sirolimus liposomal formulations for targeting of...

I. Onyesom PhD Thesis I

Sirolimus Liposomal Formulations for Targeting of Cancer Tumours

By

Ichioma Onyesom

(B.Sc. Pharm. Sci.)

A thesis submitted in partial fulfilment of the requirements of the University of

Greenwich for the Degree of Doctor of Philosophy

May, 2014

Department of Pharmaceutical, Chemical & Environmental Science

Faculty of Engineering & Science

University of Greenwich, Medway Campus,

Chatham Maritime, Kent ME4 4TB, UK

DECLARATION

II I. Onyesom PhD Thesis

DECLARATION

“I certify that this work has not been accepted in substance for any degree, and is not concurrently

being submitted for any purpose, other than that of Doctor of Philosophy being studied at the

University of Greenwich. I also declare that this work is the result of my own investigations except

where otherwise identified by references and that I have not plagiarised another’s work”.

____________________________ Ichioma Onyesom (Candidate)

………………………………………………………………………………………………………..

PhD Supervisors

____________________________ Dr Dionysios Douroumis

_____________________________ Prof. B. Z. Chowdhry

12/06/2014

ACKNOWLEDGMENTS

III I. Onyesom PhD Thesis

ACKNOWLEDGMENTS

I would like to express my sincere gratitude to my supervisors Dr Dionysios Douroumis, and

Prof B. Z. Chowdhry. I would also like to thank all my colleagues and the technical members of

staff in the School of Science for all their help and support. My thanks to the University of

Greenwich for financing this Ph.D., study.

Also, I would like to say a very big thank you to my Family, friends, Nancy James, Marco E.

Favretto and Voula Paraskevi Kallinteri for all their support.

IV I. Onyesom PhD Thesis

ABSTRACT

Sirolimus Liposomal Formulations for Targeting of Cancer Tumours

In clinical trials, sirolimus (rapamycin, a macrocyclic lactone) has been shown to exhibit antitumor

activity across a variety of human cancers by binding to and inhibiting the activation of the

mammalian target of rapamycin (mTOR), thus preventing cell cycle progression from the G1 to S

phase. Inhibitors of mTOR have received regulatory approval as immunosuppressive agents for the

treatment of allograft rejection and as antitumor agents for kidney cancer (Rapamune®). In these

clinical trials tumour cell proliferation was dramatically reduced without sirolimus being

formulated in a drug carrier. The more challenging question is whether strategies can be developed

to improve the delivery of sirolimus directly to tumour cells and maximize mTOR inhibition?

The aim of the research reported herein was to develop, characterise and evaluate the anti-cancer

activity of sirolimus loaded liposome formulations. Liposome-drug formulations were prepared

using the thin film hydration method and were characterised using particle size analysis, atomic

force microscopy (AFM), differential scanning calorimetry (DSC) and X-ray photoelectron

spectroscopy (XPS). The particle size analysis of the liposome formulations showed that the

liposome-drug formulations were stable over a 6 month period of time. Further characterization of

the liposome-drug formulations using XPS and DSC studies demonstrated the incorporation of the

drug (sirolimus) in the liposome bilayer. In order to ascertain the anti-tumour activity of the

sirolimus formulations, in-vitro studies using MTT assays were carried out on human breast cancer

cell lines (MCF-7 and BT-474). The cytotoxicity studies using pure sirolimus showed anti-

proliferative action at concentrations above 40 µg/mL and the formulated liposome formulations

also demonstrated anti-proliferative effects when incubated with the cancer cells. Parameters such

as lipid composition, incubation time and drug loading were established as important factors that

play a key role in the therapeutic efficacy of the sirolimus loaded liposomes. Fluorescent images

obtained from the cellular uptake and apoptosis studies also provided supporting data which

demonstrated the anti-proliferative effect of the liposome formulations. In addition, sirolimus was

designed to actively target breast cancer cells by conjugating transferrin on the surface of the

sirolimus loaded liposomes. Both in-vitro and in-vivo studies of the conjugated sirolimus

formulations demonstrated the formulation to be more effective in inducing anti-proliferative

effects compared to the passive formulation (non-conjugated sirolimus loaded liposomes).

Sirolimus loaded liposome anticancer activity towards prostate cancer cell lines (LNCAP and DU-

145) has also been evaluated. Similar results to the breast cancer studies were obtained; specific

parameters were also shown to influence the anti-proliferative efficacy of the sirolimus liposome

formulations in prostate cancer cells.

V I. Onyesom PhD Thesis

DEDICATION

This thesis is dedicated to:

My Family and Friends

VI I. Onyesom PhD Thesis

CONTENTS

TITLE PAGE I

DECLARATION II

ACKNOWLEDGEMENTS III

ABSTRACT IV

DEDICATION V

CONTENTS VI

LIST OF FIGURES XI

LIST OF TABLES XVI

ABBREVIATIONS XVIII

PUBLICATIONS/CONFERENCE PRESENTATIONS XXI

VII I. Onyesom PhD Thesis

1. CHAPTER 1: INTRODUCTION 1

1.0. Introduction 1

1.1.What are liposomes? 5

1.2. Classification of liposomes 11

1.2.1. Liposome classification based on composition and application 13

1.2.2. Conventional liposomes 13

1.2.3. Long circulating liposomes (Stealth) 14

1.2.4. Immunoliposomes 15

1.2.5. pH-sensitive liposomes 16

1.2.6. Cationic liposomes 17

1.3.Methods for the preparation of liposome 18

1.3.1.Mechanical dispersion method 19

1.3.1.1. Lipid hydration 19

1.3.1.4. Extrusion technique 20

1.3.1.3. French pressure cell method 20

1.3.1.2. Sonication 20

1.3.2. Solvent dispersion method 21

1.3.2.3. Reverse phase evaporation 21

1.3.2.2. Ethanol ejection 21

1.3.2.1. Ether infusion 21

1.3.3. Detergent removal method 22

1.4. Drug encapsulation in liposomal delivery systems 23

1.4.1. Characterization of liposome and liposome-drug interactions 23

1.5. Liposomes as nano drug delivery systems in cancer treatment 26

1.5.1. Clinical efficacy of liposomal formulations of anticancer agents 28

1.6. Intracellular delivery mechanisms of liposomes 33

1.7. Sirolimus (rapamycin)- a new anti-cancer agent 36

1.7.1 Mechanism of action of sirolimus (rapamycin) 36

1.8. References 38

2. CHAPTER TWO 46

Sirolimus encapsulated liposomes for cancer therapy: physico-chemical and mechanical

characterization of sirolimus distribution within liposome bilayers 46

2.0. Abstract 46

2.1. Introduction 46

VIII I. Onyesom PhD Thesis

2.2. Materials and Method 48

2.2.1. Materials 48

2.3 Methods 48

2.3.1. Liposome preparation 48

2.3.2. Particle size and zeta potential analysis 49

2.3.3. Evaluation of liposome membrane integrity 49

2.3.4. Atomic force microscopy (AFM) 50

2.3.5. Differential Scanning calorimetry (DSC) 50

2.3.6. X-ray photoelectron spectroscopy (XPS) 51

2.4. Results and Discussion 51

2.4.1. Liposome particle size, stability and membrane integrity 51

2.4.2. Atomic force microscopy of liposome formulations 53

2.4.3. Differential scanning calorimetry analysis (DSC) 57

2.4.4. X-ray photoelectron analysis (XPS) of Stealth® liposomes 64

2.5. Conclusions 71

2.6 References 71

3. CHAPTER THREE 76

Antitumor effects of sirolimus – loaded liposomes against human breast cancer cells 76

3.0. Over-view 76


3.2. Materials 78

3.3. Methods 78

3.3.1. Preparation of liposome formulations 78

3.3.2. Encapsulation efficiency of liposome formulations 79

3.3.3.Stability evaluation of formulations 79

3.3.4. Release profile studies 79

3.3.5. Quantification of sirolimus by HPLC analysis 79

3.3.6. Cytotocity test 80

3.3.7. Cellular uptake 81

3.3.8. Apoptosis study 81


3.4.1. Size, stability and encapsulation analysis 81

3.4.2. Drug release profile of sirolimus in liposome formulations 84

3.4.3. Antiproliferative effect of sirolimus versus curcumin 86

3.4.4. Antiproliferative effect of liposome formulations 88

IX I. Onyesom PhD Thesis

3.4.5. Cellular internalisation and apoptosis induced effect of liposome formulations 95

3.5. Conclusions 99

3.6. References 99

4. CHAPTER FOUR 103

Antitumor effects of sirolimus-loaded liposomes against human breast cancer cells 103

4.0. Over-view 103


4.2. Materials 104

4.3. Methods 105


4.3.2. Liposome conjugation 105

4.3.3. Methodology of transferrin assay quantification 106

4.3.4. Phosphate lipid quantification using the Bartlett Assay 107

4.3.5. In vitro studies of Stealth liposomes on BT-474 cells 107

4.3.6. Cellular uptake of conjugated stealth liposomes 108

4.3.7. In-vivo studies of transferrin conjugated and non-conjugated stealth liposomes 108

4.3.8. Statistical analysis of tumour regression 109


4.4.1 Analysis of conjugated and non-conjugated liposome formulations 109

4.5. In-vitro studies of liposomes for site specific drug delivery 112

4.5.1. Cellular uptake of stealth liposome formulations 114

4.6. In-vivo studies of stealth formulations for targeted delivery in breast cancer tumours 117

4.7. Conclusions 127

4.8. References 127

5. CHAPTER FIVE 130

Anti-cancer activity of sirolimus in prostate cancer cell lines 130

5.0. Overview 130


5.2. Materials 131

5.3. Methods 131


5.3.2. In-vitro studies of liposomes on prostate cancer cells 132

5.3.3. Cellular uptake of conjugated stealth liposomes 132

X I. Onyesom PhD Thesis

5.3.4. Statistically Analysis 133


5.4.1. In vitro study of sirolimus in LNCAP and DU-145 cells 133

5.4.2. Antiproliferative effect of liposome formulations in LNCAP prostate cells 134

5.4.3. Antiproliferative effect of liposome formulations in DU-145 prostate cells 136

5.4.4. Cellular uptake of liposomes in prostate cancer cells 139

5.5. Discussion 139

5.6. Conclusions 140

5.7. References 141

6. CHAPTER SIX 142

6.0. Conclusions and Future work 142

6.1 Conclusions 142

6.2 Future Work 144

XI I. Onyesom PhD Thesis

LIST OF FIGURES

Figures Page Number

CHAPTER ONE

Fig. 1.0 Metastatic progression in carcinoma cells. The invasion-metastasis cascade

clinically detectable metastasis represents the end products of a complex

series of cell-biological events which are collectively termed the invasion

metastasis cascade. Carcinoma cells are depicted in red

2

Fig. 1.1 The six hall marks of cancer. Other emerging hall marks include energy

metabolism and evading the immune system.

4

Fig. 1.2 Structural formula of phosphatidic acid 7

Fig. 1.3 Structure and phase transition of PC liposomes. (A) Phase transitions of PC

as a function of temperature, (B) PC configuration at different temperatures

during the phase transition

9

Fig. 1.4 (A) Schematic representation of phosphatidylcholine/cholesterol and their

association

12

Fig. 1.5 Illustration of immobilization of antibody on liposomes. Type (A) PEG-

free immunoliposomes with antibody covalently linked to the short anchor

N-glutarylphosphatidylethanolamine (NGPE); Type (B) PEG-

immunoliposomes with antibody covalently linked to NGPE; Type (C)

new type of PEG-immunoliposomes with antibody attached to the distal

terminal of DSPE-PEG-COOH, so-called pendant-type PEG

immunoliposomes

16

Fig. 1.6 Three proposed mechanisms for pH-sensitive liposome adsorption,

(a) destabilization of pH-sensitive liposomes triggers the destabilization of

the endosomal membrane, most likely via pore formation, leading to

cytoplasmic delivery of their contents, (b) upon liposome destabilization,

the encapsulated molecules diffuse to the cytoplasm via the endosomal

membrane, and (c) fusion between the liposome and the endosomal

membranes, results to cytoplasmic delivery of their contents

17

Fig. 1.7 Schematic representing the different liposome preparation methods. 19

Fig. 1.8 Size-distributions of egg PC liposome dispersions obtained using photon

correlation spectroscopy

24

Fig. 1.9 Microscopic images of liposomes using AFM, ESEM, TEM and CLSM 25

Fig. 1.10 Structures of examples of chemotherapeutic drugs that have been utilised 29

XII I. Onyesom PhD Thesis

with liposomes either in-vitro or in-vivo

Fig. 1.11 Intracellular trafficking of nanocarrier following macropinocytosis,

clathrin-mediated endocytosis and caveolae-mediated endocytosis

33

Fig. 1.12 Internalisation of nanocarrier by opsonization and phagocytosis. 34

Fig. 1.13 Vesicle formations during clathrin-mediated endocytosis. (A) The

assembly of clathrin triskelions (based on three clathrin heavy chains) into

a polygonal lattice helps deform the overlying plasma membrane into a

coated pit. (B) Dynamin is recruited at the neck of the pit to mediate the

membrane fission after assembly of the basket-like clathrin lattice. (C) This

leads to the cytosolic release of the clathrin-coated vesicle; (D) The un-

coating of the vesicle allows the recycling of clathrin triskelia.

35

Fig. 1.14 Structure and molecular target of sirolimus 37

CHAPTER TWO

Fig. 2.0 Membrane integrity of the liposome formulations using percentage latency

over a period of 24 hours

55

Fig.2.1 AFM images of sirolimus loaded DPPC/Chol/DSPE-MPEG-2000 (a,b) and

DSPC/Chol/DSPE-MPEG-2000 Stealth liposomes

56

Fig. 2.2 DSC thermograms of unloaded and loaded conventional DPPC/Chol 59

Fig. 2.3 DSC thermograms of unloaded and loaded DPPC/Chol/DSPEMPEG-2000

liposomes.

62

Fig. 2.4 DSC thermograms of unloaded and loaded DSPC/Chol/DSPEMPEG-2000

liposomes

63

Fig. 2.5 XPS wide spectra of unloaded and loaded DPPC/Chol/DSPE-MPEG-2000

and DSPC/Chol/DSPE-MPEG-2000 Stealth liposomes

65

Fig. 2.6 P2p and N1s XPS core level spectra of Stealth® liposomes 66

Fig. 2.7 C1s XPS core level spectra of DSPC/Chol/DSPE-MPEG-2000 unloaded

and loaded liposomes

67

Fig. 2.8 C1s XPS core level spectra of DPPC/Chol/DSPE-MPEG-2000 unloaded


68

Fig. 2.9 O1s XPS core level spectra of DSPC/Chol/DSPE-MPEG-2000 unloaded


69

Fig. 2.10 O1s XPS core level spectra of DPPC/Chol/DSPE-MPEG-2000 unloaded


70

XIII I. Onyesom PhD Thesis

CHAPTER THREE

Fig. 3.0 Malvern zetasizer particle size analysis of stealth liposome formulations

(A) DPPC stealth liposomes empty and loaded; (B) DSPC stealth

liposome empty and unloaded

83

Fig. 3.1 Entrapment of sirolimus in DPPC and DSPC conventional and stealth

liposome formulations

84

Fig. 3.2 HPLC chromatogram of sirolimus 84

Fig. 3.3 Drug release profile of DPPC and DSPC conventional and Stealth liposome

formulations

86

Fig. 3.4 Antiproliferative effects of pure sirolimus and curcumin on breast cancer

cells (BT-474) using the MTT assay for 24 hours incubation time

87

Fig. 3.5 Cytotoxicity of empty DPPC formulations (conventional and Stealth) on

3T3 endothelial cells. DPPC: cholesterol (molar ratio 18.6: 9) and stealth

DPPC: DSPE-Mpeg2000: cholesterol (molar ratio 12.6: 1.14: 8.0)

88

Fig. 3.6 Antiproliferative effect of empty and loaded (1.09 mM drug) conventional

liposome formulation DPPC: cholesterol (molar ratio 18.6: 9) on MCF-7

breast cancer cell line for 24 hours

89

Fig. 3.7 Antiproliferative effects of conventional formulation DPPC: cholesterol

(molar ratio 18.6:9) and stealth DPPC: DSPE-Mpeg2000: cholesterol

(molar ratio 12.6: 1.14: 8.0) (1.09 mM drug) on breast cancer cell line BT-

474 for 24 hours

90

Fig. 3.8 Antiproliferative effect of conventional formulation DPPC: cholesterol

(molar ratio 18.6: 9) and stealth DPPC: DSPE-Mpeg2000: cholesterol

(molar ratio 12.6: 1.14: 8.0) (2.18mM drug) on breast cancer cell line BT-

474 for 24 hours

92

Fig. 3.9 Antiproliferative effect of loaded DPPC conventional and stealth liposomes

(2.18 mM) on breast cancer cell line BT-474 for 72 hours

93

Fig. 3.10 Antiproliferative effect of conventional formulation DPPC: cholesterol

(molar ratio 18.6: 9) and stealth (5.46 mM drug) on breast cancer cell line

BT-474 for 72 hours

94

Fig. 3.11 Antiproliferative effect of DPPC and DSPC stealth formulations; molar

ratio (12.6: 1.14:8.0) with 2.18 mM drug on breast cancer cell line BT-474

for 72 hours

95

Fig. 3.12 Fluorescent images of the cellular uptake of empty formulated DPPC 97

XIV I. Onyesom PhD Thesis

stealth liposome localisation in the cytoplasm of the cell (A; 10x objective

magnification; B; 60x objective magnification). The nucleus of the cell

was stained blue with DAPI and liposome formulation was labelled with

rhodamine (red)

Fig. 3.13 Fluorescent microscopy images showing apoptotic cells of sirolimus loaded

DPPC stealth liposomes using Annexin V conjugate staining. Nucleus is

stained blue and apoptosis induced signal is green using Annexin V

conjugate

98

CHAPTER FOUR

Fig. 4.0 Antiproliferative effects of DPPC stealth formulations (transferrin

conjugated and non-conjugated) on breast cancer cell line BT-474 for 72

hours

112

Fig. 4.1 Antiproliferative effects of DSPC Stealth formulations (transferrin

conjugated and non-conjugated) on breast cancer cell line BT-474 for 72

hours

113

Fig. 4.2 Antiproliferative effect of Stealth liposomes of transferrin conjugated and

non-conjugated on BT-474 breast cancer cells for 72 hours. MTT assay was

access with fixed dose of 800µg/ml of each formulation with cell density of

1 million

115

Fig. 4.3 Cellular uptake of rhodamine (red) labelled stealth formulations in BT-474

cancer cells. Cell nucleus is stained with DAPI (blue)

116

Fig. 4.4 Comparative therapeutic effects of control and sirolimus anti-cancer drug

on tumour suppression of BT-474 cancer.

117

Fig. 4.5 Therapeutic effects of Stealth formulation on tumour suppression of BT-

474 tumours. (A) Efficacy of empty (unloaded) and loaded non-conjugated

stealth liposome on tumour weight; (B) Efficacy of empty (unloaded) and

loaded transferrin conjugated stealth liposome on tumour weight.

119

Fig. 4.6 Therapeutic effect of control and pure drug (sirolimus) on mice bearing

tumour.

121

Fig. 4.7 Therapeutic effect of empty and loaded stealth formulations on mice

bearing tumour

122

Fig. 4.8 Therapeutic effect of transferrin conjugated empty and loaded Stealth

formulations on mice bearing tumour.

123

Fig. 4.9 Difference in therapeutic efficiency of control, sirolimus and transferrin 124

XV I. Onyesom PhD Thesis

conjugated and non-conjugated loaded Stealth formulations in mice

bearing tumour.

Fig. 4.10 Treatment tolerance by mice bearing tumour over the treatment study

period of 14 days

125

CHAPTER FIVE

Fig. 5.0 Sirolimus antiproliferative activity in LNCAP and DU-145 prostate cancer

cells

134

Fig. 5.1 Antiproliferative effect liposomes formulations on LNCAP cells for 72

hours. (A) Cytotoxicity effect of conventional DSPC and DPPC liposome.

(B) Cytotoxicity effect of stealth DSPC and DPPC liposome

135

Fig. 5.2 Antiproliferative effect liposomes formulations on DU-145 cells for 72

hours. (A) Cytotoxicity effect of conventional DSPC and DPPC liposome.

(B) Cytotoxicity effect of stealth DSPC and DPPC liposome

137

Fig. 5.3 Differences in the antiproliferative effect of sirolimus liposomal on prostate

cancer cell lines of LNCAP and DU-145 at 72 hours

138

Fig. 5.4 Cellular internalisation of DPPC stealth liposomes in LNCAP prostate

cancer cells. Image A-F illustrate liposome uptake in the cytoplasm of

cells. The cell nucleus is stained blue and liposome nanoparticles are

labelled red

139

XVI I. Onyesom PhD Thesis

LIST OF TABLES

Tables Page Number

CHAPTER ONE

Table 1.0 Advantages of liposomal drug delivery systems 5

Table 1.1 Head group alcohols of the phosphoglycerides 6

Table 1.2 Nomenclature and approximate sizes of various liposomes 11

Table 1.3 Liposome classification based on composition and

application

13

Table 1.4 Classification of anticancer drugs 27

Table 1.5 Commercial available liposome formulations

administered via intravenous route

31

Table 1.6 Liposome formulation in clinical trials administered via

intravenous route

32

CHAPTER TWO

Table 2.0 Liposome formulation composition 49

Table 2.1 Particle size and zeta potential of extruded liposome

formulations (n= 3)

54

Table 2.2 Mechanical properties of Stealth liposomes measured by

AFM (n = 20 random particles)

55

Table 2.3 DSC transition parameters obtained for the unloaded and

loaded DPPC liposome formulations

60

Table 2.4 DSC transition parameters obtained for the unloaded and

loaded DSPC liposome formulations

61

Table 2.5 Surface atomic concentrations, and intensity ratios of

I(peak I)/I(peak II) and IP2p/IN1s for liposome

formulations

64

CHAPTER THREE

Table 3.0 Liposome formulation composition. 79

XVII I. Onyesom PhD Thesis

CHAPTER FOUR

Table 4.0 Stealth liposome formulation composition. 105

Table 4.1 Particle size and zeta potential of extruded stealth

liposome formulations (n= 3).

111

CHAPTER FIVE

Table 5.0 Liposome formulation composition 132

XVIII I. Onyesom PhD Thesis

ABBREVIATIONS

Symbol

Description

α Alpha

AFM Atomic force microscopy

β' Beta prime

CHOL Cholesterol

CME Clathrin-mediated endocytosis

CLSM Confocal laser scanning microscopy

CMC Critical micelle concentrations

DNR Daunorubicin

DNA Deoxyribonucleic acid

DAPI

4',6-Diamidino-2-phenylindole

DSC Differential scanning calorimetry

DMPC

1-α-Dimyristoylphosphatidylcholine

DOPC

1,2-Dioleoyl-sn-glycero-3-phosphocholine

DOTAP

1,2-Dioleoyl-3-trimethylammonium-propane

DPPC 1, 2-Dipalmitoylphosphatidylcholine

DSPEMPEG2000 Distearoyl-phosphatidylethanolamine-methyl-polyethyleneglycol

DSPC 1,2-Distearoylphosphatidylcholine

DXR Doxorubicin

DMEM Dulbecco’s modified Eagle’s medium

XIX I. Onyesom PhD Thesis

EPC Egg phosphatidylcholine

ESR Electron spin resonance

ECs Endothelial cells

ESEM Environmental scanning electron microscopy

EGF Epidermal growth factor

EIV Ether injection vesicles

4E-BP1 Eukaryotic initiation factor 4E binding protein-1

eIF-4E Eukaryotic initiation factor-4E

FBS Foetal bovine serum

FPV French press vesicle

HDL High density lipoprotein

HPLC High performance liquid chromatography

HCL Hydrochloric acid

HSPC Hydrogenated soy phosphatidylcholine

FTIR Infrared spectroscopy

LUV Large unilamellar vesicles

LDL Low density lipoproteins

DMPG l-α-dimyristoylphosphatidylglycerol

mTOR Mammalian target of rapamycin

MPS Mononuclear phagocyte system

MLV Multilamellar vesicles

NGPE N-Glutarylphosphatidylethanolamine

NMR Nuclear magnetic resonance

PLD Pegylated liposomal doxorubicin

PC Phosphatidylcholine

PI3K Phosphatidylinositol 3-kinase

XX I. Onyesom PhD Thesis

PHAS-1 Phosphorylated heat- and acid-stable protein–1

PCS Photon correlation spectroscopy

Akt Protein kinase B

RME Receptor mediated endocytosis

RES Reticuloendothelial system

pRb Retinoblastoma protein

REV Reversephase evaporation vesicles

RNA Ribonucleic acid

SUV Small unilamellar vesicles

SPC Soy phosphatidylcholine

SM Sphingomyelin

MTT Thiazolyl blue tetrazolium bromide

TF Transferrin

Tm Transition temperature

TEM Transmission electron microscopy

ULV Unilamellar vesicles

VCR Vincristine

XPS X-ray photoelectron spectroscopy

XXI I. Onyesom PhD Thesis

Publications and conference presentations

Publications (Peer Reviewed)

Ichioma Onyesom, Dimitrios A. Lamprou, Labrini Sygellou, Samuel K. Owusu-

Ware, Milan Antonijevic, Babur Z. Chowdhry, Dennis Douroumis, Sirolimus

Encapsulated Liposomes for Cancer Therapy: Physicochemical and Mechanical

Characterization of Sirolimus Distribution within Liposome Bilayers, Mol.

Pharmaceutics, 10 (11), 4281–4293, 2013.

Dennis Douroumis, Ichioma Onyesom, Mohammed Maniruzzaman, and John

Mitchell, Mesoporous silica nanoparticles in nanotechnology, Crit. Rev. Biotech., 33

(3), 229-245, 2013.

Book Chapters

Dennis Douroumis, Ichioma Onyesom, Mohammed Maniruzzaman, Mark Edwards

(2012). Mesoporous Silica Nanoparticles as a Drug Delivery System. In: Juan Vivero-

Escoto Nanotechnology Science and Technology. Nova. pp., 115 -146.

Dennis Douroumis and Ichioma Onyesom (2011). Novel coating strategies of

technologies of drug eluting stents. Studies in Mechanobiology, Tissue Engineering

and Biomaterials, 1, Volume 8, Active Implants and Scaffolds for Tissue

Regeneration, pp., 87-125.

Conferences (posters and podium)

July 2012, 39th Annual Meeting & Exposition of the Controlled Release Society

Conference, Quebec, Canada. Ichioma Onyesom, Samuel Owusu-Ware, Dimitris

Lamprou, Dennis Douroumis. Investigation of Sirolimus liposomes using DSC and

AFM.

July 2012, 39th Annual Meeting & Exposition of the Controlled Release Society

Conference, Quebec, Canada. Anti-tumour effect of Sirolimus Stealth liposomes on

human breast cancer cells (BT-474). Ichioma Onyesom, Dennis Douroumis.

August 2011, Academy of Pharmaceutical Sciences Conference, Nottingham, UK.

Podium presentation. Ichioma Onyesom, Dennis Douroumis. Liposomal drug

delivery for cancer treatment.


Poster presentation Samuel K. Owusu-Ware, Ichioma Onyesom, Dionysios

https://springerlink3.metapress.com/content/?Author=Dennis+Douroumis

https://springerlink3.metapress.com/content/?Author=Ichioma+Onyesom

https://springerlink3.metapress.com/content/1868-2006/

https://springerlink3.metapress.com/content/1868-2006/

https://springerlink3.metapress.com/content/978-3-642-18064-4/

https://springerlink3.metapress.com/content/978-3-642-18064-4/

XXII I. Onyesom PhD Thesis

Douroumis and Milan D. Antonijevic. DSC studies of freeze-dried DPPC and DSPC

liposome formulations in the absence of cryoprotectants.


Poster presentation A. A. Lafarga, I. Onyesom, A. Patil, D.A. Lamprou, A.J.

Urquhart, and D. Douroumis. MCM-41 nanoparticles as an anticancer drug delivery

system.

July 2011, 38th Annual meeting & exposition of the controlled release society

Conference, Maryland, USA. Poster presentation. Ichioma Onyesom, Dimitris

Lamprou, Andrew Urquhart, Dennis Douroumis. Sirolimus – Loaded Liposomes

against Human Breast Cancer Cells.

