Development of peptide-targeted gene delivery systems

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Jan 23, 2002 - 9 Investigation of affinity of selected phage clones for cellular receptors . ..... Release of plasmid DNA from pHPMA coated RPC187/DNA complexes, 127 ..... demonstrated that introduction of functional p53 into p53- cell lines can be ...... The most efficient systems for large scale production of recombinant.

Development of peptide-targeted gene delivery systems

Alan Parker

A thesis submitted to the Division of Cancer Studies of the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY

CRUK Institute for Cancer Studies The University of Birmingham June 2002

1

Abstract Gene therapy promises cures for many diseases, yet clinical efficacy is thwarted by inefficient delivery and expression of therapeutic transgenes. This project has aimed to produce a new generation of delivery vectors, combining stability within biological environments with selective, receptor mediated transgene expression in target cells.

Initial studies focused on the binding of poly(L-lysine) to plasmid DNA and the properties of the resulting nanoparticles. Although stable in HEPES, the nanoparticles aggregated rapidly in physiological salts, preventing their use for systemic gene delivery. Surface modification with the multivalent hydrophilic polymer poly-[N-(2-hydroxypropyl)methacrylamide] yielded stabilised vectors with good biodistribution properties but poor transfection activity. Introducing reducible linkages into the polycation provided a trigger mechanism that allowed combination of extracellular stability with availability of DNA for transcription within cells. Chemical linkage of targeting ligands, including the oligopeptide SIGYPLP, increased transduction of receptor positive cells. Finally phage display technology was used to iterate the basic fibroblast growth factor (bFGF) receptor-binding peptide MQLPLAT.

Phage

bearing this peptide bound bFGF receptors with a Kd of 2.51 x 10- 10 Min vitro, and showed enhanced binding to tumour tissue compared with non-tumour in surgically-resected human biopsies, raising the possibility of selective tumour-targeting of vectors for therapeutic gene delivery.

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Acknowledgements I would like to thank Dr. Len Seymour for his guidance over the last 4 years, and for the faith he has shown in me that has frequently been above and beyond the call of duty. I should also like to extend my gratitude to my good friend and colleague Dr. Fukuto Maruta for his assistance with the phage display work, particularly for his help with the clinical evaluation of the isolated samples. I would also like to acknowledge the help of Dr. David Oupicky, who was responsible for the synthesis of the polymers described here, and Dr. Kerry Fisher for his assistance with the adenovirus work.

A big "thank you" to all the members of "Team Seymour", both past and present, who have helped make my time in Birmingham both productive and fun, and to the members of "Sporting Mullet FC" - surely the finest collection of footballers the Department of Cancer Studies has ever seen. Monday and Wednesday lunchtimes will no longer be the same!

I would also like to acknowledge the part played by the best group of friends I could have possibly been blessed with.

Thanks to all my friends from the Wirral, from Sheffield

University (particularly of the Turners Lane era) and most recently from Birmingham. Especially, I would like to thank Kellie for her love and support, through good times and bad.

Lastly, but most importantly, I would like to thank my family for the help and continual support they have offered me.

Most of all thanks to Dad, without your love and

encouragement, I doubt I would be writing these words.

lll

For my parents

IV

Contents List of figures List of tables Abbreviations 1

INTRODUCTION ............................................................................................................ 1 1.1 BACKGROUND .................................................................................................................. 1 1.2 GENE TIIERAPY FOR CANCER ........................................................................................... 4

1.2.1 Gene augmentation therapy. ................................................................................... 6 1. 2. 2 Cytotoxic gene therapy ........................................................................................... 7 1. 2. 3 Anti-angiogenesis.................................................................................................... 8 1.2.-1 Targeted inhibition ofgene expression ................................................................... 9 1.2.5 Immunotherapy ..................................................................................................... 11 1.3 DELIVERY VECTORS FOR GENE TIIERAPY ....................................................................... 13

1.3.1

Viral vectors .......................................................................................................... 1-l

1.3.1.1 Retrovirus .......................................................................................................... 14 1.3.1.2 Lentivirus .......................................................................................................... 15 1.3.1.3 Adenovirus ........................................................................................................ 16 1.3.1.4 Adeno-associated virus ..................................................................................... 18 1. 3 .1. 5 Herpes simplex virus ........................................................................................ 19 1. 3 .1. 6 Other viral vectors ............................................................................................ 19 1.3.2 Non-viral vectors .................................................................................................. 20 1.3.2.1 Naked DNA ...................................................................................................... 20 1.3.2.2 Lipoplexes ......................................................................................................... 21 1.3.2.3 Polyplexes ......................................................................................................... 23 1.3.2.4 Enhancement oftransgene expression using ultrasound .................................. 26 1.3.2.5 Enhancing Gene transfer using Biolistics ......................................................... 27 1.3.2.6 Other non-viral vectors ..................................................................................... 28

1.3.3

Other DNA delivery vectors .................................................................................. 29

1.3.3.1 Non-viraVviral hybrid vectors .......................................................................... 29 1.3.3.2 Filamentous bacteriophage ............................................................................... 30 1.3.3.3 Bacteria ............................................................................................................. 31 1. 4 THE CHALLENGE OF SYSTEMIC GENE DELIVERY ............................................................. 32

1.4.1

Extracellular barriers ........................................................................................... 33

1.4.1.1 Interactions with blood serum components ...................................................... 33 1.4 .1.2 Non specific membrane interactions ................................................................. 36 1.4.1.3 Physical barriers to extravasation ..................................................................... 36 1. 4. 2 Intracellular barriers ............................................................................................ 3 7 1.4.2.1 Endosomal release ............................................................................................ 38 1.4.2.2 Nuclear uptake or expression in the cytoplasm? .............................................. .40 1.4.2.3 Vector unpackaging ......................................................................................... .42 1.5 TARGE11NG STRATEG1ES ................................................................................................ 43 1.5.1 Factors influencing suitability of targeting ligands ............................................ .44 1.5.1.1 Receptor biology in vivo .................................................................................. .44 1.5.1.2 The consequences of receptor binding .............................................................. 45 1.5.1.3 Ligand pharmacology in vivo .......................................................................... .46 1.5.1.4 Cost and ease of ligand production .................................................................. .47 1. 5. 1. 5 Receptor targeting and resistance ..................................................................... 4 7 1.5.2 Choice of targeting ligand .................................................................................... -17 1.5.3 Phage display technology ..................................................................................... -19 v

1.5.3.1 M13 biology ...................................................................................................... 50 1.5 .3 .2 p nnc1p . . 1e of panmng . usmg . ph age d"1sp1ay l"b . ........................................... 51 1 ranes

1.5.3.3 Applications of phage display technology ........................................................ 53 1.6 THESIS OBJECTIVES ........................................................................................................ 54

2

MATERIALS AND METHODS ................................................................................... 55 2.1 SUPPLIER OF MATERIALS ................................................................................................ 55 2.2 PURCHASE AND PREPARATION OF NUCLEIC ACIDS .......................................................... 55 2.2.1 Sources ofDNA ..................................................................................................... 55 2.2.2 Plasmid propagation, isolation and purification.................................................. 55 2.2.3 Quantification ofDNA .......................................................................................... 56 2.2.4 Labelling ofDNA .................................................................................................. 56 2.2.5 Preparation ofGFP mRNA ................................................................................... 58 2.3 ASSEMBLY OF PLL/DNA COMPLEXES ........................................................................... 59 2. 3.1 Calculation of charge ratio ................................................................................... 59 2.3.2 Formation ofpLLIDNA complexes ....................................................................... 60 2. 3. 3 Formation ofpHPMA surface modified pLL/DNA complexes ............................. 60 2. 4 PHYSICOCHEMICAL CHARACTERISATION METHODS ....................................................... 61 2.4.1 Gel shift assay to show complex formation .......................................................... 61 2. 4. 2 Measurement of complex formation by light scattering ....................................... 61 2. 4. 3 Monitoring particle formation by inhibition ofEtBr/DNA fluorescence .............. 61 2.4.4 Determination offluorescence excitation spectra ................................................ 62 2.4.5 Analysis ofparticle size by photon correlation spectroscopy (PCS) .................... 62 2.4. 6 Analysis ofparticle surface charge by zeta potential analysis ............................. 62 2.-1. 6 Quantification of residual reactive amino groups by fluorescamine assay.......... 63 2.4. 7 Monitoring paranitrophenol release .................................................................... 63 2.4.8 Transmission electron microscopy (TEM) ............................................................ 64 2. 5 CELL CULTURE .............................................................................................................. 64 2. 5.1 Maintenance of established cell!ines ................................................................... 65 2.5.2 Storage and resuscitation of celllines .................................................................. 65 2.5.3 Determination of viable cell number .................................................................... 66 2.5.4 Cell lines ............................................................................................................... 66 2.6 BIOLOGICAL EVALUATION OF COMPLEXES ..................................................................... 67 2. 6.1 Uptake of 32P labelled pLLlDNA complexes......................................................... 67 2.6.2 Uptake ofYOY0-1/abelledpLL/DNA complexes ................................................ 67 2. 6. 3 Transfection of cell lines in vitro .......................................................................... 68 2.6.4 Evaluation of cell viability by MTS assay............................................................. 68 2. 6. 5 Bicinchoninic acid assay ...................................................................................... 69 2.7 VIRUS PRODUCTION AND MANIPULATION ...................................................................... 70 2. 7.1 Virus propagation and purification ...................................................................... 70 2. 7.2 Polymer modification of virus............................................................................... 70 2. 7.3 Conjugation ofbFGF to pHPMA-Ad5.................................................................. 70 2. 7. 4 Infection protocol using Ad5//3-gal ....................................................................... 71 2. 7.5 Analysis of f3-Gal transgene expression using Ad-5/flgal .................................... 71 2. 8 SYNTHESIS OF POLY"MERS .............................................................................................. 71 2. 8.1 Oxidative polymerisation of Cys(Lys) 10Cys into reducible polycations (RPC) .... 71 2. 8.1. 1 Ellman's assay for measurement of residual free thiol groups ......................... 73 2.8.1.2 Size exclusion cation HPLC analysis ............................................................... 73 2.8.1.3 TNBS (2,4,6-trinitrobenzenesulphonic acid) assay for the determination of free . groups ................................................................................................................ .74 amino VI

2. 8. 2

P rod uctzon · oJ-~'peptz " ·de-p HPMA conjugate · ............................................................ 7...;

2. 9 ANIMAL EXPERIMENTS ................................................................................................... 7.f

2.9.1 Animals ................................................................................................................. 7-1 2.9.2 Preparation ofC57 black 6 mice bearing Bl6FJO tumours ................................ 75 2. 9. 3 Admznzstratzon . . · . oJ-~' samp · les to C'57 black 6 mzce · ................................................... ; ) 2.10 BIOPANNING FILAMENTOUS PHAGE AND RELATED PROCEDURES ................................ 75 2.1 0.1 Preparation of minimal agar plates ...................................................................... 76 2.1 0. 2 Strain maintenance ............................................................................................... 76 . oJJ p hage by p laque assay {;;tzterzn . . g) .............................................. 77 2. 10. 3 Quantzifi'zcatzon 2.1 0. 4 Amplification of recovered phage ......................................................................... 77 2.10.5 Iteration ofFGF receptor-binding peptide by phage display technology ............ 79 2.1 0. 6 Isolation and sequencing ofphage DNA .............................................................. 79 2.1 0. 7 Evaluation of cell binding activities of selected phage clones ............................. 80 2.1 0. 8 Immunohistochemical analysis of selected phage binding cells ........................... 81 2.1 0. 9 Investigation of affinity of selected phage clones for cellular receptors .............. 82 2.1 0.10 Competitive inhibition of selected phage clones binding FGF receptors............ 82 2.10.11 Phage binding assay on surgically resected human specimens........................... 83 2.11 EXPRESSION OF DATA ................................................................................................ 83 "7-

3. CHARACTERISATION OF COMPLEXES FORMED BY SELF ASSEMBLY OF PLASMID DNA WITH POLY(L-LYSINE) ........................................................................ 84 3.1 INTRODUCTION .............................................................................................................. 84 3.2 RESULTS ........................................................................................................................ 85 3.2.1 Influence of charge ratio on electrophoretic mobility of DNA ............................. 85 3.2.2 Influence of charge ratio on intensity of light scattering...................................... 86 3.2.3 Effect ofEtBr concentration on the fluorescence ofpLLIDNA complexes ........... 90 3.2.4 Evaluation of the mechanism of inhibition ofEtBr!DNAfluorescence ................ 92 3.2.5 Comparison of excitation spectra ofEtBr, DNA!EtBr and pLLIDNA/EtBr......... 94 3.2.6 Determination of the stability ofpLVDNA complexes to dilution in water......... 94 3.2. 7 Effect polycation molecular weight and N:P ratio on stability to dilution ........... 98 3.2.8 Effect ofphysiological salt levels on polyp/ex destabilisation. ............................. 98 3.2.9 Effect ofN:P ratio and pLL molecular weight on salt induced aggregation ....... 99 3.3 DISCUSSION ................................................................................................................. 103

4 DEVELOPMENT OF STEALTH DNA DELIVERY VECTORS CAPABLE OF RECEPTOR MEDIATED UPTAKE AND TRIGGERED INTRACELLULAR ACTIVATION ...................................................................................................................... 109 4.1 INTRODUCTION .................................................. ' ......................................................... 109 4.2 RESULTS ...................................................................................................................... 113 4.2.1 Surface modification of pLLIDNA complexes with the multivalent hydrophilic polymer pHPMA. ............................................................................................................ . 113 4. 2. 2 Zeta potential analysis ofpHPMA coated pLLIDNA complexes ........................ 115 4. 2. 3 Stability ofpHPMA coated polyp/exes to salt-induced aggregation .................. 115 4.2. 4 Stability ofpHPMA coated pLLIDNA complexes to disruption by polyanions .. 116 4.2.5 Influence of polycation molecular weight on polyanion stability of coated complexes ........................................................................................................................ 119 4.2.6 Oxidative polymerisation of low molecular weight thiolated cationic oligopeptides into high molecular weight, reducible linear polymers ........................... 121 4.2. 7 RPC mediated transgene expression of mRNA based complexes ....................... J23 .. Vll

-1.2. 7 Destabilisation ofpHPMA coated RPC/DNA complexes by reduction .............. 126 4.2.8 Effect ofpHPMA concentration on the transfection efficiency ofpLL 21 ]!'DNA and RPC1s1/DNA complexes .................................................................................................. 128 -1.2.9 Kinetics oftransgene expression ........................................................................ 130 .:/. 2.10 Incorporation of targeting ligands onto pHPMA coated complexes .................. 131 -1.2.11 Enhanced transgene activity using bFGF targeted pHPMA-RPC187 DNA complexes ........................................................................................................................ /33 4.3 DISCUSSION ................................................................................................................. 136

5 RETARGETING GENE THERAPY VECTORS USING THE PUTATIVE ENDOTHELIAL-CELL BINDING PEPTIDE SIGYPLP ............................................... 142 5.1 INTRODUCTION ............................................................................................................ 14 2 5.2 RESULTS ...................................................................................................................... 144 5.2.1 Self assembly ofSIGYPLPGGGS(K) 1 ~DNA polyp/exes .................................... 14-1

5.2.2 Uptake ofSIGYPLP targeted complexes in vitro ................................................ /46 5.2.3 Uptake ofYoYo-1/abelledplasmid DNA in vitro ............................................... 148 5.2.4 Transfection activity ofSIGYPLP targeted complexes in vitro .......................... 148 5.2.5 Comparison transgene expression using SIGYPLP targeted polyp/exes and commercially available transfection reagents ................................................................ 152 5.2. 6 Effect ofserum on transgene expression ............................................................ 153 5.2. 7 Production of a serum stable polyp/ex targeted with SIGYPLP ......................... 156 5.2.8 Transfection efficiency ofSIG-pHPMA coated complexes in vitro .................... 158 5.2.9 In vivo pharmacology of SIG-pHPMA coated complexes in B16FJO tumour bearing C57 black 6 mice ............................................................................................... 160 5. 2.10 Retargeting adenovirus using SIG-pHPMA ..................................................... .. 162 5.3 DISCUSSION ................................................................................................................. 165

6 IDENTIFICATION OF OLIGOPEPTIDES THAT BIND TO FGF RECEPTORS FOR TARGETED GENE DELIVERY .............................................................................. 170 6.1 INTRODUCTION ............................................................................................................ 170 6.2 RESULTS ...................................................................................................................... 172 6.2.1 Quantification ofphage binding FGF receptors ................................................ 172

6. 2. 2 Enhanced affinity of evolved library through rounds ofselection. ..................... 172 6.2.3 Iteration of consensus oligopeptide sequences binding FGF2 receptors ........... 173 6.2.4 Relative binding of iterated phage clones to FGF receptors on 911 cells ......... 173 6.2.5 Quantification of binding ofphage clones to FGF receptors on cells ............... 176 6. 2. 6 Immunohistochemical detection ofMQLPLAT phage binding 911 cells ........... 180 6.2. 7 Enhanced cellular association ofMQLPLAT phage at 37 CC' .......•.................•.. 180 6. 2. 8 Measurement ofaffinity of binding ofMQLPLAT phage ................................... 182 6.2.9 Quantification ofphage elution using FGF........................................................ 184 6. 2.10 Dose dependent elution ofMQLPLAT phage from 911 cells ............................. 184 6.2.11 Competitive inhibition ofMQLPLAT phage binding to 911 cells ...................... 187 6.2.12 Mitogenicity analysis ofMQLPLAT ................................................................... 189 6.2.13 Enhancement of gene expression by using MQLPLAT to target polyelectrolyte DNA complexes ............................................................................................................... 189 6.2.14 Evaluation of MQLPLAT phage binding to human tumours in surgically resected gastric cancer ................................................................................................................ . 191 6.3 DISCUSSION ................................................................................................................. 196 Vlll

7

FINAL DISCUSSION ................................................................................................... 201 7.1

CONCLUDING REMARKS.................................................................................. . .......... 20 1

7.2 FUTURE WORK ............................................................................................................. 205

8

REFEREN CES .............................................................................................................. 209

Publications

IX

List of Figures Chapter 1 Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure Figure Figure Figure

1.5 1.6 1.7 1.8

Chapter 3 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure 3.7 Figure 3.8 Figure 3.9

Chapter 4 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 Figure 4.10 Figure 4.11 Figure 4.12

Introduction In vivo and ex vivo approaches gene therapy, 2 Disease targets of gene therapy as a proportion of ongoing clinical trials, 5 Delivery vectors used for gene therapy as a proportion of ongoing clinical trials, 14 chemical structures of two commonly used cationic lipids used to form lipoplexes, 22 Chemical composition of polycations commonly used to form polyplexes, 24 Intracellular barriers to trans gene expression, 3 8 The structure of the M13 bacteriophage, 50 Identification of cellular receptor binding peptides by phage display technology, 52

