 16,

2017).




doi:10.1054/bjoc.2000.1691.

[14] B. Li, W. Du, J. Jin, Q. Du, Preservation of (-)-epigallocatechin-3-gallate

antioxidant properties loaded in heat treated β-lactoglobulin nanoparticles., J. Agric.

Food Chem. 60 (2012) 3477–84. doi:10.1021/jf300307t.





March 25, 2017).

Documents

Nanocarriers-encapsulating phytochemicals as potent