Title

Page

Chapter 1

General Introduction

1.1

General Introduction and Overall Aim

1

1.2

The Need for Targeted Therapy of Cancer

5

1.3

Targeting Therapeutics

6

1.3.1

Passive Targeting of Drugs and/or Carriers

11

1.3.2

Ligand-Targeted Therapeutics: Types of Ligand; Advantages and Limitations

14

1.3.2.1

Antibody-based Targeting

16

1.3.2.2

Proteins as Ligands in Targeted Delivery

17

1.3.2.3

Saccharide-targeted Delivery

17

1.3.2.4

Peptide-targeted Delivery

18

1.4

Rationale for the Choice of uPAR as a Target

21

1.4.1

Physiological Role and Functions of uPAR

21

1.4.2

Evidence for Over-Expression of uPAR in Cancer

26

1.4.3

Interaction of Membrane Components with uPAR

27

1.4.4

Targeting uPAR

30

1.5

Cancer Gene Therapy

32

1.5.1

Viral Vectors in Gene Therapy

32

1.5.2

Non-Viral Gene Delivery

36

1.5.2.1

Cationic Polymers in Non-viral Gene Delivery

38

1.5.2.1.1

Poly(ethylenimine) as a Non-viral Vector

38

1.5.2.1.2

Chitosans: Natural Origin Cationic Polymers as Non-Viral Vectors

39

1.5.3

Targeted Non-viral Gene Delivery

43

1.5.3.1

Targeted Lipoplexes

43

1.5.3.2

Targeted Polyplexes

44

1.6

Aims of this Research

48

Chapter 2

Materials and General Methods

2.1

Materials

50

2.1.1

Polymers/oligomers

50

2.1.2

Cell Culture

50

2.1.3

Laboratory Reagents

51

2.2

Equipment

51

2.2.1

Analytical Equipment

51

2.2.2

General Equipment

56

2.3

General Methods

56

2.3.1

Cell Culture

56

2.3.1.1

Thawing of Cryopreserved Cells

57

2.3.1.2

Maintenance of Adherent Cell lines (COS-1, COS-7, MCF-7, Caco-2, DU145 and PC3)

57

2.3.1.3

Maintenance of Cells in Suspension

57

2.3.1.4

Differentiation of U937 Cells

59

2.3.1.5

Cell Counting

59

2.3.1.6

Cell Freezing

59

2.3.2

Evaluation of Cell Growth Using the MTT Assay

60

2.3.3

Evaluation of Cytotoxicity Using the MTT Assay

60

2.3.4

Flow Cytometry: General Procedure for the Analysis of Cells

63

2.3.5

Evaluation of Protein Content Using the Bicinchoninic Acid (BCA) Assay

63

2.3.6

Luciferase Assay – Preparation of Cell Lysate

66

2.3.6.1

Luciferase Assay – Analysis of Cell Lysate

66

2.3.7

Plasmid (pGL3 luc) Amplification, Isolation and Characterisation

66

2.3.7.1

Preparation of Competent E.coli (DH5a)

66

2.3.7.2

Transformation of Competent DH5a

69

2.3.7.3

Amplification of Transformed DH5a

69

2.3.7.4

Isolation and Purification of the pGL3 luc Plasmid

69

2.3.7.5

Quantification of pGL3 luc

70

2.3.8

DNA Agarose Gel Electrophoresis

71

2.3.9

Western Blotting

71

2.3.9.1

Preparation of Cell Lysate

71

2.3.9.2

SDS-PAGE Gel Electrophoresis

73

2.3.10

Solid Phase Peptide Synthesis (SPPS)

77

2.3.11

Gel Permeation Chromatography

77

2.3.12

Photon Correlation Spectroscopy (PCS)

84

2.4

Statistics

84

Chapter 3

Synthesis and Characterisation of Modified Chitosans

3.1

Introduction

85

3.2

Methods

86

3.2.1

Preparation of N,N,N-Trimethyl Chitosan Oligomer (TMO)

86

3.2.2

Preparation of N,N,N-Trimethyl Chitosan Polymer (TMC)

89

3.2.3

Preparation of 6-O-Carboxymethyl N,N,N-Trimethyl Chitosan Oligomers (CMTMO)

89

3.2.4

Introducing Protecting Groups on the Amine of Chitosan

91

3.2.4.1

Trimethylation of Protected Chitosan

93

3.2.4.2

Trimethylated Protected Chitosan’s 6-O-Carboxymethylation

94

3.2.5

Preparation of Fluorescent Chitosan Derivatives

95

3.2.5.1

Fluorescent Derivatives (9-Anthraldehyde)

95

3.2.5.1.1

Chitosan-Anthraldehyde

95

3.2.5.1.2

Trimethyl Chitosan-Anthraldehyde

95

3.2.5.2

Fluorescent Derivatives (5/6-Carboxy Fluorescein)

97

3.2.5.2.1

TMO-Fluorescein (TMO-FAM)

97

3.2.5.2.2

CMTMO-Fluorescein (CMTMO-FAM)

97

3.2.5.3

CMTMO-Oregon Green (CMTMO-OG)