Characterisation of Complexes Formed by Self Assembly of Plasmid DNA with Poly(L-Lysine) Influence of charge ratio on electrophoretic mobility of DNA, 88 Effect of charge ratio on light scattering intensity, 89 Effect of EtBr concentration on the fluorescence profile of pLL/DNA complexes, 91 EtBr exclusion during polyplex formation, 93 Excitation spectra ofEtBr, DNA/EtBr and pLL/DNA/EtBr, 95 Effect of dilution in water on restoration of EtBr/DNA fluorescence, 96 Effect of dilution on particle size, 97 Effect of physiological salt on restoration of EtBr/DNA fluorescence, 100 Effect of N:P ratio and polycation molecular weight on salt induced aggregation of polyplexes, 101

Development of Stealth DNA Delivery Vectors Capable of Receptor Mediated Uptake and Triggered Intracellular Activation The chemical structure of the multivalent, hydrophilic polymer pHPMA, 111 Production of a laterally stabilised, receptor targeted polyplex, 112 Reactivity of poly N-(2-hydroxypropyl)methacrylamide (pHPMA-ONp )with pLL/DNA complexes, 114 Zeta potential analysis of pHPMA coated pLL/DNA complexes, 117 Effect of physiological concentrations of salt on the hydrodynamic diameter of unmodified and pHPMA coated pLL/DNA complexes, 118 pHPMA mediated polyanion stability of pLL/DNA complexes, 120 Effect of polycation molecular weight on pHPMA coated complex stability to polyanions, 120 Formation of a disulphide reducible polycation from Cys(Lys)wCys monomers, 122 Transfection activity ofmRNA based complexes, 125 Release of plasmid DNA from pHPMA coated RPC 187/DNA complexes, 127 Effect of [pHPMA] on levels of trans gene expression of pLL211/DNA and RPC181/DNA complexes, 129 Kinetics of gene expression ofpLL211 /DNA and RPC 181/DNA complexes, 132 X

Figure 4.13 Figure 4.14

Chapter 5 Figure 5.1 Figure 5.2 Figure 5.3 Figure 5.4 Figure 5.5 Figure 5.6 Figure 5.7 Figure 5.8 Figure 5.9 Figure 5.10 Figure 5.11 Figure 5.12

Chapter 6 Figure 6.1 Figure 6.2 Figure 6.3 Figure 6.4 Figure 6.5 Figure 6.6 Figure 6.7 Figure 6.8 Figure 6.9 Figure 6.10 Figure 6.11 Figure 6.12 Figure 6.13 Figure 6.14

Monitoring the incorporation of targeting ligands onto pHPMA coated complexes, 134 Enhanced transgene expression of pHPMA-RPC1s1/DNA complexes targeted with bFGF, 135

Retargeting Gene Therapy Vectors Using the Putative Endothelial-cell Binding Peptide SIGYPLP Formation ofSIGYPLP targeted polyplexes, 145 32 Uptake of P dCTP labelled complexes in vitro, 147 Fluorescence microscopy (a) and FACS analysis (b) showing cellular uptake of SIGYPLPGGGS(KhJDNA and 3.4 kDa pLL/DNA complexes, 149 Transfection activity of polyplexes targeted with SIGYPLP in (a) HUVE (b) 911 (c) SKOV-3 and (d) B 16F10 cells, 151 Enhanced transgene expression of SIGYPLP targeted polyplexes compared to commercially available transfection reagents, 154 Effect of serum on transgene expression, 155 Production of a peptide targeted, serum stable polyplex, 157 Incorporation of the peptide into pHPMA does not effect the capacity of the polymer to protect polyplexes from polyanion mediated dissociation, 157 Enhanced transgene expression of pHPMA coated complexes following incorporation of SIGYPLP into the reactive polymer, 159 Blood clearance profile for pHPMA and SIG-pHPMA coated polyplexes, 161 In vivo tumour accumulation of pHPMA and SIG-pHPMA coated polyplexes, 161 Effect of surface modification and retargeting on Ad5 mediated f3-gal trans gene expression, 164

Identification of Oligopeptides that bind to FGF Receptors for Targeted Gene Delivery Association of naive phage library with 911 cells, 174 Phage recovery from 911 cells by elution with FGF with increasing rounds of selection, 174 Iteration of consensus oligopeptide sequences binding FGF2 receptors, 175 Elution of phage clones from 911 cells, 177 Relative binding of selected phage clones to receptors on cell lines, 179 HRP detection of MQLPLAT phage binding to 911 cells, 181 Measurement of binding affinity ofMQLPLAT bearing phage, 183 Elution with 10 !J.g/ml FGF removes the majority of cell associated MXXP bearing phage, 185 Dose dependent elution ofMQLPLAT phage from 911 cells, 186 Competitive inhibition ofMQLPLAT phage binding 911 cells, 188 Evaluation of possible mitogenicity ofMQLPLAT peptide, 190 Enhancement of gene expression by using MQLPLAT to target polyelectrolyte DNA complexes, 192 Schematic showing the locality of gastric adenocarcinomas isolated from patients used in this study, 194 phage accumulation in surgically resected human tumour specimens, 195

Xl

List of Tables Chapter 1 Table 1.1 Table 1.2

Chapter 5 Table 5.1

Chapter 6 Table 6.1 Table 6.2

Introduction Familial cancers caused by tumour suppressor gene mutations~ potential targets for gene replacement strategies, 6 Examples of targeting ligands under development for gene delivery, 48

Retargeting Gene Therapy Vectors Using the Putative Endothelial-cell Binding Peptide SIGYPLP Size and monodispersity of polyplexes used in this study, 145

Identification of Oligopeptides that bind to FGF Receptors for Targeted Gene Delivery Relative binding affinities for MQLPLAT phage and insertless phage, 183 Origins of samples obtained for evaluating phage binding to human tumours in surgically resected gastric cancer, 194

.. Xll

Abbreviations AAV

Adeno associated virus type 2

ADA

Adenosine deaminase

ADEPT

Antibody direct prodrug therapy

AdS

Human adenovirus type 5

AdS/(3-Gal

Adenovirus expressing J3 -galactosidase

AFP

a-foetoprotein

AVET

Adenovirus enhanced transferrinfection

(3-Gal

E. coli J3 galactosidase

BCA

Bicinchoninic acid

bFGF

Basic fibroblast growth factor

BIV

Bovine immunodeficiency virus

bp

Base pair

BSA

Bovine serum albumin

CAR

Coxsackie and Adenovirus receptor

CB1954

5-( aziridin-1 yl)-2, 4-dinitrobenzamide

CEA

Carcinoembryonic antigen

CMV

Human cytomegalovirus

CNS

Central nervous system

Da

Dalton

DAB

3 ,3-diaminobenzidine tetrachloride

DMEM

Dulbecco's modified Eagles medium

DMSO

Dimethyl sulphoxide

DNA

2-Deoxyribonucleic acid

DOGS

Dioctadecylamidoglycyl spermine

DOTAP

N-[ 1-(2,3-dioleoyloxy)propyl]-N,N,N, trimethyl ammonium methyl-sulphate (DOTAP)

EDTA

Ethylenediamine tetra-acetic acid

EGF

Epidermal growth factor

EIAV

Equine infectious anaemia virus

EPR

Enhanced permeability and retention

EtBr

Ethidium bromide

5-FC

5-Fluorocytosine Xlll

FCS

Foetal calf serum

FGF

Fibroblast growth factor

FITC

Fluorescein isothiocyanate

FIV

Feline immunodeficiency virus

5-FU

5-Fluorouracil

GDEPT

Gene directed enzyme prodrug therapy

GFP

Green fluorescent protein (from Aequorea victoria)

GM-CSF

Granulocyte macrophage colony stimulating factor

GSH-MEE

Glutathione monoethylester

HE PES

N-[2-hydroxyethyl]-piperazine-N' -[2-ethanosulphonic acid]

DINGS

Heat inactivated normal goat serum

HIV

Human immunodeficiency virus

HPLC

High pressure liquid chromatography

HSV-tk

Herpes simplex virus thymidine kinase gene

pHPMA

Poly [N-(2-hydroxypropyl) methacrylamide]

HRP

Horseradish peroxidase

HVEM

Herpes virus entry mediator protein

HVJ

Hemagglutinating virus of Japan

IFN-y

Interferon gamma

IL

Interleukin

IPTG

Isopropyl-~-thiogalactopyranoside

ITR

Inverted terminal repeats

l.V.

Intra-venous Wavelength (in nm) Litre

LB

Liquid broth

LPD

Lipid/protamine/DNA

f.l

Micro

M

Molar

MHz

Megahertz

mm

Minute(s)

ml

millilitre

mM

millimolar

IDOl

Multiplicity of infection

XIV

MTS

(3 - (4,5-dimethlythiazol-2-yl) -5- (3-carboxymethoxyphenyl)2- (4-sulphophenyl) -2H-tetrazolium]

MW

Molecular weight

MWCO

Molecular weight cut off

ng

Nanogram

NLS

Nuclear localisation sequence

nm

Nanometre

NIP

Amino to phosphate ratio

NPC

Nuclear pore complex

OD

Optical density

ONp

4-nitrophenol

PBS

Phosphate Buffered Saline

PCR

Polymerase chain reaction

PCS

Photon correlation spectroscopy

pEl

Polyethylenimine

PEG

Poly( ethylene glycol)

pfu

Plaque forming units

pLAA

Poly (L-aspartic acid)

pLL

Poly (L-lysine)

ppc

Particles per cell (virus particles)

RES

Reticulo-endothelial system

RLU

Relative light units

RNA

Ribonucleic acid

rpm

Revolutions per minute

RF

Replicative form

RPC

Reducible polycation

RT

Room temperature

s

Seconds

scm

Severe combined immunodeficiency

SDS

Sodium dodecyl sulphate

SEC

Size exclusion cation HPLC analysis

SIV

Simian immunodeficiency virus

TEM

Transmission electron microscopy

Tf

Transferrin

TIL

Tumour infiltrating lymphocyte XV

TNBS

(2, 4, 6-trinitrobenzenesulphonic acid)

TNF-a

Tumour necrosis factor a

Tris

2-amino 2-(hydroxymethyl) propan-1 ,3 diol

Tween

Polyoxyethylene-sorbitan monooleate

USE

Ultrasound exposure

uv

Ultraviolet

VEGF

Vascular endothelial growth factor

wt

Wild-type

X-gal

5-bromo-4-chloro-3-indolyl-P-d-galactopyranoside

YOY0-1

1,1 '-[1,3-propanediylbis[( dimethyliminio) -3,1propanediyl]]bis [4-[(3-methyl-2(3H)-benzoxazolylidene) methyl]]-, tetraiodide Average diameter

XVI

1 1.1

Introduction

Background

Gene therapy can be defined broadly as the genetic modification of a patient's cells in order to achieve a therapeutic effect. This definition includes a plethora of possible approaches and can involve the transfer of numerous forms of nucleic acid from cloned human genes, double stranded gene segments, genes from other genomes, oligonucleotides, and even artificial genes such as antisense genes. Whatever the exact nature of the transferred material, the net goal is always the same- to genetically modify target cells, achieving a therapeutic endpoint. Generally, the target cells for gene therapy tend to be disease causing cells. Increasingly however, attention is being paid to targeting of immune system cells, constituting a form of genetic vaccination, or to promote enhanced immune response to the diseased cells (typically in cancer).

The appeal of gene therapy lies in its promise to provide elegant cures for serious disease, both for treatment of somatic cells and also at germ-line level. The completion of the Human Genome Project will enable correlation of specific genetic mutations with disorders to which they contribute, enabling identification of a host of new candidate agents for gene supplementation therapy. The major factor limiting successful gene therapy, however, is not identification of candidate genes, rather it is the difficulty of expressing new copies of therapeutic genes adequately within target cells and tissues.

There are two general strategies for gene therapy, ex vivo and in vivo, overviewed in Figure 1.1. Generally, ex vivo gene therapy involves the transfer of genetic information into cells expanded in culture.

In this procedure, cells are isolated from a patient and genetically

modified outside the body. Transformed cells expressing the relevant therapeutic product are

1

selected and expanded in culture before re-infusion back into the patient. Ex vivo gene therapy has undergone major breakthroughs in recent years, and the first apparently successful gene therapy trial, commenced on 14th

September 1990, involved the transfer of the

adenosine deaminase gene (ADA) gene packaged in a retroviral vector into the Tlymphocytes of a four year old boy (Ashanthi DeSilva). ADA+ T cells (selected by the coexpression of an antibiotic resistance marker gene) were expanded in culture and re-implanted into the patient (Culver eta!., 1991). More recently still, successful trials commenced for the ex vivo treatment of severe combined immunodeficiency disorder (SCID).

This disease

results from a deficiency of gamma chain (yc) of the cytokine receptor for interleukin 2, that prevents the maturation ofT and NK lymphocytes (Noguchi eta!., 1993). Correction of this defect in murine models was possible using ex vivo gene therapy to transform precursor cells using a retrovirus stably expressing the yc (Soudais et a!., 2000). The same approach has since shown considerable efficacy in the clinics, with expression of yc in up to 41 % of circulation CD34+ cells. Significantly, polyclonal T cell count was sufficient 1 year post treatment to reverse disease pathology (4800 per !J.l) (Cavazzana-Calvo eta/., 2000).

Cloned gene

in vivo

ex vivo

X t'•J·-.--·'.J',

~· Gene ';" transfer

// f_.: /

;/;'

.V

Cells removed

...

Select x+ cells • amplify

1f1"~~Ef~ . -·

r _

+. +

-~-

\I [I]

Route to target cell

release

Figure 1. 6 Intracellular barriers to transgene expression.

1.4.2.1 Endosomal release Many vectors enter cells through an acidified endosome, so one of the first major barriers to successful transgene expression is the requirement to gain exit from the endosome. Although it is not absolutely certain that entry into the cytoplasm is an obligatory step en route to the nucleus, most workers in the field assume it is so and means to enhance cytoplasmic entry are widely pursued. Lipoplexes are thought to achieve this by entering the lipid bilayer and by exchanging some cationic lipids with neutral ones, leading to release of the nucleic acid into the cytoplasm (Szoka et al., 1997).

Polyplexes, on the other hand, are generally not

intrinsically fusogenic and must employ some kind of molecular engineering to achieve this effect. The pH-dependent endosomolytic action of pEl and certain types of pH-responsive dendrimers was outlined previously. Another agent that is widely used in vitro to enhance transgene expression using polyplexes is chloroquine, although its mechanism of action is not 38

fully delineated.

Chloroquine is a lysosomotropic weak base that leads to increased

osmolarity of the endosome and may induce lysis, however it also has nuclease inhibition activity, which may be important in maintaining integrity of the DNA in both the endosome and the cytoplasm. In addition, chloroquine binds to DNA under acid conditions and may lead to release of some of the cationic polymers, perhaps increasing their interaction with the endosome membrane as the pH falls (Erbacher et al., 1996). Although its precise action is unclear, chloroquine is widely thought to promote entry of the DNA into the cytoplasm.

Another means to enhance endosome-to-cytoplasm transfer is the use of membrane active peptides, such as those based on the influenza virus haemagglutinin protein.

These N-

terminal peptides are pH dependent and adopt amphipathic configurations at low pH that are thought to insert into the endosome membrane and lead to permeabilisation. One major advantage of these peptides over chloroquine is that they can be used, at least in principle, in vivo, since the endosomolytic agent can be covalently linked to the nucleic acid delivery

vector and targeted to specific cells (Plank eta/., 1994; Wolfert and Seymour, 1998).

Another fusogenic peptide which has recently received attention is melittin, a peptide derived from the venom of the honey bee.

The membrane activity of melittin appears to be

independent of pH, unlike the peptides derived from the influenza haemagluttinin protein. The chemical incorporation of melittin into pEl via covalent linkage has been shown to dramatically improve levels of gene expression using either DNA (Ogris eta/., 2001) or even mRNA (Bettinger eta/., 2001).

39

1.4.2.2 Nuclear uptake or expression in the cytoplasm? Most present-day DNA delivery systems can achieve appreciable levels of transgene expression only in proliferating cells. This is a major limitation of the usefulness of such vectors, since the majority of target cells in vivo are non-cycling (even in proliferating tumours). The main reason for this lack of efficiency in non-proliferating cells, similar to the problems faced by retrovirus, is thought to be the barrier of the intact nuclear membrane. Whereas in proliferating cells, the nuclear membrane disintegrates during G2-M stage of the cell division cycle, allowing vectors access to the transcriptional machinery of the cell, this does not occur in non-proliferating cells. Entry into the nucleus of such non-cycling, or postmitotic cells, is restricted to passage through the nuclear pore complexes and several research groups are presently trying to devise means to achieve this efficiently (Zanta et al, 1999). It is now widely accepted that delivery of DNA from the cytoplasm to the nucleus represents one of the biggest challenges to non-viral gene delivery systems.

Mammalian cells have evolved a range of tightly regulated mechanisms for the nuclear uptake of proteins, with the nuclear pore complex (NPC) acting as the entry portal to the nucleus. The NPC complex is a multimeric structure comprising 50-100 proteins (Fontoura et a!., 1999; Ryan and Wente, 2000; Stoftler et al., 1999) with an apparent molecular weight of around 125 MDa (Reichelt et al., 1990). The pore spans the double membrane of the nucleus, and electron microscopy images suggest it contains a central transporter and so would have an effective channel diameter around 26 nm (Akey and Radermacher, 1993; Goldberg and Allen, 1996). The pore itself may exist without the central transporter, in which case it could have a central channel diameter around 45 nm in diameter. However, experimental data suggest that substances less than 9 nm in diameter can pass through the NPC by diffusion, but cargoes larger than this must enter by active transport mechanisms (Mattaj and Englmeier, 1998). The most widely used pathway for protein import appears to be mediated by basic strands of 40

amino acids known as nuclear localisation sequences (NLSs), which bind to cytoplasmic importin proteins, mediating translocation to the nucleus (Adam and Gerace, 1991 ). Transport across the NPC is energy dependent and involves the activity of a number of cofactors including neurotrophic factor 2 (NTF 2) (Moore and Blobel, 1994 ), the chromatinbound guanine-nucleotide exchange factor RCC 1 (Gorlich and Mattaj, 1996) and the small GTPase Ran (Koepp and Silver, 1996).

It is well established that the mechanisms that mediate the extremely efficient import of viral DNA to the nucleus of host cells frequently harness such pathways (Whittaker and Helenius, 1998).

Therefore considerable attention has been paid to incorporating such import

mechanisms into non-viral gene delivery systems, to increase transgene expression through an improvement in delivery of DNA to the nucleus. In principle NLS can be linked to cationic polymers, or even directly to the DNA. In one of the most convincing examples, substantial improvements in levels of trans gene expression were demonstrated using linearised DNA end modified to incorporate a single NLS sequence (Zanta eta!., 1999). Using this construct in transfection assays resulted in up to a 1000 fold increase in trans gene expression in rapidly dividing cells, but also a 10-30 fold enhancement of gene expression in slowly dividing or quiescent cells. Importantly, this enhancement of transgene expression was not observed when a mutant form of the NLS was used, confirming the specificity of the import mechanism. Whilst providing an elegant model for enhancing nuclear uptake of exogenous DNA, this synthetic method was labour intensive and gave very low yields, therefore unlikely to be useful for gene therapy in a clinical setting. A further study has suggested that NLS mediated nuclear uptake of exogenous DNA could be size dependent, with an upper size limit restricted to around 1 kb (Ludtke eta!., 1999).