1 I. Onyesom PhD Thesis

Chapter 1

Introduction

1.0 Introduction

In the past decades researchers have invested enormous efforts in gathering information on

the molecular and genetic properties/characteristics of cancer. Cancer is widely known as a

‘system biology disease’ (Bizzarri et al., 2008) and can be characterised by unregulated cell

growth, as well as the invasion and spread of cells from their primary site of origin to other

sites in the body (Pecorino, 2008). The spread of cancer cells has led to the differentiation

between a benign tumour which does not metastasize and a malignant tumour which shows

features of invasion and metastasis. Different types of cancer possess different distinct

features and thus can be classified by their tissue of origin (carcinomas). For example,

cancers derived from glandular tissue (e.g. breast cells) are referred to as adenocarcinomas

whilst those derived from the skin are referred to as basal-cell carcinomas. Evidence indicates

that carcinogenesis in humans is a multi-step process involving dynamic alterations in the

human genome. These alterations lead to the progressive transformation of normal human

cells into highly malignant and metastatic cancer cells derivatives. Cancer cell metastasis is

the adaptation of cancer cells to a tissue microenvironment at a distance from the primary

tumour. Metastases, spawned by carcinomas, are formed following a set of complex multi-

step processes driven by cellular biological events (Fig. 1.0) whereby epithelial cells in

primary tumours (1) invade locally via the surrounding extracellular matrix (ECM) and

stromal cell layers, (2) intravasate into the lumina of blood vessels, (3) survive the rigors of

transport through the vasculature, (4) arrest at distant organ sites, (5) extravasate into the

parenchyma of distant tissues, (6) initially survive in these foreign microenvironments in

order to form micrometastases, and (7) reinitiate their proliferative programs at metastatic

sites, thus giving rise to macroscopic, clinically detectable neoplastic growths. This process is

often referred to as ‘‘metastatic colonization’’ (Valastyan & Weinberg, 2011).

Metastatic cancer cells require properties that allow them not only to adapt to a foreign

microenvironment but also to convert it in a way that is conducive to their continued

proliferation and survival. Recent advanced technological concepts have contributed to the

understanding of the role of the host tissue stroma in promoting tumour cell growth and

spreading; thus they have provided new insights into the genetic make up of cancers with

high metastatic tendency (Bacac & Stamenkovic, 2008). During metastatic progression,


tumor cells exit their primary sites of growth (local invasion, intravasation), translocate

systemically (survival in the circulation, arrest at a distant organ site, extravasation) and adapt

to survive and thrive in the foreign microenvironments of distant tissues (micrometastasis

formation, metastatic colonization). Metastatic progression is not an entirely cell independent

process. Certainly, carcinoma cells enlist non-neoplastic stromal cells to aid in each step of

the invasion-metastasis cascade as illustrated in Fig. 1.0 (Valastyan & Weinberg, 2011).

Other research findings have shown that a number of molecular, biochemical and cellular

traits are shared by most if not all types of human cancerous cells. Hanahan and Weinberg in

their review ‘The hall mark of cancer’ defined the six hall marks of cancer which includes

growth signal autonomy, evasion of growth inhibitory signals, unlimited replicative potential,

angiogenesis, evasion of apoptosis, invasion and metastasis (Fig. 1.1). Energy metabolism

and evading the immune system have been discovered as two other emerging hallmarks of

the disease (Hanahan & Weinberg 2011).

Fig. 1.0 Metastatic progression in carcinoma cells. The invasion-metastasis cascade in

clinically detectable metastases represents the end products of a complex series of celular

biological events, which are collectively termed the invasion metastasis cascade. Carcinoma

cells in are depicted in red (Valastyan & Weinberg, 2011).

The discovery and development process of anti-cancer therapeutics depends on the informed

and/or anticipated mechanism of action of anti-cancer agents as well as the biological


pathways of cancer. This process is best carried out and evaluated in preclinical and clinical

studies (in-vitro and in-vivo animal models). Early therapeutic strategies against cancer

involved the surgical removal of as many cancer cells as possible. However, total surgical

removal of the affected tissue is, relatively, attainable in some types of cancer and difficult-if

not impossible- in other types. For example, surgical removal of skin cancer is more practical

than cancer of the lungs and heart. Conventional chemotherapy and radiotherapy have been

used to inhibit and eradicate the proliferation of cancer cells. Conventional chemotherapy

uses chemical entities (drugs) to target e.g. DNA, RNA or proteins in order to disrupt the cell

cycle in rapidly dividing cancer cells. This stategy is often unspecific and often induces side-

effects and cytotoxicity. As indicated by the therapeutic index, the aim with all drugs is to

achieve an effective therapeutic result with minimal side-effects. The non-specificity of

chemotherapy has further led to the development of new therapeutic strategies such as

biomarkers and site specific drug delivery systems.

Cancer targeting via the use of drug delivery system has been a theme of major therapeutic

research interest. Drug delivery systems offer the possibility of enhancing the therapeutic

effect of anticancer agents by either increased drug concentration and/or reduced exposure to

normal tissues sites. Among the variety of anti-cancer drug delivery systems that have been

developed over the past 2-3 decades (which includes microspheres, lipoprotein and

nanoparticles etc.,) the use of liposomes has attracted significant academic/industrial/clinical

interest due to their unique properties such as the ability to carry a wide variety of molecules,

and the structural versatility and biocompatibility of their components.


Fig. 1.1 The six hall marks of cancer. Other emerging hall marks include energy metabolism

and evading the immune system (Hanahan & Weinberg R, 2011).

Liposomes have been widely investigated since the 1970s as drug carriers for enhanced and

improved delivery of therapeutic agents to specific sites in biological systems. The success of

liposomes as drug carriers, especially in the treatment of cancer, has been reflected in various

liposome-based formulations which are commercially available as well as those undergoing


clinical trials. The benefit of a liposomal drug delivery system depends on the nature of the

drug, the desired pharmacological intervention and the site of application (Table 1.0)

Table 1.0 Advantages of liposomal drug delivery systems.

Solubilisation Liposomes due to their bilayer(s) may solubilise

drugs which are water insoluble and thus provide

a stable aqueous formulation.

Protection Encapsulated drugs in liposomes, in particular

those entrapped in the aqueous interior, are

protected against the action of detrimental factors

(e.g. degradative enzymes) present in the host.

Conversely, reduces systemic toxicity.(cf.

duration).

Controlled drug release Liposome membranes may provide an effective

penetration barrier for encapsulated drug; thus

controlled release effects can be obtained which

hinders the molecule from being inactivated or

being excreted prematurely.

Enhancing immune

response

Liposomes can be used to actively deliver a drug

in a preferred conformation/orientation or co-

present it together with other immune response-

enhancing structures

Internalization Liposomes can interact with target cells in various

ways by overcoming biological membranes and

are therefore able to promote the internalisation of

drug molecules which in their ‘free’ form (i.e.

non-formulated) would not be able to permeate

the cellular interior due to unfavorable

physicochemical characteristics (e.g. DNA

molecules).

1.1 What are liposomes?

Liposomes were first discovered by Bangham in the 1960’s and are defined as lyotropic

liquid crystal lipid vesicles composed of relatively biocompatible and biodegradable

materials. They consist of an aqueous core entrapped by one or more bilayers of natural

and/or synthetic lipids. Depending on their in vivo half-life, liposome encapsulated

substances are protected from exposure to the surrounding biological environment. Drugs of

varying lipophilicities can be encapsulated in liposomes, either in the entrapped aqueous core

or in the bilayer interface (Goyal et al., 2005). Liposomes are spontaneously formed as a

result of the dispersion of phospholipids in aqueous media (Bangham et al., 1974). When

phospholipids are placed in an aqueous medium the hydrophilic interaction of the lipid head


groups with water results in the formation of multilamellar and unilamellar systems

(vesicles). Fig. 1.2 shows the chemical structure of phospholipids with a glycerol backbone.

The hydroxyl moeity is esterified to phosphoric acid at the sn-3 carbon of the glycerol

molecule. The hydroxyl groups at positions 1 and 2 of the glycerol are usually esterified with

long chain fatty acids. The lipid nature of the phospholipid is due to the long chain fatty

acids. One of the remaining oxygen groups of phosphoric acid maybe further esterified to a

variety of organic molecules which include glycerol, ethanolamine, choline, inositol and

serine. The phosphate moiety together with the attached alcohol represents the head group of

the phospholipid. Table 1.1 shows various head groups and the name of the phosphatidic acid

on the head group (Vemuri & Rhodes, 1995).

Table 1.1 Head group alcohols of the phosphoglycerides.

Phosphatidic acid R-group

Phosphatidylethanolamine -CH,-CH, NH

Phosphatidylglycerol CH,-CHOH-CH,OH

Phosphatidylserine -CH,-CHNH,COOH

Phosphatidylcholline -CH,-CH, -N(CH3)

Phosphatidylinositol


Fig. 1.2 Structural formula of phosphatidic acid.

The importance of liposome as a model membrane system arises from the fact that liposome

formation could be derived from a variety of natural and synthetic phospholipids which forms

a bilayer structure similar to the lipid portion of the natural cell membrane (‘the sea of

phospholipid’ - Singer and Nicholson model).

In plants and animals the most abundant phosphatides are phosphatidylethanolamine and

phosphatidylcholine (also known as lecithin). These two components constitute the major

structural part of most biological membranes. The similarity between liposomes and natural

cell membranes can be increased by extensive chemical modification of the liposome

membrane and thus exploited in areas such as targeted drug delivery system both in-vivo and

in-vitro. In phosphatidylserine the phosphoric acid moiety is esterified to the hydroxyl group

of the amino acid L-serine and in phosphatidylinositol to one of the hydroxyl groups of the

cyclic sugar alcohol inositol. In the case of phosphatidylglycerol the alcohol that is esterified

to the phosphate moiety is glycerol (Vemuri & Rhodes, 1995).

The primary lipid used in liposomal drug delivery systems is phosphatidylcholine.


Phosphatidylcholine (PC) has a hydrophilic head group consisting of the quaternary

ammonium moiety choline, which is linked to the glycerol backbone via a phosphoric ester.

PC is zwitterionic due to the negative charge of the phosphate at physiologic pH and thus

liposomes made of it have no net charge. The remaining two hydroxyl groups of the glycerol

backbone are esterified with fatty acids of varying chain length and degree of unsaturation.

The preferred state of organisation of hydrated PC is usually in the form of a bilayer leaflet

(Fig. 1.4), whereby, the hydrophilic head groups of the lipid molecules are oriented towards

the water phase and the acyl-chains of the two mono-layers are orientated towards the middle

of the membrane. The chain length and degree of saturation of the acyl chains principally

determines the melting temperature of the liposome membrane, i.e., its transformation from a

rigid well organised “gel state’’ to a liquid crystalline, less ordered “fluid state’’ (Brandl,

2001).

Understanding the phase transition properties and fluidity of phospholipid membranes is

essential both in the formulation and exploitation of liposomes, since the phase behaviour of

liposome membranes determines properties such as permeability, aggregation and protein

binding which are important in relation to the stability of liposomes and their physiochemical

properties in biological systems. At different temperatures, phospholipid membrane can exist

in different phases. The transition from one phase to another can be identified by physical

techniques (e.g. calorimetry) in response to a change in temperature. The most frequently

observed transition phase is the one that occurs at the highest temperature, in which the

membrane passes from a tightly ordered ‘gel’ or ‘solid’ phase to a liquid crystalline phase at

increased temperatures where the mobility (e.g. lateral diffusion coefficient) of each

phospholipid molecule is significantly higher than in the gel phase.

The most commonly used phospholipids in model membrane/liposome studies are the

phosphatidycholines (PCs). The bilayer of these lipids undergoes three phase transitions

within a temperature range of 10oC to 80oC (Koynova & Caffrey, 1998). Fig. 4 illustrates

the different phase transitions of phospholipids as a function of temperature. The lowest

temperature phase is the sub-gel phase (Lc) which upon heating transforms to the gel phase

(Lβ'). In the sub-gel and gel phase the hydrocarbon tails are tilted with respect to the bilayer in

an all trans configuration. However, in the Lβ' phase the head group is more hydrated (i.e.

surrounded with water) compared to the head group in the Lc phase. This transition from Lc

to the Lβ' phase is called the subtransition. A further increase in temperature gives rise to the

formation of the of ripple phase (Pβ') which is also known as the pretransition. In the ripple


phase (Pβ') the bilayer adopts a gauche configuration (Fig. 1.3). Finally the bilayer, with a

further increase in temperature, undergoes a transition to the liquid crystalline or fluid (Lα)

phase. This phase (Lα) is generally known as the main transition phase whereby the lipids

have freedom of movement in the plane of the bilayer (Gennis, 1989).

Fig. 1.3 Structure and phase transition of phosphatidyl choline. (A) Phase transitions of PC

with response to increase in temperature, (B) PC configuration at different transition phase.

PCs are hardly ever used alone in liposomal formulations. Mixtures of other lipids with PC

are mainly used to enhance the properties and stability of the liposomes. Cholesterol on the

other hand is included in liposomes formulations in order to obtain a tighter packing of the

membrane which results in reduced leakiness of the entrapped substances and adsorption of

membrane proteins (Damen et al., 1981; Kirby et al., 1980). Cholesterol plays a vital role in

membranes as a modulator of the physiochemical functionality of the lipid bilayer (Yeagle,

1985). Cholesterol molecules insert into the liposome membrane with their hydroxyl groups

oriented towards the aqueous surface and the aliphatic chain aligned parallel to the acyl

chains in the centre of the bilayer (Fig. 1.4). The incorporation of cholesterol into liposome

vesicles increases the separation between the PC head groups and eliminates the normal

electrostatic, hydrogen bonding and Van der Waals interactions, thereby pushing the


phospholipids apart and altering bilayer membrane fluidity.

Several research studies have shown cholesterol to modify the order and mobility of

phospholipids in bilayer. Coderch et al., (2000) investigated the influence of varying amounts

of cholesterol on saturated and unsaturated phospholipids (H-PC and PC) liposomes. In their

investigation, they observed a gradual decrease in the fluidity of PC liposomes as the amount

of cholesterol was increased over all the temperature ranges studied. However, for H-PC

liposomes the opposite effect was observed whereby at low temperature, increase in the

amount of cholesterol led to an increase in fluidity but at high temperature an increase in

cholesterol led to a decrease in fluidity.

The micromechanical properties of pure and cholesterol modified egg yolk

phosphatidylcholine (EggPC) vesicles were investigated using atomic force microscopy by

Liang et al., (2004). The elastic properties obtained from the force plot analysis based on the

Hertzian model showed that Young’s modulus and the bending modulus of cholesterol

modified vesicles increased several-fold compared to the pure EggPC vesicles (without

cholesterol). This significant difference was reported to be attributed to the rigidity of the

EggPC vesicles as a result of the incorporation of cholesterol molecules.

Kirby et al., (1980) earlier reported the effect of cholesterol content of small unilamellar

liposome on their stability in-vivo and in-vitro. Cholesterol-rich (EggPC/ cholesterol, 7: 7

molar ratios) liposomes, regardless of surface charge were reported to remain stable in the

blood of intravenously injected animals for up to at least 400 min. In addition, the stability of

cholesterol-rich liposomes was also largely maintained in-vitro in the presence of whole

blood, plasma or serum for up to 90 min. However, cholesterol-poor (EggPC/cholesterol, 7:2

molar ratio) or cholesterol-free (EggPC) liposomes lost very rapidly (at most within 2 min)

much of their stability after intravenous injection or upon contact with whole blood, plasma

or serum.


1.2 Classification of liposomes

Liposomes are classified either by their structural properties (Table 1.2) or by their method of

preparation. In some cases, liposomes are also classified by their composition and application

(Fig. 1.4). Classifications of liposomes with reference to their bilayers are usually described

as unilamellar vesicles (ULV) or multilamellar vesicles (MLV). On the other hand reverse

phase evaporation vesicles (REVs), French press vesicle (FPV) and ether injection vesicles

(EIV) are descriptions based on their method of preparation. Liposomes classified based on

their size are referred to as large unilamellar vesicles (LUV) or small unilamellar vesicles

(SUV). In general liposomes are more commonly described in terms of their lamellarity and

size rather than their method of preparation (Vemuri & Rhodes, 1995; Rongen et al., 1997).

Table 1.2 Nomenclature and approximate size of liposome preparations.

Vesicle Types Abbreviation Diameter Number of lipid

bilayers

Small unilamellar

vesicles

SUV 20-100 nm One lipid bilayer

Large unilamellar

vesicles

LUV 100 nm One lipid bilayer

Multilamellar vesicles MLV 0.5 m. Five to twenty lipid

bilayers

Oligolamellar vesicles OLV 0.1-1 m Approximately five

lipid bilayers

Multivesicular vesicles MMV 1 m Multi-

compartmental

structure


(A)

(B)

Fig. 1.4 (A) Schematic representation of phosphatidylcholine/cholesterol and their

association within bilayers. (B) Classification of liposomes (Storm et al, 1998 & Brandl M,

2001).


1.2.1 Liposome classification based on composition and application

There are four major types of liposomes that can be classified on the basis of their

composition and in-vivo application. Due to the flexibility of liposomes i.e. the ability to alter

their structural and physiochemical characteristics many formulation scientists have been able

to modify the behaviour of liposomes in-vivo for various specific therapeutic requirements

(Table 1.3).

Table 1.3 Liposome classification based on composition and application.

Types of Liposome Major application

Conventional liposomes

Macrophage targeting

Local depot

Vaccination

Long-circulating liposomes

Selective targeting to pathological

areas Circulating microreservoir

Immunoliposomes Specific targeting

Cationic liposomes Gene delivery

pH-Sensitive liposomes Specific and controlled delivery of

biological active via the use of pH

sensitive lipids

1.2.2 Conventional liposomes

This is one of the early investigated liposome vesicles which comprises of different lipid

compositions that are neutral and/or negatively charged. The most commonly used lipids are

PCs and cholesterol. Conventional liposomes differ in their physicochemical properties such

as membrane packing, size and surface charge. These properties influence their

pharmacokinetics and biodistribution when administered in vivo. Drummond et al., (1999) in

their review of optimizing liposomes for delivery of chemotherapeutic agents to solid tumors,

elaborated on the effect of size, charge and membrane packing on the pharmacokinetics and

biodistribution of conventional liposomes. One of the examples cited in their review

suggested that increasing vesicle size of liposomes of similar composition results in more


rapid uptake by the reticuloendothelial system (RES). One of the major drawbacks of

conventional liposomes is their rapid uptake by macrophages after systemic administrations.

The major organs for the accumulations of conventional liposomes are the liver and spleen

which is as a result of the rich blood supply and abundance of macrophages in these organs.

This drawback has lead to the logical therapeutic translation of conventional liposomes as an

attractive candidate for drug delivery to macrophages and use in vaccination (Fidler et al.,

1989). Another limitation of conventional liposomes is their extravasation time (escaping

from the blood circulation) and poor blood circulation in solid tumours (Lasic, 1995); thus

the need for further development of more improved liposomal drug delivery system. One of

the first attempts to eliminate these problems concentrated on modification of lipid membrane

components in order to adjust bilayer fluidity. Damen et al., (2005) reported that

incorporation of cholesterol (CHOL), by causing increased packing of phospholipids in the

lipid bilayer, reduces transfer of phospholipids to high density lipoprotein (HDL); Senior

(1982) also illustrated that liposomes obtained from PC with saturated fatty acyl chains (with

a high liquid crystalline transition temperature) or from sphingomyelin (SM) are relatively

more stable in blood compared to liposomes prepared from PC with unsaturated fatty acyl

chains.

1.2.3 Long circulating liposomes (Stealth)

Liposomal formulations of several active molecules are currently in pre-clinical and clinical

trials in different fields, with promising results. Two of the key problems in drug therapy is

the bio-distribution throughout the body and the targeting to specific receptors. The

development of long circulating (Stealth) liposomes represented a step towards eliminating

these problems. The use of saturated phospholipids and cholesterol in the formulation of

liposome drug delivery systems cannot fully overcome their binding with serum components

which subsequently increases the uptake of the vesicles by the macrophage system (MPS).

Stealth liposomes are formulated vesicles with polyethylene glycol attached to the outside of

the vesicle membrane.

The first strategy employed by Gabizon and Papahadjopoulos (1988) and Allen et al (1989)

was the preparation of liposomes imitating the erythrocyte membrane; the liposome surface,

in this case, was modified with gangliosides and sialic acid derivatives, such as

monosialoganglioside (GM1). The subsequent step was to increase the hydrophilicity of the

liposomal surface by using hydrophilic polymers. The mechanism whereby steric


stabilization of liposomes increases their blood circulation time has also been extensively

discussed by Drummond et al (1999), Park & Benz (2004) and Bae & Park (2011). The

basic concept is that a hydrophilic polymer or a glycolipid, such as PEG or GM1, which

possess a flexible chain that occupies the space immediately adjacent to the liposome surface

(“periliposomal layer”), tends to exclude other macromolecules from this space (Fig. 1.4;

Immordino et al., 2006). Steric stabilization results from the local surface concentration of

highly hydrated PEG groups that generate a steric barrier against interactions with molecular

and cellular components in the biological surroundings.

1.2.4 Immunoliposomes

Stealth liposomes have been demonstrated to accumulate in tumour cells and also possess

excellent pharmacokinetic properties such as prolonged circulation time; However scientific

research has also identified the need for specificity in drug delivery to tumour cells in order

to further increase tumour accumulation and reduce systemic toxicity (Vingerhoeds, Storm

& Crommelin 1994). Immunoliposome are liposomes with antibodies coupled to their surface

(Figs. 1.4 & 1.5) which enables active tissue targeting through binding to specific receptors in

tumour cells (Maruyama, 2002). The rationale for using immunoliposomes for enhanced

antitumour therapeutic efficacy has been well demonstrated in the scientific literature (Perche

and Torchilin, 2013). Li et al., 2009 demonstrated the therapeutic efficacy of

immunoliposomes in the targeted delivery of doxorubicin using Stealth liposomes modified

with transferrin (transferrin immunoliposome). They examined the intracellular uptake,

pharmacokinetics and biodistribution of the transferrin immunoliposomes in liver cancer.

Their results demonstrated that transferrin immunoliposome enhanced intracellular uptake

and improved therapeutic efficacy in liver cancer cells compared to Stealth liposomes.

Anabousi et al., (2006) and Yamada et al., (2008) also demonstrated that immunoliposomes

with doxorubicin encapsulation improved antitumour efficacy both in vitro and in vivo via

specific binding to targeted receptor.


Fig. 1.5 Illustration of the immobilization of antibody on liposomes. Type (A) PEG-free

immunoliposomes with antibody covalently linked to the short anchor

N-glutarylphosphatidylethanolamine (NGPE); type (B) PEG-immunoliposomes with

antibody covalently linked to NGPE; type (C) new type of PEG-immunoliposomes with

antibody attached to the distal terminal of DSPE-PEG-COOH, so-called pendant-type

PEGimmunoliposomes (Maruyama, 2002).

1.2.5 pH-sensitive liposomes

The development of strategies to increase the ability of liposomes to mediate intracellular

delivery of biological active molecules has further led to the developments of fusogenic or

polymorphic liposomes (pH-sensitive liposome). pH-Sensitive liposomes are composed of

cholesteryl hemisuccinate (CHEMS), phosphatidylethanolamine (PE), oleic acid (OA) or

dioleoylphosphatidylethanolamine (DOPE) (Slepushkin et al., 1997; Garg and Kokkoli et al.,

2011). pH-Sensitive liposomes are liposomes which are stable at physiological pH (pH 7.4)

but acquire fusogenic properties and undergo destabilization under acidic conditions, thus

leading to the release of their aqueous contents (Fig. 1.6). The concept of pH-sensitive

liposomes emerged from the fact that certain enveloped viruses developed strategies to take

advantage of the acidification of the endosomal lumen to infect cells, as well as the

observation that some pathological tissues such as tumors exhibit an acidic environment as

compared to normal tissues. Similar to Stealth liposomes, incorporation of polyethylene

glycol (PEG)-conjugated lipids into pH-sensitive liposomes facilitates prolonged circulation

times of these liposomes, which are otherwise cleared rapidly. Antibodies or ligands can also

be coupled to pH-sensitive or sterically stabilized pH-sensitive liposomes for specific

targeting (Simeos et al., 2004).


Fig. 1.6 Three hypothetical proposed mechanisms for pH-sensitive liposome adsorption;

(a) destabilization of pH-sensitive liposomes triggers the destabilization of the endosomal

membrane, most likely via pore formation, leading to cytoplasmic delivery of their contents;

(b) upon liposome destabilization, the encapsulated molecules diffuse to the cytoplasm via

the endosomal membrane; and (c) fusion between the liposomes and the endosomal

membranes, result in cytoplasmic delivery of their contents (Simeos et al., 2004).

1.2.6 Cationic liposomes

These are currently the youngest members of the liposome family and are being widely

researched. They consist of positively charged lipids and are mainly utilized in the delivery of

genetic material such as vaccines. Cationic liposomes interact with and neutralize negatively

charged DNA which results in the formation of a lipid-DNA complex rather than

encapsulated DNA within the liposome. This interation and neutralization of the DNA

provides protection and promotes the cellulkar uptake and expression of the condensed DNA

plasmid (Lasic & Templeton, 1996). Several reports have demonstrated the use of cationic

liposomes in vaccines delivery (Kaur et al., 2012; Milicic et al., 2012; Carstens et al., 2011).

Perrie and co-workers have reported on the application of cationic liposomes for vaccine

delivery. One of their recently reported case studies is the investigation of the

physiochemical characteristics that dictate the function of liposomal adjuvant in vaccine

delivery. In this study, several parameters were investigated based on

dimethyldioctadecylammonium bromide and trehalose 6,6’-dibehenate cationic liposomes.


They concluded that physiochemical parameters such as a combination of high cationic

charge and electrostatic binding of the antigen to the liposome system and the use of lipids

with high transition temperatures promotes strong vaccine depot (Perrie et al., 2014).

Other report includes a recent review by Shim et al., (2013) on the application of cationic

liposomes for the delivery of nucleic acids. The review reported the engineering of different

lipoplexes of cationic liposomes and their therapeutic efficacies. It was shown that cationic

liposomes could be used to encapsulate and deliver various nuleic acids, thus presenting

cationic liposome as a potential candidate for nucleic acid therapeutics.

1.3 Methods for the preparation of liposomes

Numerous procedures have been developed for liposome preparations which can be derived

from either the use of natural and/or synthetic phospholipids. One of the vital parameters to

consider in the formulation of liposomes is the rigidity of the bilayer. Hydrated-single

component phospholipids bilayers can be in a liquid-crystalline (fluid) state or in a gel state.

An increase in temperature above the transition temperature (Tm) transforms the liposomes

from gel state into a liquid state. The transition temperature of a liposome bilayer is

dependent on many factors including (1) acyl chain length, (2) degree of unsaturation and (3)

nature of head group. Fig. 1.7 shows the different techniques or methods that can be used in

the preparation of liposomes such as mechanical or solvent dispersion and detergent removal.


Fig. 1.7 Schematic diagram representing the different liposome preparation methods.

1.3.1 Mechanical Dispersion methods

The preparation methods covered under this category start with a lipid solution in organic

solvent and ends up with lipid dispersion in water or buffer solution. This method further

incorporate some diverge processing parameters so as to modify their final properties. The

parameters includes further processing such as the post-hydration treatments of the vesicles

which comprises vortexing, freeze thawing, sonication and high-pressure extrusion (Fig. 1.7).

1.3.1.1 Lipid hydration

This is the most frequently used method for the preparation of MLVs. It involves drying a

solution of lipids to form a thin film at the bottom of round bottom flask, hydrating the film

by addition of aqueous buffer and then vortexing the dispersion. The hydration step is carried

out at a temperature above the gel-liquid crystalline transition temperature (Tm) of the lipid

or above the Tm of the highest melting component in the lipid mixture. The compounds

(drugs) to be encapsulated are added either to the aqueous buffer or to the organic solvent


containing lipids depending on the solubility of the compounds. MLVs are simple to prepare

by this method and a variety of substances can be encapsulated in these liposomes. The

disadvantages of using this method are low internal volume, low encapsulation efficiency, the

size (> 1 µm in diameter) distribution is heterogeneous (Bangham et al., 1965, 1974) and

further procedures must be employed to achieve a homogeneous population, such as

sonication or extrusion.

1.3.1.2 Sonication

This method is used in the reduction of the liposome size (MLVs) obtained via other

preparation methods, as previously discussed in the lipid hydration method. It involves the

ultrasonication of an aqueous dispersion of phospholipids using a sonicator bath or a probe

sonicator which usually yields SUVs with size diameters in the range of 15-25 nm.

1.3.1.3 French Pressure Cell Method

The method involves the extrusion of MLV at 20,000 psi through a small orifice. This

method has several advantages over the sonication method due to the fact that it is simple,

rapid, and reproducible and involves gentle handling of unstable materials (Hamilton & Guo,

1984). The resultant liposome vesicles are usually larger (> diameter) than sonicated SUVs.

1.3.1.4 Extrusion techniques

Liposome suspensions are gradually extruded through polycarbonate membranes (e.g.