97

3.2.6

Conjugation of Peptides to CMTMO or Fluorescent CMTMO

101

3.3

Results

104

3.3.1

Trimethylation of Chitosan

104

3.3.2

TMO 6-O-Carboxymethylation

109

3.3.3

Protecting the NH2 of Chitosan

109

3.3.3.1

Trimethylation of Protected Chitosan

116

3.3.3.2

Trimethylated Protected Chitosan’s 6-O-Carboxymethylation

116

3.3.4

Fluorescent Derivatives of Chitosan

121

3.3.4.1

Chitosan-Anthraldehyde

121

3.3.4.2

Trimethyl Chitosan-Anthraldehyde

121

3.3.4.3

TMO-FAM

121

3.3.4.4

CMTMO-FAM

123

3.3.4.5

CMTMO-OG

123

3.3.4.6

Conjugation of Peptides

123

3.4

Discussion

128

3.5

Conclusions

132

Chapter 4

Trimethylated Chitosans as Non-Viral Gene Delivery Vectors: Cytotoxicity and Transfection Efficiency

4.1

Introduction

135

4.1.1

Mechanism of Polycation Induced Toxicity

135

4.1.2

Cytotoxicity Assessment

136

4.1.3

Transfection Efficiency Assessment

137

4.1.4

Study Aims and Objectives

137

4.2

Materials and Methods

138

4.2.1

Materials

138

4.2.2

Evaluation of Cytotoxicity

138

4.2.3

Preparation and Characterisation of Polyplexes

140

4.2.4

Transfection of COS-7 and MCF-7 cells

140

4.3

Results

141

4.3.1

Cytotoxicity of Quaternised Chitosan Derivatives

141

4.3.2

Effect of Polyplex Formation on Cytotoxicity

149

4.3.3

Confirmation of Quaternised Chitosan : pGL3 luc Polyplex Formation

149

4.3.4

Transfection of COS-7 and MCF-7 Cells Using Chitosan Derivatives

152

4.4

Discussion

152

4.5

Conclusions

159

Chapter 5

Characterisation of uPA-Expressing Cell Lines and Use of the Prostate Cancer (DU145) and Leukaemic (U937) Cell Lines to Study the Binding and Uptake of Free and Conjugated u7 and u11 Peptide Constructs

5.1

Introduction

161

5.2

Methods

163

5.2.1

Effect of pH and Concentration on FAM, OG and FITC Fluorescence

165

5.2.2

Synthesis and Characterisation of Activated mPEG

165

5.2.2.1

Synthesis and Characterisation of SC-mPEG

165

5.2.2.2

Synthesis and Characterisation of PNP-mPEG

167

5.2.3

Reaction of PNP-mPEG with Peptides

169

5.2.4

Investigation of Ligand-Conjugate Interaction with uPAR Expressing Cells Using Flow Cytometry

171

5.2.4.1

Flow Cytometric Assessment of the Binding of uPA-FITC

171

5.2.4.2

Displacement of uPA-FITC from U937 Cells by uPA, Peptides and Peptide Conjugates

172

5.2.4.3

Flow Cytometry Assessment of the Binding/Uptake of Fluorescently Labelled Peptide-CMTMO Conjugates by U937 Cells

172

5.2.4.4

Flow Cytometry Assessment of the Binding/Uptake of Fluorescently Labelled Peptide-CMTMO Conjugates by DU145 Cells

172

5.2.5

Epifluorescent Microscopy

173

5.2.5.1

Visualisation of Trimethylchitosan 9-Anthraldehyde Cell Association and Uptake

173

5.2.5.2

Visualisation of u11-CMTMO-FAM and u11-CMTMO-OG

174

5.3

Results

174

5.3.1

Effect of pH and Concentration on Probe Fluorescence

174

5.3.2

Peptide conjugation to mPEG

176

5.3.3

Determination of uPAR Content in a Panel of Cell Lines by Western Blotting

181

5.3.4

Development of Flow Cytometry to Detect uPAR

186

5.3.5

Determination of Ligand Affinity Using Displacement Experiments

190

5.3.6

Uptake of u7- and u11- Containing CMTMO-FAM Conjugates

193

5.3.7

Displacement of u11-CMTMO-FAM by uPA or Gu11G

199

5.3.8

Fluorescence Microscopy of DU145 Cells Incubated with u11-CMTMO-FAM and u11-CMTMO-OG

201

5.4

Discussion

204

Chapter 6

Development of a Novel Synthetic Gene Delivery System Using a uPAR Targeted Conjugate

6.1

Introduction

214

6.1.1

Targeted Synthetic Gene Delivery

214

6.1.2

Factors Affecting Polyplex Formation and Methods of Polyplex Characterisation

215

6.1.3

Ideal Characteristics of Polyplexes

216

6.1.4

Parameters Considered and Actions Taken in Preparation of u11-Targeted Polyplexes

218

6.2

Methods

219

6.3

Results

222

6.3.1

Characterisation of TMO51 : pGL3 luc and PEI : pGL3 luc Polyplexes

222

6.3.2

Characterisation of u11-CMTMO : pGL3 luc Polyplexes

222

6.3.3

Gu11G-Coated PEI : pGL3 luc Polyplexes

228

6.3.4

Transfection of COS-7, DU145 and MCF-7 with TMO51 Polyplexes and  u11-CMTMO-Coated TMO51 Polyplexes

228

6.3.5

Characterisation of u11-CMTMO-coated TMO51 Polyplexes

228

6.3.6

Transfection of COS-7, DU145 and MCF-7 with u11-CMTMO-Coated PEI Polyplexes

235

6.3.7

Characterisation of u11-CMTMO-Coated PEI Polyplexes

235

6.4

Discussion

235

Chapter 7

General Discussion

7.1

Non-Viral Gene Therapy: a Perspective

243

7.2

Trimethylated Chitosan as a Non-Viral Vector

245

7.3

Is uPAR the Correct Target for Cancer Gene Therapy?

246

7.4

Developing a Targeted Non-Viral Vector

247

7.5

uPAR Targeted Drug Delivery

248

7.6

Conclusions

249

7.7

Future Work

249