41

An interesting alternative to devising means of gaining nuclear entry is to employ cytoplasmic transgene expression systems.

Some authors have demonstrated the feasibility of using

plasmids encoding transgenes under control of the phage T7 polymerase, which is able to mediate cytoplasmic transcription in mammalian cells (Brisson et al., 1999; Deng and Wolff, 1994 ). Encoding the T7 polymerase itself as an "autogene" shows good ability to achieve cytoplasmic expression that may be effective in post-mitotic cells. More intriguing, however, is the use of mRNA as the exogenously-applied genetic material. Historically mRNA has been regarded as being too labile for application in gene therapy strategies, although recently it has emerged as a powerful means to gain substantial transgene expression in post-mitotic cells (Bettinger et al., 2001 ). Cytoplasmic expression systems offer a promising and safe alternative to simple plasmid DNA, without the need for nuclear entry.

Although these

approaches, however, are effective in non-proliferating cells, the selectivity of expression that can be conferred by using carefully chosen promoters or other transcriptional regulatory elements is essentially lost.

1.4.2.3 Vector unpackaging

It has been suggested that further barriers downstream of endosomal escape and into the cytoplasm and localisation within the nucleus may also be important. For example, only a small fraction of B 16F 10 mouse melanoma cells expressed a GFP trans gene delivered by a cationic lipid, even when tens of thousands of intact plasmids were apparent intracellularly after 24 hours of incubation, despite the cells containing more than 100 plasmids within the nucleus (Zelphati et al., 1999).

This suggests a further possible barrier of plasmid

unpackaging, i.e. for gene expression to occur, the cargo DNA must dissociate from its protective vehicle. Complex dissociation can occur in the endosome network, allowing for movement from the endosome to the nucleus. However, should nuclear targeting of the vector/DNA complex occur, then complex dissociation must occur within the nucleus. In 42

fact, nuclear co-localisation of fluorescently labelled DNA and polycation has already been demonstrated (Godbey et al., 1999; Schaffer et al., 2000). Therefore nuclear delivery of vector/DNA complexes may well be insufficient to guarantee transgene expression unless dissociation occurs.

For polyplexes, it is considered that polycation size could influence

complex dissociation. For example, one study showed that nuclear co-localisation of DNA and polycation occurred when complexes were formed with high molecular weight polycations, whilst naked DNA alone was found in the nucleus of cells when complexes were formed with relatively short polycations (Schaffer et al., 2000). Interestingly, maximal gene expression was observed with complexes formed using intermediate length polycations. This perhaps represents an optimal balance between protection from degradation (conferred by larger polycations) and the ability to unpackage the DNA in a transcriptionally active form (conferred by smaller polycations).

1.5

Targeting strategies

For many envisioned applications of gene therapy, the gene delivery vector is required to provide efficient expression of the encoded transgene within a selected cell type, without stimulating significant immune, inflammatory or cytotoxic responses. One such approach involves the use of cell or tumour specific promoter elements to regulate the expression of the transgene in a selective manner (Nettelbeck et al., 2000). Considerable attention has been paid towards the deployment of tumour specific promoters for cancer gene therapy. Such promoters tend to be embryonic promoters which become switched on during the development of some tumours. Examples include carcinoembryonic antigen (CEA) (Schrewe et al., 1990) and a-foetoprotein (AFP) (Godbout et al., 1986), and have been used to guide

the specific expression oftransgenes within tumour cells (Cao et al., 1999; Ido et al., 1995). However, the use of tumour specific promoters in cancer gene therapy is likely to be limited by a number of crucial factors.

For example, uptake into non-target cells may result in 43

depletion of the pool of administered vector available for transducing the target cells, and simultaneously can promote unwanted toxicities that could be dose limiting. A more rational approach therefore is to identify suitable targeting ligands that might be unique or upregulated within the target organ or tumour endothelium and microenvironment, providing a level of targeted gene delivery. This would result in decreased vector loss, reduced side effects and maximise the effect of the administered vector.

1.5.1

Factors influencing suitability of targeting ligands

The use of cell surface receptors to enable the binding and intemalisation of DNA delivery vectors has seen significant development over the last 15 years. Numerous ligand/receptor interactions have been characterised and are under development for targeting DNA vectors (see table 1.2). When making an informed choice of targeting ligand for delivering genes in vivo, there are numerous considerations which ought to be addressed.

Some of the pivotal

considerations are outlined below.

1.5.1.1 Receptor biology in vivo

When choosing a suitable ligand for targeting trans genes to tumours (or organs) in vivo, the most obvious primary consideration is in the biodistribution of the ligand receptor. Ideally, the ligand would bind to receptors expressed uniquely on the target (tumour) cells, and not expressed on the cell surface of any other non-target cells. A number of candidate receptors have been iterated that were considered to be unique to the tumour microenvironment, however on closer inspection, expression of the receptor was also demonstrated on normal, non-malignant cells at low level, e.g. CEA (Kinugasa et a/., 1998) and the receptor for fibroblast growth factor (FGFRl/2) (Luqmani et al., 1992). Superficially, targeting receptors which are upregulated in tumours might seem less appealing than targeting uniquely

44

expressed receptors. Such receptors however, might be useful for the targeted delivery using systems which require high avidity (rather than relying on affinity) i.e. the uptake of the vector is dependent on multiple binding events occurring, and therefore is dependent on a high density of surface receptor to mediate receptor mediate cell uptake.

Another crucial consideration when deciding on suitable targeting ligands for gene delivery in vivo is that the receptor should be anatomically accessible given the route of administration of the vector. The polarised nature of cells in vivo means that promising results achievable in vitro will not necessarily be mirrored by in vivo efficacy. For example, whilst the transfer of transgenes to airway epithelial cells using Ad5 vectors has been shown to be extremely efficient in vitro, efficacy in vivo was minimal, since the adenovirus CAR receptor is localised to inaccessible baso-lateral surfaces of the airway epithelial cells in vivo (Walters et al., 1999). Similarly, when developing ligand targeted complexes for systemic delivery of DNA vectors to tumours, the target receptor must be accessible from the bloodstream, therefore must be expressed on the luminal surface of the polarised endothelial cells. Consequently attention is being paid to the identifying ligands which will bind to endothelial cells in an organ or tumour specific manner (Arap et al., 2002; Arap et al., 1998; Essler and Ruoslahti, 2002).

1.5.1.2 The consequences of receptor binding A further key issue when selecting a suitable targeting ligand for in vivo targeting is what are the consequences of a successful ligand/receptor interaction? From the perspective of gene delivery, the desired outcome would be to stimulate receptor mediated uptake into the cell. However, cellular receptors have not evolved merely as tools for delivery scientists, rather receptor binding and stimulation results in activation of cell signalling pathways to promote a physiological response. For example, some commonly deployed ligands such as fibroblast 45

growth factors (FGFs) and vascular endothelial growth factor (VEGF), have been shown to be mitogenic, and so may be far from ideal for targeting delivery to tumours. Similarly, another commonly used targeting agent is the tripeptide RGD, which initiates apoptosis through activation of procaspase 3 following the successful binding and activation of av~ 3 or av~ 5 integrins (Buckley et al., 1999).

Whilst this might not be disadvantageous from the

perspective of cancer gene therapy, it could pose difficulties from the point of view of correctional gene therapy strategies, where apoptosis could be initiated within cells successfully expressing the therapeutic transgene. The efficacy of such systems for some envisaged applications is likely therefore to require a careful balance between delivery of the therapeutic transgene and toxicity mediated through cellular apoptosis.

1.5.1.3 Ligand pharmacology in vivo

Incorporation of targeting ligands, which might be bulky, charged or of a hydrophobic nature could result in rapid vector clearance following systemic administration in vivo due to interactions with blood serum components (Unger et al., 2000). Soluble heparans are known to bind to FGFs and VEGF, so incorporation of such agents into delivery constructs might well mediate opsonisation and clearance through liver Kupffer cells. Ideally, the targeting ligand of choice would have good receptor affinity and specificity, as well as being small, charge neutral and would not initiate significant levels of downstream intracellular signalling following receptor binding.

Furthermore, investigation should be pursued to examine

whether the administration of biologically active targeting ligands could be dose limiting, i.e. whether there any toxic or immunological effect are observed.

46

1.5.1.4 Cost and ease of ligand production

The production of some ligands, particularly those which are large biologically active molecules is both labour intensive and costly. Such ligands also tend be readily denatured and difficult to handle. The most efficient systems for large scale production of recombinant proteins are prokaryotic systems, and therefore there are no guarantees that the protein produced will be correctly folded and post translationally modified, which will affect their biological properties. Refolding of recombinantly produced proteins is again costly and time consuming, and is frequently unsuccessful.

The identification of small, synthetically

produced peptides for targeting purposes is therefore likely to reduce production cost and is likely to result in the formulation of better defined better tolerated gene delivery constructs.

1.5.1.5 Receptor targeting and resistance

Previous studies investigating the targeted delivery of anticancer agents have shown that receptor down-regulation within the tumour microenvironment can result in rapid resistance to the administered agent. For example, resistance to methotrexate therapy can be mediated through down regulation of the folic acid receptor from a population of tumour cells (Saikawa et a!., 1993 ). It will be important to identify multiple ligands which bind to deliver genes to

tumour endothelia via numerous receptors, decreasing the opportunity for down regulation or clonal selection within the tumour population.

1.5.2

Choice of targeting ligand

To date, there have been numerous studies regarding ligand targeting of gene delivery vectors using many different receptor/ligand interactions. Such targeting systems are versatile, and can be applied to viral, non-viral and hybrid vectors appropriately (Table 1.2). The large majority of studies have focused on the use of antibodies (or fragments of), physiological

47

ligands, or small peptides as tools for vector retargeting. Each ligand type has advantages and disadvantages over the others, for example, antibodies are available against a whole range of cell surface receptors, however, the use of whole antibodies may be limited by interactions with scavenging macrophages carrying Fe receptors. Physiological ligands on the other hand

Targeting Moiety

Antibodies/SCFV

Galactose

EGF

bFGF

VEGF

Receptor

Vector Type

Reference

Viral

(Somia et al., 1995)

Synthetic

(Chen et al., 1994)

Hybrid

(Merwin et al., 1995)

Asialoglycoprotein

Synthetic

(Plank et al., 1992)

receptor

Hybrid

(Cristiano et al., 1993)

Viral

(Hatziioannou et al., 1999)

Synthetic

(Schaffer et al., 2000)

Viral

(Goldman et al., 1997)

Synthetic

(Hoganson et al., 1998)

Viral

(Fisher et al., 2000)

Synthetic

(Fisher et al., 2001)

Various

EGFRs

FGFR2

VEGFR 1/2/4

NGF

NGFR

Synthetic

(Chevalier et al., 1994)

Erythropoietin (EPO)

EPOR

Viral

(Laquerre et al., 1998)

Folic acid

hFAR

Synthetic

(Ward, 2000)

Transferrin

CD31

Synthetic

(Kircheis et al., 1997)

Hybrid

(Wagner et al., 1992)

Viral

(Han et al., 1995)

Viral

(Nicklin et al., 2000)}

Synthetic

(Hart et al., 1997)

Heregulin

ErbB-3/4

Peptides

Various

Table 1.2 Examples oftargeting ligands under development for gene delivery.

tend to be extremely efficient in stimulating their target receptors since they are generally of higher affinity than antibodies, but their bulky and (frequently) charged nature often results in short serum half lives. They are also expensive and difficult to produce on a therapeutically

48

significant scale. Peptide fragments of targeting ligands or peptides iterated by phage display technology are generally a great deal smaller than both physiological ligands and antibodies (generally smaller than 3 kDa, compared with 150 kDa for antibodies). Generally, smaller, simple ligands are preferable to larger ones since by and large they tend to have less biological baggage. Peptides can also be produced on relatively large scales and are often robust to chemical handling, facilitating their incorporation into delivery constructs. Although individual peptides are generally lower affinity than antibodies or physiological ligands, presentation of multiple copies on the surface of delivery vector may provide an avidity effect, enabling uptake into target cell types presenting an upregulated amount of the target receptor on their cell surface.

1.5.3

Phage display technology

Phage display describes a selection technique in which a protein or peptide is expressed as a fusion with a coat protein of a filamentous bacteriophage, resulting in the display of the fused protein on the virion surface, whilst the DNA encoding the fusion resides within the virion. Phage display has been used to create a physical linkage between a vast library of random peptide sequences to the DNA encoding each sequence, allowing the rapid determination of peptide ligands for any given target molecule (antibodies, enzymes, cell surface receptors etc) by a selection process known as panning (Parmley and Smith, 1988~ Smith and Scott, 1993). There are a wide variety of bacteriophage vectors currently under development as tools for phage display although the vast majority of vectors are based on the filamentous bacteriophage M13.

49

1.5.3.1 M13 biology The Ml3 phage particle (shown schematically in Figure 1.7) is approximately 7 nm in diameter and 900-2000 nm in length. The single stranded DNA is encased within a flexible cylinder of around 2700 pVIII, constituting the major coat protein. At one end of the particle there are 5 molecules of pVII and piX, whilst at the opposite end there are 5 molecules of pili

pill Coat Protein: Attach tcpili of E. Coli

.

~

pVI Coat Protein E.Coli is infected with phage SSDNA 900nm

pVIII Coat Protein

Vll & piX Coat Protein

Figure 1. 7: The structure of the M13 bacteriophage.

and pVI. Propagation ofM13 bacteriophage occurs by infection and replication within gram negative bacteria. Their infection process is dependent upon the interaction of the pili with the F-pilus of the bacterium, therefore replication is limited to F+ bacteria. Interaction of pili with the F-pilus results in retraction of the pilus by depolymerisation of the pilin subunits bringing the phage particle to the membrane surface. The next step requires the integration of the pVIII coat proteins into the inner membrane and the corresponding transfer of the genetic material into the cytoplasm, involving the bacterial TolQ, R and A proteins. Once in the cytoplasm, bacterial enzymes synthesise the complementary strand, resulting in the production of a supercoiled, double stranded replicative form (RF) molecule. This molecule serves as a template for transcription and translation from which all phage proteins are synthesised. Some of the phage products, in concert with bacterial enzymes, direct the further

50

synthesis of single stranded DNA which are subsequently converted to additional RF molecules.

Capsid proteins, and other proteins involved in particle assembly become

integrated in the cell envelope, whilst DNA replication proteins remain cytoplasmic. When the concentration of pV, a phage specific single stranded DNA binding protein, reaches a critical concentration, it sequesters newly synthesised single stranded DNA into a complex, preventing its conversion to RF molecules, for assembly into new phage particles.

Phage assembly occurs at the bacterial envelope where, following the selective displacement of the pV single stranded binding protein, the capsid proteins assemble around the DNA as it is extruded from the envelope. Assembly continues, driven by three phage specific noncapsid assembly proteins and bacterial thioredoxin, until the end of the DNA is reached. Assembly is then terminated by the addition of pVI and pill, and the infectious phage particle is released into the media.

1.5.3.2 Principle of panning using phage display libraries

The display of random peptides on the surface of bacteriophage represents a powerful technology for carrying out the molecular evolution in the laboratory. This technology was first described over 15 years ago following the successful expression of a portion of the gene encoding the endonuclease EcoRI to the minor coat protein pill of bacteriophage M13 (Smith, 1985), and the subsequent demonstration of selective enrichment of phage containing the

EcoRI-piii fusion compared to wild type.

Within a few years of Smith's original

experiments, the first random peptide libraries were being assembled (Cwirla et al., 1990), along with reports that properly folded and functional proteins could be displayed on the surface of M13 (McCafferty et al., 1990).

This technology has numerous possible

applications for generating extremely high affinity peptide agonists for protein-protein interactions or, from the persepective of targeted gene therapeutics, receptor-ligand 51

interactions, by the process of "biopanning" (Figure 1. 8).

In this procedure, synthetic

oligopeptides, fixed in length but with unspecified codons are cloned as fusions to pili or pVIII. The library can then be incubated with a target molecule or cells, followed by capture

Phage library, each displaying a random peptide, is exposed to cells.

Unbound phage washed away.



Receptor

Specifically-bound phage eluted using excess of a known ligand ( FGF2)

Cell

Next Round of ..6.... Biopanning .,.... Amplification . .

•••• , •••

Figure 1.8: Identification of cellular receptor binding peptides by phage display technology

of the target molecule together with the associated phage, or by collection of associated phage using an excess of known ligand for the target molecule. Recovered phage can then be amplified and used for further rounds of selection for binding to the target molecule. Thus, rare phage that bind efficiently can be selected from greater than 109 different individuals in one experiment In some cases the iterated peptides resemble the primary structure of the receptor ligands, and in other cases the peptide mimics the binding of nonpeptide ligands.

52

1.5.3.3 Applications of phage display technology

Random peptide libraries displayed on phage have been used for numerous applications, including epitope mapping (Scott and Smith, 1990), mapping protein-protein interactions (Hong and Boulanger, 1995), and identification of peptide mimics of non-peptide ligands (Devlin et al., 1990; Hoess eta!., 1993; Scott eta!., 1992). Bioactive peptides have been identified either by panning against purified, immobilised receptors, (O'Neil eta!., 1992), or against intact cells (Barry eta!., 1996; Binetruy-Tournaire eta!., 2000; Doorbar and Winter, 1994 ). In addition to random peptides, larger proteins such as antibodies (Barbas et a!., 1994), hormones (Lowman et al., 1991), protease inhibitors (Roberts et al., 1992), enzymes (Soumillion et a!., 1994) and DNA binding proteins (Choo and Klug, 1995) have been displayed on phage and variants with altered affinity or specificity isolated from libraries of random mutants.

Recent studies have demonstrated the feasibility of using phage display libraries for in vivo selection of targeting peptides. One such study describes the successful isolation of tumourbinding peptides directed against integrins, by injecting peptide phage libraries in tumourbearing mice, and eluting tumour associated phage from the excised tumour from the sacrificed animal (Arap et a!., 1998; Pasqualini et a!., 1997). Phage displaying specific peptides were shown to target to the tumour vasculature in vivo, and furthermore, chemical conjugation of the phage derived peptides to doxorubicin showed significantly increased levels of efficacy and less side effects compared to unconjugated doxorubicin in the treatment of tumours grafted in mice (Arap eta!., 1998; Pasqualini eta!., 1997).

Such has been the success of screening phage libraries both in vitro and in vivo that in February 2002, the first in vivo screening of a peptide library was reported in a human patient (Arap et a!., 2002).

This study attempted to identify molecular markers of the human

53

vasculature by the mass screening of motifs localising to different organs. In this preliminary study (in which only 1 round of biopanning was possible) the tissue distribution of circulating peptides was found to be non-random. High throughput analysis demonstrated that some of the identified motifs showed similarities to ligands of differentially expressed cell surface proteins.

Whilst this study may be preliminary, the data present initial steps towards the

construction of molecular maps of the human vasculature, which, if feasible, will have broad implications for the development of targeted therapies.

1.6

Thesis objectives

Chapter 3 develops methods for evaluation of polyplex formation and investigates their stability under physiological conditions, including an evaluation of possible effects of charge ratio and polycation molecular weight.