Nucleopore) of different pore sizes according to the desired final size of the vesicles. In this

technique the MLV dispersions are extruded starting from 0.8 µm to the desired liposomes

size e.g 0.2 µm. Liposomes can also be sized with several pressure extrusion devices

(Amselem et al., 1993; Olson et al., 1979). It has been demonstrated by several researchers

that when MLVs are repeatedly passed through very small pore polycarbonate membranes

(0.8 to 0.1 nm) under high pressure the average diameter of the liposomes become

progressively smaller reaching a minimum of 60-80 nm after 5-10 passes. As the average size

is reduced the liposome vesicles tend to become unilamellar. Similar findings have also been

reported for MLVs when passed through a microfluidizer (Hope et al., 1985; Mayhew et al.,

1985; Vemuri et al., 1990). A microfluidizer is an instrument that forces the feed material

under high pressure through a narrow orifice. It appears that when MLVs are forced through

the small orifice, layers of bilayers are removed from the liposome structure, in the same way

that layers of onion skin are separated when an onion is peeled. It was also suggested, that the


mechanism of layer separation is only applicable to liposome vesicles made with positively

charged phospholipids and vesicles that are greater than 70 nm in diameter.

1.3.2 Solvent Dispersion Method

This method requires the lipids to be dissolved in organic solvents and then brought into

contact with an aqueous phase at a set temperature or under reduced pressure. Liposomes

prepared using these methods always have high internal volume and encapsulation efficiency.

Two types of solvent are commonly used in this preparation technique: ether or ethanol.

1.3.2.1 Ether infusion

Liposomes are formed by the injection of solutions of lipids (dissolved in diethyl ether or

ether/methanol mixture) slowly into an aqueous solution of the material to be encapsulated at

a set temperature or under reduced pressure. The subsequent removal of ether under vacuum

leads to the formation of liposomes. The major setbacks of this method are that the liposome

population obtained is heterogeneous (usually ranging from 70-190 nm) and the compounds

to be encapsulated are exposed to organic solvents or high temperature (Deamer and

Bangham, 1976; Schieren et al., 1978).

1.3.2.2 Ethanol ejection

A lipid solution of ethanol is rapidly injected into a large excess of buffer; MLVs are formed

immediately. The drawbacks of the method are that the population is heterogeneous

(30-110 nm), liposomes are very dilute, it is difficult to remove all the ethanol because it

forms an azeotrope with water and the possibility of inactivation of various biologically

active macromolecules in the presence of even low amounts of ethanol (Batzri & Korn,

1973).

1.3.2.3 Reverse phase evaporation

This technique comprises of two steps. The step requires the preparation of a water-in-oil

emulsion of phospholipids and buffer in excess organic phase. Secondly, removal of the

organic phase under reduced pressure. The two phases (phospholipids and water) are usually

emulsified by mechanical methods or by sonication. Removal of the organic solvent under

vacuum causes the phospholipid coated water droplets to come together to form a gel like


matrix. Further removal of organic solvent, under reduced pressure, causes the gel like matrix

to form into a paste of smooth consistency. This paste is a suspension of LUVs (Szoka &

Papahadjopoulos, 1978). Drug entrapment efficiencies up to 60-65% can be achieved using

this technique. The key disadvantage of this method is the exposure of the drug to be

encapsulated to organic solvents and to mechanical agitation such as sonication. In this

procedure, phospholipids are dissolved in organic solvents such as chloroform and/or

isopropylether. In order to promote conditions for good emulsification a mixture of two

organic solvents may be required to adjust the density so as to be closer to the density of the

aqueous phase. Biologically active molecules such as enzymes, protein pharmaceuticals, and

DNA type molecules may undergo conformational changes, protein denaturation, or breakage

of DNA strands due their exposure to the harsh conditions of organic solvent and mechanical

agitation (Vemuri & Rhodes, 1995).

1.3.3 Detergent removal method

In this method phospholipids are brought in contact with the aqueous phase via detergent,

which associate with phospholipids molecule and serve to screen the hydrophobic portion of

the molecules from water. Detergents at their critical micelles concentrations (CMCs) have

been utilised to solubilize lipids. Upon dissolving the detergent in water at concentration

higher than the CMC, micelles form in more and more numbers. As the detergent is removed,

the micelles become progressively richer in phospholipid and therefore combine to

spontaneously form LUVs. The detergents can be removed by dialysis, or column

chromatography (e.g. Sephadex G-25 column). The advantages of using the detergent dialysis

method are excellent reproducibility and production of liposome populations which are

homogenous in size. However, the main drawback of this method is the retention of traces of

detergent(s) within the liposomes. Using gel chromatography for detergent removal involves

a column (e.g. Sephadex G- 25) by which the liposome vesicles are eluted while the detergent

(e.g. Triton X-100) is retained or binds to the Bio-Beads (SM-2) in the column (Schubert,

2003; Szoka, & Papahadjopoulos, 1980).

1.4 Drug encapsulation and characterization of liposomal delivery systems

The choice of suitable technology used in the encapsulation of drugs into liposomes depends

primarily on the physiochemical characteristics of the drug which can be classified into two

major categories: (i) water soluble substances, which upon loading are entrapped or


incorporated within the aqueous compartment in the core of the liposomes or the aqueous

compartment between the liposome lamellae; (ii) amphiphilic or lipophilic substances which

upon loading are incorporated into the liposome bilayer(s) (Chonn & Cullis, 1995).

Drug loading can be achieved either passively (i.e. the drug is encapsulated during the

formation of the liposomes e.g. lipidic films) or actively (i.e. after the formation of liposome

vesicles). Hydrophobic drugs, such as amphotericin B, taxol or annamycin, can be directly

incorporated into liposomes during vesicle formation, and the extent of uptake and retention

is governed by drug-lipid interactions. Trapping efficiencies of 100% are often attainable, but

these depend on the solubility of the drug in the liposome membrane. Passive encapsulation

of water-soluble drugs relies on the ability of liposomes to trap aqueous buffer containing a

dissolved drug during vesicle formation. Trapping efficiencies are generally low and are

limited by the trapped volume contained in the liposomes and drug solubility. Alternatively,

water-soluble drugs that have protonizable amine functions can be actively entrapped by

employing pH gradients, which can result in trapping efficiencies approaching 100% (Mayer

et al., 1993).

1.4.1 Characterization of liposomes and liposome-drug interactions

An increase in the therapeutic application of liposomes has led to the development of

analytical and technological approaches to characterise liposomes in terms of size,

morphology, polydispersity index, charge, bilayer fluidity, encapsulation efficiency as well as

liposome-drug interactions. In most laboratories routine liposome size analyses are carried

out by photon correlation spectroscopy (PCS) using commercial instruments such as the

Malvern zeta-sizer (Ingebrigtsen et al., 2002). PCS is the analysis of time dependence

intensity fluctuations of scattered laser light due to Brownian motion of particles in solution

or suspensions.

In PCS, a correlation function is calculated from the intensity versus time profile. Since small

particles diffuse faster than large particles, the rate of fluctuation of scattered light intensity

varies accordingly. Therefore the translational diffusion coefficient can be measured which in

turn can be used to determine the mean hydrodynamic radius (or diameter) of the particles

using the Stoke-Einstein equation. Generally, PCS is regarded as being suitable for obtaining

the particle size distribution for samples with particles ranging from a few nanometres to

several microns depending on instrumental characteristics. Ingebrigtsen et al (2002) and

Hupfeld et al (2006) both reported the study of particle size distribution of size range

liposome dispersion (Fig. 9) prepared by high pressure homogenisation using PCS combined


with other various analytical techniques such as size exclusion chromatography. The particle

size obtained for the liposome dispersion indicated that the combination of PCS with other

analysis techniques can give a more detailed and more reliable insight into particle size

distribution of small liposome than PCS alone.

Fig. 1.8 Size-distributions of egg PC liposome dispersion as obtained by photon correlation

spectroscopy (PCS) (Hupfeld et al., 2006).

Nuclear magnetic resonance (NMR), X-ray photoelectron spectroscopy (XPS), infrared

spectroscopy (FTIR) and electron spin resonance (ESR) spectroscopy are very useful tools to

study the structural and motional features of designated liposome segments. The use of such

techniques provides a better understanding of lipid morphology, detailed insights into the

molecular structure as well as predictions of in-vitro and in-vivo behaviour of liposomes.

Fluorescence spectroscopy on the other hand also provides complementary approaches for

studying bilayer re-arrangements that includes vesicle leakage, membrane fusion and

hydrophobic exposure (Ulrich, 2002).

The development of microscopy techniques has also made the study of the surface and

systems structure more attractive. Microscopic techniques such as atomic force microscopy


(AFM), environmental scanning electron microscopy (ESEM), transmission electron

microscopy (TEM) and confocal laser scanning microscopy (CLSM) have been reported to

provide vital information on size stability and bilayer organization. Rouzi et al (2005)

employed AFM and PCS in the investigation of liposome size and physical stability during

their storage. AFM images acquired were reported to show the spherical shape of the

liposome vesicles and the average size of the liposomes evaluated using the two techniques

were comparable. In addition, AFM confirms the liposome loss of stability (aggregates) after

several months which was strengthened by the increased polydispersity index value obtained

using PCS. These findings illustrated the use of AFM as a valuable tool for the technological

control of the size distribution profile and, moreover, for formulation batch to batch

reproducibility.

Fig. 1.9 Microscopy images of liposomes evaluated using AFM, ESEM, TEM and CLSM

(Ruozi et al., 2011).

Comparative microscopic images of liposomes using different techniques were also evaluated

by Ruozi et al (2011). Their investigation provided detailed information about shape and

morphology (AFM, TEM), dimensions (AFM, ESEM, TEM, and CLSM), surface properties

(AFM), and internal structure (CLSM) of the liposomes as illustrated in Fig. 1.9.

Differential scanning calorimetry (DSC) is another valuable technique used to evaluate the

onset temperature of lipid chain-melt and phase transitions in liposomes. DSC is a very

sensitive means of investigating changes in the bilayer packing and thus can be used to


evaluate the presence of added entities (e.g. drug) in the bilayer interfering with the chain

packing thus causing a reduction in the main transition temperature (Winden et al., 1998).

DSC has been used to measure the incorporation of hydrophobic drugs in liposome

formulations. DSC thermograms of liposomes incorporated with hydrophobic drugs have

generally been reported to cause a broadening and/or reduction in the main transition

endotherm (Jain et al., 1977; Winden et al., 1998).

Perrie and co-workers (2013) recently reported on the investigation of cholesterol packing in

cationic liposomes using DSC and surface pressure isotherms of lipid monolayers. In their

report they suggested that small changes in bilayer mechanics such as cholesterol

incorporation can impact both cellular interactions and immune responses. Findings in their

investigations demonstrated that incorporation of cholesterol into liposomal membranes

promotes the formation of a liquid-condensed monolayer and removed the main phase

transition temperature of the system; thereby resulting in increased bilayer fluidity and altered

immune response

1.5 Liposomes as nano drug delivery system in cancer treatment

Most anticancer drugs are chosen for chemotherapy based on the type of cancer. Tumour

cells, generally, can be targeted at the DNA, RNA or proteins level in a time or concentration

dependent manner. Despite much research in chemotherapy most cancer patients with

advance solid tumours still die of their disease. Due to this reason new effective drugs are

needed and thus potentially new anti-cancer agents are needed. A drug classification serves

two major purposes (1), the achievement of a comprehensive view of available drugs and (2)

the design of combination therapy. Generally, anticancer drugs are simply grouped as

chemotherapy, hormonal therapy and immunotherapy (Table 1.4).


Table 1.4 Classification of anticancer drugs.

Cancer drug groups Sub-classification groups Example drugs

Chemotherapy

Alkylators

Antibiotics

Antimetabolites

Topoisomerases inhibitors

Mitosis inhibitors

Doxorubicin, Mitoxantrone

Cladribine,

Fluoropymidine(5FU)

Topotecan, Rubitecan

Vincritin, Paclitaxel

Hormonal therapy Steroids

Anti-estrogens

Anti-androgens

LH-RH analogs

Anti-aromatase agents

Prednisolone, Dexamethasone

Tamoxifen, Fulvestrant

Flutamide, Bicallutamide

Buserelin, Goserelin

Exemestane,

Immunotherapy Interferon

Interleukin 2

Vaccines

The chemotherapy group comprises a number of families defined by their chemical structure

and mechanism of action; for example, alkylating agents. Alkylating agents were the first set

of compounds identified to be useful in cancer treatments. Their mode of action is that they

bind to DNA forming a variety of interstrand cross-links termed adducts, thereby altering

DNA structure or function. Examples of anticancer alkylating agents are cisplatin,

carboplatin and chlorambucil. Hormonal therapy class of anticancer agents comprise of

steroids hormones that bind to steroid receptors. Hormonal therapy is clinically useful in the

treatments of breast and prostate cancer (Espinosa et al., 2003).

The classification of anticancer drugs have been reviewed by many researchers e.g., Espinosa

et al (2003) who in their classical review of anticancer drug classification proposed a new

drug classification system based on drugs directed at tumour cells or other elements involved

in carcinogenesis, i.e., the endothelium matrix, extra-cellular matrix and the immune system.

Due to the expanding discovery of new anticancer agents and their mode of actions the

classification of other groups of anticancer agents has greatly expanded. Mitochondria have

recently emerged as an exciting target for anticancer drugs. This potential arises from the

discouraging findings that even same tumours or tumours of the same type but from different

individual patients differs in a number of mutations (Gerlinger et al., 2012; Jones et al.,


2008). Therefore therapies involving the target of single gene or a single pathway that may

alter amongst cancer patients or subject to mutations are most unlikely to sufficiently

suppress cancer. Mitochondria apart from being the “powerhouse” of cell are also the

reservoirs of a number of apoptosis promoting proteins that are vital for apoptosis induction.

Neuzil et al (2012) recently reviewed the classification of mitochans, anticancer drugs acting

on mitochondria. In their review, they defined mitochans and classified them into several

class based on their molecular mode of action.

1.5.1 Clinical efficacy of liposomal formulations of anticancer agents

Many barriers hinder the effective delivery of a drug in its active form to solid tumours. Most

chemotherapeutic agents have a large volume of distribution upon intravenous (i.v)

administration and subsequently narrow the therapeutic index due to a high level of toxicity

in healthy tissues. Via encapsulation of drugs in a drug delivery system, such as liposomes,

the volume of distribution can be significantly reduced and the concentration of drug in the

tumour site potentially or actually increased (Speth et al., 1988; Chabner & Longo, 1996).

Liposome formulations of various anticancer agents (Fig. 1.10) have been shown to be less

toxic than the free drug. Several research investigations are concentrated in improving the

safety profile of the anthracycline cytotoxic drugs (such as doxorubicin (DXR) and

daunorubicin (DNR)), or vincristine (VCR), which is associated with severe cardiotoxic side

effects, although acute gastrointestinal effects and other toxicities may also occur. Liposomal

encapsulation of these drugs showed reduced cardiotoxicity, dermal toxicity and better

survival of perimental animals compared to the controls receiving free drugs (Gregoriadis,

1991).


Fig. 1.10 Structures of some chemotherapeutic drugs that have been utilised with liposomes

either in-vitro or in-vivo (Drummond et al., 1999).

Anthracyclines are drugs that terminate the growth of dividing cells by intercalating into the

DNA and therefore predominantly kill quickly dividing cells including other non-cancerous

cells, thus making this class of drugs very toxic. The most used and studied is adriamycin

(commercial name for doxorubicin HCl) which is a potent antineoplastic agent active against

a wide range of human cancers including lymphomas, leukemia and solid tumours. In

addition to the above mentioned acute toxicities, its dosage is limited by its cumulative

cardiotoxicity (Lasic, 1995). However clinical trials and reports on administration of

liposomal doxorubicin have shown significant reduction of the drug associated cardiotoxicity.

This is because cardiac uptake of liposome-encapsulated doxorubicin is substantially reduced

compared to the free drug (Storm et al., 1987; Lasic, 1995).

These drugs (Fig. 1.10) operate via a variety of different mechanisms; some have different

mechanisms of drug resistance and varying physical characteristics that make them more or


less compatible for encapsulation in liposomes. At the present time, some formulations of

anthracyclines (DNX and DXR) have been sufficiently developed for use in the clinic.

Vincristine (VCR) has just been recently, 2012, approved by the FDA for the treatment of

leukaemia and is being marketed as “marqibo”.

Liposome formulations have made successful progress from being a simple conventional

vesicles to a more targeted and new generation drug delivery system as seen in the

commercially available liposome products (Table 1.5) and those (Table 1.6) undergoing

clinical trials (Chang & Yeh, 2012; Gregoriadis & Yvonne, 2010). Doxurubicin

hydrochloride comprises the first liposomal product (DoxilTM) to be licensed in the United

States. Moreover, surface modified methoxypolyethylene glycol (MPEG) provides the

hydrophilic Stealth coating, which allows the DoxilTM liposomes to circulate in the blood

stream for prolonged periods for up to 52 hours in humans (Gabizon et al., 1994). Vail et al

(2004) in their pre-clinical model study of pegylated liposomal doxorubicin (PLD)

demonstrated that pegylated liposomal doxorubicin produces a cure against many cancers

which includes tumors of the breast, ovaries, lung, bladder, prostate, colon and pancreas, as

well as lymphoma, sarcoma, and myeloma. In their study, encapsulation of doxorubicin in

polyethylene glycol-coated liposomes was developed to enhance the safety and efficacy of

conventional doxorubicin. The PEG coated doxorubicin liposomes was reported to alter

pharmacologic and pharmacokinetic parameters associated with conventional doxorubicin so

that drug delivery to the tumor is enhanced while toxicity normally associated with

conventional doxorubicin is decreased. In animals and humans, pharmacokinetic advantages

of PLD include an increased area under the plasma concentration-time curve, longer

distribution half-life, smaller volume of distribution, and reduced clearance. In addition, PLD

was found to cross the blood-brain barrier and induce remission in tumors of the central

nervous system. Increased potency over conventional doxorubicin was observed and, in

contrast to conventional doxorubicin, PLD was equally effective against low- and high-

growth fraction tumours. The combination of PLD with vincristine or trastuzumab was also

reported to result in additive effects and possible synergy. Their findings illustrated the

ability of PLD to overcome multidrug resistance, possibly as a result of increased

intracellular concentrations and an interaction between the liposomes and P-glycoprotein

function. Thus on the basis of pharmacokinetic and preclinical studies PLD, either alone or as

part of combination therapy has potential applications to treat a variety of cancers. Lorusso

et al (2004) also demonstrated the use of pegylated liposomal doxorubicin as an emerging

option for patients with recurrent ovarian carcinoma.


Clinical observation trials of metastatic breast cancer treated with single agent of PLD was

recently reported. The study outcome illustrated PLD to be a very valuable option in the

treatment of advance breast cancer especially for patients with cardiac risk therapies (Fiegl et

al., 2011). However, recent phase I clinical trial of dose combination of PDL and docetaxel

in patients with advance solid tumours investigated by Igbal et al. (2011) also found the

combination of PLD and docetaxel to be feasible and tolerable. Therefore, further dosing of

the drug combination was recommended for phase II clinical trials.

Table 1.5 Commercially available liposome formulations administered via intravenous

route.

Product

name

Drug Lipid composition /Storage

time

Approved indication Reference

Daunoxome Daunorubicin Liposome (Emulsion/12

months)

DSPC and cholesterol (2:1

molar ratio)

Blood tumours Tomkinson

et al.,2003

Doxil Doxorubicin PEGylated liposome

(Suspension/20 months)

HSPC, cholesterol, and PEG

2000-DSPE (56:39:5 molar

ratio)

Kaposi’s sarcoma,

Ovarian/breast cancer

Park JW

2002

Lipo-dox Doxorubicin Pegylated liposome

(Suspension/36 months)

DSPC, cholesterol, and PEG

2000-DSPE (56:39:5 molar

ratio)

Kaposi’s sarcoma,

ovarian/breast cancer

Hong RL

2004

Myocet Doxorubicin Liposome (Powder/18

months) EPC and

cholesterol (55:45 molar

ratio)

Combination therapy

with

cyclophosphamide in

metastatic breast

cancer

Gardikis

2010

Marqibo Vincristin

sulphate

cholesterol and egg

sphingomyelin (45:55)

molar ratio

Acute lymphoblastic

leukemia

Fan et al.,

2013;

Silverman et

al., 2013

Abbreviations: I.V, intravenous HSPC, hydrogenated soy phosphatidylcholine; EPC, egg

phosphatidylcholine; DSPC, distearoylphosphatidylcholine; PEG 2000-DSPE.


Table 1.6 Liposome formulations in clinical trials, administered via the intravenous route.

Product

name

Drug Lipid composition Approved indication Trial

phase

Reference

LEP-ETU

(powder/12

months)

Paclitaxel DOPC, cholesterol, and

cardiolipin (90:5:5 molar

ratio)

Ovarian, breast, and lung

cancers

Phase

I/II

Immordino et al. 2006

LEM-ETU Mitoxantrone DOPC, cholesterol, and

cardiolipin in 90:5:5 molar

ratio

Leukemia, breast, stomach,

liver, ovarian cancers

Phase

I

Cattaneo et al., 2010

EndoTAG-1

(powder/24

months)

Paclitaxel DOTAP, DOPC, and

paclitaxel (50:47:3 molar

ratio)

Anti-angiogenic properties,

breast cancer, pancreatic

caner

Phase

II

Robert et al., 2009

LE-SN38 SN-38, the

active

metabolite of

irinotecan

DOPC, cholesterol, and

cardiolipin

Metastatic colorectal cancer Phase

I/II

Pal et al., 2005

Aroplatin Cisplatin

analog (L-

NDDP)

DMPC and DMPG Metastatic colorectal

carcinoma

Phase

II

Dragovich et al., 2006

Lipoplatin

(suspension/3

6 months)

Cisplatin SPC, DPPG, cholesterol, and

mPEG 2000-DSPE

Pancreatic cancer, head and

neck cancer, mesothelioma,

breast and gastric cancer, and

non-squamous non-small-cell

lung cancer

Phase

III

Stathopoulous et al.,

2012

Abbreviations: DOPC, 1,2-Dioleoyl-sn-glycero-3-phosphocholine; DPPC, dipalmitoylphosphatidylcholine; DOTAP, 1,2 dioleoyl-3-

trimethylammonium-propane; SPC, soy phosphatidylcholine; mPEG 2000-DSPE, methoxy-polyethylene glycol-distearoyl

phosphatidylethanolamine; DMPC, 1-α-dimyristoylphosphatidylcholine; DMPG, l-α-dimyristoylphosphatidylglycerol.


1.6 Intracellular delivery mechanisms of liposomes

One of the unique properties of liposomes as drug delivery agents in cancer treatment is the

ability to overcome cellular barriers in order to improve the delivery of various

chemotherapeutic agents. Depending on the cellular uptake and intracellular trafficking,

different therapeutic applications may be considered. There are various mechanisms of

liposome cellular internalization which are much influenced by their physiochemical

properties. A common strategy for intracellular delivery of liposome encapsulated materials

is via the use of pH-sensitive liposome which upon acidification along the endosomal

pathway from early endosome to lysosome releases its encapsulated cargo. This process is

known as endocytosis (clathrin-mediated pathway). Several alternative pathways (Fig. 1.11)

in addition to the clathrin-mediated pathway have been explored and this includes caveolae

mediated endocytosis, phagocytosis, macropinocytosis and non-clathrin-non-caveolae-

dependent endocytosis (Pollock et al., 2010).

Fig. 1.11 Intracellular trafficking of nanocarrier following macropinocytosis, clathrin-

mediated endocytosis and caveolae-mediated endocytosis (Hillaireau & Couvreur, 2009).

In phagocytosis a specially designed nanocarrier (such as liposomes) undergoes extensive

opsonisation in the bloodstream i.e. adsorption of immunoglobulin, complement components

and other proteins. Fig. 1.12 depicts the process of phagocytosis whereby the opsonised

nanoparticles specially bind to the cell surface via specific recognition and the particles are


then engulfed leading to phagosome formation. The resulting phagosome matures, fuses with

the lysosomes and becomes acidified thus leading to the enzyme-rich phagolysosomes (which

facilitates particle degradation) then releases the drug (Hillaireau & Couvreur, 2009).

Fig. 1.12 Internalisation of nanocarrier by opsonization and phagocytosis (Hillaireau &

Couvreur, 2009).

Endocytosis mainly occurs in a membrane region that is rich in clathrin, a main cytosolic coat

protein. Clathrin-mediated endocytosis (CME) occurs in all mammalian cells. CME via

specific receptor mediated endocytosis (RME) is very well described in literature. CME

either receptor-dependent or receptor-independent causes the internalised material (such as

liposome vesicles) to enter the cell and end up in the degradative lysosome and thus the

encapsulated drug or material is released due to biodegradation of the liposome vesicle

membrane. Liposome surface coated with ligands including low density lipoproteins (LDL),

epidermal growth factor (EGF) and transferrin have been described in literature to internalise

via receptor-dependent CME (Bareford & Swaan, 2007).

Another CME mechanism pathway involving non-specific adsorptive pinocytosis (meaning

cell drinking) is the receptor-independent CME. Compounds are adsorbed via this pathway

by display of non-specific charges and hydrophobic interactions with cell membrane. Similar

to every other endocytosis pathway compounds (fluids) enters the cellular compartments via

the clathrin coated vesicles (Fig. 1.13) This process internalization rate is usually slow

compared to receptor dependent CME (Hillaireau & Couvreur, 2009; Stromhaug et al.,

1997).


Fig. 1.13 Vesicle formations during clathrin-mediated endocytosis. (A) The assembly of

clathrin triskelions (based on three clathrin heavy chains) into a polygonal lattice helps

deform the overlying plasma membrane into a coated pit; (B) Dynamin is recruited at the

neck of the pit to mediate the membrane fission after assembly of the basket-like clathrin

lattice; (C) This leads to the cytosolic release of the clathrin-coated vesicle; (D) The

following uncoating of the vesicle allows the recycling of clathrin triskelia (Hillaireau &

Couvreur, 2009).

Caveolae are flask-shaped invaginations of the plasma membrane which participate in

regulating many cellular functions. Caveolae occur at different densities in different cell

types including vascular endothelial cells (ECs), fibroblasts and epithelial cells. As

specialised forms of lipid rafts (assembled of sphingolipids and cholesterol) contained within

the plasma membrane, they function as cell signalling platforms and regulate the kinetics of

vesicle transport (Chidlow & Sessa, 2010). Caveolae mediated endocytosis differs

mechanically from clathrin dependent endocytosis and other clathrin independent

endocytosis. These differences comprises of a highly regulated processes involving complex

signalling which may be driven by the cargo itself. Upon binding of the cargo to the cell

surface, the cargo is conveyed along the plasma membrane to caveolae (from membrane

invagination) where they may be maintained via receptor-ligand interactions. Fission of the

caveolae from the membrane generates cytosolic caveolae vesicles which do not contain any

enzymatic cocktail (Chidlow & Sessa, 2010; Hillaireau & Couvreur, 2009). The uptake and

trafficking of liposomes are not fully understood. However, it is generally perceived that the

majority of liposomes are internalised into cells via endocytotic pathways. This possibility of

liposome internalisations via this pathway is been supported by fluorescent microscopic

analysis as well as competition and inhibitor assays (Tseng et al., 2002; Pullock et al., 2010;

Mock et al., 2012).


1.7 Sirolimus (rapamycin) - a new anti-cancer agent

Advances in the understanding of cancer has made it clear that even tumours of the same

pathological classification can have distinctive (different) signaling-pathway profiles and

gene expression patterns. Thus the use of biological markers to distinguish the various

signaling pathways in different patients has shown the significant role of some growth

pathways in relation to cancer treatment. Biomarkers indicate that the mTOR (mammalian

target of rapamycin) growth pathways are hyperactive in certain cancers, thereby suggesting

mTOR as an attractive target for cancer therapy (Bjornsti et al., 2004; Chan, 2004).

The identification of mTOR as a potential strategic target for anticancer therapeutic agents

occurred serendipitously after the natural product sirolimus (rapamycin) demonstrated unique

antineoplastic properties especially in drug eluting stents for the treatment of percutaneous

coronary intervention. Sirolimus (rapamycin) is a lipophilic macrolide identified more than

30 years ago during antibiotic screening from a strain of Streptomyces hygroscopicus found

in the soil of Easter Island (Rapa Nui). The drug (Fig. 1.14) is a white crystalline solid that is

virtually insoluble in aqueous solutions, but readily soluble in organic solvents (Vezina et al.,

1975; Baker et al., 1978). Although investigations showed sirolimus to lack antibacterial

activities, it was, however, found to be a potent fungicide and subsequently demonstrated to

reverse acute allograft rejection and enhance long-term donor-specific allograft tolerance

(Morris et al., 1991). Further investigations of sirolimus found the drug to inhibit the growth

and function of many other cell types besides lymphocytes and yeast, which led to efforts to

evaluate its growth inhibitory effects on malignant cells and demonstrated impressive and

unique antiproliferative actions in a diverse range of experimental tumours (Eng et al., 1984;

Seufferlein et al., 1996).