Chapter 4 involves the concepts of "steric" and

"lateral" stabilisation to endow these polyplexes with stability sufficient to enable their systemic delivery. However, since surface modification of polyplexes with the multivalent hydrophilic polymer pHPMA yields complexes with little intrinsic transfection activity in

vitro, attention is also paid to the design of a "trigger mechanism" capable of activating the stabilised complexes for transcription following their entry into target cells.

Chapters 5

examines the possibility of utilising small receptor-binding peptides iterated by phage display technology to confer receptor-tropism to gene delivery vectors (including both non-viral and viral based vectors), whilst chapter 6 investigate the use of phage display to iterate FGF receptor-binding heptapeptides that could have potential applications for the targeted delivery of drugs or genes to angiogenic vasculature in vivo. Overall the thesis aims to establish a new type of peptide-targeted polyplex vector, capable of delivering nucleic acids (including both DNA and mRNA) to specific target cells in relevant physiological systems.

54

2 Materials and methods

2.1

Supplier of materials

All materials were purchased from Sigma, Dorset, UK unless otherwise stated.

2.2

Purchase and preparation of nucleic acids

2.2.1

Sources of DNA

The expression vector used for the majority of this study was the pGL3 - Control Vector (Promega, UK), a 5.3 kb vector containing a single modified copy of the firefly luciferase gene under the transcriptional control of the SV40 promoter and enhancer regions.

For

formulation studies discussed in chapter 3, the 6 kb expression vector pSV2BCL2 was used (gift from Dr Aris Eliopolous, Institute for Cancer Studies, University of Birmingham, UK),

comprising the BCL2 gene under the transcriptional control of the SV40 promoter and enhancer regions.

2.2.2

Plasmid propagation, isolation and purification

Transforming E. coli: Competent E. coli (50 !-!1, kept on ice) were transformed with pGL3 - Control Vector or pSV2BCL2 (10 ng total) by addition of the plasmid tothe bacteria followed by heat shock at 42°C for 45 seconds. The sample was subsequently cooled on ice for 30 min, added to sterile LB broth (900 1-!1) and incubated at 37° C for 1 hour on an orbital shaker. The resulting cells (200 !-!1) were spread onto antibiotic selection plates (ampicillin, sodium salt, 100 J.lg/ml) and incubated overnight at 3 7°C. Plates were stored at 4 oc for up to 2 weeks.

55

Propagation and purification of plasmid DNA: Plasmid DNA was prepared using Qiagen Gigaprep Kits (Crawley, West Sussex, UK) according to the manufacturer's guidelines.

2.2.3

Quantification of DNA

Concentration and purity of the resultant DNA were checked using a spectrophotometer at OD260 nm I OD280 nm absorbance wavelengths, where an OD 260 reading of 1. 0 corresponds to a concentration of approximately 50 !J.g/ml of double stranded DNA, and a ratio between OD260 and OD28o between 1. 8 - 2 was used to indicate pure DNA.

2.2.4

Labelling of DNA

(i) Cy-3 labelling of DNA expression vectors: The Cy-3 labelling kit was purchased from Panvera (Madison, WI, USA) and 10 !J.g of plasmid DNA labelled using the manufacturer's guidelines. Briefly, DNA (0.1 mg/ml) was reacted with 10 !J.l of Cy-3 at room temperature for 1 hour. Umeacted dye was removed by centrifugation through a G-50 microspin purification column (3000 rpm, 2 min).

Cy-3

labelled DNA was analysed following agarose gel electrophoresis using a Typhoon Fluorimager (Molecular dynamics, High Wycombe, UK) (Aex

=

550 nm, Aem

=

570 nm).

Aliquots were stored in the dark at -20°C.

32 (ii) P dCTP labelling of DNA expression vectors: 32

Plasmid DNA was radiolabelled by random prime incorporation of P-dCTP using a Readyto-Go DNA Labelling Kit (Amersham Pharmacia, UK).

The DNA to be labelled was

denatured by boiling for 3 minutes and mixed with small fragments of single stranded DNA (oligodeoxyribonucleotides) of random sequence. These oligomers anneal to random sites on 56

the DNA and then serve as primers for DNA synthesis by a DNA polymerase. The presence of labelled

32

P-dCTP nucleotides during this synthesis result in the production of labelled

DNA.

The reagents in the Ready-to-Go kit were reconstituted by the addition of 20 Jll of water (without mixing) and then left on ice for 5-30 minutes. The DNA (50 ng in 25 Jll) was denatured by heating for 5 minutes at 95-1 00°C and then placed on ice for 2 minutes before being briefly centrifuged. The DNA was added to the reaction mixture, along with 5 Jll of [a32P]dCTP (50 J.1Ci) and distilled water to make the volume up to 50 Jll. The labelling reaction was started by mixing the components by gentle pipetting, followed by a 15 minute incubation at 37°C. After this time the reaction was stopped by the addition of 5 Jll of 0.2 M EDTA (pH 8.0).

The DNA was purified away from unincorporated nucleotides usmg MicroSpin DNA purification columns (Amersham Pharmacia, UK). The columns were pre-equilibrated in TE buffer (pH 7.6) and pre-packed with Sephacryl S-200 resin. The resin was resuspended by vortexing briefly, and placed in a microcentrifuge tube and spun for 1 minute at 3000 rpm in order to remove the TE buffer and avoid any dilution of the sample. After centrifugation the column was placed in a new microcentrifuge tube and the labelled plasmid sample added to the top of the column. The column was then centrifuged for 2 minutes at 3000 rpm; the purified sample eluted into a microcentrifuge tube with unincorporated oligos and oligonucleotides retained in the resin. Finally, the purity of the labelled DNA was checked following agarose gel electrophoresis and quantitative analysis with a Typhoon Fluorimager (Molecular Dynamics, UK). (iii) YOY0-1 labelling of DNA expression vectors: Plasmid DNA labelled with the cyanine dimer nucleic acid stain YOY0-1 (Molecular Probes, Eugene, USA) was prepared by reaction of 20 J..lg of plasmid DNA with 1 Jll of 100 J.!M

57

YOY0-1 (in 10 mM HEPES, pH 7.4) in the dark for 5 hours on ice in a total volume of 10 f..ll. The volume was increased to 1 ml and free YOY0-1 was removed by dialysis overnight against 10 mM HEPES, pH 7.4 at 4°C, protected from light, in snake-skin dialysis tubing with a molecular weight cut-off of 10 kDa (Pierce & Warriner, Cheshire, UK).

2.2.5

Preparation of GFP mRNA

10 J.tg of the mRNA template plasmid pGEM4Z-5 '/GFP/3 '-Ao4 (kind gift from Dr. David Baczkowski, Duke University Medical Centre, Durham, USA) encoding the GFP protein under the control of the T7 promoter was linearised using the 20 units of the restriction endonuclease Spel for 4 hours at 37°C (total reaction buffer 50 J..ll, containing 0.1 mg/ml BSA).

Linearisation of the plasmid was confirmed by agarose gel electrophoresis ( 1 %

agarose, 1 hour, 120 V).

In vitro transcription to produce a capped GFP mRNA was performed using the RibomaxTM large scale mRNA production system- T7 (Promega, Southampton, UK). Stock solutions of 7

rNTP (25 mM rATP, rCTP, rUTP, 5 mM rGTP), and of Ribo m G Cap Analog (40mM) (Ambion, Huntingdon, UK) were prepared.

The following reagents were then mixed to

produce the mRNA transcript 5x Transcription Buffer Stock rNTP solution Linear DNA Template (lug) plus Nuclease Free Water 7 Ribo m G Cap Analog (40mM) Rnasine Enzyme Mix

4 J.tl

Final Volume

20 J.!l

6 J.!l 3.75 J.tl

3.75 J.!l 0.5 J.tl 2 J.tl

The contents were gently mixed with a pipette and incubated at 37

oc for 3 hours.

To remove

the template DNA, RQl RNase-free DNase (1 unit) was added to the reaction mixture, and subsequently incubated for a further 20 minutes at 3 7°C.

58

The mRNA was then

phenoVchloroform extracted, and in 1/lOth volume of sodium acetate and 2.5 x volume of 100 % ethanol (overnight, -20°C). mRNA solution was then pelleted by centrifugation for 1 hour

at 13000 rpm and 4°C, and washed with 70 % ethanol. The solution was again centrifuged for a further 2 minutes to pellet the mRNA, and the ethanol was removed and discarded. The mRNA pellet was dried in a heat block (55°C) for

approximately 2 minutes finally

resuspended in nuclease free water (40ul). Typically, greater then 200 J.lg ofmRNA could be produced from 1 J.lg of DNA template. The concentration of the mRNA solution was calculated using the following formula: Absorbance (@260nm) x Dilution x 40 =Concentration (J.lg/ml) Size of mRNA transcripts generated was determined by samples may be run on a denaturing agarose gel, along with a set of mRNA markers (Ambion's RNA Millenium Size Markers). The mRNA produced was found to be of the correct size (894 nt) with minimal degradative products observed.

2.3

Assembly of pLL/DNA complexes

2.3.1

Calculation of charge ratio

An average mass per phosphate group of 325 Da was used for DNA (hydrochloride salt) and an average mass per amino group of 209 was used for pLL (hydrobromide salt), thus to achieve a theoretical charge ratio of 1:1 between pLL and DNA, a weight/weight (w/w) ratio of 1:0.64 is required. For pEl, where only a proportion of the amino groups are fully ionised

N:P (defined as N, the number of amino groups in the polycation, and P, the number of DNA phosphate groups) ratios were used, rather than charge ratio, since the charge ratio is dependent on pH and therefore difficult to calculate accurately.

59

2.3.2

Formation of pLL/DNA complexes

Addition of poly(L-lysine) (pLL) to a solution of DNA results in an electrostatic interaction between the oppositely charged molecules, resulting in charge neutralisation and hydrophobic collapse of the DNA into discrete nanomolecular sized complexes (referred to as polyplexes, or pLL/DNA complexes).

Unless otherwise stated in the figure legend, pLL (of varying

average molecular weight) was added to a DNA solution (20 J..Lg/ml DNA in 10 mM HEPES, pH 7.4) to a charge ratio of 1, 2 or 4 (see method 2.3.1), mixed thoroughly and left to stand at room temperature for 1 hour.

2.3.3

Formation of pHPMA surface modified pLL/DNA complexes

Polyplexes were formed between pLL or reducible polycation (RPC) (Chapter 4) and DNA to a charge ratio of 2 as described in 2.3.2 to a DNA concentration of 26 J..Lg/ml. Subsequent surface modification of the polyplexes was achieved by buffering the solution by the addition of HEPES pH 7.8 to a final concentration of 50 mM before addition of poly N-(2hydroxypropyl)methacrylamide,

(pHPMA-ONp)

(prepared

by

the

Institute

for

Macromolecular Chemistry, Prague) to achieve a final concentration of between 0.1 - 10 mg/ml. The polymer was left to react with the polyplexes for 12 hours at room temperature, after which any remaining reactive esters were removed by aminolysis using 0.01% amino ethanol. Targeted pHPMA modified polyplexes were produced by the addition of targeting ligands (1 00 J..Lg/ml) two hours after the addition of the pHPMA with the mixture left to react for a further 10 hours on ice, after which any remaining reactive esters were amino lysed using 0.01% amino ethanol. Production of SIGYPLP targeted complexes (Chapter 5) was achieved

by prederivatising the pHPMA-ONp to incorporate the SIGYPLP peptide (see method 2.8.2), and then surface modifying polyplexes as described above.

60

2.4

Physicochemical characterisation methods

2.4.1

Gel shift assay to show complex formation

Plasmid DNA was labelled with Cy-3 (see method 2.2.4 (i)), and subdivided appropriately. Subsequently, pLL/DNA complexes were prepared in water (N:P ratios 0 - 1.4) and electrophoresed on agarose gels (1 % w/v, 120 min, 100 V). The DNA in the complexes was analysed by following the Cy-3 dye on agarose gel electrophoresis using a Typhoon Fluorimager (Molecular dynamics, High Wycombe, UK) (Aex = 550 nm, Aem = 570 nm). In some studies, EtBr/DNA fluorescence was detected rather than Cy-3/DNA. In these studies, the gels were incubated, post electrophoresis, in heparin sulphate ( 10 mg/ml) solution to disturb the DNA-cationic polymer complexes, and restore access of subsequently added EtBr (1 mg/ml).

2.4.2

Measurement of complex formation by light scattering

Complex formation was also investigated by measuring changes in light scattering intensity using a fluorimeter. Plasmid DNA (20 !J.g/ml) in HEPES (10 mM pH 7.4) was incubated in cuvette and the intensity of scattered light (Aex = 600 nm, Aem = 600 nm) was set to zero. pLL

(111 kDa) was added at 0.2 N:P increments and the changes in scattering intensity monitored. Data are presented as the relative scattered intensity, with the maximal signal detected at any N:P ratio set to 1.0.

2.4.3

Monitoring particle formation by inhibition of EtBr/DNA fluorescence

The EtBr/DNA fluorescence (Aex = 510 nm, Aem = 590 nm) of plasmid DNA (20 !J.g/ml) in ultrapure water containing EtBr (0.4 - 3 !J.g/ml) was measured and set to 100 % using a fluorimeter (Perkin Elmer LS50B), with DNA fluorescence (without EtBr) set to 0 o/o.

61

Aliquots of pLL (or SIGYPLPGGGS(K)16 (Chapter 5)) were added sequentially and the fluorescence was measured after each addition until minimal further change in fluorescence was observed. In some experiments, poly(L-aspartic acid) (pLAA) was subsequently added incrementally to the complexes to sequester the pLL and restore the EtBr/DNA fluorescence.

2.4.4

Determination of fluorescence excitation spectra

Fluorescence excitation spectra for EtBr (0.4 J.Lg/ml), DNA (20 J.Lg/ml)/EtBr and pLL/DNA!EtBr (N:P excitation (Aex

=

=

2) (all in water) were determined using 2 ml solution and scanning

350 - 560 nm) with Aem fixed at 590 nm using a fluorimeter (Perkin Elmer

LS 50B)

2.4.5

Analysis of particle size by photon correlation spectroscopy (PCS)

Light scattering analysis was performed using a Malvern Instruments light scattering spectrophotometer in order to determine the size of DNA/polycation complexes. Measurements were taken at 25°C at an angle of 90° to the incident light using a disposable poly-methacrylate cuvette. To obtain accurate readings, buffers were degassed and filtered before use.

The machine was calibrated using latex spheres (204 nm) before and after

experimental samples using Contin software with monomodal deconvolution for analysis. Samples for PCS measurement were prepared according to method 2.3.2 and 2.3.3.

2.4.6

Analysis of particle surface charge by zeta potential analysis

Measurement of surface charge was performed using microelectrophoresis. Combining laserDoppler velocimetry with electrophoresis gives a direct measurement of particle mobility in an electric field, the specific mobility of an object in an electric field is determined by its surface charge or 'zeta potential'. The machine used was a Malvern Instruments Zetasizer 4 62

with a series 7032 multi 8 correlator. Polyplexes were formed according to method 2.3.2 and 2.3.3 for measurement in the Zetasizer instrument.

2.4.6

Quantification of residual reactive amino groups by fluorescamine assay

The reagent fluorescamine was used to monitor free amino groups in order to follow the reaction between DNA and polycations The assay was also adapted to permit determination of residual amino groups by reaction with pHPMA. Complexes were formed between pLL and DNA and surface modified with pHPMA (0 - 2 mg/ml, in 50 mM HEPES, pH 7.8 overnight at room temperature). 100 Jll of each sample was diluted into 1.4 ml of assay buffer (100 mM boric acid- NaOH, pH 9), prior to the addition of 500 Jll 0.01 o/o fluorescamine (in acetone). The samples were inverted several times to mix, and incubated at room temperature for 10 min. The fluorescence (Fx) was measured using a Perkin - Elmer LS 50B fluorimeter (Aex = 392 nm, Aem = 480 nm, 10 nm slit widths). Free polycation in solution was used to determine a 100 % value (Fpc, and the background fluorescence determined using assay buffer alone (Fbg). The % free amino groups was subsequently determined using the formula: %Fag= (Fx- Fbg) /(Fpc- Fbg) X 100.

2.4. 7

Monitoring paranitrophenol release

Release of the reporter molecule paranitrophenol (ONp) is an alternative method for monitoring the reaction between the ester groups on the polymer and accessible amino groups on the polymer. Polyplexes were formed (30 min, room temperature) prior to the reaction with pHPMA (0- 2 mg/ml). Release of ONp was monitored by measuring the increase in absorbance at 450 nm using a Victor2 platereader (EG&G Ltd, Massachusetts, USA), and reaction of the polymer with amino groups determined by correcting for the degree of hydrolysis of the polymer (also resulting in the release of ONp) as measured by the increase

63

in absorbance at 450 nm observed with the polymer in free solution. In some experiments, the subsequent incorporation of targeting ligands was also monitored by measuring the further release of ONp following the addition of ligand and the and corresponding increase in Abs 450 nm. These data have been corrected for any increase observed when the buffer (in which the ligand was dissolved) was added to pHPMA coated complexes.

2.4.8

Transmission electron microscopy (TEM)

An aliquot (approx 10 J..Ll) of solution containing polyplexes (prepared as described in 2.3.2) was added to 200 mesh carbon coated copper grids and left to air dry. Uranyl acetate stain (10 Jll, 0.5 %in water) was added for 1 min at room temperature. The strain was drawn off using filter paper, the grid washed by bathing in distilled water and left to air dry. Complexes were viewed in a JOEL 1200EX transmission electron microscope.

2.5

Cell Culture

All cell culture was preformed using a biological safety class II laminar flow cabinet swabbed with 70 o/o ethanol before and after use. Cells were maintained in a Galaxy R incubator (Ayrshire, Scotland) at 37°C with a constant C0 2 level at 5 %. All procedures were carried out in Dulbecco's modified Eagles medium (DMEM, Gibco-BRL, Paisley, UK) supplemented with 2 mM L-glutamine and 10% foetal calf serum (FCS), with the exception of HUVE cells which were maintained on gelatin coated· plates containing M911 media supplemented with 2 mM L-glutamine, 20 o/o FCS, 1 o/o penicillin/streptomycin solution and 20 ng I ml of the growth factor basis FGF (FGF2) (kind gift from Selective Genetics, San Diego, USA).

64

2.5.1

Maintenance of established cell lines 2

Cells were grown as monolayers in 75 cm flasks containing 20 ml of culture media. Media was replenished every 3 days to avoid depletion of essential nutrients and increases in toxic metabolites. Flasks were routinely sub-cultured at 80 % confluence at a ratio of 1: 10 to prevent overgrowth and loss of surface contact. To sub-culture, media were discarded, and the cell monolayer washed briefly with pre-warmed PBS (10 ml) and the cell-surface adherence proteolytically cleaved by the addition of trypsin I EDTA (4 ml; 0.05/0.02 % respectively) for 1 min followed by incubation at 37°C for 5 min. Cells were resuspended in fresh media and aliquoted into fresh flasks. Antibiotics were not used in established cell cultures unless otherwise stated, to reduce the possibility of antibiotic-resistant infections. Cells were kept for approximately 30 sub-cultures and then discarded.