1.7.1 Mechanism of action of sirolimus (rapamycin)

The mammalian target of rapamycin (mTOR) is a downstream effector of the

phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signalling pathway that mediates

cell survival and proliferation and is a prime strategic target for anticancer therapeutic

development. Sirolimus (immunosuppressant and antiproliferative agent) by targeting mTOR,

inhibits signals required for cell cycle progression, cell growth and proliferation.

Rapamycin interferes with critical elements of signal transduction pathways, particularly


those that link mitogenic stimuli to the synthesis of specific proteins required for G1-to-S

phase traverse by binding to FKBP12.

Sirolimus (rapamycin)

Molecular weight:

914.18 Da

Fig. 1.14 Structure and molecular target of sirolimus (Mita et al., 2003).

The resultant FKBP1–rapamycin complex inhibits mTOR kinase activity, which, in turn,

blocks the activation of critical downstream signaling elements (Fig. 1.14) by inhibiting the

phosphorylation of mTOR. The activation of the 40S ribosomal protein S6 kinase (p70s6k) is

blocked, which reduces the translation of 5'-terminal oligopyrimidine messenger RNAs

(mRNAs) that encode for essential components of the protein synthesis machinery. The

inhibition of mTOR in turn blocks phosphorylation of the eukaryotic initiation factor 4E

binding protein-1 (4E-BP1), also known as phosphorylated heat- and acid-stable protein–1

(PHAS-1) (Mita et al., 2003). In its dephosphorylated state, 4E-BP1 binds tightly to the

eukaryotic initiation factor-4E (eIF-4E) translational complex, thereby inhibiting the

translation of mRNAs with regulatory elements in their 5'-untranslated regions (5'- UTRs).

These mRNAs encode for critical regulatory proteins including growth factors, onco proteins,

and cell cycle regulators (Jefferies et al., 1997). Other downstream targets that are affected by

inhibiting mTOR include cyclin D, p27, and retinoblastoma protein (pRb). By inhibiting

translation of the aforementioned proteins, some of which are essential for cell cycle traverse,

cell growth, survival, and cell division, sirolimus may produce profound antiproliferative and

immunosuppressive effects. The precise role of each of these effectors in the antitumor

properties of sirolimus is still yet to be fully clarified (Mita et al., 2003).

http://2.bp.blogspot.com/_E_1zwNw-esM/S28bhBwoLGI/AAAAAAAAAFs/v79bUCeSf0c/s1600-h/Sirolimus1.gif


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CHAPTER TWO

Sirolimus encapsulated liposomes for cancer therapy: physico-chemical and mechanical

characterization of sirolimus distribution within liposome bilayers

2.0 Overview

Sirolimus has recently been introduced as a therapeutic agent for breast and prostate cancer.

In the current study, conventional and Stealth liposomes were used as carriers for the

encapsulation of sirolimus. The physicochemical characteristics of the sirolimus liposome

nanoparticles were investigated including the particle size, zeta potential, stability and

membrane integrity. In addition atomic force microscopy was used to study the morphology,

surface roughness and mechanical properties such as elastic modulus deformation and

deformation. Sirolimus encapsulation in Stealth liposomes showed a high degree of

deformation and lower packing density especially for DPPC Stealth liposomes compared to

unloaded. Similar results were obtained by differential scanning calorimetry (DSC) studies;

sirolimus loaded liposomes were found to result in a distorted state of the bilayer. X-ray

photon electron (XPS) analysis revealed a uniform distribution of sirolimus in multilamellar

DPPC Stealth liposomes compared to a non-uniform, greater outer layer lamellar distribution

in DSPC Stealth liposomes.

2.1 Introduction

Sirolimus (rapamycin) is a potent immunosuppressive agent clinically approved for the

prevention of organ transplant rejection and restenosis (Saunders et al., 2001; Sousa et al.,

2007). Other sirolimus related compounds are in phase I-III clinical trials for the treatment of

cancer (Bjomsti & Houghton 2004). Sirolimus inhibits a subset of mammalian target of

rapamycin (mTOR) signalling functions leading to a growth-inhibitory effect against a wide

range of human cancers. mTOR is a 289-kDa serine/threonine kinase, termed PI3K-related

kinase (Tibbetts & Abraham, 2000), which is regulated by the upstream molecules PI3K/Akt,

and subsequently phosphorylates two downstream substrates leading to initiation of protein

translation (Chen & Fang, 2002; Mita et al., 2003). Furthermore, it has been shown that

mTOR inhibition is an effective approach for the treatment of ErbB2-positive breast cancers.

Human breast cancers often over-express mutationally activated epidermal growth factor

(RGF) receptors such as ErbB2 (Edinger et al., 2003) and sirolimus decreases the coupling

efficiency between these receptors and the PI3K signalling cascade leading to tumour

suppression (Liu et al., 2005).


Liposome drug delivery formulations for cancer treatment have been extensively investigated

for the last 30 years for the improved delivery of a wide range of pharmaceutically active

agents including antibiotics (doxurobicin, daunorobicin), alkaloids (paclitaxel, vincristine),

pyrimidine antagonists (fluorouracil) or alkylating drugs (cisplatin) (Lian et al., 2001). The

aim of any drug carrier is to modulate the pharmacokinetics and/or the tissue distribution,

achieve controlled release or enable drug targeting to specific tumour sites and subsequently

to increase the therapeutic index of the drug while minimizing its side-effects (Storm &

Crommelin, 1998; Immordino et al., 2006). Liposomes are the most commonly used carriers

for both hydrophilic and hydrophobic anticancer drugs due to their versatile in vitro and in

vivo physicochemical behaviour. The use of conventional (first generation) or long

circulating (second generation, Stealth) liposome-drug formulations results in a wide variety

of liposome shapes and sizes with improved pharmacokinetic properties (AUC, t1/2, Cmax,

plasma clearance), decreased toxicity and immunogenicity, less fluctuation in plasma drug

concentrations and increased chemical stability of the drug(s) used relative to the drug alone

(Reddy, 2000). It is known that the encapsulation of the anthracycline doxorubicin into

liposomes with attached polyethylene glycol (PEG) substantially alters it’s in vivo

performance (Bakker-Woudenberg et al., 1994; Gabizon & Martin, 1997). The half-life of

liposome encapsulated drugs depends on the size and the composition of the liposomes, with

Stealth®liposomes showing slower uptake by the reticuloendothelial system and reduced

leakage while in circulation. Nevertheless, considerable progress has been made in

engineering liposome based drug formulations with acceptable patient compliance and

therapeutic effectiveness of drugs by improving the aforementioned features (Rouf et al.,

2009; Elbayoumi & Torchilin, 2009; Musacchio et al., 2009).

Sirolimus liposome formulations (Castelli et al., 2006) can be used as an alternative for the

treatment of breast cancer compared to the free drug preparation (Zeng et al., 2010) and have

demonstrated significant efficacy in suppressing tumour growth and metastasis. For such

purposes it is critical to engineer suitable liposome formulations with the appropriate

encapsulated drug loading, particle size distribution and lipid composition. Thus, the

physicochemical characterization of liposomes incorporating sirolimus can provide useful

insights in relation to liposome stability, fluidity, morphology, drug-model biomembrane

interactions and drug distribution/incorporation within the lipid bilayers. A detailed

understanding of the foregoing parameters, using contemporary analytical techniques, will

facilitate further engineering of the sirolimus liposomal formulations prior to clinical trials,

thus maximizing the future clinical performance of sirolimus for chemotherapy of breast


cancer patients.

In the current study a variety of techniques such as differential scanning calorimetry (DSC),

atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) have been

used to determine the characteristics of empty and sirolimus loaded

conventional/Stealth®liposomes formulations. AFM measurements of the shape, morphology

and mechanical properties can provide important information in relation to liposome

elasticity and successful drug incorporation (Ramachandran et al., 2006). DSC was used to

investigate lipid thermotropic phase behaviour and possible drug-liposome interactions (Zhao

& Feng, 2006). Finally, XPS is introduced as an approach to confirm sirolimus–liposome

interactions and characterize the distribution of the drug within the lipid bilayers.

2.2 Materials and Methods

2.2.1 Materials

Sirolimus was obtained as a gift from Phoqus Pharmaceuticals Ltd (West Malling, UK).

Distearoylphosphatidylethanolamine-methyl-polyethylene glycol (DSPE-MPEG-2000),

distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC) and

cholesterol (Avanti Lipids Inc) were kindly donated by Lipoid GmbH (Ludwigshafen,

Germany). Calcein, Triton X-100 and serum FBS were purchased from Sigma (Gillingham,

UK).

2.3 Methods

2.3.1 Liposome preparation and encapsulation

Oligolamellar (OLV) liposome vesicles were prepared following the thin film hydration

method, as previously described by Bangham et al (1965). In brief, conventional and

Stealth®liposomes formulations were produced by dissolving the appropriate amounts of

lipid mixtures (DPPC, DSPC, DSPE-MPEG-2000 and cholesterol) in the presence or absence

of sirolimus in chloroform. The lipid mixtures were subsequently evaporated under a vacuum

in a round-bottomed flask connected to a rotor evaporator to obtain thin lipid films. The

lipidic films were hydrated with deionised water and then passed through an extruder

(LipexTM extruder by Northern lipids Inc.) 20 times via a 400 and then a 200 nm

polycarbonate filter (Nucleopore) at a set temperature of 5°C above the phase transition

temperature of the lipid mixtures. The liposme formualations used in this study are depicted

in Table 2.0.

The sirolimus encapsulation efficiency (EE) was estimated by using the following equation:


EE = amount of drug encapsulated in the NPs

amount of the drug added in the process X 100

Briefly, the drug amount encapsulated in the liposome dispersions was measured by high

performance liquid chromatography (HPLC). An Agilent Technologies 1200 series with a

quaternary pump, an autosampler and a detector used at 278 nm was used for HPLC assay. A

Lichrocart 250-4-RP 18 5 μm (Merck) column was used with mobile phase 60:40

acetonitrile:water. The column was maintained thermostatically at 50 °C. The flow rate was

1.0 mL/min and the injection volume 10 μL. Calibration curves were constructed using

standard solutions of known concentrations (3−50 ng/mL). The Chemstation software

calculated the peak area of each standard solution and sample automatically. Freeze-dried

liposome formulations (3 mg) were dissolved in 1 mL of DCM followed by solvent evaration

and the addition of 3 mL of mobile phase. The solution was filtered by 0.45 mm PVDF

syringe filter for HPLC analysis.

Table 2.0 Liposome formulation composition.

Formulation Molar Ratio

DPPC: Cholesterol (DPPC conventional) 18.6: 9.0

DPPC: DSPE-MPEG2000: Cholesterol (DPPC Stealth) 12.6: 1.14: 8.0

DSPC: Cholesterol (DSPC conventional) 18.6: 9.0

DSPC: DSPE-MPEG2000: Cholesterol (DSPC Stealth) 12.6: 1.14: 8.0

2.3.2 Particle size and zeta potential analysis

The particle size distribution and the zeta potential of the liposome preparations were

determined by dynamic light scattering (PCS) using a Malvern Zetasizer Nano-ZS (Malvern,

UK). Measurements were carried out at 25°C at a fixed angle 90°. The sizes quoted are the

z-average means (dz) of the liposomal hydrodynamic diameters (nm). The Nano-ZS

instrument can be used for particle size determination in the 0.6 nm-6 µm range. All samples

were stored at 4°C and the particle size/zeta potential was measured at one, three and six

month time intervals in order to determine their stability.


2.3.3 Evaluation of liposome membrane integrity

The membrane integrity of liposome formulations, comprising different lipid compositions

after incubation at 37°C, was evaluated by calculating the percentage retention latency of the

liposome encapsulated calcein formulations. Briefly, calcein encapsulated liposomes

formulation fractions (1 ml) were incubated with serum proteins (80% FBS) at 37°C in a

shaker bath and samples were collected at different time intervals for 24 hours. The retention

of calcein in the liposomes formulations were estimated by mixing 20 µl of the collected

samples and 4 ml of PBS, pH 7.4. The calcein fluorescence of the samples was measured (at

excitation and emission wavelengths of 490 nm and 520 nm, respectively) before and after

the addition of Triton X-100 (final concentration of 1%). The percentage latency of the

calcein was determined using the expression.

% 𝐿𝑎𝑡𝑒𝑛𝑐𝑦 = 1.1 (𝐹𝐴𝑇 − 𝐹𝐵𝑇)𝑋 100

1.1 𝐹𝐴𝑇

Where FBT and FAT represent the fluorescence intensities of calcein before and after the

addition of Triton X-100, respectively.

2.3.4 Atomic force microscopy (AFM)

For AFM experiments, the Stealth® loaded and unloaded DPPC and DSPC samples were

diluted 50 times with saturated drug and then 3 µL of the liposome sample was deposited

onto a freshly cleaved mica surface (G250-2 mica sheets 1" x 1" x 0.006"; Agar Scientific

Ltd, Essex, UK), and left to dry for 1 h before AFM imaging. The images were obtained by

scanning the mica surface, in air, under ambient conditions using a PeakForce QNM

Scanning Probe Microscope (Digital Instruments, Santa Barbara, CA, USA; Bruker

Nanoscope analysis software Version 1.40). The AFM measurements were obtained using

ScanAsyst-air probes, and the spring constant (0.67 N/m; nominal 0.4 N/m) and deflection

sensitivity were calibrated, but not the tip radius (a nominal value of 2 nm was used). Surface

roughness (Ra) values were determined by entering surface scanning data into a digital

levelling algorithm (Bruker Image Analysis Nanoscope Analysis software V 1.40). AFM

images were collected from two different samples by random spot surface sampling (at least

five areas).


2.3.5 Differential Scanning calorimetry (DSC)

A Mettler-Toledo 823e (Greifensee, Switzerland) differential scanning calorimeter (DSC)

was used to conduct DSC scans of sirolimus as well as unloaded and loaded liposome

formulations. Each sample (2-5 mg) was placed in a sealed aluminium pan and heated at

10°C/min from 0°C to 220°C under an atmosphere of dry nitrogen. An empty pan was used

as a reference. All DSC data was normalized to a sample mass of 1 g.

2.3.6 X-ray photoelectron spectroscopy (XPS)

The surface analysis studies were performed in a UHV chamber (P < 10-9 mbar) equipped

with a SPECS LHS-10 hemispherical electron analyzer. The XPS measurements were carried

out at room temperature using a non-monochromatized AlKα radiation source under

conditions optimized for maximum signal (constant ΔΕ mode with a pass energy of 97 eV

giving a fullm width at half maximum (FWHM) of 1.7 eV for the Au 4f7/2 peak). The

analyzed area was a rectangle with dimensions of 2.5 x 4.5 mm2. To improve the energy

resolution the O1s and C1s spectra were also obtained with pass energy of 36 eV giving a

FWHM of 0.9 eV for the Au 4f7/2 peak. The XPS core level spectra were analyzed using a

fitting routine, which can decompose each spectrum into individual mixed Gaussian-

Lorentzian peaks after a Shirley background subtraction. The liposome samples were in

freeze-dried form, pressed on a freshly cut indium support.

2.4 Results and discussion

2.4.1 Liposome particle size, stability and membrane integrity

The particle size and zeta potential of the produced conventional and Stealth liposomes

formulations was measured immediately after preparation and for a period of six months after

storage at 4°C (Table 2.1). All formulations showed very good polydispersity less than 0.2

and the zeta potential was approximately zero for the non-drug loaded liposome samples. The

formulations all showed good encapsulation efficiency of more than 90%. The incorporation

of sirolimus induced a negative zeta potential leading to improved particle stability. For

conventional liposome formulations no substantial difference in the particle size was

observed between unloaded and loaded dispersions, with only a slight increase due to

sirolimus addition. The particle size obtained for unloaded conventional and Stealth

liposomes showed a small particle size decrease for the latter of approximately 22 nm. This

could possibly be attributed to the different molar ratios of DSPC:chol, DPPC:chol and the


presence of DSPE-MPEG-2000. More importantly, the data in Table 2.1 shows that sirolimus

loaded conventional and Stealth liposomes display size reductions of around 35 nm compared

to the respective unloaded liposome formulations. The additional particle size reduction was

initially attributed to possible drug interactions with the lipid bilayers, as previously reported

(Souza &Teschke, 2003; Castelli et al., 2005). However, further characterization of the

dispersions revealed that drug-liposome interactions are negligible and the size reduction is

related to the drug distribution within the liposome bilayers.

Particle size measurements of the stored liposomes demonstrated excellent stability even after

six months with only a slight increase in the size (2-12 nm) for some of the formulations.

Similarly, the zeta potential remained unchanged for the same time period. The overall

stability of both the conventional and Stealth formulations was attributed not only to the

composition of the formulations but also to the optimized sirolimus loading at 2 mg/ml. The

formulations were also evaluated in terms of drug loading (data not shown) for 1-5 mg/ml

where particle aggregation was noticed for loadings above 2 mg/ml. These results are in

agreement with studies conducted by Rouf et al., (2009) who observed similar stability

properties of sirolimus using different liposome formulations.

Calcein encapsulation and release from liposomes was used to study the integrity of

conventional and Stealth formulations. The possibility of increasing the membrane integrity

and physical stability of the liposome dispersions by coating their surface with polyethylene

glycol molecules was also evaluated. Calcein release across the phospholipid bilayer

membrane is a simple but efficient assay often used to elucidate the influence of different

factors on membrane permeability or rigidity (Benachir & Lafleur, 1996; Gubernator et al.,

1999). The data in Fig. 2.0 demonstrates that conventional DPPC-Chol liposome membranes

possesses a less rigid membrane followed by DSPC-Chol liposomes as assessed by the

percentage of encapsulated calcein after 24 hours. In contrast, Stealth liposomes appear to be

more rigid compared to the conventional formulations. Piperoudi et al., (2006) reported that

pegylation of liposome formulations increases membrane rigidity and this is observed in the

membrane integrity difference between the Stealth and conventional formulations. Previous

investigations have also demonstrated that the lipid composition has a significant effect on

liposome membrane permeability due to the differences in their phase transition temperatures

(Shimanouchi et al., 2009). In addition to these findings, a difference in membrane rigidity is

also found between the two different Stealth formulations of which DPPC-Stealth liposomes

demonstrated decreased membrane integrity compared to DSPC-Stealth liposomes. It is,

however, important to consider the permeability of the liposome formulations in the context


of the encapsulation of the drug for delivery into cancer cells. A very permeable liposome

membrane formulation suggests greater release of the drug from the liposome matrix but this

could also mean a less stable formulation.

2.4.2 Atomic force microscopy of liposome formulations

Scanning probe microscopy (SPM) has long been recognized as a useful tool for measuring

mechanical properties of materials, but until recently it has been impossible to achieve truly

quantitative high-resolution mapping. The atomic force microscope (AFM) is one kind of

scanning probe microscope (SPM), and with the mode PeakForce QNM™ AFM instrument it

is possible to identify material properties at high resolution across a topographic image such

as surface roughness, density, adhesion, DMT modulus, deformations and dissipation. The

mechanical values for the individual spherical lipid nanoparticles (all the particles are

separated from each other and do not form aggregates) were calculated by AFM QNM mode

(Lamprou et al., 201), and the results are shown in Table 2.2. For all liposome formulations

investigated by AFM the overall particle diameter (estimated from the width of the peak at

the baseline in sectional height profiles) was estimated, as shown in Fig. 2.1. The AFM

studies showed spherically shaped vesicles, which were flattened as a result of their

adsorption on the mica surface. However the average diameters of the liposomes are slightly

larger than those determined by DLS, which may be attributed to the slight deformation of

the liposomes caused by adsorption (Jung et al., 2006). The height (≈ 13 nm) is usually

underestimated due to the high surface density of the liposomes preventing the tip from

accessing the substrate and the width overestimated because of tip broadening effects

(Tarasova et al., 2008). The surface roughness, determined from the images, for all the

liposomes was ≈ 5 nm (rms), which means the liposomes are very smooth and without any

detectable pores Furthermore, the individual liposomes appear to be located in different

planes, which may be caused by rearrangements of the liposomes during imaging, causing a

reduction in image resolution (Serro et al., 2012). The DMT modulus of the unloaded

liposomes was approximately 5 times larger than the drug loaded liposomes. The force of

adhesion (Fad) of the drug-loaded liposomes was approximately two times larger, and the

deformation was approximately 30 times larger than the unloaded liposomes. The energy

dissipation does not show significant differences (≈ 700 eV) between the loaded/unloaded

liposomes and the mica surface at the imaging set point employed.


Table 2.1 Particle size and zeta potential of extruded liposome formulations (n= 3), polydispersity for all formulations was less than 0.2.

Months

Particle Size (nm) Zeta Potential (mV)

Empty Drug Loaded Empty Drug Loaded

DPPC/Chol

1 177.0 ±5.1 190.8 ± 7.4 -0.87 ± 1.11 -6. 14 ± 7.74

3 181.4 ±6.0 195.6 ± 6.8 0.17 ± 0.49 -7.25 ± 4.69

6 192.8 ±5.8 202.1 ± 8.3 0.01 ± 0.96 -5.36 ± 6.31

DSPC/Chol

1 161.1 ±3.2 128.1 ± 4.5 0.73 ± 0.21 -8.02 ± 5.20

3 166.4 ±4.7 132.7 ± 4.8 0.55 ± 0.13 -7.46 ± 6.73

6 174.1 ±4.3 132.4 ± 5.1 0.70 ± 0.10 -7.98 ± 4.61

DPPC/Chol/

DSPE-

MPEG-2000

(Stealth®)

1 197.0 ±4.3 160.8 ± 5.8 0.91 ± 0.45 -13.15 ± 5.45

3 201.4 ±3.7 161.6 ± 5.5 -0.08 ± 0.01 -13.78 ± 3.72

6 204.8 ±4.1 163.1 ± 6.3 0.70 ± 0.13 -12.01 ± 3.09

DSPC/Chol/

DSPE-

MPEG-2000

(Stealth®)

1 180.5 ±3.0 138.2 ± 3.0 0.59 ± 0.78 -18.22 ± 3.10

3 181.7 ± 4.8 138.8 ± 4.0 -0.47 ±0.52 -19.66 ± 4.55

6 182.6 ± 3.1 141.3 ± 2.7 1.71 ±0.12 -19.44 ± 4.88


Fig. 2.0 Membrane integrity of the liposome formulations using percentage latency over a

period of 24 hours.

Table 2.2 Mechanical properties of Stealth liposomes measured by AFM (n = 20 random

particles).

Mechanical properties DSPC - Empty DSPC - Drug DPPC - Empty DPPC - Drug

Height (nm) 12.0 ± 1.0 14.0 ± 3.0 11.0 ± 1.0 12.0 ± 2.0

Diameter (nm) 176.0 ± 26.0 200.0 ± 45.0 185.0 ± 35.0 187.0 ± 44.0

Surface Roughness / nm 5.0 ± 1.0 5.0 ± 2.0 5.0 ± 1.0 5.0 ± 1.0

Density (µm-2) 3.97 0.63 3.67 0.85

Force (pN) 179.0 ± 31 348 ± 36 158.0 ± 58.0 292.0 ± 82.0

DMT modulus (MPa) 235.0± 27 49.0 ± 24.0 230 ± 27 51.0 ± 30.0

Deformation (pm) 88.6 ± 36.0 2648.0 ± 715.0 97.0 ± 45.0 3747.0 ± 661.0

Dissipation (eV) 721.0 ± 76.0 701.0 ± 85.0 744.0 ± 73.0 737.0 ± 51.0


Fig. 2.1 AFM images of sirolimus loaded DPPC/Chol/DSPE-MPEG-2000 (a,b) and

DSPC/Chol/DSPE-MPEG-2000 Stealth liposomes.


The mechanical data clearly shows that sirolimus significantly influences liposome stiffness

(or perhaps ‘solidness’). Liposomes produced from either DSPC or DPPC without sirolimus

loading show similar mechanical properties (force, DMT modulus and deformation), which is

unsurprising given the similarity in chemical structure (a difference of two CH2 groups in the

phospholipid acyl chain). Loading liposomes with sirolimus results in a significant reduction

in liposome stiffness (particularly seen in the DMT modulus and the deformation data). This

can be potentially explained by two phenomena. Firstly, sirolimus loading may result in

liposomes with a lower phospholipid packing density (PPD) due to the molecular dimensions

of sirolimus. The lower PPD results in less stabilising intermolecular forces between

phospholipids and therefore a lower stiffness. Secondly, the intermolecular forces between

sirolimus-phospholipid and sirolimus- sirolimus are not as significant as phospholipid-

phospholipid, phospholipid-cholesterol and cholesterol-cholesterol interactions and as such

liposome stiffness is decreased.

2.4.3 Differential scanning calorimetry analysis (DSC)

It is well accepted that upon dehydration of liposome formulations, the main transition

temperature (Tm), which represents the transition from the gel-to-liquid crystalline state or

the acyl chain melt, increases. This change in Tm has been attributed to the decrease in

phospholipid head group spacing in the bilayer, resulting in an increase in Van der Waals

interaction between phospholipid molecules (Crowe et al., 1996). In this study the Tm

observed for freeze-dried DPPC liposomes is 99.7±0.6C, which is comparable with

previously reported data for a similar system (Ohtake et al., 2006). There were no significant

differences observed in the Tm for conventional DPPC and DSPC liposomes (Tm = 99.7±0.6

and 100.4±0.3C, respectively).

By incorporating cholesterol into the DPPC liposomes (conventional liposome formulation),

the Tm decreased to 90.4± 0.1C (Table 2.3). In addition, significant differences in the

enthalpy change associated with this chain melting transition (∆H) were observed (19.3±1.8

and 2.2±0.1 J/g for DPPC only and DPPC conventional formulations, respectively). Again

the observed ∆H for the DPPC conventional formulation is very similar to that reported

previously (Ohtake et al., 2005). Similar behaviour is observed for the DPPC and DSPC

conventional liposome formulations i.e. the Tm for the DSPC liposome decreases to 94.6

±0.7C in the presence of cholesterol and the ∆H decreases from 17.6±1.5 J/g (DSPC


liposome) to 8.1±1.7 J/g (DSPC conventional). The change in temperature and magnitude of

the chain melting transition in the presence of cholesterol has been attributed to weakened

intermolecular interactions between the phospholipids and increased motional freedom (Chen

et al., 2010). This suggests that in the presence of cholesterol the liposomal bilayer is in a

more fluid state in comparison to the bilayer without cholesterol. This can be explained by

considering the packing properties of the phospholipids in liposome formulations. In the

absence of cholesterol the phospholipids in the liposome bilayers are able to pack tightly

together, hence restricted motional freedom. However, when cholesterol is added to the

formulation, this packing ability is hindered because of the multi-ring, rigid and non-linear

structure of cholesterol, which wedges between phospholipid molecules and prevents tight

packing of lipids molecules.

When comparing the DPPC and DSPC conventional liposomes, it is clear that the DSPC

conventional liposome formulations exhibits better stability, as both the Tm and ∆HTm is

greater (Tm = 94.6±0.7C, ∆H = 8.1±1.7 J/g) than the conventional DPPC formulation

(Tables 2.3 and 2.4). This is due to the differences in the carbon chain length and hence

molecular weight of the DSPC (C18 and 789 g/mol) in relation to the DPPC (C16 and 733

g/mol) i.e. DSPC has greater acyl chain interactions due to the extra (CH2)4 groups. The

presence of cholesterol has a relatively lower disruptive influence on the packing of DSPC

molecules, hence the need for more energy to disrupt the phospholipid interactions in the

bilayer, resulting in the observed higher Tm and ∆H values, in addition to the differences in

Tm. There are also significant differences in the pre-Tm transitions observed for DPPC and

DSPC. DPPC exhibits two pre-Tm transitions at 46.0 ±2.2C and 70.2±0.4°C (Table 2.3),

while DSPC exhibits only one pre-Tm transition at 65.1±0.1°C.

The origins of the endothermic events observed below the Tm values described above for the

unloaded DPPC and DSPC conventional liposome formulations are not entirely clear.