2.5.2

Storage and resuscitation of cell lines

Cells were stored by resuspending a cell pellet (from 75 cm2 culture flask) in cryopreservation solution (comprising 10% DMSO, 90% FCS). The cell solution was aliquoted into cryovials (1 ml) and stored overnight at -84°C wrapped in paper towels in a polystyrene box. Cells were subsequently frozen and stored in liquid nitrogen. Cells were resuscitated by thawing at 37°C and transferring, drop by drop, into warm DMEM (10 ml supplemented with 2 mM Lglutamine and 10% FCS). The solution was centrifuged at 1000 rpm for 5 min to pellet the cells, media was discarded and the cells resuspended in fresh DMEM and transferred to a fresh 75 cm2 culture flask at 37°C, 5% C02 for 24 hours. Media were replenished daily until the cells reached confluence.

65

2.5.3

Determination of viable cell number

An aliquot of cell suspension (50 ~1) was added to PBS (100 ~1) and trypan blue solution (50 f.ll) and left at room temperature for 5 min. Only non-viable cells take up the trypan blue dye, thereby allowing the number of viable cell numbers to be determined microscopically by discounting blue cells using a haemocytometer of fixed volume (chamber volume= 0.001 ml)

2.5.4

Celllines

The following cell lines were used in this study and were obtained from ATCC unless otherwise stated: 911: human embryonic retinoblasts transformed with Ad5 E1 region (Fallaux eta!., 1996),

maintained in DMEM 10% FCS, 2 mM L-glutamine. AB22: asbestos induced murine mesothelioma cells (kind gift from Katerine Lucas, National Heart and Lung Institute, London, UK), maintained in DMEM 10% FCS, 2 mM L-glutamine. HUVEC: primary human umbilical vein endothelial cells used up to passage 3, prepared from umbilical cords, Women's Hospital, Birmingham UK, maintained in M199 HEPES 20% FCS, 2 mM L-glutamine, 20 ng/ml FGF2. B16F10: murine melanoma cells (kind gift from Prof Ernst Wagner, IMC, Vienna, Austria), maintained in DMEM 10% FCS, 2 mM L-glutamine. SKOV-3: human ovarian carcinoma cells, maintained m DMEM 10% FCS, 2 mM Lglutamine.

A549: human lung carcinoma cells, maintained in DMEM 10% FCS, 2 mM L-glutamine. PC3: human prostatic carcinoma (Kaighn eta!., 1979), maintained in DMEM 10o/o FCS, 2

mM L-glutamine. COS-7: African green monkey SV40 transformed kidney fibroblasts, maintained in DMEM 10% FCS, 2 mM L-glutamine.

66

2.6

Biological evaluation of complexes

2.6.1

Uptake of 32 P labelled pLL/DNA complexes

Cells were plated out in 24-well plates ( 50000 cells/well, in I ml/well DMEM supplemented with 2 mM L-glutamine and IO o/o FCS).

The following day,

32

P labelled polyplexes

(prepared as described in sections 2.2.4 (ii) and 2.3.2) were incubated on cells (in DMEM) in the presence or absence of IOO J..LM chloroquine for 4 hours (37°C, 5 % C0 2 ). Following incubation, the media were removed, the cells washed with acid saline (I 50 mM NaCl, pH 3) to remove surface bound complexes, and finally dissolved in NaOH (0.5 ml, 2 M). The solutions were diluted in Ultima Flo AF scintillation fluid (4 ml, Packard) and assayed for radioactivity in a Packard I900TR liquid scintillation analyser. Known quantities of

32

P

labelled DNA were also assayed to provide a standard curve, and the results corrected for background radioactivity.

2.6.2

Uptake of YOYO-llabelled pLL/DNA complexes

Polyplexes were formed (as described in 2.3.2) using plasmid DNA labelled with YOYO-I (as described in method 2.2.4 (iii)). The resulting complexes were subsequently incubated on cells (70-80% confluent in 6-well plates in I ml DMEM) for 4 hours at either 4°C or 37°C. Media were subsequently discarded and replaced with PBS. Uptake of fluorescently labelled complexes was visualised using a Zeiss Axioscope microscope (Zeiss, Welwyn Garden City, UK). Trypsin was then used to remove the cell monolayer from the plate, and the cells were

resuspended and fixed using 2 % paraformaldehyde (in PBS). The cells suspensions were analysed for fluorescence using a Perkin Elmer Coulter XL flow cytometer.

67

2.6.3

Transfection of cell lines in vitro

Cells were plated out into 48 well plates on the day prior to transfection (20000 cells /well in 500 111 of DMEM supplemented with 2 rnM L-glutamine and 10 °/o FCS) and incubated at 37°C overnight. The following day, media were discarded and replaced with 175 11-l of fresh DMEM in the presence or absence of FCS ( 10 %) and also in the presence or absence of chloroquine (100 J..LM). Polyplexes prepared as described in methods 2.3.2 and 2.3.3 were added to the cells with 500 ng I 25 J..tl of plasmid DNA per well in a total volume of 200 11-L After 4 hours, media were removed, the cells washed twice in PBS, and replenished with fresh DMEM supplemented with 2 mM L-glutamine and 10 o/o FCS and incubated at 3 7°C, 5 % C02 for a further 20 hours.

Following incubation the media were discarded, the cells

washed twice in PBS, and the celllysates harvested by incubation of cells for 30 min at room temperature in 200 11-l of 1x lysis reagent (Promega, Madison, WI, USA).

50 11-l of the

remaining lysate was assayed for luciferase expression by the following method: Luciferin (500 111 of a 10 mM stock solution consisting of: 10 mg beetle luciferin, 0.47 ml of 1 M glycoglycine pH 8.0, 15.3 ml water) was diluted into 10 ml of luciferase reaction buffer (20

mM glycoglycine, 1 mM MgCh, 0.1 mM EDTA, 3.3 mM DTT, 0.5 mM ATP, 0.27 mM coenzyme A lithium salt). 100 11-l of this luciferin/luciferase reagent was added to 50 111 of the cell lysate, and the resulting luminescence integrated over 10 sin a Lumat LB 9507 (EG & G Berthold, Bundoora, Australia). Results are expressed as relative light units (RLU) per mg of cell protein, determined by BCA assay (see method 2.6.5) (Pierce, Chester, UK).

2.6.4

Evaluation of cell viability by MTS assay

This assay provides a colorimetric method for determining the number of viable cells in proliferation and cytotoxicity assays. The active agent, [3 - ( 4,5-dimethlythiazol-2-yl) -5- (3carboxymethoxyphenyl) -2- (4-sulphophenyl) -2H-tetrazolium] (Owen's reagent or MTS) is 68

bioreduced by cells by NADPH and NADH produced by dehydrogenase enzymes m metabolically active cells, producing a yellow/orange formazan product.

The assay was performed by plating cells at a density of 1. 5 x 104 per well in 12-well plates and incubating at 37oc in M199 medium in the presence or absence of 1 nM of FGF2, MQLPLAT peptide or VR WEMNL peptide (an irrelevant peptide control). After 48, 72 and 96 hrs, viability of the cell culture was assessed using the MTS assay. Media were replaced with 600 !J.l of FCS-free Ml99 containing 100 1-11 of CellTiter 96 AQueous One Solution Reagent (Promega Corporation, Madison, WI, USA). Culture plates were incubated at 37°C for 2 hrs. Following incubation, 100 1-11 of medium was transferred to new 96 well plates and the quantity of formazan product present was determined by measuring the absorbance at 490

nm using a Bio-Tek Instruments Microplate Autoreader EL311(Bio-Tek Instruments INC., Winooski, VT, USA).

2.6.5

Bicinchoninic acid assay

This assay is based on the observation that proteins reduce alkaline Cu (II) to Cu (I) in a concentration dependent manner. Determination of Cu (I) in a protein solution is possible using bicinchoninic acid, a highly specific chromogenic reagent for Cu (I), forming a purple complex with an absorbance maximum at 562 nm. Aliquots of cell lysate solution (50 1-1l) were transferred into round bottom Rohren tubes. 1 ml ofBCA assay solution (1:50 dilution of Cu (II) solution in BCA) was added to each tubes and incubates at 60oc for 30 min. The level of Cu (I) was measured at 562 nm using a UV mini 1240 UV-VIS spectrophotometer (Shimadzu, Duisburg, Germany), and the total protein concentration determined by comparison of the samples with known standards ofbovine serum albumin (BSA).

69

2.7

Virus production and manipulation

2.7.1

Virus propagation and purification

The virus used in these studies was the E1 deleted adenovirus vector Ad5/f3Gal~ 1 . All virus was propagated, purified and obtained from Dr. Kerry Fisher according to previously published protocols (Stallwood et al., 2000).

2.7.2

Polymer modification of virus

Frozen stocks of adenovirus were thawed at room temperature, vortexed and centrifuged for 1 9

min (5000 rpm). 10 -10

10

virus particles in 25 J.!l were adjusted to pH 7.8 by addition of 5 J.!l

of 0.4 M HEPES pH 7.8.

The desired amount of polymer (pHPMA) or polymer-peptide

conjugate (SIG-pHPMA) (1-10 mg) was weighed out and diluted in distilled water to obtain a final concentration of 20 mg/ml pHPMA. 10 J..Ll of the polymer solution was then added to the virus, vortexed and spun at 1000 rpm for 1 minute to obtain a final pHPMA concentration of 5 mg/ml.

The reaction was allowed to proceed at 4°C for 12h to form polymer coated

adenovirus (pHPMA-Ad5).

The reaction was terminated by the addition of aminoethanol

(final concentration 0.01% v/v) to effect aminolysis of remaining reactive ester groups.

2.7.3

Conjugation of bFGF to pHPMA-Ad5

bFGF was incorporated onto the surface of pHPMA modified particles by addition 2 hours after the initiation of the coating reaction was added to a final concentration of 80 J..Lg/ml, and incubation continued for a further 10 hours , before termination of the reaction with aminoethanol.

70

2.7.4

Infection protocol using Ad5/f3-gal

For assays of gene expression, 96-well plates were seeded at a density of 1xl04 cells/well. Cells were infected with polymer modified, retargeted or parental Ad5/b-gal at 104 ppc for 2 hours in DMEM supplemented with 2 mM L-glutamine and 2 % FCS. Expression of the

f3-

Gal transgene was quantified 48 hours post infection.

2.7.5

Analysis of f3-Gal transgene expression using Ad-5/f3gal

Following infection of cells using Ad5-f3gal, expression of the transgene was analysed using the Galacto-Light Plus chemiluminescent assay system (Tropix, Perkin-Elmer, Massachusetts, USA). Cells were washed briefly twice with PBS before the addition of 200 1-11 of 1 x prom ega lysis buffer to each well. The Galacton-Plus substrate was diluted 100-fold with Galacto-Light reaction buffer and warmed to room temperature.

Samples of cell lysate (5-50 Jll) were

placed in a luminometer tube and 200 J.!l of the reaction buffer was added and gently mixed before being incubated at room temperature for 60 minutes. 300 J.!l of accelerator was added prior to measurement of f3-gal expression in the luminometer.

2.8

Synthesis of polymers

All polymers used in this study were synthesised by, or with the help of Dr. David Oupicky in our laboratory.

2.8.1

Oxidative polymerisation of Cys(Lys) 10Cys into reducible polycations (RPC)

The C(K) 10C peptide (prepared by standard Fmoc/tBoc peptide chemistry) was purchased from Alta Biosciences as TFA salt. 20 mg of C(K) 10C (33 mM) was dissolved in 150 1-11 of 71

0.5 x PBS and 70 Jll DMSO (~70 fold molar excess in respect to thiol groups). The reaction was left to proceed at room temperature and the progress was monitored by measuring increase in molecular weight of resulting polymer and the concentration of unreacted thiol groups by Ellman's assay (method 2.8.1.1). The reaction was stopped after 48 hrs when no further changes in 1nolecular weight were observed.

RPC with reduced molecular weight was synthesised by introducing 8 mol % of aminoethanethiol into the reaction mixture.

The polymers (RPC) were purified on centrifugal concentrators with MWCO 10,000 (cellulose membrane) in swing-bucket centrifuge at 3000 rpm. The recovery of pLL was first tested on commercial pLL (20 kDa). Briefly, 20 mg pLL in 150 Jll 0.5 x PBS and 70 IJ.l DMSO was mixed with 12 ml of 5 mM HEPES pH 7.4; spun down to 750 IJ.l, then another 12 ml of HEPES was added and spun again to 1.2 ml. The removal of DMSO was verified on size exclusion cation analysis (SEC, method 2.8.1.2). TNBS assay (method 2.8.1.3)- showed 84% and 78% pLL recovery after the first and second spin respectively. The reaction mixture containing RPC was added to 15 ml of 5 mM HEPES pH 7.4 and spun down to 2 ml (sample taken for SEC and TNBS). 13 ml of HEPES was then added to the 2 ml of RPC and spun again to final volume of 700 111 to which 13 ml of HEPES was added (sample taken for SEC and TNBS). The concentration of RPC in the final solution was determined by TNBS assay using calibration curve constructed with commercial pLL. (29 kDa and 205 kDa).

The

concentrations of both RPC was further verified by ethidium bromide condensation which showed same transition point for both RPC a.nd their commercial pLL controls. molecular weights of both purified RPC was measured by SEC.

72

The

2.8.1.1 Ellman's assay for measurement of residual free thiol groups The decreasing concentration of free thiol groups during polymerisation was determined by Ellman's assay. Briefly, 2 J..tl of reaction mixture was diluted to the final volume 150 J..tl with SEC eluting buffer, frozen immediately and kept in -80C freezer. All samples were then analysed after the polymerisation had finished as follows: 0.5 ml of Ellman's reagent [5,5'Dithiobis(2-nitrobenzoic acid)] (4 mg/ml in 250 mM phosphate pH 8.0) was added to 1-ml plastic cuvette followed by 0.060 ml of each sample (or SEC buffer for the background) from SEC analysis followed by 0.440ml of250 mM phosphate pH 8.0. Absorbance at 412 nm was measured 15 min after mixing. Fresh solution of starting C(K) 10 C peptide was used to obtain 100% value.

2.8.1.2 Size exclusion cation HPLC analysis The changes in molecular weight distributions during progress of the polymerisation were monitored as follows: 2 J..tl of reaction mixture was diluted to the final volume 150 Jll with SEC eluting buffer (0.3 M NaCl + 0.1% v/v trifluoroacetic acid).

The sample was then

analysed on CATSEC-300 column (0.25 ml/min- 220 nm- 45 min run time- 50 !J.l injection volume) with Catsec guard column and Kontron Instruments HPLC 332 UV detector. Immediately after injection the remaining sample was frozen and kept in -80C freezer for Ellman's assay (see method 2. 8.1.1 ). SEC was also used to monitor the purification of RPC solution from DMSO.

To determine molecular weights (weight averages) of resulting RPC the column was calibrated with commercially available pLL samples with molecular weights in the range 3400- 205000. The calibration curve constructed then provided the following values of Mw for RPC: 187000 and 45000.

73

2.8.1.3 TNBS (2,4,6-trinitrobenzenesulphonic acid) assay for the determination of free amino groups Aliquots of peptide solution were added to reaction buffer (1.0 ml; 100 mM di-sodium tetraborate, pH 7.3). 30 ~1 of TNBS (1 Min water) was diluted with 970 ~1 reaction buffer, and 25 ~1 of the resulting solution was added to each solution and left at room temperature for 30 min. Absorbance of the samples was measured at 420 nm and the available amino groups determined using calibration curve constructed with commercial pLL.

2.8.2

Production of peptide-pHPMA conjugate

The conjugate between SIGYPLP and pHPMA described in Chapter 5 was produced as follows: SIGYPLP peptide (3 mg) dissolved in 5 added to 35 mg of pHPMA in 200

~1

~1

of anhydrous dimethylformamide was

dimethylformamide. Diisopropylethylamine (1.9

~1)

was then added and the solution was stirred for 5 hrs at room temperature. The polymer was then precipitated into 20 ml of acetone-diethylether ( 1:1) mixture. Precipitated polymer was isolated by filtration and dried in vacuo overnight (yield 94 and 88% ). The amount of remaining reactive groups was quantified from absorbance at 274 nm using molar extinction coefficient 9600.

2.9

Animal experiments

2.9.1

Animals

Male C57 black 6 mice were purchased (Charles River UK Ltd, Thanet, UK) and kept inhouse according to home office guidelines (Animals (Scientific Procedures) Act 1986).

74

2.9.2

Preparation of C57 black 6 mice bearing B16F10 tumours

Bl6Fl0 cells were grown to sub-confluency and suspended in PBS at a concentration of 10 7 cells/ml on the day of use. C57 mice were anaesthetised, shaved and inoculated with B 16F 10 cells (100 f.ll total volume) by subcutaneous injection into the rear flank. Tumour appeared 7-

14 days post inoculation.

2.9.3

Administration of samples to C57 black 6 mice

Mice were anaesthetised prior to administration of samples by intravenous tail vein injection (100 Jll total injected volume) and the animals were sacrificed after various lengths of time by cervical dislocation. Organs for radioactive quantification were dissolved in NaOH ( 10 M, 80°C for 1 hour).

Blood for radioactive quantification was placed into EDTA-containing

tubes (5 mM total concentration). Samples were diluted in Ultima Flo AF scintillation fluid (20 ml) and analysed for radioactivity in a liquid scintillation analyser (1900TR, Packard, USA). Organs for quantification of phage association were homogenised using ceramic beads in a Fastprep FP120 homogeniser (Qbiogene-Alexis Ltd, Nottingham, UK), and phage association quantified by titering dilutions of the resulting solutions on Xgal IPTG agar plates, as described in method 2.10.3.

2.10

Biopanning filamentous phage and related procedures

The phage display library used in all the procedures described here and in this thesis is the New England Biolabs Ph.D-7 M13 phage display library (New England Biolabs (UK) Ltd., Hitchin, Hertfordshire, UK). All procedures were performed using aerosol resistant tips to minimise the possibility of wild type infection of the library occurring.

75

2.10.1

Preparation of minimal agar plates

To prepare minimal agar the following autoclaved solutions were prewarmed to 60°C and mixed under sterile conditions (per litre): 500 ml 3 % bactoagar, 500 ml 2 x M9 minimal salts, 2 ml MgS04, 1 ml filter sterilised thiamine (50 mg/ml) and 100 f.ll CaCh. The resulting agar solution was aliquoted into petri dishes, flame sterilised and allowed to set (by cooling to room temperature). Minimal agar plates were stored at 4 oc until required or for a maximum of 2 weeks. Similarly agarose top was prepared, consisting of (per litre): 20 g LB media, 7 g agarose, 1 g MgCh.6H20. The solution was autoclaved and aliquoted into 50 ml tubes and stored solid at room temperature, melting using a microwave as required. Agarose top (50 ml) was supplemented with 480 f.!l of

5-bromo-4-chloro-3-indolyl-~-d-galactopyranoside;

(Xgal, Bioline Ltd., London, UK, 50 mg/ml in N,N dimethylformamide stored at -20°C wrapped in foil) and 120 f.ll of isopropyl-~-thiogalactopyranoside (IPTG Bioline Ltd, 1 Min autoclaved distilled water, stored at -20°C) prior to use to allow detection of wild type infection (by blue I white colour selection).