However, previous studies have suggested that these transitions (in addition to the main

transitions described earlier) may be due to uneven distribution of cholesterol in the liposome

formulations (Ohtake et al., 2005). Ohtake et al (2005) explained that the lower temperature

transitions might actually be chain-melting transitions, resulting from “cholesterol-rich”

domains. For example, the first event observed at 46.0± 2.2°C and 65.1±0.1°C for DPPC and

DSPC conventional liposomes, respectively, arises from the chain melt of “cholesterol-rich”

domains in the formulation. This implies that the Tm values at 90.4±0.1°C and 94.6±0.7°C

result from the chain melt of “cholesterol-poor” domains for conventional DPPC and DSPC


liposomes, respectively.

The incorporation of molecules (drugs) into liposomes has been reported to affect the thermal

behaviour of its phase transitions, which results in reduction in the main transition

temperature. However studies have shown that a more pronounce effect can also be observed

in the pre-Tm transitions and in some cases effect can only be observed in the enthalpy

change (Taylor & Craig, 2003). Papahajopoulos et al (1975) classified the interaction of

molecules with phospholipids into three major groups which comprises (i) surface adsorption

only, (ii) partial embedding into the bilayer of the lipid and (iii) penetration into the core of

the anionic or zwitterionic lipids bilayers. Depending on the molecule (drug) interaction with

the lipid bilayer the following observations are seen in DSC with the first interaction resulting

in an increase in the enthalpy change accompanied by either an increase or no change in the

transition temperature (Tm). The other two interaction processes result in the reduction of

enthalpy with a decrease or no change in the transition temperature.

Fig. 2.2 DSC thermograms of unloaded and loaded conventional DPPC/Chol.

In this study the encapsulation of sirolimus into different liposome formulations resulted in

either the decrease of Tm or the enthalpy change depending on the drug distribution and

interaction with the lipid bilayers. The addition of sirolimus to the conventional DPPC


liposome formulation showed significant differences in the temperature and enthalpy changes

of the pre-Tm transitions (Fig. 2.2). This is not surprising if we consider these pre-Tm

transitions to result from “cholesterol rich” domain phospholipid chain melts. In theory, the

incorporation of the drug into the bilayer increases the distance between the phospholipids in

the bilayer (this is conditional on the fact that the drug molecules occupy sites between

phospholipid molecules, rather than between the lipid monolayers of the bilayer) and

therefore causes a further increase in motional freedom of the phospholipid molecules. This

in turn results in a decrease in transition temperatures and enthalpies of the phospholipid

molecules in the bilayer. What is observed here, however, is that a decreased fraction of

DPPC exists in the drug loaded formulations, hence in the “cholesterol rich” domains there

are relatively lower amounts of DPPC molecules in a liposomal bilayer in comparison to the

bilayer which gives a Tm of 90.4°C. As a result the presence of both the cholesterol and drug

causes greater disruption of the DPPC packing in the bilayer and this further result in a

greater motional freedom. This causes the reduction of the enthalpy change associated with

the transition and a slight decrease in temperatures (Table 2.3). Interestingly there were no

significant differences in the temperatures and enthalpies changes of the Tm for loaded and

unloaded DPPC conventional formulations (Table 2.3). It can only be assumed that the

disruption of packing of the DPPC molecules by the cholesterol and drug was negligible due

to, again if we consider cholesterol distribution in the bilayer, a lower proportion of

cholesterol. These observations indicate two things: (1) sirolimus is incorporated in the lipid

bilayers of the conventional DPPC formulations, and (2) attention need not be focused only

on the Tm values when analysing liposome formulations. The pre-Tm transitions are also

good indicators of drug incorporation into lipid bilayers, particularly when studying nano size

liposome formulations.

Table 2.3 DSC transition parameters obtained for the unloaded and loaded DPPC liposome

formulations.

DPPC conventional (empty) DPPC Stealth (empty)

Transition

1st event 2nd event 3rd event

(Tm)


(Tm)

Peak (°C) 47.6 ± 0.2 70.2 ± 0.4 90.4 ± 0.1 51.3 ± 0.4 - 89.5 ± 0.4

∆H (J/g) 15.1 ± 4.0 7.9 ± 0.3 2.2 ± 0.4 102.9 ±15.6 - 2.2 ± 1.1

DPPC conventional (loaded) DPPC Stealth (loaded)

Transition


(Tm)

1st event 2nd event

3rd event

(Tm)

Peak (°C) 46.0 ± 2.2 67.8 ± 0.1 90.4 ± 0.4 51.5 ± 1.1 - 83.5 ± 0.4

∆H (J/g) 12.3 ± 0.8 5.7 ± 0.5 2.8 ± 0.2 30.3 ± 1.0 - 0.8 ± 0.1


Table 2.4 DSC transition parameters obtained for the unloaded and loaded DSPC liposome

formulations.

There are no significant differences in phase transition temperatures and the enthalpy changes

of the Tm between the DSPC conventional loaded and unloaded formulations (Table 2.4).

However, there were significant differences in the enthalpy change of the pre-Tm transitions

(22.4±2.8 J/g for DSPC conventional unloaded and 17.6±7 J/g for DSPC conventional

loaded). This is also due to the reduction in the fractional composition of the DSPC in the

conventional loaded formulation. Similar findings have also been recently reported by

Tabbakhian and Rogers (2012) where by the incorporation of insulin and polymers into

DPPC liposome cause no significant change in the phase transition temperature of the

liposome but however decrease enthalpy of the transition was observed in some cases.

The addition of DSPE-MPEG 2000 in the unloaded Stealth formulation for DPPC showed no

significant difference in the characteristics of the Tm when compared with the unloaded

DPPC conventional liposome formulation i.e. the Tm for unloaded DPPC Stealth formulation

is 89.5 ± 0.4°C and ∆H of 2.2±1.1 J/g and that observed for the unloaded DPPC conventional

formulation is 90.4±0.1°C and ∆H of 2.2 J/g. However, below this chain melt transition

significant differences exist between the conventional and Stealth formulations. Only one

transition (at 51.3±0.4°C with ∆H of 102.9±15.6 J/g) is observed below the Tm for the

Stealth formulation. This is a broad transition spanning a temperature range from 40 to 80°C

that consists of several events, which may include transitions resulting from “cholesterol-

rich” domains of the liposome formulations.

DSPC conventional (empty) DSPC Stealth (empty)

Transition


(Tm)


(Tm)

Peak (°C) 65.1 ± 0.1 - 94.6 ± 0.7 46.2 ± 0.5 58.9 ± 0.6 78.5 ± 0.4

∆H (J/g) 22.4 ± 2.8 - 8.1 ± 1.7 16.8 ± 2.3 18.7 ± 1.0 2.4 ± 0.1

DSPC conventional (loaded) DSPC Stealth (loaded)

Transition


(Tm)

1st event 2nd event

3rd event

(Tm)

Peak (°C) 65.6 ± 0.2 - 94.4 ± 0.1 46.8 ± 0.6 59.2 ± 0.5 74.6 ± 0.6

∆H (J/g) 17.6 ± 1.7 - 8.6 ± 0.1 6.5 ± 1.5 18.1 ± 0.4 0.6 ± 0.3


Fig 2.3 DSC thermograms of unloaded and loaded DPPC/Chol/DSPEMPEG-2000 liposomes.

The most important contribution, however, is that from the melting of PEG2000 which in the

pure form occurs at ~ 53°C (Taylor & Craig, 2003). It is important to remember that in the

Stealth formulations there is a mixture of phospholipids (DPPC and DSPE which is

conjugated with MPEG2000). The differences in the head groups could be causing the small

differences in the Tm values observed for the DPPC conventional and Stealth formulations

(Table 2.3). Drug loading of the DPPC Stealth formulation (Fig. 2.4) causes a significant

change in the temperature and magnitude of the chain melt transition. The Tm for this

transition decreases from 89.5± 0.4°C to 83.5±0.2°C and the ∆H decreases from 2.2±1.09 J/g

to 0.8±0.1 J/g when the DPPC Stealth formulation is loaded with sirolimus. The foregoing

parameters confirm the interaction of sirolimus in the lipid bilayer and thus the incorporation

of the drug into the lipid bilayer. Interestingly the peak temperature of the transition observed

at 51.3C for the Stealth formulation (Table 2.3) does not change significantly when the

formulation is loaded with sirolimus. However, the ∆H for this transition decreases from

102.9±15.6 J/g to 30.3±1.0 J/g. This is likely to be the result of the reduction in the fractional

composition of the DPPC and DSPE-MPEG 2000 in the loaded formulation. However, this

behaviour also supports the notion that the formulation exists in a state of increased motional


freedom driven by the lipid bilayer, rather than the external PEG2000 molecules. As a result

the liposomes in the loaded DPPC Stealth formulation are in a relatively more disordered

state in comparison to the unloaded Stealth formulation.

Significant differences were observed in the Tm transition between the conventional and

Stealth DSPC formulations (94.6±0.7°C, ∆H of 8.1±1.7J/g and 78.5±0.4°C, ∆H of 2.4±0.1

J/g, respectively). In addition, the DSPC Stealth formulation exhibits two pre-Tm transitions

at 46.2±0.5 and 58.9 ± 0.6°C. This is very interesting because it is the reverse of what is

observed for the DPPC i.e. in the DPPC the conventional formulation exhibited two pre-Tm

transitions while the Stealth formulation exhibited only one. No explanation can be offered

for this observation, apart from the differences in interaction of the DSPE-MPEG2000 with

the DPPC and DSPC molecules.

Fig. 2.4 DSC thermograms of unloaded and loaded DSPC/Chol/DSPEMPEG-2000

liposomes.

Drug loading of the DSPC Stealth formulation (Fig. 2.4) causes a significant shift in the Tm

transition temperature and enthalpy change (Table 2.4). On the other hand, the temperature

and ∆H of the pre-Tm at 58.9±0.6°C remains similar. The 1st pre-Tm transition exhibits no


significant difference in temperature, but the ∆H changes from 16.8±2.3 J/g to 6.5±1.5 J/g

when the DSPC Stealth liposomes are loaded with drug. However, the ∆H reduction of the

1st pre-Tm transition of the DSPC Stealth liposomes is lower compared to the DPPC Stealth

suggesting a farther disordered state of the latter. The differences observed with the addition

of DSPE-MPEG 2000 for both the DPPC and DSPC Stealth formulations are most likely due

to steric hindrance caused by the molecules of the MPEG 2000 near the conjugation site of

the DSPE-MPEG 2000. This could prevent closer packing of the DSPE and the DPPC or

DSPC head groups, which in turn affects the packing ability of the lipid acyl chains (Serro et

al., 2012). Hence, the lower temperatures and ∆H changes observed for the Stealth

formulations in comparison to the conventional. This could also be an additional explanation

as to why greater differences were observed between loaded and unloaded Stealth®

formulations in comparison to the conventional formulations.

2.4.4 X-ray photoelectron analysis (XPS) of Stealth® liposomes

Fig. 2.5 shows the XPS spectra recorded for all samples, where the presence of the following

elements (hydrogen is not accessible for analysis by XPS) was detected: In, O, C, P and N.

Based on the binding energy (BE) values of a strong photoelectron peak, information about

the chemical state for each element can be obtained.

Table 2.5 Surface atomic concentrations, and intensity ratios of I(peak I)/I(peak II) and

IP2p/IN1s for liposome formulations.

C : O : N : P I(peak I)/I(peak II) P/N

Stealth DSPC empty 1: 0.19 : 0.012 : 0.014 0.62 1.17

Stealth DSPC loaded 1: 0.17 : 0.0092 : 0.010 2.1 1.09

Stealth DPPC empty 1: 0.19 : 0.014 : 0.016 0.55 1.14

Stealth DPPC loaded 1: 0.18 : 0.014 : 0.016 0.76 1.14

A correction to account for binding energy shift due to electrostatic charging was applied

based on In3d BE at 443.6 eV (Hofmann, 2013). The indium substrate is composed of a

superficial thin indium oxide layer and carbon contamination due to exposure to the

atmosphere. Fig. 2.6 shows the P2p and N1s core level spectra of all samples. The BE of P2p

is 133.8±0.1eV which was identified as the P5+ state resulting from the PO4 moiety of the


lipid head group (Eighmy et al., 1993). The BE of N1s is 402.8±0.1 eV which is assigned to

positively charged nitrogen atoms (Kumar et al., 1990). In addition, Fig. 2.7 shows the C1s

peaks for unloaded and loaded Stealth DSPC samples while Fig. 2.8 shows the C1s peaks for

unloaded and loaded Stealth DPPC samples, respectively. The peak for the unloaded samples

and for the Stealth DPPC loaded is analyzed into 3 components corresponding to the C–C/C–

H component at 285.0±0.1 eV, C-O(H) groups at 286.6±0.1 eV and O-C=O acrylate or ester

groups at 289.5±0.1 eV.

Fig. 2.5 XPS wide spectra of unloaded and loaded DPPC/Chol/DSPE-MPEG-2000 and

DSPC/Chol/DSPE-MPEG-2000 Stealth liposomes.


Fig. 2.6 P2p and N1s XPS core level spectra of Stealth® liposomes.

In Stealth DSPC loaded samples the main peak for the (C–C/C–H) component centred at

284.5 eV shows a significant shift from the 285.25 eV of the unloaded liposomes. This shift

is attributed to the presence of increased C-O/C-C(Η) moieties on the liposome surface (e.g.

sirolimus). Furthermore, the data shown in Fig. 2.9 depict the O1s peaks for unloaded and

loaded Stealth DSPC samples while Fig. 2.10 depicts the Stealth DPPC unloaded and loaded

samples, respectively. The O1s peak is divided into three components where the first is

centred at 530.6 eV corresponding to O2− of In2O3. The second at 532.5±0.1 eV is due to

C=O groups (ketones) peak I, which are present in sirolimus and the third at 533.8±0.1 eV

corresponds to C-O-C or C-OH bonds (peak II) which are present in the liposomes. Using the

total peak area of N1s, P2p and C1s peaks, the sum of peak I and peak II of O1s peak in each

case, the appropriate sensitivity factors (based on Wagner’s collection and adjusted to the

transmission characteristics of analyser EA10) and physic-chemical parameters, the average

relative atomic concentration in the analysed region, normalized with respect to carbon, can

be determined. The peak intensities are corrected in relation to the thickness of liposome

samples. The data in Table 2.5 shows the relative atomic concentrations, the P/N ratio and the

intensity ratio of the two O1s peaks I(peak I)/I(peak II).


Fig. 2.7 C1s XPS core level spectra of DSPC/Chol/DSPE-MPEG-2000 unloaded and loaded

liposomes.


Fig. 2.8 C1s XPS core level spectra of DPPC/Chol/DSPE-MPEG-2000 unloaded and loaded

liposomes.

The theoretical atomic concentrations for the unloaded liposome samples, (based on the

molar ratios) are: C:O:N:P = 1: 0.19 : 0.016 : 0.015 for the Stealth DSPC and C:O:N:P = 1:

0.20 : 0.017 : 0.016 for the DPPC. The experimental atomic ratios and the P/N ratio are,

within the experimental error of the XPS technique (10%), close to the theoretical (0.93)

value. As shown in the data in Table 2.5the P/N ratio of Stealth DPPC loaded and unloaded


nanoparticles remain identical. This means that the distribution of N near or at the surface did

not increase with the presence of the drug.

Fig. 2.9 O1s XPS core level spectra of DSPC/Chol/DSPE-MPEG-2000 unloaded and loaded

liposomes.


Fig. 2.10 O1s XPS core level spectra of DPPC/Chol/DSPE-MPEG-2000 unloaded and loaded

liposomes.

As a result it can be concluded that sirolimus is concentrated inside the nanoparticles with an

even and random distribution. This is also confirmed by the small increase of the I(peak

I)/I(peak II) ratio which is related to the increase in the C-OH(R) groups of sirolimus. In

contrast, the decrease in the P/N ratio for the Stealth


outer lamellar layer of sirolimus on the liposome surface. This becomes more evident by the

significant increase of the I(peak I)/I(peak II) ratio of the Stealth DSPC liposomes. The

significant increase in the C-OH groups of sirolimus suggests the drug distribution on the

liposome surface during nanoparticles formation. The results of sirolimus co-localization are

very interesting as the drug was expected to distribute within the liposome bilayers due to its

high lipophilicity. However, this was observed only for the Stealth DPPC liposomes and not

for the Stealth DSPC liposomes.

The XPS analysis provided a comprehensive understanding of sirolimus distribution in

Stealth liposomes in complement with the AFM and DSC studies. The analysis of the

mechanical properties (AFM) and the thermal behaviour of the sirolimus Stealth DPPC

liposomes showed a lower packing density and more disordered state compared to the Stealth

DSPC liposomes which is attributed to the different drug distribution along the lipid bilayer.

Stealth DPPC liposomes showed lower packing density and higher bilayer distortion due to

the more uniform distribution of sirolimus within the nanoparticles.

2.5 Conclusions

Conventional and Stealth liposome nanoparticles were loaded with the anticancer drug

sirolimus and characterized using a range of analytical techniques. All formulations were

found to be stable over six months with Stealth liposomes demonstrating better membrane

integrity. It was shown that sirolimus encapsulation in liposomes has a significant effect on

the packing density of the bilayers leading to a distorted state of the nanoparticles. Indeed,

the incorporation of sirolimus into liposomes resulted in a uniform distribution of sirolimus in

multilamellar DPPC Stealth liposomes compared to a non-uniform outer layer lamellar

distribution in DSPC Stealth liposomes. The techniques employed in this study can be a

valuable tool for the characterization and development of anticancer liposome formulations.

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CHAPTER THREE

Antitumor effects of sirolimus-loaded liposomes against human breast

cancer cells

3.0 Overview

The rationale for using sirolimus (rapamycin) as an anticancer agent was based on findings

that identify tumour sensitivity to sirolimus by its inhibition of the mTOR signalling

pathway. This finding has stimulated both preclinical and clinical studies of the

antiproliferative and inhibition effects of sirolimus in different tumour cells. In this study the

antitumour effect of liposome formulations of sirolimus were evaluated on human breast

cancer cell lines. Different parameters affecting the accumulation of the formulated sirolimus

liposomes in the breast cancer cells were evaluated and these parameters include the effects

of drug loading, liposome composition and the incubation time of the liposomes in the

cancer cells. Data obtained from this study shows sirolimus to induce antiproliferative action

at concentrations above 40 µg/ml without however being able to suppress cell viability below

30% when the concentration exceeded 200 µg/ml. Factors such as lipid composition,

incubation time and drug loading were found to play crucial roles in the therapeutic

efficiency of the sirolimus liposome formulations. In addition, the cytoxicity of empty

liposomes formulations was also assessed using 3T3 endothelial cells, which in turn also

demonstrated negligible cytotoxicity even at liposome concentrations of 1 mg/ml.

3.1 Introduction

The use of drug delivery systems to enhance the therapeutic effect of anticancer agents and

reduce systemic toxicity has been widely investigated (Allen & Cullis, 2013). Liposomal

drug delivery systems currently serve as a useful tool for the delivery of active anticancer

agents, such as doxorubicin and paclitaxel. However, research investigations have shown

sirolimus to be an active anticancer agent (Cloughesy et al., 2008 & Guertin et al., 2007)

apart from being used in drug eluting stents for treatments in percutaneous coronary

intervention (Abizaid & Costa, 2010; Douroumis & Onyesom, 2011). Sirolimus is a

macrocyclic lactone immunosuppressive agent that inhibits the cell division cycle and

cellular proliferation by facilitating kinase activation and stopping the cellular growth phase

(Eng et al., 1984). The inhibitors of mTOR as anticancer agents, such as sirolimus, are

undergoing active evaluation in various malignancies (Yu and Sato 1999; Zeng et al., 2010).

About 20 to 30 percent of human breast cancers associated with poor clinical prognosis have


been reported to have amplification and/or overexpression of the HER2/ErbB2 oncogene

(Slamon et al.,1987; Slamon et al., 1989; Vernimmen et al., 2003). ErbB2 also known as

HER2/Neu belongs to a sub-class of the tyrosine kinase epidermal growth factor (EGF)

receptor family (Badache & Hynes, 2004). HER2/ErbB2 signals via the Akt/PI3-K pathway

and leads to the activation of mTOR, a critical mRNA translation regulator that controls cell

growth via translational control of an array of proteins, as previously discussed in Chapter 1.

Several research studies have demonstrated that amplification and/or overexpression of

HER2 in breast cancers results in sensitivity to rapamycin; therefore evaluation of

overexpression of HER2 in breast cancer patient could serve as a prediction of rapamycin

sensitivity in breast cancer patients. Mosley et al., (2007) reported that rapamycin inhibits

multiple stages of c-Neu/ErbB2 tumour progression in a transgenic mouse model of HER2

positive breast cancer. In their study, treatment of MMTV-c-Neu transgenic mice with

rapamycin caused growth arrest and regression of primary tumours with no evidence of

toxicity or weight loss. The observed effect was proposed to be due to decreased proliferation

associated with reduced cyclin D1 expression (an essential regulator of proliferation in

HER/ErbB2 cells) and increased cell death in primary tumours. The data from this preclinical

study of ErbB2/Neu induced breast cancer models suggests that HER2/ErbB2 positive breast

cancer may be particularly sensitive to the effects of rapamycin (sirolimus).

Noh et al., (2004) also reported that the activation of the AKT/PI3-K pathway, which is

associated with HER2/ErbB2 activity, is associated with increased rapamycin sensitivity. In

their study breast cancer cell lines representing a spectrum of aberrations in the mTOR

signaling pathway were treated with rapamycin to test for sensitivity. Cell lines such as

MDA-MB-453 cells overexpressing HER2 demonstrated more sensitivity to rapamycin

compared to cell lines with no HER2 expression (NIH3T3 cells). Breast cancer cell lines such

as MCF-7 and BT-474 have also previously been reported to have p70S6K amplification and

protein over-expression which is a proposed indication of rapamycin sensitivity in breast

cancer patient (Barlund et al., 2000; Barlund et al., 2000) In these studies, the antitumor

effects of sirolimus-loaded conventional and Stealth liposomes were investigated in breast

cancer cell lines. The in vitro cellular uptake, apoptosis and cytotoxicity of sirolimus loaded

liposomes were determined using BT-474 and MCF-7 cancer cells while the cytotoxicity of

empty liposomes was also determined in 3T3 endothelial cells. Sirolimus loaded liposomes

showed substantial cytotoxicity after 24 hours while the qualitative determination of cellular

uptake showed that the liposome formulation localised in the cytoplasm. The developed

formulations were characterized by their particle size and zeta potentials. The preliminary


findings of this study provide promising results for the development of sirolimus liposomal

formulations.

3.2. Materials

Rapamycin was obtained as a gift from LC laboratories (Woburn, Massachusetts, USA).

Distearoyl-phosphatidylethanolamine-methyl-polyethyleneglycol (DSPE-MPEG-2000),

distearoylphosphatidylcholine (DSPC) and dipalmitoyl-phosphatidylcholine (DPPC) were

obtained from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol and curcumin was

purchased from Sigma Aldrich (Dorset, UK). The BT-474 cell line was purchased from the

American Type Culture Collection (ATTC: Manassa, Virginia, USA). Dulbecco’s modified

Eagle’s medium (DMEM), thiazolyl blue tetrazolium bromide (MTT), L-glutamine,

penicillin, streptomycin and heat inactivated fetal bovin serum (FBS) and trypsin were all

purchased from Sigma-Aldrich (UK).

3.3 Methods

3.3.1 Preparation of liposome formulations

Liposome vesicles were prepared by the thin film hydration method (Bangham et al., 1965).

In brief, various appropriate amounts of lipid mixtures (DPPC, DSPC, and DSPE-MPEG-

2000), cholesterol and/or no drug (sirolimus) were weighed out and dissolved in chloroform.

The lipid mixtures (Table 3.0) were subsequently evaporated under a vacuum in a round

bottom flask connected to a rotor evaporator to obtain thin lipid films. The resultant lipidic

films were hydrated with 1 ml of deionised water pre-heated to a set temperature of 5C

above the lipid transition temperatures. After hydration, size reduction of the liposome

vesicles was obtained by extruding (LipexTM extruder by Northern lipids INC) the hydrated

liposome suspension 20 times through a 400 nm, 200 nm and then 100 nm polycarbonate

filter (nucleopore), respectively at a set temperature of 5C above the lipid transition

temperature. For drug encapsulated liposomes, the liposomes were then passed through a

Sephadex G50 column to remove free drug. The particle size of the liposome vesicle was

determined using a particle size analyser (Zetasizer Nanoseries, Malvern Instrument, Malvern

UK). For cellular uptake, an empty liposome formulation was prepared by the addition of

rhodamine (10 µg/10 mg of lipid) to the lipid mixture to form a lipid film followed by

hydration and then extrusion. Excess rhodamine was removed by passing the extruded

liposome formulation through a PD-10 column.


Table 3.0 Liposome formulation compositions.

Formulations Molar Ratio





3.3.2 Encapsulation efficiency of liposome formulations

After the removal of unbound drug, the remaining drug in the liposomes was considered as

encapsulated drug. Encapsulation efficiency (EE) was measured by dissolving liposomes in

the mobile phase and analyzing them by HPLC. Encapsulation efficiency (EE) was calculated

by the following equation:

EE (%) = (𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑎𝑓𝑡𝑒𝑟 𝑐𝑜𝑙𝑢𝑚𝑛 𝑓𝑖𝑙𝑡𝑟𝑎𝑡𝑖𝑜𝑛

𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑏𝑒𝑓𝑜𝑟𝑒 𝑐𝑜𝑙𝑢𝑚𝑛 𝑓𝑖𝑙𝑡𝑟𝑎𝑡𝑖𝑜𝑛) x 100%

3.3.3 Formulation stability evaluations

The formulated liposomes were visually observed and analysis studies which included

particle size and zeta potential measurements were carried out (prior to incubation in the

cells). The formulations were stored in the refrigerator at 4C throughout the evaluation

period.

3.3.4 Release profile studies

One milliliter of liposome formulation was transferred into a cellulose dialysis bag (molecular

weight cut-off 10 kDa), which was then suspended in a beaker containing 20 ml of deionised

water. The beaker was then placed in a shaker bath at a set temperature of 37C. At various

time intervals, the whole content of the beaker was emptied and replaced with another 20 ml

of deionised water. Drug content was then analysed using HPLC.


3.3.5 Quantification of sirolimus by HPLC analysis

HPLC analysis of sirolimus content in liposome formulations were performed using a HPLC-

UV system (Agilent technologies, United Kingdom). Chromatographic separation was

obtained using a Hichrome C18 column (150 mm x 4.6 mm, 5 µm). The mobile phase

consisted of 60% acetonitrile and 40% deionised water. The temperature of the column was

set at 50C and the wavelength was set at 278 nm and pump rate of 1 ml/min. The injection

volume for all samples was 50 µl and elution time was 6 minutes.

3.3.6 Cytotocity test

3T3 (endothelial cells), BT-474 and MCF-7 breast cancer cell lines were cultured using

DMEM culture medium (supplemented with 10% serum, 1% L-glutamine and 1% penicillin

and streptomycin) in an incubator maintained at 37C and 5% CO2. The culture medium was

changed every three days. The cytotoxicity of sirolimus encapsulated liposomes and pure

sirolimus (drug dissolved in ethanol) was determined in MCF-7 and BT-474 breast cancer

cell line using the MTT assay. Cells were seeded in a 24 well flat-bottom plate at a cell

density of 1 × 106 cells/well and incubated for 24 hr. After 24 hr the liposome formulations

were added to the 24 well plates at various concentrations and incubation time. 100 µL of

MTT solution (5 mg/ml) was added to each well plate at the end of the incubation time and

incubated at 37C for another 2 hours. The culture medium was discarded, followed by

addition of 200 µl of acidified isopropanol to dissolve the MTT formazan crystals. 100 µL of

the dissolved MTT formazan crystals was then transferred into a 96 well flat-bottom plate

and the absorbance was read at 492 nm using a microplate reader. Controls included non-

treated cells and fixed volume of ethanol for the pure drug cytotocity assay. Sirolimus was

used at concentrations of 20, 60, 100 200 and 500 µg/ml. Empty (unloaded ) and loaded (1, 2

and 5 mg of drug) liposomes formulations were incubated at liposome concentrations of 20,

100, 200,400 and 800 µg/ml. Cytotoxicity of empty liposomes was carried on MCF-7,

BT-474 and 3T3 cell lines. Empty and loaded liposome formulations were also tested on

MCF-7 and BT-474 breast cancer cell lines, respectively. The cytotoxicity of pure curcumin

drug was also evaluated on BT-474 breast cancer cells for direct anti-proliferative efficacy

evaluation with sirolimus pure drug. Curcumin was dissolved in ethanol.


3.3.7 Cellular uptake

The cellular uptake of the empty liposome formulations was determined using a Nikon

fluorescent microscope. 20 × 10³ cells/well were seeded on cover slips in a 24 well flat-

bottom plate. Empty rhodamine (10 µg/10mg of lipid) stained liposome formulations were

incubated with the cells for 24 hr at various concentrations. The cell medium was discarded

from the well after 24 hr incubation time and washed three times with PBS. 1ml of 4% para-

formaldehyde was added to each of the wells to fix the cells on the cover slips and left in the

dark for 15 min. The p-formaldehyde solution was discarded from the well and cells were

washed three times with PBS and then mounted on a glass slide using vectashield mounting

medium containing DAPI. The cover slips were sealed on a glass slide with nail polish and

left to dry. Images of the liposome localization in the cell were acquired using a fluorescence

microscope (Nikon, Eclipse Ni-E).

3.3.8 Apoptosis study

Apoptosis of the loaded Stealth liposome formulations (DPPC, MPEG2000, cholesterol and

5 mg of drug) was determined using annexin V (Alexa Fluor 488) conjugate staining. Loaded

DSPC Stealth liposome formulations (2 mg drug) were incubated with BT-474 cells, seeded

at 20 × 103 cell density on a cover-slip for 24 hr. Fluorescent images of the apoptotic cells

were observed using a Nikon fluorescent microscope. Briefly 20 µl of sirolimus loaded (2 mg

drug) Stealth liposomes were incubated with BT-474 cells for 24 hr, non- treated cells were

used as a negative control. After incubation, cells were washed twice with cold PBS and 1ml

of annexin binding buffer was added to the cells on the coverslip followed by 10 ul of

annexin V conjugate and 5 ul of propidium iodide. Cells were further incubated at room

temperature for 15 min and then washed three times with annexin binding buffer. The cells

on the cover slips were then mounted on slides using mounting medium containing DAPI and

images were acquired using Nikon fluorescent microscope.

3.4. Results and Discussion

3.4.1 Size, stability and encapsulation analysis

The particle size and zeta potential of liposomes formulations were measured over a period


of six months (as previously described in chapter two). All liposome formulations showed an

average particle size less than 200 nm (Fig. 3.0) and the polydispersity of all formulations

were less than 0.2 which indicates that the liposome population was homogenous in size. The

encapsulation efficiency of the various liposome formulations is shown in Fig. 3.1 Liposome

formulations show a good encapsulation efficiency; higher than 90%. However Stealth

liposome formulations diplayed a slightly higher encapsulation efficiency of up to 3% higher

than conventional liposome and this is most probably due to the composition of the

formulation i.e. the addition of MPEG200 to the Stealth formulations. Previous reports on

liposome drug delivery system investigation attributed high encapsulation efficiency of drug

in liposomes to a number of factors such as formulation composition (e.g. amount of

cholesterol, MPEG2000), preparation method and drug solubility (Nii & Ishii., 2005;

Ramana et al., 2010; Kumar et al., 2010). Nii & Ishii (2005) evaluated the encapsulation

efficiency of both lipophilic and amphiliphilic drugs in three kinds of egg lecithin with

different degrees of saturation. Their studies attributed high encapsulation to lipid

composition, drug solubility in terms of logP and preparation method. The most saturated

egg lecithin liposome formulation was found to show a higher encapsulation efficiency

compared to others. This observation was reported to be probably due to the differences in

the packing geometry of the hydrophobic carbon chains in the liposomes membrane. It was

also reported that the LogP of a drug affects its encapsulation in liposome. Drugs with a

higher lipophilicity were also demonstrated to have higher encapsulation efficiency. They

also found that amphiphilic drugs resulted in better encapsulation when dissolved in water

than chloroform. Ramana et al., (2010) on the other hand also demonstrated the effect of

ratios of egg phospholipid to cholesterol as well as drug to total lipid. Their study showed egg

phospholipid liposome formulation to be optimised with high encapsulation efficiency of

approximately 80% of niverapine when the phospholipid to cholesterol ratio was 9:1. In

addition to this, a significant increase in the amount of drug loading was also reported with

increasing drug-lipid ratios of up to 1 : 5, but not beyond these ratios. However, in this study

the high encapsulation of drug achieved for the liposome formulations cannot be exclusively

attributed to the preparation method but also could be associated with the high lipophilicity of

sirolimus (LogPo/w of 5.77).


A

B

Fig. 3.0 Malvern zeta-sizer particle size analysis of Stealth liposome formulations (A) DPPC

Stealth liposomes empty and loaded; (B) DSPC Stealth liposomes empty and unloaded.


Fig. 3.1 Encapsulation of sirolimus in DPPC and DSPC conventional and Stealth liposome

formulations.

3.4.2 Drug release profile of sirolimus in liposome formulations

Sirolimus formulation release study was carried out over a period of 72 hr at a controlled

temperature of 37C. Liposome formulation samples placed in the beaker were sealed tightly

with parafilm to prevent water loss. HPLC assay validation of sirolimus was achieved using

the linear regression method and the calibration curve established showed good linearity

(with R2 value of 0.9993) of concentrations ranging from 10-100 µg/ml. The HPLC

chromatogram is shown in Fig 3.2.

Fig. 3.2. HPLC chromatogram of sirolimus.

88

89

90

91

92

93

94

95

96

DPPC

Conventional

DSPC

Conventional

DPPC Stealth DSPC Stealth

En

cap

sula

tio

n E

ffie

cien

cy (

%)

Sirolimus


Fig. 3.3 shows the cumulative drug release profile of loaded sirolimus liposome formulations.

In this study, there was no initial burst release observed which indicates that drug molecules

were well encapsulated in the bilayer of the liposomes and no/very little free drug was

present on the liposome surface. As expected for a lipophilic drug, the release rate of the drug

was very slow. After 24 hr, only about 13% of the encapsulated drug was released from the

formulation. A significant difference was observed in the release profile between

conventional and Stealth liposomes. Conventional liposomes (DPPC, DSPC) show a higher

drug release profile (13%) compared to Stealth liposomes (DPPC, 10%; DSPC, 5%) after

24 hr. Similar finding have previously been reported by Panwar et al., (2010) and Hioki et

al., (2010) where pegylation of liposomes (Stealth liposomes) was demonstrated to retard

drug release compared to the non-pegylated counterpart (conventional liposomes). In the in

vitro release study of the encapsulation of a hydrophobic drug (albendazole) in nanosized

liposomes reported by Panwar et al., (2010) it was observed that the drug release rate showed

a decrease in albendazole release in descending order of free drug, drug loaded conventional

liposomes and least with drug loaded Stealth liposomes. Hioki et al., (2010), on the hand,

also reported the effect of temperature and serum on the release profile of both conventional

and Stealth liposomes. Their study illustrated higher drug release with conventional

liposomes with increase in temperature and the presence of serum. The drug release study of

sirolimus liposome formulation in Fig.4 showed a maximum drug release after 72 hr of 14%

(DSPC) and 16% (DPPC) for conventional liposome and 10% (DSPC), 12% (DPPC) for

Stealth liposomes, respectively.

For Stealth liposomes, the effect of lipid with different phase transition temperature on drug

release was also demonstrated in the in vitro drug release of sirolimus. DSPC Stealth

liposomes showed only about 5% release after 24 hr while DPPC Stealth liposomes showed

10% drug release. This result is expected due to the higher phase transition temperature (Tm)

of DSPC compared to DPPC. This result, however, indicates that the liposome formulations

are very stable with rigid membranes, which is important especially when formulations are

intended for in-vivo applications. The rigidity of liposome membranes plays an important role

in increasing the chances of the formulation not being rapidly disrupted before it reaches its

sites of action. In other words it prevents or reduces the chances of pre-mature drug release

and increases its circulation time. Chen et al., (2012) reported on the effect of Stealth

formulations prepared using different lipids with different phase transition temperatures on

drug release. Brucine was encapsulated in four different PC Stealth liposomes (SPC, HSPC,

DPPC and DSPC). The in vitro release study revealed that the rate of brucine release


increased with decrease phase transition temperature of the PC (SPC: 13.2%, DPPC: 10.5%,

DSPC: 6.4%, HSPC: 6.1%), especially in the presence of rat plasma with maximum drug

release of approximately 80.9% for SPC Stealth and 15.5% for HSPC Stealth liposomes after

10 hr duration.

Fig. 3.3 Drug release profile of DPPC and DSPC conventional and Stealth liposome

formulations.

3.4.3 Antiproliferative effect of sirolimus versus curcumin

The antiproliferative effects of sirolimus loaded liposome compared to an alcoholic solution

of the pure drug and empty liposome formulations were investigated. The antiproliferative

effect of pure sirolimus on HER-2 overexpressing breast cancer BT-474 cells was also

compared to the antiproliferative effect of pure curcumin a more potent anticancer drug.

Sirolimus and curcumin were dissolved in ethanol. Various concentration ranges of both

sirolimus and curcumin were incubated in cells. It is however imperative to first determine

the maximum tolerable limit of the solvent in cells. Thus different amounts of the solvent

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ethanol were also incubated with cells in order to determine the tolerable amount of solvents

in the cells. Experimental findings for this study indicated that the maximum tolerable

amount of ethanol was 2%. Therefore the drugs (sirolimus and curcumin) were dissolved in

ethanol and incubated with cells at solvent concentration below 2%. Each experiment were

repeated in triplicate and the variation in each data set were represented by the standard

deviation of the mean for each data set (standard deviation, n=3).

Fig. 3.4 Antiproliferative effects of pure sirolimus and curcumin on breast cancer cells (BT-

474) using the MTT assay for 24 hours incubation time.

The cytotoxicity studies of pure sirolimus (Fig. 3.4) showed sufficient antiproliferative

activity at concentrations above 40 µg/ml without, however, being able to suppress cell

viability below 20% when the concentration exceeded 200 µg/ml. Curcumin, on the other

hand, a well-known potent anticancer agent showed a more pronounced reduction (Fig. 3.4)

in the cell viability percentage of breast cancer cells at a much lower concentration

(40 µg/ml) reducing the cell viability to almost 0% compared to sirolimus. These findings can

sometimes be regarded as being advantageous in chemotherapy as various reports of other

anticancer agents with high potency have always highlighted its drawbacks with increased

systemic toxicity. Carboplatin, for example, contains active fragments of cisplatin; while it is

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effective in ovarian cancer, it is less potent than cisplatin and correspondingly the platinum

side-effects or systemic toxicities are less severe. Although cisplatin remains the agent of

choice, carboplatin on the other hand has been reportedly utilised in the clinic when there is a

clinical need to minimize platinum drug side effects (Hannon, 2007).

3.4.4 Antiproliferative effect of liposome formulations

Initial studies of the cytotoxicity of unloaded (empty) conventional and Stealth liposomes

were assessed on both fibroblast endothelial cells (3T3) and breast cancer cells (MCF-7)

using MTT assay. The cell viability data shows that both liposomes formulations are not

cytotoxic.

Fig. 3.5 Cytotoxicity of empty DPPC formulations (conventional and Stealth) on 3T3

endothelial non-cancerous cells. DPPC: cholesterol (molar ratio 18.6: 9) and Stealth DPPC:

DSPE-MPEG2000: cholesterol (molar ratio 12.6: 1.14: 8.0).

In-vitro cytotoxicity studies of empty DPPC Stealth and conventional liposome formulations

were evaluated on normal endothelial cells (3T3) in order to evaluate their cytotoxicity. The

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Stealth unloaded


MTT assay of the non-cancerous 3T3 cell line incubated with DPPC Stealth and conventional

liposomes showed a negligible cell viability of 92% (Stealth) and 90% (conventional) at a

maximum concentration of 800 µg/ml (Fig. 3.5).

Fig. 3.6 Antiproliferative effect of empty and loaded (1.09 mM drug) conventional liposome

formulation DPPC: cholesterol (molar ratio 18.6: 9) on MCF-7 breast cancer cell line for

24 hr.

This finding demonstrates that both liposome formulations are non-toxic even at a very high

concentration and therefore suggests systemic biocompatibility in other non-cancerous site

when administered. Earlier studies have also reported the biocompatibility of both

conventional and Stealth liposomes in vitro. Pitrubhakta et al., (2012) in their investigation of

gematasine hydrochloride liposome formulations in human lung carcinoma cells reported a

78% and 87% cell toxicity of empty Stealth and conventional liposomes, respectively. Earlier

studies carried out by Ahmad & Allen (1992) on doxorubicin liposome delivery to lung

cancer cells demonstrated that conventional liposomes elicited no toxic effects; but, they

observed a reduction in cell proliferation with empty Stealth liposomes (IC50 = 68 µM)

compared to the free drug (IC50 = 8 µM). However, they also stated that previous studies of

empty Stealth liposomes in cultured bone marrow macrophages showed no cytotoxic effect.

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Therefore suggesting that cytotoxicity of empty liposomes formulations could vary from cells

to cells.

The aim of this study was to evaluate sirolimus on BT-474 HER-2 overexpressing breast

cancer cells. Although studies have shown sirolimus to have antiproliferative activity on a

number of breast cancer cells but the sensitivity of sirolimus has been associated with the

presence of high levels of phospho-AKT or phospho-S6K1 and expressed wild-type levels of

PTEN (Phosphatase and tensin homolog) abberations. Noh et al (2004) reported that S6K1

overexpression in breast cancer results is more sensitive to sirolimus compared to other

aberrations. Their data of sirolimus sensitivity on a panel of breast cancer cells (which

includes MCF-7, BT-474 and MDA-MB-231) showed BT-474 to be most sensitive to

sirolimus thus indicating S6K1 association with sirolimus sensitivity. In this study the

cytotoxic effect of sirolimus was first evaluated on MCF-7 breast cancer cells. The data in

Fig. 3.6 shows the cytotoxic effect of sirolimus loaded DPPC conventional liposomes on

MCF-7 breast cancer cells for 24 hr. It can be seen that sirolimus loaded DPPC conventional

liposome formulation (1.09 mM drug) reduced cell viability up to 75% after 24 hr compared

to the unloaded (empty) DPPC conventional liposomes (92%).

Fig. 3.7 Antiproliferative effects of conventional formulation DPPC: cholesterol (molar ratio

18.6: 9) and Stealth DPPC: DSPE-MPEG2000: cholesterol (molar ratio 12.6: 1.14: 8.0)

(1.09 mM drug) on breast cancer cell line BT-474 for 24 hr.

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In-vitro studies of the liposome formulations were further carried out on a different breast

cancer cell line (BT-474). Fig. 3.7 illustrates the effect of sirolimus liposome formulations on

cancer cell proliferation. Conventional and Stealth liposomes encapsulated with 1.09 mM

drug were tested in-vitro on BT-474 breast cancer cells for 24 hr. The DPPC conventional

liposome formulations showed significantly higher antiproliferative activity (68% cell

viability) in a dose dependent manner compared to the Stealth liposome formulation (75%).

A reduction in the cell viability was observed for both Stealth and conventional liposomes at

liposome dose of 20 µg/mL, reducing cell viability to 80 %. A difference in the cytotoxic

effect of DPPC conventional and Stealth liposomes is associated with the formulation

composition. As expected, addition of MPEG2000 to the liposome formulation creates a

steric hindrance in the membrane of the Stealth liposome results in a decrease interaction

with blood borne components (lack of recognition by macrophages), increased circulation

time and enhanced resistance to serum degradation (Perche & Torchillin, 2013). In in vitro

studies, due to the steric hindrance created by pegylation, it has been shown that Stealth

liposomes tend to show lower antiproliferative effects compared to conventional liposomes.

This observation has been associated with slower drug release and slow interactions of

Stealth liposomes with tumour cells compared to a conventional liposomes (a more leaky

membrane hence faster drug release profile). Righeschi et al., 2014 demonstrated a higher

cytotoxicity effect in vitro of conventional liposomes compared to Stealth liposomes. In the

aforementioned study dihydroartemisinin was encapsulated in EggPC conventional and

Stealth liposome and its therapeutic efficacy was evaluated in breast cancer cells (MCF-7).

The cytotoxicity effect of the empty liposome formulations showed no significant

cytotoxicity effect with cell viability above 92%. However a significant cytotoxic effect was

observed for the loaded liposome formulations with conventional liposome (IC50= 48 µM)

exhibiting a higher toxicity of 1.6 times higher than the Stealth formulation (IC50= 77 µM).

Similar findings were also reported by Yang et al., (2007) and Rouf et al., (2009).


Fig. 3.8 Antiproliferative effect of conventional formulation DPPC: cholesterol (molar ratio

18.6:9) and Stealth DPPC: DSPE-MPEG2000: cholesterol (molar ratio 12.6:1.14:8.0) (2.18

mM drug) on breast cancer cell line BT-474 for 24 hr.

The effect of various factors such as lipid composition, drug loading and particle size was

also explored for the developed liposome formulations. In order to investigate the effect of

drug loading, different amounts of sirolimus (1.09 mM and 2.18 mM) were encapsulated into

the liposome formulations and their antiproliferative effects were studied. The increase in

amount of sirolimus encapsulated resulted in a further significant reduction in cell viability

for both formulations as shown in Figs. 3.7 and 3.8, respectively. Stealth liposome

formulations showed a further reduction of more than 10% in cell viability when the drug

amount was increased and hence the cell viability was reduced to 75% and 66%,

respectively. On the other hand conventional liposomes showed a cell viability of 61% when

drug amount increased to 2.18 mM.

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Fig. 3.9 Antiproliferative effect of loaded DPPC conventional and Stealth liposomes (2.18

mM drug) on breast cancer cell line BT-474 for 72 hr.

Fig. 3.9 shows the effect of liposome formulations on cell proliferation at prolonged

incubation time of 72 hr. In this study the incubation time of the liposome formulations with

the breast cancer cells (sirolimus 2.18 mM) was increased from 24 hr to 72 hr. Significant

anti-proliferative effect is observed for DPPC liposome formulation with maximum reduced

cell viability of 48% for conventional liposomes and 50% for Stealth liposomes. A further

reduction in cell viability was observed for both DPPC Stealth and conventional liposomes

but significant difference in cell viability was not observed in the formulations at liposome

concentration of 800 µg/ml. The mechanism of this effect is not fully understood and a

possible explanation for this observation may be attributed to the slower rate of drug release

in Stealth liposomes compared to conventional liposomes. But, a high dose of the liposomes

formulations and longer incubation time in vitro could lead to destabilization of the

membrane of the Stealth liposomes and results in an antiproliferative effect similar to the

conventional liposomes.

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DPPC Stealth Loaded


Fig. 3.10 Antiproliferative effect of conventional formulation DPPC: cholesterol (molar ratio

18.6: 9) and Stealth (5.46 mM drug) on breast cancer cell line BT-474 for 72 hr.

Further increases in drug amount per formulation did not show any significant difference in

the antiproliferative effect of the liposome formulations with further cell viability reduction

of only 3% for Stealth liposomes as observed in Fig. 3.10. The cytotoxicity of the liposome

formulations remains significantly unchanged even with an increase in the amount of drug

from 2.18 mM to 5.46 mM. Similar findings were also reported by Zeng et al., (2010) who

illustrated that in vivo sirolimus at a high drug dose of 5 mg/ml shows no significant

difference in antiproliferative effect compared to a low dose of 1.5 mg/ml.

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DPPC Stealth Loaded


Fig. 3.11 Antiproliferative effect of DPPC and DSPC Stealth formulations (molar ratio 12.6:

1.14:8.0) with 2.18 mM drug on breast cancer cell line BT-474 for 72 hr.

The length of the acyl chain of phospholipids plays an important role in the membrane

rigidity of liposome formulations (Liu et al.,2001). Lipids with longer acyl chain length used

in liposome formulations tend to have a more rigid membrane packing and higher transition

temperature compared to lipids with shorter acyl chain length. The acyl chain length of DSPC

comprises of 18 carbons compared to that of DPPC which is 16 carbons (Jullien et al., 1989).

Fig. 3.11 shows the cytotoxicity effect of DSPC Stealth liposome using MTT assay.

Compared to the result obtained for DPPC Stealth liposomes, DSPC Stealth liposomes

showed less cytotoxic effects, with cell viability of 58% compared to DPPC Stealth

liposomes (50%).

3.4.5 Cellular internalisation and apoptosis induced effect of liposome formulations

Liposomes are known to enter cells via endocytosis mechanism pathway. In order to confirm

the internalization of the liposomes formulation, 800 µg of unloaded DPPC Stealth liposomes

were incubated with breast cancer cell line (BT-474) at different time intervals of 2, 5 and 24

hr. The qualitative cellular uptake studies were investigated with fluorescent microscopy after

staining the nucleus of the cells with DAPI while rhodamine was used to label liposomes.

Free rhodamine was removed from the surface of the liposome by column filtration of the

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DSPC Stealth Loaded

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formulation through a PDL 10 column. According to the cellular uptake images (Fig. 3.12)

internalization of the liposome formulation was observed after after 5 hr but significant

cellular uptake of liposome was seen after 24 hr. It can also be seen that liposome particles

are localised in the cytoplasm around the cell nucleus as illustrated in Fig. 3.12.

Apoptosis is defined as a physiologically programmed cell death, which occurs during

cellular development. Research evidence has shown that DNA repair is activated early in p53

gene induced apoptosis (Geske et al 2001). Moreover, Kao et al (2009) also demonstrated

that sirolimus increases p53 dependent apoptosis in translational inhibition of mdm2 in

cancer cells. Sirolimus on its own as an administered drug has also been reported to induce

apoptosis in HER2 over-expressing breast cancer cell lines (Zeng et al., 2010). Fig. 3.13

shows that sirolimus formulated Stealth liposomes induce apoptosis. During apoptosis

phosphatidylserine (PS; usually located on the cytoplasmic side of a normal cell bilayer) is

translocated from the inner to the outer leaflet of the plasma membrane thus exposing PS to

the external cellular environments. Annexin V labelled with a fluorophore or biotin can

identify apoptotic cells by binding to PS exposed on the outer leaflet. Fig. 3.13 illustrates

significantly high levels of surface labelling (green) at the end of the 72 hr incubation (i.e.

intense membrane staining for externalized PS), which suggests that sirolimus Stealth

liposome induces apoptosis.

Most anticancer drugs are necrotic which leads to inflammation in other cells compartments

and as a result cause various adverse side effects in patients when administered. This finding

demonstrated that sirolimus could be a potential valuable drug candidate for cancer treatment

due to its apoptosis inducing ability in cancer cells which is a more favourable form of

inducing death in cancer cells compared to necrotic processes.

Yellen et al., (2011) and Dai et al., (2013) reported rapamycin (sirolimus) to induce apoptosis

via inhibition of mTOR. High dose rapamycin treatment in breast cancer cells (MDA MB

231) was demonstrated to result in apoptotic effect due to suppressed phosphorylation of the

mTOR complex (Yellen et al., 2011).


Fig. 3.12 Fluorescent images of the cellular uptake of empty formulated DPPC Stealth liposome localisation in the cytoplasm of the cell (A; 10x

objective magnification; B; 60x objective magnification). The nucleus of the cell was stained blue with DAPI and the liposome formulation was

labelled with rhodamine (red).


Fig. 3.13 Fluorescent microscopy images showing apoptotic cells of sirolimus loaded DPPC Stealth liposomes using Annexin V conjugate

staining. The nucleus is stained blue and apoptosis induced signal is green using Annexin V conjugate.


To further facilitate the uptake of liposomal drugs into targeted cancer cells, cell penetrating

antibodies or proteins could be conjugated on the surface of the Stealth liposomes in order to

enhance the therapeutic efficiency of the liposomes. Although conventional liposomes may

demonstrate more cytotoxic effect in-vitro, their pharmacokinetic properties such as short

circulation time are limiting factors in-vivo.

3.5. Conclusions

In this study sirolimus was encapsulated in both conventional and Stealth liposome

formulations and introduced as an anticancer drug delivery system against BT-474 and MCF-

7 breast cancer cells. The particle size characterization of both Stealth and conventional

liposomes showed Stealth liposomes to have a smaller particle size. The cytotoxicity studies

of both pure and liposome loaded sirolimus provided encouraging results for further

development of an effective sirolimus liposomal formulation. The Stealth formulation

although showed less antiproliferative effect compared to conventional liposomes, it however

remains a better choice of formulation when considering application in in-vivo studies due to

its prolonged circulation time. Findings in this study also demonstrated that drug loading and

incubation time plays a vital role in the efficiency of the formulations. Liposomes with higher

drug loading and longer incubation time showed higher antiproliferative effect.

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CHAPTER FOUR

In vivo Evaluation of Conjugated Stealth Liposomes for Targeted Delivery in HER-2

Overexpressing Breast Cancer Cells (BT-474)

4.0 Overview

Liposomal Stealth formulations were conjugated with transferrin for specific (active) delivery

of sirolimus into breast cancer cells. In this study, the conjugated Stealth liposomes were

characterised in terms of particle size, zeta potential and drug loading. In vitro and in vivo

studies of Stealth liposomes for passive and active delivery of sirolimus in BT-474 breast

cancer cells were evaluated. Intravenous administration of sirolimus and sirolimus liposomes

showed Stealth formulations to be more effective in suppressing tumour growth compared to

the free drug. In addition, transferrin conjugated Stealth liposomes demonstrated even higher

anti-tumour activity. These findings demonstrate the potential usefulness of sirolimus Stealth

liposomes for cancer cell targeting treatment.

4.1 Introduction

Stealth liposomes have been widely utilized in site-specific delivery of anticancer drugs in

vivo and subsequently reduce the toxicity of the drugs by altering non-specific distribution

(Nag & Awasthi, 2013). In site-specific delivery, a recognition ligand is usually attached to

the surface of liposomes in other to achieve controlled and sustained release of the drug in the

target site. One of the physiochemical properties of Stealth liposomes is their ability to

accumulate in cancer cells due to increased vascularization in cancer cells compared to

normal healthy cells (Park et al., 2000; Li et al., 2009). However, the presences of specific or

unique receptors/ligands on the surface of tumour cells have been shown to improve the

therapeutic effects of designed drug carriers (Lu et al., 2013). Several research findings have

not only shown a number of tumours to express high level of transferrin receptors but also

shown transferrin endocytosis uptake in tumours. These findings have led to more focus in

the development of drug carriers conjugated with transferrin on their surface for specific site

delivery of drugs (Joo & Kim, 2002; Anabousi et al., 2006; Kobayashi et al., 2007; Yhee et

al., 2013). Transferrin is a serum glycoprotein that transports ferric ions and is known to be

internalized by cells via transferrin reception mediated endocytosis (Huebers & Finch, 1987;

Derycke et al., 2004). After binding to the cell surface, transferrin is rapidly internalized as a

ligand-receptor complex via coated pits. The protein ligand-receptor complex formed by


transferrin binding enters into the cellular compartment followed by the delivery of

encapsulated drug.

Perche & Torchilin (2013) recently reported the trends in multifunctional liposomal

nano-carriers for enhanced tumour targeting. Their review highlighted the developmental

stages and current strategies used for cancer detection and therapy using liposomes with

attached ligands such as transferrin. Li et al. (2009) also demonstrated that transferrin

conjugated Stealth liposomes when administered in-vivo in tumour bearing mice were able to

show significant increase in the concentration of the loaded drug doxorubicin in tumours and

a decrease of the drug in the heart and kidney. Although several reports have shown BT-474

cells to express high levels of transferrin receptor and sensitivity to sirolimus due to mTOR

(Del Bufalo et al., 2006; Anabousi et al., 2006) only a few studies have been reported on

liposomal drug delivery systems for site specific targeting. Rouf et al. (2009) reported the

antiproliferative effect of rapamycin (sirolimus) on MCF-7 cells. In their investigation, both

conventional and Stealth liposomes were incubated with MCF-7 breast cancer cells and the

antiproliferative effects were examined in-vitro. The findings showed both conventional and

Stealth liposomes encapsulated with sirolimus to be effective against MCF-7 breast cancer

cells. In this study transferrin mediated endocytosis pathway and the mammalian target of

rapamycin (mTOR) approach in liposomal drug delivery was evaluated on BT-474 breast

cancer cells both in vitro and in vivo. Since sirolimus is not as potent as most anti-cancer

agents, the concept of using conjugated Stealth dispersions is proposed to enhance the

therapeutic efficacy of the drug and hence reduce systemic toxicity usually associated with

most potent anticancer drugs.

4.2 Materials

Phosphorus standard solution, perchloric acid, 4-Amino-2-naphthyl-4-sulfonic acid reagent,

ammonium molybdate solution and transferrin were purchased from Sigmal-Aldrich, UK.

Rapamycin was obtained from LC laboratories (Woburn, Massachusetts, USA).


distearoylphosphatidylcholine (DSPC) and dipalmitoyl-phosphatidylcholine (DPPC) was

obtained from Lipoid GmbH (Ludwigshafen, Germany). DSPE-MPEG-2000-NH2 was

purchased from Avanti Lipids, USA. Cholesterol was purchased from Sigma Aldrich, UK.