2.10.2

Strain maintenance

The supplied E. coli host strain ER2537 was used in all the studies. The strain was induced to grow by streaking out supplied glycerol stock onto minimal plates followed by incubation overnight at 37°C and wrapped in parafilm and stored at 4 °C. The strain was kept for a maximum of one month before being replaced to prevent significant loss ofF-factor and maintaining maximal infectivity.

76

2.10.3

Quantification of phage by plaque assay (titering)

To quantify phage, agarose top was melted, supplemented with Xgal and IPTG and aliquoted into 3 ml culture tubes equilibrated to 50°C (one tube per expected phage dilution). Minimal plates were pre-warmed to 3 7oc until ready for use. Multiple dilutions of solution containing phage were prepared in LB, and 10 !J.l of each dilution dispensed into a labelled microfuge tube. 200 !J.l of mid log phase (OD6oo

=0.5) ER2537 culture (grown in LB media using an

orbital shaker, 37°C, 200 rpm) was then dispensed into each tube, vortexed briefly and incubated at room temperature for 5 min. One at a time, the infected cells were transferred into a culture tube containing Xgal IPTG supplemented agarose top, vortexed briefly and immediately poured onto a pre-warmed minimal plate, spreading the agarose top evenly over the plate by gently tilting the plate.

Plates were allowed to cool for 5 minutes at room

temperature to allow the agarose top to set, then inverted and incubated overnight at 37°C. The following day, the number of plaques on the plates were counted. Phage were quantified by multiplying the observed number of plaques by the dilution factor for that plate to get phage titer in plaque forming units (pfu) per 10 !J.l original solution.

2.10.4

Amplification of recovered phage

20 ml ofLB media was inoculated with a single colony ofER2537 and incubated in an orbital shaker (37°C, 200 rpm), and allowed to grow to early log phase growth. The recovered phage for amplification was added to the bacterial solution, retUrned to the orbital shaker and allowed to grow for a further 4.5 hours. The culture was then transferred to a centrifuge tube and spun for 10 min at 10000 rpm (using a Sorvall SS-34 centrifuge) at 4°C. The supernatant was then transferred to a fresh centrifuge tube and re-spun.

The upper 80 % of the

supernatant was then drawn off and transferred to a fresh sterile tube and phage in solution precititated by the addition of 1/6 of PEG/NaCl solution (comprising 20 % w/v polyethylene 77

glycol-8000, 2.5 M NaCl). Phage were allowed to precipitate overnight at 4°C. The next day, the phage precipitation was pelleted by centrifugation at 10000 rpm for 15 min at 4°C, supernatant discarded, respun briefly and any residual supernatant removed with a pipette. The pellet was resuspended in 1 ml of PBS, transferred to a microfuge tube and spun to pellet any residual cells ( 13000 rpm, 5 min, 4 °C). The phage containing supernatant was transferred to a fresh microfuge tube and re-precipitated with 1/6 volume PEG/NaCl solution, and incubated on ice for 60 min.

Precipitated phage was then re-pelleted using a microfuge

( 13000 rpm, 10 min, 4 °C), supernatant discarded, re-s pun briefly and any residual supernatant removed with a micropipette. Finally the pellet was resuspended in PBS, spun briefly to remove any remaining insoluble matter (13000, 1 min) and the supernatant transferred to a fresh tube. The concentration of the amplified eluate was calculated by titering multiple dilutions on Xgal IPTG agar plates, as described in method 2.10.3. The amplified phage were stored at 4oc for no longer than 2 weeks. For preparation of high titer homogeneous glycerol stocks of individual selected phage clones, 1 ml of an overnight culture ofER2537 was diluted 1:100 inLB and dispensed into individual culture tubes. Individual phage clones were isolated by stabbing a relevant plaque with a sterile pipette tip, and transferring to the culture tube. Tubes were incubated for 4.5-5 hours in an orbital shaker (200 rpm, 37°C) before being transferred to microfuge tubes.

The

cultures were centrifuged ( 13000 rpm, 1 min, room temperature) and the phage containing supernatant withdraw to a fresh tube. The supernatant was re-spun briefly, and the upper 80 %recovered and diluted 1:1 with sterile glycerol. The concentration of phage in the glycerol stocks was evaluated by plaque assay as described in method 2.10.3. Homogeneous glycerol stocks could be stored indefinitely at -20°C with little discemable loss of infectivity

78

2.10.5

Iteration of FGF receptor-binding peptide by phage display technology

Candidate peptides which bind to FGF receptors on cells were identified by growing 911 cells to confluency in 6 well plates. The cells were then acclimatised to 4°C for 30 min before being washed briefly twice with PBS.

Phage library (Comprising 2 x 10 11 pfu/ml/well)

diluted in DMEM containing 1 % (w/v) bovine serum albumin was then added to the cells. Phage were allowed to bind to cellular receptors for 1 hr at 4

oc with gentle agitation, then

medium containing unbound phage was removed and discarded.

Cells were washed four

times for 5 min in PBS containing 1 % (w/v) BSA. Finally, phage bound to FGF-receptors were specifically eluted by the addition of 10 J.lg/ml FGF2 (in PBS) to each well for 1 hr at 4°C with gentle agitation. Numbers of eluted phage were established by titering on X-gal, IPTG agar plates, as described in method 2.10.3). The phage eluted by FGF2 were amplified (as described in method 2.10.4) for use in further rounds ofbiopanning. In total, five rounds ofbiopanning were performed in triplicate.

2.10.6

Isolation and sequencing of phage DNA

Following each round of selection, individual phage clones were picked, expanded and their

DNA isolated and sequenced. Individual plaques were picked and expanded as described in method 2.10.3. After the amplification step, the culture was centrifuged and the upper 50% of the phage containing supernatant transferred to a fresh microfuge tube.

Phage were

precipitated by the addition of 200 J.!l of PEG I NaCl solution, mixed and allowed to precipitate for 10 min at room temperature. Precipitated phage was then centrifuged (13000 rpm, 10 min, room temperature) and the resulting pellet resuspended in 100 J.!l iodide buffer (comprising 10 mM Tris-HCl (pH 8), 1 mM EDTA, 4 M Nal (stored at room temperature protected from light))to which was added 250 J.!l of absolute ethanol. Short incubation at room temperature results in the preferential precipitation of single stranded phage DNA, 79

whilst leaving most phage protein in solution. Phage DNA was allowed to precipitate for 1o min at room temperature before being centrifuged ( 13000 rpm, 10 min, room temperature). The pellet was then washed briefly with 70 % ethanol and dried briefly on a hot block (65°C , .

5 min) the resulting DNA pellet was resuspended in 30 J..tl ofTE buffer (10 mM Tris-HCI (pH 8), 1 mM EDTA).

100 ng of the resulting DNA was used for sequencing using 40 ng of the supplied -96 primer together with the BigDye Terminator Cycle Sequencing Kit (Biosystems, Perkin Elmer). Chain terminating PCR was performed using a GeneAmp PCR system 9700 PCR machine set to 25 cycles of 96°C for 10 seconds (denaturing), 50°C for 5 seconds (primer annealing) and 60°C for 4 min (chain elongation).

DNA sequences were determined using an automated ABI PRISM 3700 sequencer (Perkin Elmer).

2.10.7

Evaluation of cell binding activities of selected phage clones

To evaluate the binding of selected phage clones for cell surface receptors, cells were grown to confluence in 6 well plates. Cells were acclimatised to 4 oc for 30 min, then washed briefly in PBS prior to the addition to each well of 5 x 107 pfu of each selected phage clone diluted into 1 ml DMEM, 1 o/o BSA. Phage were allowed to bind to cells for 1 hr at 4 oc with gentle agitation. Media containing unbound phage were discarded and the cells washed four times for 5 min in PBS containing 1 % BSA. Bound phage were subsequently eluted from the cell surface by the addition of0.01-10 J.lg/ml FGF2 for 1 hr at 4

oc with gentle agitation. In some

experiments, residual phage that remained bound to the cells following elution with FGF2

80

were quantified by scraping the cells from the 6 well plate. Recovery was determined by plaque infection assay (see method 2.10.3) on Xgal IPTG agar plates at 37°C, with plaques counted the following morning. To discount the possibility that selected phage clones might be binding to cell surface heparans, heparin (0.4 - 400 J.lg/ml) was added to the elution medium, cells were incubated for 1 hr and phage recovery was determined by titration as described above.

2.10.8

Immunohistochemical analysis of selected phage binding cells

To investigate binding of phage to 911 cells, 8 x 10 5 of cells were plated in 100 mm dish and grown to a confluency. Cells were then washed twice with PBS, and incubated with 5 x 10 11 pfu of selected or insertless phage diluted in 5 ml ofDMEM containing 1 % w/v BSA for 1 hr at 37°C. The cells were washed 3 times for 5 min with 10 ml of PBS containing 1 % w/v BSA), and recovered by scraping the bottom of the dishes. 2 drops of solution containing 5 x 5

10 cells/ml were centrifuged for 5 min at 1500 rpm onto a glass slide using a Cytospin 2 centrifuge (Shandon, Waltham, MA, USA). Cells were fixed with acetone for 10 min at room temperature. The slides were then washed, blocked with 5 % heat inactivated goat serum (IDNGS) in PBS for 10 min and incubated with anti-M13 monoclonal antibody (Amersham Pharmacia, Piscataway, NJ, USA) at a 1:100 dilution for 1 hr at room temperature. After several washes with PBS, slides were incubated with StreptABComplex Duet HRP mouse/rabbit secondary antibody (DAKO, Ely, U.K.) at a 1:200 dilution for 30 min at room temperature. Signal was revealed by the addition of 3,3-diaminobenzidine tetrahydrochloride

(DAB, DAKO, UK). Slides were analysed with a Zeiss Axiovert 25 microscope.

81

2.10.9

Investigation of affinity of selected phage clones for cellular receptors

Affinity analysis of selected phage clones was investigated by incubating cells grown to contluency in 6-well plates with selected phage clones (prepared in PBS as described in method 2.10.4) of increasing concentrations from 107 pfu to 10 12 pfu. Briefly, cells were acclimatised to 4 oc for 30 min prior to the experiment, and the cells were washed twice with PBS. Selected phage were then diluted into 1 ml DMEM containing 1 % w/v BSA, and applied to the cells. Phage were allowed to bind to cellular receptors for 1 hour at 4°C with gentle agitation, before unbound phage in free solution were discarded. Cells were then washed with PBS containing 1 % w/v BSA, before the cell associated phage were recovered and tittered, as described in method 2.10.3. Phage input was plotted against bound phage, and the data were analysed by fitting the general logistic equation: [bound]= Bmax. [freet I ([free]n + Kdn)

2.10.10 Competitive inhibition of selected phage clones binding FGF receptors Selective inhibition of phage binding was achieved by preincubation of acclimatised cells with FGF2 (0.5

~M),

MQLPLAT peptide or VRWEMNL peptide (each 0.5

~M,

synthesised

by Alta Biosciences, Birmingham, UK) for 1 hr at 4 °C. Medium was discarded and the cells washed briefly with PBS prior to addition of 5 x 107 pfu of selected phage (diluted into 1 ml

DMEM containing 1 % BSA). Phage were allowed to bind to cellular receptors for 1 hr at 4

oc with gentle agitation.

Medium containing unbound phage was discarded, the cells washed

four times for 5 min in PBS containing 1 % BSA, and bound phage eluted using 2 ~g/ml FGF2. Eluted phage were quantified by titering, as described in method 2.10.3.

82

2.10.11 Phage binding assay on surgically resected human specimens To investigate the possibility that selected phage clones might bind preferentially to human tumours in a clinically relevant setting, we developed an ex vivo human organ system using surgically resected specimens. This work was performed in collaboration with Dr. Fukuto Maruta in our laboratory who performed all the surgical manipulations. Ethical permission for the study was obtained in advance from the South Birmingham Health Authority local research ethics committee prior to the surgical procedures described here. Patients selected for this study were suffering from operable gastric adenocarcinoma. Immediately following surgical resection of the stomach, canulae were inserted into the left gastric artery and vein. The vessels were then washed with 100 ml of PBS. 2 x 109 pfu of selected phage, diluted in 5 ml PBS, were then injected into the left gastric artery through the canula and allowed to bind for 5 min. Unbound phage were washed away using 100 ml of PBS injected via the canula into the left gastric artery. The stomach was then opened along the greater curvature and samples of tumour and adjacent normal gastric tissue were collected. Samples were weighed and homogenised using a motor-driven teflon-on-glass homogeniser.

Phage were then

quantified by titering multiple dilutions of the resulting homogenate, as described in method 2.10.3. Results are presented as pfu of associated phage per milligram of tissue.

2.11

Expression of data

All error bars shown represent the mean and standard deviation of the data from three independent experiments.

83

3.

Characterisation of Complexes Formed by Self Assembly of Plasmid DNA with Poly(L-Lysine)

3.1

Introduction

Polyelectrolyte complexes (polyplexes) provide a simple yet highly versatile non-viral vector for delivery of nucleic acids for genetic therapies (Hwang and Davis, 200 1). Polyanionic DNA can be complexed with simple cationic polymers in salt free conditions, to undergo selfassembly reaction that yields discrete nanoparticles suitable as gene delivery vectors. Previous studies have examined the influence of the cationic polymer on the biophysical properties of the complex produced with DNA, and rationally designed or ligand-modified polymers are being increasingly used in sophisticated vectors developed to meet specific biological objectives (Kabanov eta!., 1998; Kircheis eta!., 2001; Wolfert eta!., 1999).

A range of techniques are routinely employed to monitor the self-assembly process and to characterise properties of the polyelectrolyte complexes produced.

Gel retardation is a

method that monitors the loss of electrophoretic mobility of DNA when it binds a critical amount of cationic polymer (Lane eta!., 1992). However, the mechanistic basis of the assay is unclear, specifically whether it represents nanoparticle formation or simply neutralisation of DNA. Similarly, inhibition of ethidium bromide (EtBr)/DNA fluorescence resulting from DNA condensation by cationic polymers is a convenient method to monitor the polyelectrolyte interaction (Xu and Szoka, 1996); again, however, it is not known whether the assay measures particle formation or simply neutralisation of the DNA. In addition, possible influences of EtBr concentration have not been characterised, neither is it known whether EtBr is expelled from the DNA structure during condensation or merely quenched.

84

ln this study we have assessed fundamental aspects of the interpolyelectrolyte self assembly reaction of plasmid DNA with poly(L-lysine) (pLL), and have compared the analytical techniques commonly used to monitor particle formation.

Different analytical techniques

measure different parameters~ hence results obtained must be interpreted carefully in light of the specific question addressed. The two preferred techniques - light scattering (Konak et a/., 1998) and EtBr/DNA fluorescence- are then used to characterise the stability of nanoparticles formed using polycations of varying molecular weight, to address the possibility that complexes formed with lower molecular weight polycations may be more susceptible to disruption under physiological conditions.

This would impose limitations on their

physiological use, but has not been systematically examined before.

3.2

Results

3.2.1

Influence of charge ratio on electrophoretic mobility of DNA

The influence of charge (N:P) ratio of cationic polymer (bearing positively charged amino (N) groups) to DNA (bearing negatively charged phosphate (P) groups), on electrophoretic mobility of DNA is outlined in Figure 3.1. At low N:P ratios of 0.2 and 0.4, over 92% of the total DNA showed bands corresponding to supercoiled and relaxed forms of plasmid DNA that migrated as free Cy-3 pDNA with only trace amounts of DNA were retained at the origin. Complexes formed at slightly higher N:P ratios of 0.6 and 0.8 displayed intermediate behaviour with 59% and 28% migrating as free DNA into the gel and 41% and 72°/o respectively retained in the well as complexed or semi-complexed DNA. At such N:P ratios the DNA appeared to show a shift from banding to a rather more smeared profile (Figure 3.1a, and 3.1 b) suggesting that non-neutralising binding of pLL to Cy-3 DNA can affect its migratory capacity. Complexes formed at N:P ratios 0.8 and higher displayed almost total retention (greater than 95% at all N:P ratios) at the origin indicating the formation of non-

85

migratory, positively charged nanoparticles.At certain N:P ratios e.g. 0.4, there was also evidence for DNA migrating in the gel even faster then supercoiled DNA ("gel acceleration"). This is thought to represent partially condensed plasmid DNA, still bearing a strong net negative charge but whose more compacted structure allows a more efficient penetration through the gel.

Indications of disproportionation, whereby DNA with full electrophoretic mobility exists alongside DNA which is totally retained at the origin representing fully condensed DNA was also apparent, though this only appears to occur at intermediate N:P ratios (0.4 and 0.6) with little or no evidence for any DNA condensation at lower N:P ratios (i.e. 0.2), where less than 2% of the Cy-3 DNA retained at the origin. If pLL does indeed undergo any electrostatic interaction with DNA at such low N:P ratios, it appears to be readily shed during the electrophoresis procedure.

3.2.2

Influence of charge ratio on intensity of light scattering

An alternative technique to monitor the formation of polyplexes is to observe the increase in

intensity of scattered light resulting from the sudden increase in apparent molecular weight during hydrophobic collapse of the DNA into small solid nanoparticles. Such an experiment is outlined in Figure 3.2, where the intensity of scattered light was measured following each sequential addition of pLL (lllkDa). The intensity of scattered light was found to increase up to N:P = 1.0, with a particularly steep increase in scattering intensity between N:P

=

0.6-

1.0. At N:P ratios greater than 1, signal intensity plateuing was observed indicating that complex formation was completed by N:P

=

1. These data suggest few changes in DNA

morphology at low N:P ratios, with the self assembly of discrete nanoparticles occurring between N:P 0.6 and 1.0.