The BT-474 cell line was purchased from the American Type Culture Collection (ATTC:

Manassa, Virginia, USA). Dulbecco’s modified Eagle’s medium (DMEM), thiazolyl blue


tetrazolium bromide (MTT), L-glutamin, penicillin, streptomycin and fetal bovin serum

(FBS) and trypsin were all purchased from Sigmal -Aldrich (UK).

4.3 Methods

4.3.1 Liposome preparation

Stealth liposomes (Table 4.0) were prepared as previously discussed in Chapter 2. Briefly,

pegylated Stealth liposomes were prepared by the thin film hydration method (Bangham et

al., 1965). In brief, various appropriate amounts of lipid mixtures (DSPC and DSPE-MPEG-

2000), cholesterol and/or no drug (sirolimus) were weighed and dissolved in chloroform. For

the Stealth liposomes to be conjugated, DSPE-MPEG-2000-NH2 from Avanti lipids was

utilised instead in other to facilitate coupling of the transferrin on the liposome surface via the

NH2 terminal of the DSPE-MPEG-2000-NH2 lipid. The lipid mixtures were subsequently

evaporated under vacuum in a round bottom flask connected to a rotor evaporator to obtain

thin lipid films. The resultant lipidic films were hydrated with 1 ml of deionised water pre-

heated to a set temperature of 5C above the lipid transition temperatures. After hydration,

size reduction of the liposome vesicles was obtained by extruding (LipexTM extruder by

Northern lipids INC) the hydrated liposome suspension 20 times through a 400 nm and then a

200 nm polycarbonate filter (nucleopore) at a set temperature of 5oC above the lipid transition

temperature. For drug encapsulated liposomes, the liposomes were then passed through a

Sephadex G50 column to remove free drug. The particle size of the liposome vesicle was

determined using a particle size analyser (Zetasizer Nanoseries, Malvern Instrument, Malvern

UK).

Table 4.0 Stealth liposome formulation composition.




4.3.2 Liposome conjugation

Transferrin (Tf) from Sigma Aldrich was coupled on the surface of the liposomes using

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in a one-step reaction. 10 mg of

EDC was dissolved in 0.3 ml of deionised water and added to the pegylated (Stealth)

liposome suspension. The mixture was stirred and allowed to react for 1 hr. 10 mg of


transferrin was then added to the mixture, further stirred and allowed to react for an

additional 4 hr. At the end of the reaction, excess EDC and transferrin was excluded from the

transferrin conjugated liposome suspension by size exclusion technique using a Sepharose

CL-4B column. The diluted Tf-Stealth liposome fractions collected after column filtration

were then re-concentrated by using a viva spin concentrator and liposomes were centrifuged

for 60 min at 11000 rpm and temperature of 4oC. The resultant Tf-Stealth liposomes

suspension was quantified by HPLC to confirm drug loading efficiency as well as Bradford

and Bartlett assays for protein. HPLC methodology for drug loading efficiency was

performed as previously discussed, in chapter three.

4.3.3 Methodology of transferrin assay quantification.

Transferrin is a globular glycoprotein with molecular weight of 80 kDa and high aqueous

solubility. The average amount of transferrin conjugated to liposome was quantified by the

Bradford assay. Bradford protein assay is a commonly used procedure for determination of

protein concentrations in solutions that depends on the change in absorbance of Coomassie

Blue G-250 upon binding of protein. Because the assay is a colorimetric assay, as the

concentration of the protein increases, the colour of the test sample becomes darker. The

protein concentration in a sample (e.g. Tf-Stealth liposome) is determined in comparison to

that of a series of protein standards known to reproducibly exhibit a linear absorbance profile.

In this study the Bradford assay reagent was purchased from Bio-Rad, UK and the provided

protocol for protein determination was followed. Bovine serum albumin (BSA) was utilised

as a protein standard win concentration range of 0.0, 0.125, 0.5, 1.0 and 2.0 mg/ml. 100 µl

of each standard was pipetted in glass tube and 5 ml of the Bradford reagent was added. The

mixture was vortex and allowed to stand at room temperature for 5 minutes before reading at

595 nm using a UV spectrophotometer (Varian Cary, 5000). For the blank 100 µl of

deionised water was used and 5 ml of Bradford reagent was added followed by vortex and

UV reading. For Tf-Stealth liposome, 100 µl of the liposome sample was used and the same

procedure as the BSA standard was followed. All tests were carried out in triplicate and a

linear regression calibration curve was obtained for BSA standards. The amount of transferrin

conjugated on Stealth liposomes surface was determined using the BSA calibration curve.


4.3.4 Phosphorous lipid quantification using the Bartlett assay

The amount of lipids present in liposomes are commonly quantified by two methods known

as the Bartlett and Stewart assay, Nicolaas et al. (2003). The Bartlett assay was used in lipid

quantification in this study. The principle of the Bartlett assay is based on the colorimetric

determination of inorganic phosphate. The phospholipid content of liposomes is quantified

after conversion of the phospholipid with perchloric acid to inorganic phosphate. The

inorganic phosphate is converted to phospho-molybdic acid by the addition of ammonium

molybdate, which is then reduced to a blue colour complex during heating with 4-amino-2-

naphthyl-4-sulfonic acid. Similar to most colorimetric assay, the blue colour complex is

determined at 830 nm using a UV spectrophotometer (Barenholz & Amselem, 1993). The

protocol for phospholipid determination was as described by Torchilin & Weissig (2003). A

concentration range of 0, 25, 50, 100 and 125 µl of 0.65mM phosphorus standard solution

and 20 µl of Tf-Stealth liposomes were added in separate glass tubes. Two boiling stones

were added to each glass tube followed by the addition of 0.4 ml of 70% perchloric acid. The

tubes were covered with marbles and heated for 30 min at 180C in a heating block then

cooled to room temperature. 1.2 ml of double deionised water, 0.2 ml of 5% (w/w)

ammonium molybdate solution (2.5 g dissolved in 50 ml double deionised water) and 50 µl

of amino-naphthyl-sulfonic acid reagent (0.8 g in 5 ml double deionised water) were added to

each test tube, vortexed and placed in boiling water for 7 min. The tubes were allowed to

cool to room temperature and absorbance of the standard and liposomes samples were

measured at 830 nm. Analysis of both the standards and liposome samples were carried out in

triplicate. A linear calibration curve of absorbance plotted against phosphate concentration

was obtained and the concentration of the phospholipids in the liposome samples was

determined.

4.3.5 In vitro studies of Stealth liposomes on BT-474 cells

BT-474 breast cancer cell lines were cultured using DMEM culture medium (supplemented

with 10% serum, 1% L-glutamine and 1% penicillin-streptomycin) in an incubator

maintained at 37C and 5% CO2. The culture medium was changed every three days. The

cytotoxicity of 2 mg of sirolimus encapsulated conjugated and non-conjugated DPPC and

DSPC Stealth liposomes were determined in-vitro. Cells were seeded in a 24 well flat-bottom

plate at a cell density of 1 × 106 cells/well and incubated for 24 hr. After 24 hr the liposome

formulations were added to the 24 well plates at various concentrations for 72 hr. 100 µl of


MTT solution (5 mg/ml) was added to each well at the end of the incubation time and

incubated at 37C for another 2 hr. The culture medium was discarded, followed by addition

of 200 µl of acidified isopropanol to dissolve the MTT formazan crystals. 100 µl of the

dissolved MTT formazan crystals was then transferred into a 96 well flat-bottom plate and

absorbance was read at 492 nm using a microplate reader.

4.3.6 Cellular uptake of conjugated Stealth liposomes

The cellular uptake of empty liposome formulations was determined using a Nikon

fluorescent microscope. 20 × 10³ cells/well were seeded on cover slips in a 24 well flat-

bottom plate. Rhodamine (10 µg/10 mg of lipid) stained transferrin conjugated Stealth

liposome formulations were incubated with the cells for 24 h. The cell medium was

discarded from the well after 24 hr incubation time and washed three times with PBS. 1 ml of

4% p-formaldehyde was added to each well to fix the cells on the cover slips and left in the

dark for 15 min. The p-formaldehyde was discarded from the wells and the cells were washed

three times with PBS and then mounted on a glass slide using vectashield mounting medium

containing DAPI. The cover slips were sealed on a glass slide with nail polish and left to dry.

Images of the localisation of the liposomes in the cell were acquired using a fluorescence

microscope (Nikon, Eclipse Ni-E).

4.3.7 In-vivo studies of transferrin conjugated and non-conjugated Stealth liposomes

In-vivo studies were carried out following treatment protocol previously reported by Zeng et

al., (2010) with slight modification. Breast cancer xenografts were established by

subcutaneously injecting cultured BT-474 breast cancer cells (2 x 106) into female nude mice.

Tumours were allowed to grow without any treatments and monitored for tumour volume

daily by measuring two perpendicular tumour diameters with a calliper [tumour volume

[mm3] = (length [mm]) x (width [mm])2 x 0.5]. The mice were divided into groups of six

with each group comprising 6 mice as listed below (n=6).

Group A =DSPC Stealth liposome (non-conjugated) unloaded

Group B= DSPC Stealth liposome (TF-conjugated) unloaded

Group C= DSPC Stealth liposome (non-conjugated) loaded

Group D= DSPC Stealth liposome (TF-conjugated) loaded

Group E= Sirolimus

Group F= Control (PBS)


Treatments of each formulations and control were administered by intravenous injection via

the mice tail vein. Injection volume was 100 µl of formulations, injected in mice for 5

consecutive days, stopped for 1 day and then the cycle repeated for 14 days. Mice body

weights were recorded daily before and after administration of treatments.

4.3.8 Statistical analysis of tumour regression

Results were expressed as means and standard deviation of the mean (n=3). T-test analysis

(p < 0.05) was used to confirm statistical differences between two treatment groups.

4.4 Results and Discussion

4.4.1 Analysis of conjugated and non-conjugated liposome formulations

The physical properties of the liposomes in terms of size and zeta potential before and after

the transferrin conjugation are shown in Table 4.1. All measurements showed good

polydispersity of less than 0.2 and good stability over six months with a slight increase in

particle size of between 2 nm to 5 nm. Furthermore, the zeta potential of all formulations

showed negligible increase also suggesting excellent stability. The average particle size of

empty (unloaded) DPPC and DSPC Stealth liposomes before and after conjugation were

197 nm and 180 nm, respectively. After the addition of transferrin the particle size of the

liposome dispersions increased to between 6 nm and 14 nm. However, sirolimus loaded

formulations both conjugated and non-conjugated show reduction in particle size of between

37 nm to 76 nm, respectively compared to the empty formulations. An explanation for the

reduction in size (as previously given in Chapter 2) where sirolimus incorporation into

liposomal membranes was found to have different distribution and/or possible interaction in

liposome bilayers thus resulting in particle size reduction (Souza & Teschke, 2003; Castelli et

al., 2005). Similar findings of the reduction in particle size of loaded Stealth liposomes have

been previous reported by Yang et al., (2007). The drug encapsulation efficiency for both the

Tf-conjugated and non-conjugated Stealth dispersions showed high entrapment efficiency

above 90%. Sirolimus incorporation efficiencies were found to be 93.8±0.5 % and 94.1±0.4%

for DSPC-Stealth and DSPC-Stealth-Tf liposomes, respectively. The average amount of


transferrin (assessed by Bradford assay) in correlation to the amount of phospholipids

(assessed by Bartlett assay) was determined to be 4200 µg/10 mg of phospholipid.


Table 4.1 Particle size and zeta potential of extruded Stealth liposome formulations (n= 3). Polydispersity were all less than 0.2.

Months

Particle Size (nm) Zeta Potential (mV)

Empty Drug Loaded Empty Drug Loaded

DPPC/Chol/DSPE

-MPEG-2000

(Stealth)

1 197.0 ± 4.3 160.8 ± 5.8 0.91 ± 0.45 -13.15 ± 5.45

3 201.4 ± 3.7 161.6 ± 5.5 -0.08 ± 0.01 -13.78 ± 3.72

6 204.8 ± 4.1 163.1 ± 6.3 0.70 ± 0.13 -12.01 ± 3.09

DPPC/Chol/DSPE

-MPEG-2000

(TF-Conjugated)

1 203.3 ± 3.6 136.4±4.28 2.77 ± 0.11 -10.24 ± 3.11

3 203.8 ± 2.9 136.1±3.19 2.13 ± 0.58 -10.01 ± 5.73

6 205.2 ± 2.2 140.2±3.42 3.26 ± 0.22 -11.45 ± 4.21

DSPC/Chol/DSPE-

MPEG-2000

(Stealth)

1 180.5 ± 3.0 138.2 ± 3.0 0.59 ± 0.78 -18.22 ± 3.10

3 181.7 ± 4.8 138.8 ± 4.0 -0.47 ± 0.52 -19.66 ± 4.55

6 182.6 ± 3.1 141.3 ± 2.7 1.71 ± 0.12 -19.44 ± 4.88

DSPC/Chol/DSPE-

MPEG-2000

(TF-Conjugated)

1 194.6 ± 3.6 118.2 ± 2.6 -3.19 ± 0.43 -24.14 ± 3.41

3 197.1 ± 2.1 120.1 ± 1.1 -3.72 ± 0.28 -24.67 ± 4.09

6 198.8 ± 1.3 120.6 ± 1.9 -3.63 ± 0.77 -27.95 ± 2.44


4.5 In-vitro studies of liposomes for site-specific drug delivery

In order to evaluate the efficiency of both conjugated and non-conjugated DPPC and DSPC

Stealth formulations the viabilityof BT-474 cells was investigated for 72 hr.

Fig. 4.0 shows that DPPC Tf-conjugated Stealth liposomes induces higher antiproliferative

effect in a dose dependent manner with cell viability reduced to 37% compared to non-

conjugated DPPC Stealth (50%) liposomes. Similar findings were also found with

Tf-conjugated DSPC Stealth liposomes presenting higher antiproliferative effect of up to

11% more than DSPC non-conjugated Stealth liposomes. This observation strengthens the

notion that liposome surface modification with antibody or protein improves the therapeutic

effects of designed drug delivery systems, as previously reported in literature.

Fig. 4.0 Antiproliferative effect of DPPC Stealth formulations (transferrin conjugated and

non-conjugated) on breast cancer cell line BT-474 for 72 hr.

Nevertheless, it is not unusual to find that the conjugation of Tf on the surface of Stealth

liposomes improves the delivery of sirolimus in cancer cells by inhibiting cell proliferation

(Li et al., 2009; Zhuo et al., 2013); however, it was also demonstrated in this study that cell

proliferation effect of TF-conjugated Stealth is affected by lipid composition. Fig. 4.1 shows

30

40

50

60

70

80

90

100

0 100 200 300 400 500 600 700 800 900

Cel

l V

iab

ilty

(%

)


DPPC Stealth TF conjugated Loaded

DPPC Stealth Loaded


that DPPC Tf-conjugated Stealth induces higher cytotoxicity (37%) in B-T474 cancer cells

when compared to the DSPC Tf-conjugated Stealth dispersions (46%).

Fig. 4.1 Antiproliferative effects of DSPC Stealth formulations (transferrin conjugated and

non-conjugated) on breast cancer cell line BT-474 for 72 hr.

Lipids with a higher transition temperature (DSPC) are known to have a more rigid

membrane and thus a higher drug retention profile which significantly influences formulation

behaviour in vitro.

The data in Fig. 4.2 also shows the in vitro cytotoxicity of unloaded non-conjugated and

Tf-conjugated Stealth liposomes of two different compositions. In all cases, the unloaded

(empty) Tf-conjugated and non-conjugated Stealth formulations showed negligible

cytotoxicity with values above 90% .

The experimental findings obtained from the in vitro studies demonstrated that the designed

formulations were effective against BT-474 breast cancer cells. It is however important to

state that several factors such as serum protein and formulation circulation times play

significant role in determining the in vivo performance of a drug delivery system. The aim of

30

40

50

60

70

80

90

100

0 100 200 300 400 500 600 700 800 900

Cel

l V

iab

ilit

y (

%)


DSPC Stealth TF conjugated Loaded

DSPC Stealth Loaded


this study was to evaluate the antiproliferative effect of sirolimus loaded Stealth formulations

and to compare the efficacy of both Tf-conjugated and non-conjugated Stealth liposomes.

The results obtained from this comparison, suggest that DSPC Stealth formulations both

conjugated and non-conjugated are most likely to perform better in vivo due to increased

membrane rigidity. As a result of these observations, it was anticipated that DSPC Stealth

formulations would demonstrate enhanced prolonged circulation time and improved

accumulation of the liposomes at the site of action without premature release of the drug

content.

4.5.1 Cellular uptake of Stealth liposome formulations

In order to confirm the internalization of Tf conjugated Stealth liposome, rhodamine labelled

DSPC Stealth-Tf formulations were incubated with BT-474 cells for different time intervals

of 2, 5 and 24 hr. The data in Fig. 4.3 illustrates significant internalisation and localisation in

the cytoplasm of the cells of the Tf Stealth formulation after 24 hr incubation with BT-474

found at 5 hr.


Fig. 4.2 Antiproliferative effect of Stealth liposomes of transferrin conjugated and non-conjugated on BT-474 breast cancer cells for 72 hr. MTT

assay was access with fixed dose of 800 µg/ml of each formulation with cell density of 1x106 per well.

DPPC Stealth

Unloaded

DPPC Stealth

TF-

conjugated

Unloaded

DPPC Stealth

Loaded

DPPC Stealth

TF

conjugated

Loaded

DSPC Stealth

Unloaded

DSPC Stealth

TF-

conjugated

Unloaded

DSPC Stealth

Loaded

DSPC Stealth

TF

conjugated

Loaded

Liposome dose

(800µg/ml)92.27 91.58 50.45 37.54 91.16 90.011 58.15 46.83

0

20

40

60

80

100

Cel

l V

iab

ilit

y

(%)


Fig. 4.3 Cellular uptake of rhodamine (red) labelled Stealth formulations in BT-474 cancer cells. Cell nucleus is stained with DAPI (blue).

Images on the left (Fig. 4.3) shows the

cellular internalisation of Stealth-TF

liposomes in BT-474 cancer cells.

Liposome vesicles (block C) are well

localised in the cytoplasm of the cells

around the nucleus. Images marked

(A) illustrate DAPI staining of the

nucleus. Images (block B) show

liposome rhodamine vesicles

internalization in the cell. The hollow

middle shown in the image represents

the nucleus of the cells while the red

dots represent liposome nanoparticles.


4.6 In- vivo studies of Stealth formulations for targeted delivery in breast cancer tumour

The BT-474 HER-2 overexpressing breast cancer cells were implanted in mice at a cell

density of 2 x 106 and tumour cells were allowed to grow to a substantial palpable tumour

volume. The developed mice xerographs of BT-474 breast cancer tumour were injected with

a sirolimus ethanolic solution and various sirolimus encapsulated Stealth formulations at dose

of 20 mg/kg. All formulations and drug solution were administered intravenously via the tail

of the mice. The therapeutic effect of drug solution and drug encapsulated formulations were

evaluated alongside with the effects on the weight of the mice.

Fig. 4.4: Comparative therapeutic effects of control and sirolimus anti-cancer drug on tumour

suppression of BT-474 cell cancer.

In order to evaluate the differences in the therapeutic effect of the administered liposome

formulations, an unpaired t-test was used to identify statistical significant difference in the

mean of the tumour tissue mass. Fig. 4.4 shows therapeutic efficiency of sirolimus compared

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Tum

ou

r w

eight

per

mou

se (

g)

Control Sirolimus


to the control. The data are given in terms of the mean of total tumour tissue weight in each

group tother with the standard deviation.

According to the t-test analysis, the effect of sirolimus on total tumour tissue weight was

significantly higher (p < 0.0001) than the control. Sirolimus suppressed total tumour tissue

mass by 53% compared to the control at the end of the treatment. The comparative anti-

tumour efficiency of sirolimus encapsulated liposome formulations is given in Fig. 4.5.

Empty formulations of DSPC Stealth non-conjugated liposome showed no significant

difference compared to the control with a p-value equal to 0.5333. The comparative data of

pure sirolimus and Stealth loaded liposomes showed that the latter induced higher therapeutic

efficiency activity in the tumour bearing mice. DSPC Stealth loaded formulation induce

further reduction in tumour mass of 66% compared to the sirolimus solution. The t-test

analysis showed a significant difference in the anti-tumour efficacy between bulk sirolimus

and encapsulated DSPC Stealth liposomes with a p-value equal of 0.0014 (Figs. 4.4 and 4.5).

Fig. 4.5 illustrates the differences in the therapeutic efficacy of Tf-conjugated and non-

conjugated Stealth loaded formulations. Although DSPC Stealth loaded formulation induced

significant tumour suppression compared to pure sirolimus in tumour bearing mice, the Tf-

Stealth liposomes clearly presented a superior activity in comparison to un-conjugated

liposomes and the pure drug. An increase in the therapeutic efficacy can be seen in the

following ascending order; drug (53%) < DSPC Stealth (63%) < Tf-DSPC Stealth (81%).

The therapeutic effect examined by measuring the suppression of tumour growth given in

terms of tumour volume confirms that both sirolimus and its encapsulated Stealth

formulations inhibit tumour growth (Figs. 4.6-4.8). As expected, free sirolimus treatment

produces higher tumour inhibition as compared to the control (Fig. 4.6). Mice bearing tumour

showed regression in tumour growth six days after treatments compared to the control.

The tumour volume for mice treated with sirolimus was 411 mm3 which is two fold smaller

than the tumour volume of the control (886 mm3) at the end of the treatments. Statistical

analysis using t-test shows that sirolimus effect on tumour growth regression is significantly

different from the control treatment (p <0.0001).


Fig. 4.5 Therapeutic effects of Stealth formulation on tumour suppression of BT-474

tumours. (A) Efficacy of empty (unloaded) and loaded non-conjugated Stealth liposome on

tumour weight; (B) Efficacy of empty (unloaded) and loaded transferrin conjugated Stealth

liposome on tumour weight.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Tum

our

wei

ght

per

mouse

(g)

DSPC/Chol/DSPE-MPEG2000 Stealth (empty)

DSPC/Chol/DSPE-MPEG2000 Stealth (loaded)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Tum

our

wei

ght

per

mouse

(g)

DSPC/Chol/DSPE-MPEG2000/Tf Stealth (empty)

DSPC/Chol/DSPE-MPEG2000/Tf Stealth (loaded)

A

B


The data in Figs. 4.7 and 4.8 also showed that empty DSPC Stealth liposomes did not show

any significant decrease in tumour growth suppression, similar to the control treatment

(p = 0.078). However, loaded DSPC Stealth and DSPC Tf-Stealth liposomes exhibited a

significant decrease in tumour growth compared to pure sirolimus, empty formulations and

control. The DSPC Tf-Stealth loaded presented the highest tumour growth inhibition up to

92% when compared to the control. The tumour volume of mice treated with loaded DSPC

Tf-Stealth was only 74 mm3 compared to loaded DSPC Stealth (411 mm3), sirolimus (411

mm3), empty formulations (808 mm3) and control (886 mm3). The percentage difference

between conjugated and non-conjugated loaded Stealth liposome is 37% with conjugated

Stealth liposome demonstrating higher growth inhibition efficacy. Non-conjugated DSPC

Stealth liposome showed a percentage growth inhibition of 46% while conjugated DSPC Tf-

Stealth showed 8.3% growth inhibition.


Fig. 4.6 Therapeutic effect of control and pure drug (sirolimus) on mice bearing tumour.

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12 14 16

Tum

our

Volu

me

(mm

3)

Time (days)

Control Sirolimus


Fig. 4.7 Therapeutic effect of empty and loaded Stealth formulations on mice bearing tumour.

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12 14

Tum

our

Volu

me

(mm

3)

Time (days)

DSPC/Chol/DSPE-MPEG2000 Stealth (empty)

DSPC/Chol/DSPE-MPEG2000 Stealth (sirolimus)


Fig. 4.8 Therapeutic effect of transferrin conjugated empty and loaded Stealth formulations on mice bearing tumour.

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12 14

Tum

our

Volu

me

(mm

3)

Time (days)


DSPC/Chol/DSPE-MPEG2000/Tf Stealth (sirolimus)


Fig. 4.9 Difference in therapeutic efficiency of control, sirolimus and transferrin conjugated

and non-conjugated loaded Stealth formulations in mice bearing tumour.

The data in Fig. 4.9 shows a summary graph of tumour regression efficiency of sirolimus and

loaded Stealth formulations . The results of this study illustrates that sirolimus Stealth

liposome formulations are effective against BT-474 tumours especially the Tf-conjugated

liposomes. As observed in Fig. 4.10 the liposome formulations were well tolerated by the

mice with no seemly signs of formulation or vehicle related toxicity as seen in the relative

animal weight % which is one of the indications for formulation or vehicle tolerance.

0

100

200

300

400

500

600

700

800

900

1000

Control Sirolimus DSPC Stealth

loaded

DSPC TF-

Stealth loaded

Tum

our

Volu

me

(mm

ᵌ)


Fig. 4.10 Treatment tolerance by mice bearing tumour over the treatment study period of 14

days.

-15

-10

-5

0

5

10

15

0 2 4 6 8 10 12 14

Rel

ativ

e an

imal

wei

ght

(%)

Time (days)

Control

DSPC/Chol/DSPE-MEP2000 Stealth (empty)


-15

-10

-5

0

5

10

15

0 2 4 6 8 10 12 14

Rel

ativ

e an

imal

wei

ght

(%)

Time (days)

Sirolimus

DSPC/Cho/DSPE-MPEG2000 Stealth (loaded)

DSPC/Chol/DSPE-MPEG2000/Tf Stealth (loaded)

A

B


Both empty and drug loaded Stealth formulations showed no significant weight loss (Fig.

4.10) after injection. Empty Stealth formulations and the control vehicle showed a maximum

increase in body weight of about 7%; while mice injected with drug loaded Stealth

formulations showed less than 5% increase in body weight.

Breast cancer is the most common malignancy in women and a major cause of death. One of

the challenges with chemotherapy in cancer treatment is the lack of specificity, which results

in reduced therapeutic index and thus compromises clinical prognosis. Nanothechnology

presents significant tool for combatting these challenges by enabling large amount of various

therapeutic drugs to be embedded into nanoparticles. This consequently increases the half-life

of drugs and minimise adverse systemic toxicity by enhancing therapeutic and

pharmacokinetic profile of the drug.

Targeted delivery of drugs to tumour has become a major trend in chemotherapy

advancement. Nanotechnology comprising of liposome drug delivery system have been

reported severally to show effective delivery of anti-cancer drug by targeting specific ligand

in tumours (Mamot et al., 2005; Li et al., 2009). In this study transferrin was attached on the

surface of Stealth liposome to actively target breast tumours with sirolimus. In-vivo studies

show that sirolimus effectively inhibits tumour growth as previously reported by Liu et al.,

(2005) and Zeng et al., (2010). Sirolimus, which is known to act on mTOR, has also been

reported to be more effective in some type of breast cancers such as HER-2 over expressing

breast cancers. This study also illustrates that sirolimus when encapsulated in Stealth

liposome is able to significantly suppress tumour growth in HER-2 over expressing breast

cancer (BT-474). Moreover, when sirolimus loaded Stealth formulation was conjugated with

transferrin; a higher therapeutic efficacy was observed with the formulation compared to the

non-conjugated loaded Stealth. In other words transferrin conjugation on Stealth surface

dramatically suppresses tumour growth. Sirolimus Tf-conjugated Stealth liposomes could

efficiently suppress tumour growth after administration for a total of 14 days study compared

to the control treatment with only a slight increase in tumour volume after day 10. In general

the sirolimus Stealth liposomes significantly slowed tumour growth and prolonged mice

survival but did not eliminate tumour growth. Similar findings were also reported by Liu et

al., (2005) and Zeng et al., (2010) were sirolimus was demonstrated to have an effective

therapeutic effect on breast cancer cells but was however not able to completely suppress or

eliminate tumour.


4.7 Conclusions

In summary sirolimus was successfully encapsulated into Stealth liposome formulations

which were stable over the period of six months. In-vitro cellular uptake of DSPC Tf-Stealth

demonstrated accumulation of the formulation in BT-474 breast cancer cells. This study has

demonstrated that Stealth liposomes enhanced cytotoxicity effect on tumour cells by

liposome encapsulated sirolimus especially in the presence of transferrin. Stealth liposome

properties such as increased circulation time and dose dependent pharmacokinetics may have

very little influence on the therapeutic outcome of an in-vitro study but will have substantial

benefits when the liposomes are administered in-vivo. Based on the data obtained in this

investigation it can be concluded that Stealth formulations of both transferrin conjugated and

non-conjugated liposomes enhance the delivery of sirolimus in tumour bearing mice

compared to the free drug.