86

0.0 0.2

-~

~~~

a 0.4

9-. 5

t··.·

0.6

f

0.8

t

N:P

1.0 1.2 1.4

1 •, I

'~ _,

H

N:P

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

b

Fig 3.1 Cont. ..

87

c

100 90 ~ ....... rFl 80 ~ ([) 70 ~ ([) 60 u ~ ([) 50 u rFl ([) 40 1-< 0 ......;::::$ 30 ~ 20 ~ 10 ~

~

0 0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

N:P Ratio

Figure 3.1 Influence of charge ratio on electrophoretic mobility of DNA. Plasmid DNA was labelled with Cy-3 and purified to remove free label. DNA was then complexed by addition ofpLL (111kDa) in water at various N:P ratios and allowed to stabilise for 1 hour at room temperature. Finally, the complexes were loaded onto a 1% agarose gel and subjected to electrophoresis (100 V, 2 hr) and the DNA was visualised by planar imaging (Figure 3.1a). Individual lanes were integrated (Figure 3.1b) and the relative quantities of migratory (black squares) vs. non migratory DNA (open squares) was quantified (Figure 3.1c), with the total DNA in each lane set to 100o/o. See methods 2.2.4 (i) and 2.4. 1.

88

1.2

·-.c V)

~

.....~

1.0

(].)

~

'"0

0.8

(].)

1-


0.4

~ ........ (].)

~

0.2

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

N:P Ratio

Figure 3.2 Effect of charge ratio on light scattering intensity. pLL was added in increments of 0.2 N:P ratio to plasmid DNA (20 J.lg/ml) in Hepes (10 mM, pH 7.4) and mixed thoroughly. Following each addition of pLL, the scattering intensity of the resulting solution was measured using a fluorimeter set to Aex=Aem= 600run (slit widths 5 run, cut off filter open, integration time 3 seconds), and the maximum recorded signal set to 1 to give a relative scattering intensity for each sample. Data represents the mean and standard deviation of3 independent experiments. See method 2.4.2.

89

3.2.3

Effect of EtBr concentration on the fluorescence of pLL/DNA complexes

The most commonly used technique for monitoring particle formation is the ethidium bromide (EtBr) exclusion assay. The principle of this assay is based on the fact that EtBr fluorescence in enhanced 10-15 fold when intercalated into DNA. However during particle formation, there is a drop in the signal intensity, though whether this represents EtBr exclusion from DNA, or merely a quenching of the signal will be considered in greater detail later.

Figure 3.3 shows the profile of EtBr/DNA fluorescence observed at increasing

concentrations of EtBr. At low, sub-saturation levels of EtBr (400 ng/ml, representing 1 ethidium molecule per 60 DNA phosphate groups) the profile of EtBr/DNA fluorescence showed a sigmoidal curve, dropping sharply over the N:P range 0. 75-1. However the profile of fluorescence was observed to shift from sigmoidal towards linearity as the EtBr concentration approached saturation levels (up to 3 1-1g/ml, representing 1 ethidium molecule per 8 DNA phosphate groups). Fluorescence observed using the highest concentrations of EtBr did not fall as low in percentage terms as was observed with the lowest concentrations of EtBr even at high N:P ratios.

Sequential addition of the polyanion poly(L-aspartic acid)

(pLAA) resulted in a complete restoration of fluorescence following particle disruption of complexes formed of the lowest concentration of EtBr indicating that sufficient numbers of sites were available as to allow 100 % intercalation of the EtBr, however fluorescence was only partially restored at higher EtBr concentrations, indicating that 100 % re-intercalation of EtBr into the DNA was not possible. This suggests that addition of pLAA to pLL/DNA complexes results in a fraction of the EtBr intercalation sites being exposed - sufficient to restore the total fluorescence to 100% for low EtBr concentrations, but greatly less so at EtBr concentrations approaching saturation (3 1-1g/ml). This also suggests the formation of a ternary

90

120.00

100.00 Q) (.)

c

Q) (.) C/)

80.00

Sequential addition of polyanion

Q)

.._ 0

..2 LJ_

60.00

1

Q)

>

:;::;

ro

Q)

40.00

0:: ;::R

0

20.00

0.00

0

1.25

2.5

N:P Ratio

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Charge RatiopLAA/pLL

Figure 3.3 Effect of EtBr concentration on the fluorescence profile of pLL/DNA complexes. EtBr was dissolved in Hepes (10mM, pH 7.4) to concentrations of 0.4(•), 1( +), 1.5(o), 2(x), 2.5(.&.) or 3(o) Jlg/ml and the fluorescence measured using a fluorimeter (Aex = 510nm, Aem=590nm, 10 nm slit widths, cut off filter open, integration time 3 seconds) and set to 0%. Plasmid DNA was then added to the solutions to a final concentration of20Jlg/ml and resulting fluorescence set as 100%. Aliquots of pLL (111kDa) were then added sequentially to the solutions (0.25:1 N:P ratio increments), mixed thoroughly and the fluorescence measured and correlated to the 100% figure. Following the formation of complexes to N:P = 2.5, restoration ofEtBr/DNA fluorescence was monitored by titrating in the polyanion pLAA, until equimolar equivalents of the polycationic pLL and the polyanionic pLAA were present in the solution. Data represents the mean and standard deviation of 3 independent experiments. See method 2.4.3.

91

pLAA/pLL/DNA complex in which only a small proportion of the DNA is available for EtBr binding.

The formation of such a ternary complex has been confirmed by agarose gel

electrophoresis (data not shown).

3.2.4

Evaluation of the mechanism of inhibition of EtBr/DNA fluorescence

In order to establish whether the mechanism of inhibition of DNA/EtBr fluorescence during particle formation involves the exclusion of EtBr from the DNA or merely a quenching of the signal, DNA was preincubated with EtBr (400 ng/ml), complexed with pLL (111KDa, N:P 2.0) and electrophoresed on an EtBr-free agarose gel (1 %, 100 V, 120 min). Controls were plasmid DNA alone, with and without preincubation with EtBr. Post electrophoresis, the gel was visualised and photographed on a UV transilluminator. The only detectable fluorescence on the gel was from the free plasmid with EtBr (figure 3.4 (i)), indicating that EtBr is not stripped from the DNA during electrophoresis through agarose.

This did not change

following subsequent polyplex dissociation in situ by exposure of the gel to heparin solution (Figure 3.4 (ii)). Subsequent incubation of the gel in a solution containing EtBr produced fluorescence from all DNA samples (Figure 3.4 (iii)). Free plasmid DNA showed mobility unaffected by preincubation in EtBr, whereas the pLL/DNA complexes showed fluorescence only in the well, as expected.

Given that DNA and EtBr remain associated during

electrophoresis, and restoration of fluorescence was not observed following the in situ dissociation of pLL/DNA/EtBr complexes with heparin, the mechanism of inhibition of EtBr/DNA fluorescence during complex formation is expulsion of the EtBr rather than signal quenching of EtBr/DNA complexes.

92

(i)

(ii)

(iii)

1.

2. 3.

Figure 3.4 EtBr exclusion during polyplex formation. DNA!EtBr (1), DNA (2) and pLL(lll kDa)/DNA!EtBr (N:P = 2) (3) were electrophoresed on a 1% EtBr free agarose gel and photographed on a UV transilluminator (i). The gel was then soaked in a solution containing heparin ( 10000 units, 30 min, room temperature) to dissociate the complexes in situ, and again photographed on a UV transilluminator (ii). Finally the gel was soaked in a solution containing EtBr (0.5J.!g/ml, 30 min, room temperature) to reveal the undetected DNA and photographed again on a UV transilluminator (iii). See method 2.4.1.

93

3.2.5

Comparison of excitation spectra of EtBr, DNA!EtBr and pLL/DNA/EtBr.

The fluorescence excitation spectra for EtBr are also consistent with its expulsion from DNA during condensation (Figure 3.5). EtBr in free solution shows an excitation peak at 480 run approx., shifting to 520 nm following intercalation into DNA. Following complexation with pLL, the maximum shifts back to about 490nm, suggesting loss of the intercalated species. Low level fluorescence from residual intercalated EtBr (15 % approx., Figure 3.3) is thought to combine with the fluorescence of EtBr free in solution to give the intermediate spectrum observed.

3.2.6

Determination of the stability of pLL/DNA complexes to dilution in water.

To investigate the possibility that complexes formed of lower molecular weight pLL might display a reduced stability with respect to electrostatic reversibility to complexes formed of higher molecular weight pLL, complexes were formed by sequential addition of either low (3.97 kDa), medium (29.3 kDa) or high (111 kDa) molecular weight pLL to N:P = 2.8 (Figure 3.6). Complex formation was monitored by EtBr/DNA fluorescence inhibition as previously described. Complexes were then subject to dilution with ultra pure water and any decrease in fluorescence monitored. Dilution in water did not result in a quenching effect on EtBr signal since dilution of EtBr/DNA in water results in a linear fall in fluorescence, proportional to the dilution factor, i.e. the signal remains at 100% when corrected for the dilution factor (data not shown). Polyplexes formed of all molecular weight pLL displayed a greater than expected decrease in relative fluorescence following dilution in water, indicating that complex stability was independent of the molecular weight of the polycation and also indicating that complexes formed to this N:P ratio (2.8) appear to be stable to dilution in water. Indeed, since the corrected relative fluorescence value actually drops away towards zero rather than remaining

94

7000

850

BOO

DNA/EtBr

550 5llO

450

§

....... Q

500

-~ 400

..s~

300

0

1-o

~ 200

::r:

100

0

0.1

0.2

0.5

1.0

2.0

[pHPMA] I mg/ml

* =p< 0.01

Figure 4.5. Effect of physiological concentrations of salt on the hydrodynamic diameter of unmodified and pHPMA coated pLL/DNA complexes. pLL/DNA complexes (20 f..!g/ml DNA; 29.3 kDa pLL; 2:1 charge ratio in 50mM HEPES, pH 7.8) were reacted overnight at room temperature with various concentrations of pHPMA (0 to 2 mg/ml). Hydrodynamic radius of the resulting complexes was then measured by light scattering (white bars). The concentration of salt in the solution was subsequently raised to physiological levels by addition of 1110 v/v of lOx PBS, mixed thoroughly and the effect on hydrodynamic radius again monitored by light scattering after a further 15 min (black bars). Data represents the mean and standard deviation of3 independent experiments. See methods 2.3.3 and 2.4.5.

118

4.2.5

Influence of polycation molecular weight on polyanion stability of coated

complexes

A series of experiments was designed to address the proposal that pHPMA coated polyplexes formed using polycations of different molecular weight might display differential levels of stability to polyanions. Accordingly pLL/DNA complexes (NIP = 2) were formed using low (4.0 kDa), medium (29.3 kDa) and high (111 kDa) molecular weight poly(L-lysine). The resulting complexes were then surface modified with pHPMA (2 mg/ml in 50 mM HEPES, pH 7.8), or polyethylene glycol- N-hydroxysuccinimide ester (PEG, 10 mM HEPES, pH 7.4) overnight at room temperature. The resultant solution was subdivided, with half exposed to pLAA ( 1 mg/ml, 30 min, room temperature) with the other half remaining unexposed to pLAA. Finally the samples were electrophoresed (45 min, 100 V, 1% agarose, 0.5

~g/ml

EtBr) and the location of the DNA analysed by planar imaging (Figure 4.7). As previously observed, uncoated polyplexes were readily dissociated following exposure to pLAA, and the DNA migrated into the gel (Figure 4.7(a)). In contrast, pHPMA coated complexes formed using medium (29.3 kDa) and high (Ill kDa) molecular weight pLL were resistant to polyanion dissociation, with the DNA retained at the origin (Figure 4.7(b) and 4.7(c)). However coated complexes formed of the low molecular weight pLL (4.0 kDa) behaved as uncoated complexes, with the DNA migrating freely into the gel (Figure 4.7(d)).

Similarly,

complexes that had been coated with the monovalent hydrophilic polymer PEG were also unstable to pLAA mediated dissociation (Figure 4.7(e)). These results indicate two key parameters which determine stability of such complexes to polyanions. The first parameter is valency of the hydrophilic protective polymer, since complexes stabilised with the monovalent hydrophilic polymer PEG were readily dissociated by pLAA, in contrast to pHPMA coated complexes. The second parameter is the molecular weight of the polycation

119

[pLAA] (mf!/ml)

0.0 1.0

+ pHPMA (2 mg/ml)

0.5

I 1 :f l

pLL/DNA

0.25

Figure 4.6. pHPMA mediated polyanion stability of pLL/DNA complexes. pLL/DNA complexes (NIP = 2) or pHPMA coated pLL/DNA complexes (2 mg/ml pHPMA, 50mM HEPES pH 7.8) were incubated with a range of concentrations of pLAA (0 to 1 mg/ml, 30 min, room temperature) and electrophoresed (IOOV, 45 min, 1 % agarose, 0.5 1-1g/ml EtBr). The resulting gel was visualised by planar imaging using a Typhoon gel imaging system (Aex = 532, Aem = 610 nm, 450V). See methods 2.3.3 and 2.4.1.

+1-pLAA

(a)

+

'

~...

pLL (29.3 kDa)/DNA

',·fl.·
250

Figure 5.8 Incorporation of the peptide into pHPMA does not effect the capacity of the polymer to protect polyplexes from polyanion mediated dissociation. Complexes were formed between pLL (29.3 kDa) and plasmid DNA to N:P = 2. Resulting complexes were subdivided and either unmodified (a) or surface modified with SIG-pHPMA (b) or pHPMA (c) (200 1-!g/ml, 50 mM HEPES, pH 7.8) prior to polyanion mediated dissociation with pLAA (0-1 000 1-1g/ml). The resulting gel was visualised by planar imaging using a Typhoon gel imaging system (Aex = 532, Aem = 610 nm, 450V). See methods 2.3.2, 2.3.3 and 2.4.1.

157

dissociation, polyplexes were formed between poly(L-lysine) and pGL3 plasmid DNA, and surface modified with either unmodified pHPMA (200 ~g/ml), or the pHPMA prederivatised to incorporated the SIGYPLP peptide (SIG-pHPMA). Complexes were then challenged with increasing (0-1000 ~g/ml) concentrations of the polyanion poly(L-aspartic acid) for 30 min. The resulting complexes were subject to electrophoresis and planar imaging to detect EtBr/DNA fluorescence (Figure 5.8). Simple polyplexes complexes were readily dissociated by polyanions, and hence the plasmid DNA migrated freely into the gel. However, complexes which had been subject to surface modification with either pHPMA or SIG-pHPMA were resistant to pLAA mediated dissociation at all concentrations tested, indicating that the polymer-peptide conjugate retains its ability to stabilise polyplexes to polyanions.

5.2.8

Transfection efficiency of SIG-pHPMA coated complexes in vitro

To address whether the incorporation of the targeting peptide into the hydrophilic polymer might result in a restoration of levels of trans gene expression in vitro, complexes were formed between pGL3 plasmid DNA and pLL 29.3 kDa. Complexes were subsequently subdivided and surface modified with either pHPMA or SIG-pHPMA at concentrations of either 2 mg/ml or 0.2 mg/ml. The resulting complexes were using in a transfection assay using B16F10 cells (20000 cells/well) in the presence or absence of 100 ~M chloroquine (Figure 5.9). Simple polyplexes mediate high levels of trans gene expression in the absence of serum through non specific electrostatic interaction with the cellular membrane, with their efficiency of trans gene expression is improved up to 100 fold in the presence of chloroquine.

As previously

demonstrated, surface modification with pHPMA reduces the efficiency of transgene expression in a dose dependent manner, although the low levels of trans gene expression could still be augmented by the presence of chloroquine in the transfection media, indicating low level uptake of complexes are achieved, possibly due to local regions still bearing a slight net

158

0.2 mglml SIG-pHPMA

1

I

2 mg/ml SIG-pHPMA

0.2 mglml pHPMA 2mglml pHPMA

f--"

pLL/ DNA I

Figure 5.9 Enhanced transgene expression of pHPMA coated complexes following incorporation of SIGYPLP into the reactive polymer. Complexes were formed between pGL3 plasmid DNA and pLL 29.3 kDa (N:P = 2), subdivided and surface modified with either pHPMA or SIG-pHPMA (2 or 0.2 mg/ml). Complexes were use in a transfection assay using B16F10 cells (20000 cells/well) in the presence of 10 °/o serum, except for pLL/DNA complexes which were transfected in the absence of serum. Transfections were performed in the presence (white bars) or absence (black bars) of 100 J..LM chloroquine in the transfection media. Levels of reporter gene expression were monitored 24 hours post transfection. Data represents the mean and standard deviation of3 independent experiments. See methods 2.3.2, 2.3.3, 2.6.3 and 2.6.5.

159

positive charge or through non-specific uptake of complexes during cell division. Prederivatisation of the polymer to incorporate the SIGYPLP targeting peptide resulted in increased in levels of trans gene expression in all the formulations tested compared with nontargeted controls, with levels of gene expression enhanced 60.3 x and 3.2 x for complexes coated at 2 mg/ml in the presence and absence of chloroquine respectively, and 8.4 x and 4.2 x for complexes formed at 0.2 mg/ml in the presence and absence of chloroquine respectively.

5.2.9

In vivo pharmacology of SIG-pHPMA coated complexes m B16F10 tumour bearing C57 black 6 mice

To evaluate whether serum stable SIGYPLP targeted complexes might be able to deliver DNA to B16F10 tumours in vivo,

32

P dCTP labelled DNA was used to form pHPMA or SIG-

pHPMA coated complexes. Complexes were injected i.v. into the tail vein ofB16F10 tumour bearing C57 black 6 mice.

Mice were sacrificed at 30 min, 5 hours and 24 hours post

injection, and the biodistribution of the injected complexes determined by dissolving the resected organs in NaOH, diluting the resultant solution in scintillation fluid and assaying for radioactivity.

Complexes surface modified with SIG-pHPMA were removed from the

bloodstream more rapidly than the untargeted complexes, with less than 20 % of the injected dose remaining in the bloodstream 30 min post injection, compared to greater than 40% with untargeted pHPMA coated complexes (Figure 5.10), as would be predicted if the targeting peptide were binding to receptors expressed on endothelial cells. After 24 hours, minimal levels of

32

P were detectable in the blood of either pHPMA or SIG-pHPMA coated

complexes. Complexes surface modified with SIG-pHPMA showed significantly (at the 10 % level) elevated levels of tumour accumulation 5 hours post injection compared to the

untargeted controls (p = 0.057), with 6.4 +/- 0.65 % and 3.9 +/- 1.3 % (per g) respectively of the recovered dose associated with the tumour (Figure 5.11). However, 24 hours post

160

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~ T .:))Y Self-Assembly of DNA with Poly(L-Iysine) \Ian L. Parker, David Oupicky, Philip R. Dash, and Leonard W. Seymour 1 J?C Institute for Cancer Studies, University of Birmingham, BJ5 2TA, United Kingdom

~eceived

July 12, 2001; published online January 23, 2002

DNA self-assembly with polycations produces nano>articles suitable for gene delivery, although there is 10 standard methodology to measure particle formajon and stability. Here we have compared three comnonly used assays, namely, light scattering, inhibition >f ethidium bromide fluorescence, and modified elec:rophoretic mobility of DNA. Analysis by light scattering and loss of ethidium bromide fluorescence both ;bowed poly(L-lysine) (pLL)/DNA nanoparticles form >ver the lysine/phosphate ratio range 0.6-1.0, although retardation of DNA electrophoretic mobility :ommenced at lower lysine/phosphate ratios. This ~robably indicates that the first two assays monitor llNA coll~pse into particles, while the electrophoresis itSSa.Y measures neutralization of the charge on DNA. Gel analysis of the complexes showed disproportioniltion during nanoparticle formation, probably reflectIng cooperative binding of the polycation. The assays were used to examine stability of complexes to dilution in water and physiological salts. Whereas all pLL/ llNA nanoparticles were stable to dilution in water, the presence of physiological salts provoked selective [tisruption of complexes based on low-molecularweight pLL. Polyelectrolyte complexes for targeted ilpplication in vivo should therefore be based on highnolecular-weight polycations, or should be stabilized to prevent their dissociation under physiological salt mnditions. e 2002 Elsevier Science (USA) Key Words: gene therapy; poly(L-lysine); condensation; ethidium bromide.

.

Polyelectrolyte complexes provide a versatile nonvi~al vector for delivery of nucleic acids in genetic theripies (1). Polyanionic DNA can be complexed with simlle cationic polymers, undergoing a self-assembly