4.8 References

1. Anabousi S, Bakowsky U, Schneider M, Huwer H, Lehr CM, Ehrhardt C (2006) In

vitro assessment of transferrin-conjugated liposomes as drug delivery systems for

inhalation therapy of lung cancer. Eur J Pharm Sci, 29:367–374.

2. Barenholz Y, Amselem S (1993) Liposome Technology, 2nd edition, CRC Press,

Boca Raton, FL. Vol. I, pp. 527.

3. Bufalo D, Ciuffreda L, Trisciuoglio D (2006) Antiangiogenic Potential of the

Mammalian Target of Rapamycin Inhibitor Temsirolimus. Cancer Res, 66:5549 -

5554.

4. Castelli F, Raudino A, Fresta MJ (2005) A mechanistic study of the permeation

kinetics through biomembrane models: Gemcitabine–phospholipid bilayer interaction

Colloid Interface Sci, 285:110-117.

5. Derycke AS, Kamuhabwa A, Gijsens A, Roskams T, De Vos D, Kasran A, Huwyler

et al. ( 2004) Transferrin-conjugated liposome targeting of photosensitizer AlPcS4 to

rat bladder carcinoma cells. J Natl Cancer Inst, 96:1620–1630.

6. Grabarek Z, Gergely J (1990) Zero-length crosslinking procedure with the use of

active esters. Anal Biochem, 185:131–135.

7. Huebers HA, Finch CA (1987) The physiology of transferrin and transferrin

receptors. Physiol Rev, 67:520–582.

8. Joo SY, Kim JS (2002) Enhancement of gene transfer to cervical cancer cells using

transferrin-conjugated liposome. Drug Dev Ind Pharm, 28:1023–1031.


9. Kobayashi T, Ishida T, Okadaa Y, Ise S, Harashima H, Kiwad H (2007) Effect of

transferrin receptor-targeted liposomal doxorubicin in P-glycoprotein-mediated drug

resistant tumor cells. Int J Pharm, 329: 94–102.

10. Li XM, Ding L, Xub Y, Wang Y, Pinga Q (2009) Targeted delivery of doxorubicin

using Stealth liposomes modified with transferrin. Int J Pharm, 373:116–123.

11. Li XM, Ding LY, Xu Y et al. (2009) Targeted delivery of doxorubicin using Stealth

liposomes modified with transferrin. Int J Pharm, 373:116–123.

12. Lu RM, Chen MS, Chang DK, Chiu CY, et al. (2013) Targeted drug delivery systems

mediated by a novel peptide in breast cancer therapy and imaging. PLoS One, 8(6):

e66128.

13. Mamot C, Drummond D, Noble C, et al. (2005) Epidermal growth factor receptor–

targeted immunoliposomes significantly enhance the efficacy of multiple anticancer

drugs in vivo. Cancer Res, 65(24):11631-11638.

14. Nag KO, Awasthi V (2013) Surface engineering of liposomes for stealth behavior.

Pharmaceutics, 5:542-569.

15. Park, JW, Hong K, Kirpotin DB, Colbern G, Shalaby R, Shao Y, et al. (2002) Anti-

HER2 immunoliposomes: enhanced efficacy attributed to targeted delivery. Clin

Cancer RES, 8(4):1172-1181.

16. Perche F, Torchilin VP (2013) Recent Trends in Multifunctional Liposomal

Nanocarriers for Enhanced Tumor Targeting. J Drug Deliv, 2013:1-32.

17. Souza EF, Teschke O (2003) Liposome stability verification by atomic force

microscopy. Rev Adv Mater Sci, 5(1):34-40.

18. Torchilin VP, Weissig V (2003) Liposomes: A Practical Approach. Oxford University

Press: Oxford.

19. Yang T, Choi MK, Cui F, Kim JS, et al. (2009) Preparation and evaluation of

paclitaxel-loaded PEGylated immunoliposome. J Control Release, 120:169-177.

20. Yhee JY, Lee SJ, Lee S, Song S, Min HS, Kang SW (2013) Tumor-targeting

transferrin nanoparticles for systemic polymerized siRNA delivery in tumor-bearing

mice. Bioconjugate Chem, 24(11):1850–1860.

21. Zeng Q, Yang Z, Gao Y, et al. (2010) Treating triple-negative breast cancer by a

combination of rapamycin and cyclophosphamide: An in vivo bioluminescence

imaging study. Eur J Cancer, 46:1132-1143.


22. Zhuo H, Peng Y, Yao Q, et al. (2013) Tumor Imaging and Interferon-γ–Inducible

Protein-10 Gene Transfer Using a Highly Efficient Transferrin-Conjugated Liposome

System in Mice. Clin Cancer Res, 19:4206-4217.


CHAPTER FIVE

Sirolimus anti-cancer activity in prostate cancer cell lines

5.0 Overview

The therapeutic efficacy of sirolimus liposomes was assessed in two different prostate cancer

cell lines (LNCAP and DU-145). Both conventional and Stealth liposomes demonstrated

antiproliferative effect against LNCAP and DU-145 cells. Factors such as incubation time

and lipid composition were demonstrated to play a role in the antiproliferative efficacy of the

liposome formulations with conventional liposome showing a higher antiproliferative effect.

The cellular uptake study also showed that the liposome formulation was internalized in the

cytoplasm of the prostate cancer cells. This study provides encouraging results for further

development of sirolimus liposomes for improved therapeutic efficacy such as specific drug

delivery to cancer sites using a commercial antibody.

5.1 Introduction

Cancer is a global burden that is one of the leading causes of death worldwide especially in

economically developed countries (WHO, 2004 & 2008). Globally it is estimated that about

7.6 million cancer deaths have occurred by 2008 with prostate cancer being rated as the

second most common cause of death in men (Newschaffer et al., 2000; Jemal et al., 2011).

Several research studies have employed various strategies in the development of diagnosis

and treatments to combat prostate cancer (Deshmukh et al., 2014).

The concept and success of molecular targeting strategy of mTOR has been demonstrated in

breast cancer especially in the treatment of HER-2 positive disease. Molecular targeting in

prostate cancer is currently being explored in the clinic and one of the proposed targets is

mTOR (Dufour et al., 2011). A major signalling pathway common to prostate cancer is the

phosphoinositide-3 kinase pathway (PI3K). This pathway involves a number of vital

downstream cellular signalling components one of which is the mammalian target of

rapamycin (mTOR). Activation of mTOR gives rise to serial activation of downstream

molecules which eventually results in cell division. Rai et al., (2010) reported the rationale

for pursing mTOR as a therapeutic target in prostate cancer. In their review, they elaborated

and summarise preclinical and clinical studies of mTOR inhibition in prostate cancer. Several

reports have demonstrated anti-tumour activity of sirolimus in variety of prostate cancer cells.

However, there are currently no reports of a designed carrier system for sirolimus in the

treatment of prostate cancer. In the current study, sirolimus was encapsulated both in

conventional and Stealth liposomes. The efficiency of the designed liposome drug delivery


system was evaluated in-vitro. Cellular uptake studies confirmed the internalisation of the

liposomes in the cytoplasm of the prostate cancer cells.

5.2 Materials

Phosphorus standard solution, perchloric acid, 4-Amino-2-naphthyl-4-sulfonic acid reagent,

ammonium molybdate solution and transferrin were purchased from Sigmal-Aldrich, UK.

Rapamycin was obtained from LC laboratories (Woburn, Massachusetts, USA).


distearoylphosphatidylcholine (DSPC) and dipalmitoyl-phosphatidylcholine (DPPC) were

obtained from Lipoid GmbH (Ludwigshafen, Germany). DSPE-MPEG-2000-NH2 was

purchased from Avanti Lipids, UK. BT-474 cell line was purchased from the American Type

Culture Collection (ATTC: Manassa, Virginia, USA). Cholesterol, Dulbecco’s modified

Eagle’s medium (DMEM), thiazolyl blue tetrazolium bromide (MTT), L-glutamine,

penicillin-streptomycin, fetal bovin serum (FBS) and trypsin were all purchased from Sigmal-

Aldrich (UK).

5.3 Methods

5.3.1 Liposome preparation

Conventional and Stealth liposomes were prepared in the same way as discussed previously

Chapter three; Table 5.0). Briefly, pegylated (Stealth) liposomes were prepared by the thin

film hydration method (Bangham et al., 1965). In brief, various appropriate amounts of lipid

mixtures (DSPC, DPPC and DSPE-MPEG-2000), cholesterol and/or no drug (sirolimus)

were weighed out and dissolved in chloroform. Lipid films were hydrated with deionised

water followed by extrusion. The particle size of the liposome formulations were measured

using a Malvern Zetasizer.


Table 5.0 Liposome formulation compositions.






5.3.2 In-vitro studies of liposomes on prostate cancer cells

LNCAP and DU145 prostate cancer cell lines were cultured using RPMI-16 and DMEM

culture medium (supplemented with 10% serum and 1% penicillin/streptomycin) in an

incubator maintained at 37°C and 5% CO2. The culture medium was changed every three

days. The cytotoxicity of sirolimus drug solution and sirolimus encapsulated (2 mg drug)

DPPC and DSPC liposomes formulations were determined in-vitro. Cells were seeded in a 24

well flat-bottom plate at a cell density of 1 × 106 cells/well and incubated for 24 hr. After 24

hr the liposome formulations were added to the 24 well plates at various concentrations for

72 hr. 100 µl of MTT solution (5 mg/ml) was added to each well plate at the end of the

incubation time and incubated at 37°C for another 2 hr. The culture medium was discarded,

followed by addition of 200 µl of acidified isopropanol to dissolve the MTT formazan

crystals. 100 µl of the dissolved MTT formazan crystals was then transferred into a 96 well

flat-bottom plate and the absorbance was read at 492 nm using a microplate reader.

5.3.3 Cellular uptake of conjugated Stealth liposomes

The cellular uptake of the empty liposome formulations was determined using a fluorescent

microscope (Nikon, Eclipse Ni-E). 20 × 10³ cells/well was seeded on cover slips in a 24 well

flat-bottom plate. Rhodamine (10µg/10mg of lipid) stained Stealth liposome formulation was

incubated with the cells for 24 hr. The cell medium was discarded from the well after 24 hr

incubation time and washed three times with PBS. 1 ml of 4% p-formaldehyde was added to

the well to fix the cells on the cover slips and left in the dark for 15 min. The p-formaldehyde

was discarded from the well and cells were washed three times with PBS and then mounted

on a glass slide using vectashield mounting medium containing DAPI. The cover slips were


sealed on a glass slide with a nail polish and left to dry. Images of the liposome localisation

in the cell were acquired using a Nikon fluorescence microscope.

5.3.4 Statistical Analysis

Results are expressed as means and standard deviation of the mean (n=3). Statistical

significance was also determined by t-test analysis (p < 0.05) for comparison in sirolimus

activity between LNCAP and DU-145 cells. Differences were considered as significant when

the p-value was less than 0.05.

5.4 Results and Discussion

5.4.1 In-vitro study of sirolimus in LNCAP and DU-145 cells.

The particle size of the formulations prepared were all less than 200 nm in size (Table 5.1)

with polydispersity also less than 0.2. The MTT assay results are depicted in Fig. 5.0 showing

the cytotoxicity effect of pure sirolimus in LNCAP and DU-145 cells. Sirolimus induced

antiproliferative effect in both cell lines in a dose dependent manner with cell viability

reaching 10% for LNCAP and 29% for DU-145 at drug concentration of 500 µg/ml. LNCAP

cells shows better sensitivity to sirolimus compared to DU-145 after 24 hr incubation with

sirolimus. An unpaired two tailed t-test statistical analysis carried out at 500 nM drug

concentration shows that there is a significant different in the antiproliferative activity of

sirolimus in both cell lines (p < 0.0006).

Table 5.1 Liposome particle size and zetapotential (n=3).

LIPOSOME FORMULATIONS DPPC

Conventional

Empty

DPPC

Conventional

Loaded

DPPC

Stealth

Empty

DPPC

Stealth

Loaded

DSPC

Conventional

Empty

DSPC

Conventional

Loaded

DSPC

Stealth

Empty

DSPC

Stealth

Loaded

Size

(nm)

182.1±3.4

187.6±5.3

195.2± 2.4

158.6±6.9

170.2±3.1

137.7±2.6

193.7±6.2

144.2±1.7

Zeta

potential

(mV)

0.29±0.6

-10.17± 2.3

0.94±0.23

-15.27± 4.1

0.94±11

-11.38±4.18

-2.47±0.66

-23.46±2.1


Fig. 5.0 Sirolimus antiproliferative activity in LNCAP and DU-145 prostate cancer cells.

5.4.2 Antiproliferative effect of liposome formulations in LNCAP cells

In order to evaluate sirolimus activity in LNCAP cells using the drug carrier system

(liposome) sirolimus was encapsulated in conventional and Stealth liposomes and their

antiproliferative profile were tested in-vitro using the MTT assay.

Both conventional and Stealth liposomes demonstrated induced antiproliferative effect on

LNCAP cells. However conventional liposomes showed a higher cytotoxicity when

compared to the Stealth liposomes. Differences were observed in the cell viability of both

DPPC and DSPC liposome formulations. DPPC liposome formulations of both conventional

and Stealth liposome illustrated a higher antiproliferative effect than DSPC formulations.

The data in Fig. 5.1 shows the cytotoxicity effect of conventional and Stealth liposomes of

various lipid compositions on LNCAP cells. DSPC conventional liposomes reduced cell

viability up to 48% while DPPC conventional liposomes resulted in a further 8% reduction in

cell viability at the maximum liposome dose concentration. A similar trend was also observed

in the Stealth formulations with the DPPC Stealth liposomes inducing a higher cytotoxic

effect.

0

10

20

30

40

50

60

70

80

90

100

0 100 200 300 400 500 600

Cel

l V

iab

ilit

y (

%)

Drug concentration (µg/ml)

LNCAP DU-145

*

*


Fig. 5.1 Antiproliferative effect liposomes formulations on LNCAP cells for 72 hours. (A)

cytotoxicity effect of conventional DSPC and DPPC liposomes and (B) cytotoxicity effect of

Stealth DSPC and DPPC liposomes.

30

40

50

60

70

80

90

100

0 100 200 300 400 500 600 700 800 900

Cel

l V

iab

ilit

y (

%)


LNCAP Prostate cancer cells

DPPC Conventional

DSPC Conventional

30

40

50

60

70

80

90

100

0 100 200 300 400 500 600 700 800 900

Cel

l V

iab

ilit

y (

%)


LNCAP Prostate cancer cells

DPPC Stealth

DSPC Stealth

B

A


5.4.3 Antiproliferative effect of liposome formulations in DU-145 prostate cells

Sirolimus liposomal formulations were also evaluated on DU-145 cells. Similar to

observations for the LNCAP MTT assay, DPPC formulations of both conventional and

Stealth liposome demonstrated higher antiproliferative effect than the DSPC liposome

formulations.

The data in Fig. 5.2 shows the effect of the various liposome formulations on DU-145. DPPC

conventional liposomes reduced cell viability to 46 %, while DPPC Stealth cause a 53% cell

viability reduction. On the other hand, reduction in cell viability between DSPC conventional

and Stealth was only 5% at maximum liposome dose of 800 µg. DSPC conventional induced

cell viability of 54% and DSPC Stealth 59%. A logical explanation for this observation in

DSPC formulations would be the membrane rigidity properties induced by DSPC lipid as a

result of the long acyl chain length hence the higher transition temperature.

Again observations in this study demonstrates liposomes membrane stability and rigidity thus

increase its chances of long circulation when administered in vivo since formulations are

intended for in vivo administrations.


Fig. 5.2 Antiproliferative effect liposomes formulations on DU-145 cells for 72 hours. (A)

Cytotoxicity effect of conventional DSPC and DPPC liposomes; (B) Cytotoxicity effect of

Stealth DSPC and DPPC liposomes.

30

40

50

60

70

80

90

100

0 100 200 300 400 500 600 700 800 900

Cel

l via

bil

ity (

%)


DU-145 Prostate cancer cells

DPPC Conventional

DSPC Conventional

30

40

50

60

70

80

90

100

0 100 200 300 400 500 600 700 800 900

Cel

l via

bil

ity (

%)


DU-145 Prostate cancer cells

DPPC Stealth

DSPC Stealth

A

B


The data in Fig. 5.3 shows a summary of the differences in the antiproliferative effect of

sirolimus liposome formulations on LNCAP and DU-145 prostate cancer cells at a liposome

dose of 800 µg/ml. Sirolimus encapsulated liposomes induce antiproliferative effect on the

prostate cancer cells compared to the empty formulation. It was also observed that all the

liposome formulations of both Stealth and conventional liposomes showed a higher

antiproliferative effect in LNCAP cells (Fig. 5.3) compared to DU-145 cells. This

observation corresponds to results obtained in the MTT assay of the pure drug in Fig 1 which

illustrated higher sensitivity of LNCAP cells to sirolimus.

Fig. 5.3 Differences in the antiproliferative effect of sirolimus-liposomal preparations on

prostate cancer cell lines of LNCAP and DU-145 at 72 hr.

0

10

20

30

40

50

60

70

80

90

100

Cel

l via

bil

ity (

%)

LNCAP DU-145


5.4.4 Cellular uptake of liposome in prostate cancer cells

The data in Fig. 5.4 shows the qualitative cellular uptake of DPPC Stealth liposome

nanoparticles in LNCAP cells. Empty rhodamine stained DPPC Stealth liposomes were

incubated with the LNCAP prostate cancer cell line for 24 hr. According to images A-F in

Fig. 5.4 significant uptake of liposomes were observed after 24 hr incubation. It can also be

seen that liposome nanoparticles are localised in the cell cytoplasm around the nucleus of the

cell.

Fig. 5.4 Cellular internalisation of DPPC Stealth liposomes in LNCAP prostate cancer cells.

Images A-F illustrate liposome uptake in the cytoplasm of cells. The cell nucleus is stained

blue and liposome nanoparticles are labelled red.

5.5 Discussion

Genetic determinant of prostate cancer sensitivity to rapamycin show that the PTEN

(Phosphatase and tensin homolog) status of established prostate cancer cell lines affects it’s

sensitivity to rapamycin (Neshat et al., 2001; Wahdan-Alaswad et al., 2012). PTEN wild type

cells (DU-145) have been previously reported to be less sensitive to rapamycin than PTEN-

null cell lines such as LNCAP (Balakumaran et al., 2009).


In this study antitumour activity of sirolimus in LNCAP and DU-145 was determined in vitro

using the MTT assay. Cell viability data obtained from this study show LNCAP cells to be

more sensitive to sirolimus than the DU-145 cell line. Although several studies have shown

sirolimus to be effective against prostate cancer, one of the drawbacks in clinics is the

increased systemic toxicity of the drug after administration. Drug delivery systems are

proposed intervention for improving the therapeutic effect of anticancer drugs. In this study

sirolimus was incorporated into liposome nanoparticles and were evaluated both in DU-145

and LNCAP prostate cancer cells. Data obtained from this investigation show conventional

formulations to be more effective against prostate cancer cells compared to Stealth

formulations due to the more leaky membrane associated with conventional liposomes. This

observation is due to the formulation composition with mPEG in the Stealth formulation

creating a steric hindrance in the Stealth formulation; thereby resulting in a more stabilised

membrane and drug retention. Although conventional liposomes show higher toxicity effects

against prostate cancer cells, in the clinic Stealth liposomes remain a better choice of

formulation due to their long circulatory time after administration, as previously explained. In

addition, the DPPC Stealth formulation also demonstrated a higher antiproliferative effect

compared to the DSPC Stealth formulation. This is, however, expected due to the lower

transition temperature of the DPPC lipid. The low transition temperature resulting from a

shorter acyl chain length also influences the membrane properties and drug retention profile

of the liposome formulation. Fluorescent images obtained for DPPC Stealth formulations

also confirms the cellular internalisation of the liposomes nanoparticles.

5.6 Conclusions

This study is the first report of the effect of various liposome formulations encapsulated

with sirolimus on both LNCAP and DU-145 prostate cancers cells with different PTEN

status. Although several reports have shown sirolimus therapeutic efficiency in both LNCAP

and DU-145 cells, no research publications have reported on the delivery of sirolimus to

such cells using liposomes. Findings from this study demonstrated liposomes as a potential

carrier for the delivery of sirolimus to prostate cancer cells especially those with mTOR as

the molecular target. Formulations of stealth liposome illustrated uptake of the nanoparticle

by the LNCAP cells. As previously reported in literature (Balakumaran et al., 2009) LNCAP

in this study demonstrated more sensitivity to sirolimus in-vitro than DU-145 when

sirolimus was incubated in the cells both as pure drug or sirolimus liposomal.


5.7 References

1. Balakumaran BS, Porrello A lessandro, Hsu DS, et al. (2009) MYC activity mitigates

response to rapamycin in prostate cancer through eukaryotic initiation factor 4E-

binding protein 1- mediated inhibition of autophagy. Cancer Res, 69(19):7803-7810.

2. Deshmukh RR, Schmitt SM, Hwang C, Dou QP (2014) Chemotherapeutic inhibitors

in the treatment of prostate cancer. Expert Opin Pharmaco, 15(1):11-22.

3. Dufour M, Dormond-Meuwly A, Demartines N, Dormond O (2011) Targeting the

Mammalian Target of Rapamycin (mTOR) in Cancer Therapy: Lessons from Past and

Future Perspectives. Cancers, 3:2478-2500.

4. Jemal A, Bray F, Center MM, Ferlay J, et al. (2011) Global Cancer Statistics. CA

Cancer J Clin, 61:69–90.

5. Jiao J, Wang S, Qiao R, Vivanco I, et al. (2007) Murine cell lines derived from PTEN

in hormone refractory prostate cancer development. Cancer Res, 67:6083-6091.

6. Majumder PK, Febbo PG, Bikoff R, Berger R, et al. (2004) mTOR inhibition

reverses Akt-dependent prostate intraepithelial neoplasia through regulation of

apoptotic and HIF-1 dependent pathways. Nat Med, 10:594-601.

7. Newschaffer CJ, Otani K, McDonald MK, Penberthy LT (2000) Causes of Death in

Elderly Prostate Cancer Patients and in a Comparison Nonprostate Cancer Cohort. J

Natl Cancer Inst, 92(8):613-621.

8. Podsypanina K, Lee RT, Politis C, et al. (2001) An inhibitor of mTOR reduces

neoplasia and normalizes p70/S6 kinase activity in PTEN+/- mice. Proc Natl Acad

Sci, 98:10320-10325.

9. Rai JS, Henley MJ, Ratan HL (2010) Mammalian target of rapamycin: A new target

in prostate cancer. Urol Oncol- Semin Ori, 28:134–138.

10. Wahdan-Alaswad RS, Bane KL, Song Kyung, et al. (2012) Inhibition of Mtorc1

Kinase activates smads 1 and 5 but not smad8 in human prostate cancer cells,

mediating cytostatic response to rapamycin. Mol Cancer Res, 10(6):821-833.

11. World Health Organization. The Global Burden of Disease: 2004 Update.

Geneva:World Health Organization; 2008.


CHAPTER SIX

6.0 Conclusions and Future Work

6.1 Conclusions

Clinical and research studies have recently introduced sirolimus as an anticancer therapeutic

agent for both breast and prostate cancer. The incentive for using sirolimus as an anticancer

agent was based on investigations that recognize the sirolimus-sensitive function of mTOR

(mammalian target of rapamycin) in regulating the cell survival pathway that is hyperactive

in many cancers. Research investigations have also reported the trends in multifunctional

liposomal nanocarriers for enhanced tumour targeting one of which comprises of transferrin

mediated endocytosis pathway. With these findings and knowledge of the clinical limitations

associated with administering drug solutions in mind it is imperative that drug carriers are

developed in order to improve therapeutic effects of therapeutic agents.

In this study transferrin mediated endocytosis pathway and the mammalian target of

rapamycin (mTOR) approach in liposomal drug delivery was evaluated on both breast and

prostate cancer cells. This study investigated the physiochemical properties of sirolimus in

liposome drug delivery systems was initially evaluated using various analytical tools and

then followed by the evaluation of their therapeutic efficiency against breast and prostate

cancer cells. Liposome drug delivery system of both conventional and stealth and was

developed using different lipids with varying transition temperatures (Tm). The formulations

were characterised in terms of particle size and zetapotential for 6 months as an indication

for stability assessment. Conventional and Stealth liposomes were used as carriers for the

encapsulation of sirolimus. The development of sirolimus liposome formulations using the

thin film hydration method followed by extrusion resulted in the formulation of stable

liposome formulations for the effective delivery of the anti-cancer agent sirolimus. The

particle size analysis of both conventional and Stealth liposomes were measured over the

course of 6 months and the results indicated good stability of the formulation with a slight

increase in size of maximum 12nm. However Stealth liposomes both loaded and unloaded

was found to give a smaller particle size than the conventional lipososome. An explanantion

for the difference in size was attributed to the attributed to the addition of MPEG2000 to the

stealth formulation. Sirolimus on the other hand when encapsulated into the liposome

formualtions (conventional and Stealth) was also found to further to reduced the particle size

of the formulations. The observed particle size reduction in the loaded liposomes

formualtions was suggested to be as a result of possible interactions of sirolimus with the


lipid bilayer. Various analytical tools have been previously described to study drug

interactions/encapsulation in liposomes bilayer. Hence further analytical tools was employed

to study the incorporation of sirolimus in the liposome bilayer using AFM, XPS and DSC.

These techniques confirmed sirolimus encapsulation and the parameters such lipid

composition influencing the physiochemical properties of the sirolimus formulations. AFM

and XPS analysis revealed a uniform distribution of sirolimus in the liposome formulations.

DSC studies carried out demonstrated sirolimus incorporation in the liposomes as observed in

the change in the transition temperatures of the liposomes upon sirolimus loading.

Sirolimus liposomal therapeutic efficacy after characterisation was assessed invitro and

invivo both in breast and prostate cancer cells. Factors such as incubation time, lipid

composition and drug loading was studied to evaluate their influence on the the efficacy of

the liposomes formulations. In vitro studies of both conventional and stealth liposomes in

breast and prostate cancer cells showed conventional liposomes to have a better

antiproliferative effect due to the more leaky membrane associated with conventional

liposomes compared to stealth liposomes. Incubating the sirolimus liposomes in the cancer

cells for longer time (72 hr) was also found to improve the antiproliferative effect of the

sirolimus liposome formulations. However increase in the sirolimus drug concentration from

2mM to 5mM was found not to further or significantly improve the antiproliferative effect of

the formulation. Quantitative analysis of liposomes cellular uptake was also evaluated using

a florouscent microscope and the images obtained demonstrated liposomes internalization in

the cytoplasm of cells. Invitro data obtained showed promising results for further

development of the liposomes formulation. Further invitro studies of the Stealth liposomes

comprising transferrin conjugated onto their surface was carried to study the therapeutic

effect of using the active targeting approach in drug delivery to cancer cells. Sirolimus

Stealth liposomes conjugated with transferrin was incubated with breast cancer cells (BT-

474) for active targeting. The MTT assay revealed a further improvement of up to 10% in the

antiproliferative effect of the sirolimus Stealth liposome conjugated with transferrin.

Additional studies involving invivo studies in mice was carried out in mice bearing BT-474

cancer cells. Although conventional liposomes showed a better antiproliferative effect invitro,

it is however not not suitable for drug delivery invivo due to the it’s rapid clearance from the

circulation by the reticulum endothelial system (RES). Stealth liposomes both transferrin

conjugated and non-conjugated was studied invivo and the results demonstrated transferrin

conjugation on the Stealth liposome surface to have the best therapeutic effect compared to

the Stealth liposome (non-conjugated) and the pure drug.


In summary, this study demonstrated that liposome drug delivery could potentially serve as a

carrier of sirolimus for both passive and active delivery of the drug to tumours sites. There

are currently no studies of sirolimus liposomes on prostate cancer cells and very few on

breast cancer cells.

6.2 Future work

The proposed future work for this study includes further characterisation of the liposome

formulations using other techniques such as Raman spectroscopy, NMR and FTIR in order

to evaluate physiochemical properties of the liposomes such as interactions between

sirolimus and the liposome nanoparticles. NMR, FTIR alongside molecular modelling could

also give insights into possible interactions and chemical structures of sirolimus encapsulated

liposomes; hence better understanding of the liposome properties which could give more

insight to their possible therapeutic behaviour. Bioligical studies could also include

evaluation of the precise mechanism of uptake into the cells using various biological cell

probes. In addition, further investigations in prostate cancer using a commercially available

growth factor hormone conjugated on the liposome surface for active delivery to prostate

tumours in-vivo for improved therapeutic efficiency of the formulation. Moreover, a

combination of sirolimus with other anticancer agents in liposome nanoparticles could also be

investigated, particulary in multi-resistance breast and prostate cancers.

sirolimus liposomal formulations for targeting of...

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