reaction to produce discrete nanoparticles suitable as gene delivery vectors. Previous studies have examined the influence of the cationic polymer on the biophysical properties of the complex produced with DNA, and rationally designed or ligand-modified polymers are being increasingly used in sophisticated vectors developed to meet specific biological objectives (2- 4). A range of techniques have been employed to monitor the self-assembly process and to characterize properties of the polyelectrolyte complexes produced. Gel retardation is a method that monitors the loss of electrophoretic mobility of DNA when it binds a critical amount of cationic polymer (5). However, the mechanistic basis of the assay is unclear, specifically whether it represents nanoparticle formation or simply neutralization of DNA. Similarly, inhibition of ethidium bromide (EtBr) 2/DNA fluorescence resulting from DNA condensation by cationic polymers is a convenient method to monitor the polyelectrolyte interaction (6); again, however, it is not known whether the assay measures particle formation or simply neutralization of the DNA. In addition, possible influences of EtBr concentration have not been characterized; neither is it known whether EtBr is expelled from the DNA structure during condensation or merely quenched. In this study we have assessed fundamental aspects of the interpolyelectrolyte self-assembly reaction of plasmid DNA with poly(L-lysine) (pLL). and have compared the analytical techniques commonly used to monitor particle formation. The two preferred techniques-light scattering (7) and EtBr/DNA fluorescence-were then used to characterize the stability of nanoparticles formed using polycations of varying molecular weight, to address the possibility of differential Abbreviations used: EtBr, ethldium bromide; pLL, poly(L-lysine); pLAA, poly(L-aspartic acid); PBS, phosphate-buffered saline; pHPMA, poly(N[2-hydroxypropyl]methacrylamide. 2

1

To whom correspondence should be addressed. Fax: 44+ 121 414

1263. E-mail: L.Seymour@bham.ac.uk. 003-2697/02 $35.00 ) 2002 Elsevier Science (USA) ~I rights reserved.

75

76

PARKER ET AL.

stability und:r physiological conditions, enabling more informed destgn of vectors for gene delivery in vivo. MATERIALS AND METHODS

Sources of DNA. The 6-kb expression vector pSV2BCL2 containing the BCL2 gene driven by the SV40 promoter {8) was grown in Escherichia coli and purified using Qiagen Gigaprep Kits (Crawley, West Sussex, UK). Concentration and purity of the DNA were checked on a spectrophotometer at A 260 /A 280 absorbance wavelengths. Formation ofpLL/DNA complexes. DNA (20 11-g/ml) was added in ultrapure water to a polypropylene Eppendorf and mixed thoroughly. pLL (2.5 mg/ml stock in water) was added to give an N:P ratio of 2.0 (defined as the molar ratio of amino groups in the pLL to phosphate groups in the DNA), unless otherwise stated, and gently mixed. Complexes were allowed to form for 30 min prior to use. The N:P ratio of 2.0 was selected to ensure efficient complex formation without a great excess of free pLL. Although some pLL will remain unbound following complex formation, this is unlikely to interfere with the assays performed. Cy-3 labeling of DNA expression vectors. The Cy-3 labeling kit was purchased from Panvera (Madison, WI), and 10 11-g of plasmid DNA labeled using the standard protocol. Briefly, DNA (0.1 mg/ml) was reacted with 10 11-l of Cy-3 in appropriate buffer at room temperature for 1 h. Unreacted dye was removed by centrifugation through a G-50 microspin purification column. Cy-3-labeled DNA was analyzed following agarose gel electrophoresis using a Typhoon Fluorimager {Molecular Dynamics, High Wycombe, UK) (Aex 550 nm, Aem 570 nm). Agarose gel electrophoresis. pLL/DNA complexes were prepared in water (N:P ratios 0.2-2.0) and electrophoresed on agarose gels (1% w/v, 120 min, 100 V). In some studies the gels were incubated, postelectrophoresis, in heparin sulfate solution to disturb the DNA-cationic polymer complexes, and restore access ofEtBr. Measurement of complex formation by light scattering. Complex formation was investigated by measuring changes in light scattering intensity using a fluorometer. Plasmid DNA (20 ,..,gJml) in Hepes (10 mM, pH 7.4) was inctibated in a cuvette and the intensity of scattered light (Aex 600 nm, Aem 600 nm) was set to zero. pLL (Ill kDa) was added at 0.2 N:P increments and the changes in scattering intensity monitored. Data are presented as the relative scattered intensity, with the maximum signal detected at any N :P ratio set to 1.0. Inhibition of EtBr/DNA fluorescence by poly{L-lysine). The EtBr/DNA fluorescence (Aex 510 nm, Aem 590 nm) of plasmid DNA (20 JLg/ml) in ultrapure water

containing EtBr (400 ng/ml-3 ~-tg/ml) was measured and set to 100%. Aliquots of pLL (2.5 mg/ml stock in water, 111 kDa) were added sequentially and the fluorescence was measured after each addition, until the N:P ratio reached 2.5. In some experiments poly(L-aspartic acid) (pLAA) was added incrementally to the complexes to sequester the pLL and restore EtBr/DNA fluorescence. Determination of fluorescence spectra. Fluorescence excitation spectra for EtBr (0.4 JLg/ml), DNA (20 ILgl ml)/EtBr and pLUDNA/EtBr (N:P = 2) (all in water) were determined using 2 ml solution and scanning excitation (Aex 350-560 nm) with Aem fixed at 590 nm. Evaluation ofstability ofcomplexes to dilution. Stability of pLLIDNA complexes was assessed by measuring restoration of EtBr/DNA fluorescence on dilution. Complexes were formed by sequential addition of pLL (4.0, 29.3, or 111 kDa) to DNA (20 ILg/ml) in water containing EtBr (400 ng/ml), to a final N:P ratio of 2.8. EtBr/DNA fluorescence was measured after each addition. Ten percent (v/v) lOX PBS solution (without EtBr) was then added. After 15 min the change in fluorescence was measured and the complexes were subject to a series of dilutions in PBS (without EtBr). Alternatively, complexes were diluted in pure water. Fluorescence was measured after each dilution and corrected for volume changes and for salt-mediated inhibition of EtBr/DNA fluorescence (using matched EtBr/DNA controls without pLL). RESULTS

Influence of Charge Ratio on Electrophoretic Mobility of DNA Self-assembly of pLL/DNA complexes was examined by agarose electrophoresis, using plasmid DNA trace labeled with Cy-3. Complexes were formed in water, using the single-step procedure, allowed to stabilize for 1 h, and then subjected to agarose gel electrophoresis (Fig. 1A). At N:P ratios of 0.2 and 0.4, more than 92% of the total DNA showed bands corresponding to supercoiled and relaxed forms that comigrated with free Cy-3-labeled DNA. In contrast, complexes formed at N:P ratios of 1.0-1.4 all showed fluorescence that was almost entirely retained in the well (more than 93% in each case, Fig.lB). Complexes formed at N:P ratios of 0.6 and 0.8 showed intermediate behavior, with 74.1 + 13.3 and 33.5 :t 6.5% behaving as free DNA, respectively, and 25.9 :t 13.3 and 66.5 :t 6.5% retained in the well. Complexes formed at such N :P ratios showed no obvious signs of banding, rather evidence of smearing suggesting that nonneutralizing binding of pLL to the Cy-3-DNA can affect its migratory capacity. At certain N:P ratios, e.g., 0.4 and 0.6, there was also evidence for DNA migrating in the gel even faster than supercoiled

MONITORING NANOPARTICLE FORMATION BY SELF-ASSEMBLy OF DNA

A

N:P 0 0.2 _}__ _ _ _ _ _ _ _ _ --"---- 0.4

decreased mobility. If pLL does interact electrostaticall!' with DNA at these low N :P ratios it appears to be easily released under electrophoresis conditions.

0.6

Influence of Charge Ratio on Intensity of Light Scattering

0.8 1.0

1.2

Plasmid DNA was mixed with pLL (111 kDa) by single-step additions, at N:P ratios 0-2. The intensity of scattered light was measured and found to increase up to N:P 1.0, with particularly steep rise between N:P 0.6 and 1.0 (Fig. 1C). Above N:P 1.0 there was minimal further increase. These data suggest few changes in DNA morphology at low N:P values, with self-assembly of discrete nanoparticles occurring between N:P 0.8 and 1.0.

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Effect of EtBr Concentration on the Fluorescence Observed with pLl/DNA Complexes

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N:P Ratio: FIG. 1. Cy3-DNA (10 p.g) was complexed with pLL (111 KDa, N:P ratio 0-1.4) and electrophoresed, and the signal in individual lanes was quantified using a Typhoon Fluorimager (A). The distribution of DNA retained in the well (D) or entering the gel (•) is shown in (B). Complex formation was also monitored by light scattering (C), with polycation titrated into a DNA solution (20 p.g/ml) in pure water, and the intensity of scattered light measured.

DNA (so called;gel acceleration"). This is thought to represent semicondensed DNA, still bearing a strong net negative charge but with a more compact structure that enables easier penetration through agarose. Some preparations (e.g., N:P 0.4) contain both DNA with full electrophoretic mobility and DNA that is retained in the well, suggesting that disproportionation occurs during complex formation. Despite this possibility for disproportionate binding, there was very little evidence for DNA condensation at very low N:P ratios; for example, at N:P 0.2, only 2% of the DNA shows

The profile of EtBr/DNA fluorescence observed during pLL binding to DNA was dependent on the concentration of EtBr used. Low EtBr concentrations (400 ng/ml) showed a sigmoidal curve, falling sharply over the N:P ratios 0.75-1.0. In contrast, high EtBr concentrations (3 ~J.g/ml) produced a more linear fall across the entire N:P range. Intermediate EtBr concentrations showed a gradual shift from sigmoidal to linear profile (Fig. 2A). These data are compatible with a finite number of EtBr binding sites in DNA that become gradually unavailable with increasing pLL binding. For high EtBr concentrations (where most of the sites are occupied in free DNA) this results in a linear fall in fluorescence. In contrast, for low EtBr concentrations (where 100% fluorescence corresponds to occupation of only a fraction of the sites available in DNA) fluorescence remains high during the early part of the condensation but is lost rapidly toward the end of condensation. Fluorescence observed using the highest concentrations of EtBr (2.5-3 11-g/ml) never fell as low (in % terms) as those obtained using low EtBr concentrations. This suggests that high levels of intercalated EtBr stabilize the DNA structure against complete inhibition of fluorescence by pLL. Addition of the polyanion pLAA restored fluorescence to 100% when a low EtBr concentration was used, although higher EtBr concentrations allowed a lower maximum percentage restoration. This supports the model of a finite number of EtBr/DNA sites and suggests that pLAA is able to release only a fraction of them, presumably resulting in the formation of a ternary pLAA/pLlJDNA complex wherein some of the DNA is available for EtBr binding. The fonnation of a ternary complex was confirmed by agarose gel analysis, where addition of pLAA to pLL/ DNA complexes led to a restoration of fluorescence,

78

PARKER ET AL.

A

caused by fluorescence quenching or by expulsion of EtBr from the DNA structure. In the first study, DNA ; was preincubated with EtBr {400 ng/ml), complexed i with pLL (111 kDa, N:P 2.0), and electrophoresed on an EtBr-free agarose gel (1 %, 45 min, 90 V). Controls included plasmid DNA alone, with and without EtBr. The only fluorescence visible on the gel was from the free plasmid with EtBr (Fig. 2B). This did not change following dissociation of the complexes in situ by incubation of the gel, postelectrophoresis, in heparin solution. Subsequent incubation of the gel in a solution containing EtBr produced fluorescence from all DNA samples. Free plasmid DNA showed mobility unaffected by preincubation in EtBr, whereas the pLUDNA 1.25 2.5 0.1 e.2 t.l ... Cl.5 Q.6 0.7 u 0,9 1.0 complexes showed fluorescence only in the well, as ___../ ....... / ·---v y - - - - expected. N:I"Ibtio (pLUDNA) Nil" ..... (pLM/pLL) Since EtBr is not removed from free DNA during electrophoresis (Fig. 2B), and complexes formed in the A B c presence of EtBr did not fluoresce even following disruption with heparin, this indicates that EtBr is expelled during complexation. The fluorescence excitation spectra for EtBr are also consistent with its expulsion from DNA during condensation (Fig. 2C). EtBr in free solution shows an excitation peak at 480 nm approximately, shifting to 520 nm following intercalation into DNA. Following pLL complexation the maximum shifts back to about 490 nm, suggesting loss of the intercalated species. Low-level fluorescence from residual intercalated EtBr (15% approx, Fig. 2A) is thought to combine with the fluorescence of EtBr free in solution to give the intermediate spectrum observed. ·- ···---·-··-·· ------ ····---------------

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FIG. 2. Complexes were formed (N:P 2.5) as described in the text, in the presence of EtBr, monitoring inhibition of EtBr fluorescence to indicate particle formation (A). EtBr concentrations were 0.4 (II), 1.0 (+).1.5 (0). 2.0 (x). 2.5 (.&.),and 3.0 (D) p.g/ml. Subsequently, complexes were destabilized by the sequential addition of pLAA, and restoration of fluorescence was monitored. Expulsion of EtBr from DNA during particle formation was demonstrated by electrophoretic analysis (B(A)) of DNNEtBr (lane 1), DNA alone (lane 2), and pLUDNA/EtBr (lane 3) on an EtBr-free agarose gel. The gel was then incubated in heparin solution (B(B)), and DNA in the gel visualized with EtBr (B(C)). Excitation spectra for EtBr, DNA/EtBr, and pLUDNA/EtBr were also analyzed (C) using a fixed emissi~ wavelength (590 run).

although the DNA was unable to enter the agarose gel (data not shown).

Evaluation of the Mechanism of Inhibition fJf EtBr/DNA Fluorescence Two assays were used to determine whether pLLmediated inhibition of EtBr/DNA fluorescence was

Determination of the Stability ofpLI./DNA Complexes at High Dilution pLLIDNA complexes were formed (N:P ratio of 2.8, pLL molecular weights 4.0K, 29.3K, and 111K) in the presence of EtBr (400 ng/ml). All complexes were found to be stable to serial dilutions in water, judged by a lack of restoration of EtBr fluorescence. Analysis using photon correlation spectroscopy of the sizes of complexes based on pLL 29.3 and 111 kDa showed no changes during dilution of complexes (data not shown). with hydrodynamic radius unaffected after up to 16fold dilution. These dilutions represent the highest dilutions that could be tested subject to the sensitivity limitations of the analytical techniques. To determine stability in physiological salts, complexes were formed in water (as above) and then the salt concentration was raised to physiological levels. EtBr/DNA fluorescence was measured and corrected for the slight quenching effect of PBS. Complexes formed between medium (29.3K)- and high (111K)molecular-weight pLL showed a relative increase in fluorescence to 50% of the initial value (Fig. 3).

MONITORING NANOPARTICLE FORMATION BY SELF-ASSEMBLy OF DNA

---------------l

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~rG. 3. To investigate the effect of dilution in physiological salts, omplexes were fonned by sequential addition of pLL to DNA in rater containing EtBr (0.4 J-A.gfml), using pLL of molecular weight verage 4K (x), 29.3K (+). and lllK (0). Ten percent (v/v) of lOX BS was then added. After 30 min the change in EtBr/DNA fluoresence was measured and complexes were subjected to serial dilutions 1 PBS. Changes in fluorescence were monitored and corrected for 1e dilution factor and the slight quenching effect of PBS on EtBr/ INA fluorescence.

rhereas complexes formed using low-molecular-weight LL (4.0K) were more susceptible to destabilization, rith fluorescence increasing to around 80% of the alue for DNAIEtBr alone. Effects of further dilution in tBS showed no progressive destabilization of the comlexes. The slight fall in fluorescence at higher dilulons is thought to be an artifact resulting from loss of ignallinearity due to proximity to the machine limit of uorescence detection. These data suggest that pLL/ INA complexes are generally stable to dilution in wa~r or 10 mM Hepes buffer; however, complexes formed sing lower-molecular-weight polycations are more rone to destabilization when introduced into physiolgical concentrations of salt. ISCUSSION

In this study we have compared three different aslJS that are often used to study self-assembly of DNA ith polycations, namely, particle formation by light :attering, loss ~f EtBr fluorescence, and changed elecophoretic mobility of DNA on agarose gels. Probably 1e simplest measure is the light scattering approach, hich indicates physical formation of nanoparticles tpable of reflecting light. However, this approach reIires relatively pure samples, and it is difficult to nploy it productively in typical biological samples. [ormation gained using light scattering was therere compared with that obtained using the other, more bust, approaches to determine which is a better incator of particle formation.

79

Analysis of DNA mobility changes showed evidence fo~ disproportionation of pLL binding to plasmid DNA. WI~ some plasmids binding large amounts of pLL and losmg electrophoretic mobility, and others binding littie or no pLL and behaving as free plasmid on electrophoresis. Such disproportionate behavior of plasmid DNA when mixed with polycations at low N:P ratios is in concordance with recently published articles (9). This effect could result from cooperative binding of cationic polymers to plasmids, possibly promoted by the formation of locally charge-neutral, relatively hydrophobic, regions that facilitate the binding of further cationic polymers to adjacent regions of the DNA. Considering the gel shift information itself, substantial amounts of DNA could be retained in the well at relatively low NIP ratios (e.g., 25% at NIP 0.6) when particle formation (as gauged by light scattering} was very limited; this suggests that the gel shift assay determines formation ofplasmids that bind a certain critical amount of polycation, assuming modified charge or structural properties that change their electrophoretic behavior, but may not actually reflect formation of discrete nanoparticles. Monitoring loss of EtBr/DNA fluorescence can also be used to determine DNA condensation; however, the fluorescence profile obtained is strongly dependent on the concentration of EtBr used. This partly reflects the ability of EtBr to stabilize DNA against polyelectrolyte condensation, but also results from the ability of small EtBr concentrations to give 100% fluorescence signals even when the majority of binding sites are no longer available. The assay must therefore be used with caution, including when it is used to monitor destabilization of complexes since, at low EtBr concentrations, 100% restoration of fluorescence may represent availability of only a fraction of the binding sites in DNA. Nevertheless, carefully interpreted, this assay generates results that are similar to those from light scattering, and provides a simple and sensitive means to monitor particle formation. Each ofthe assays reported was also performed using linear calf thymus DNA, with the results generated being virtually identical; hence any role played by the constrained topology of circular DNA on influencing condensation properties is thought to be minor. A range of techniques show that loss of fluorescence coincides with- expulsion of EtBr from the DNA, rather than quenching. In addition to the two assays shown, we have also formed pLL/DNA complexes in the presence of limiting amounts of EtBr and surface modified them with a multivalent reactive polymer based on poly(N [2-hydroxypropyl] methacrylamide) (pHPMA), as described elsewhere (1 0). The resulting polymercoated complexes are stable to disruption by polyanions, and show no increase in EtBr/DNA fluorescence following addition of pLAA. However, addition of free

80

1

PARKER ET AL

DNA results in return of fluorescence, indicating that the EtBr is free in solution and not trapped within the coated complex. Finally, previous studies using atomic force microscopy showed that low-molecular-weight pLL produced very small (25-nm) complexes at high NIP ratio with nNA (11), spawning the hypothesis that the small size !fleeted a dynamic structure capable of continual rerrangement. We therefore evaluated the ability of 1lts to destabilize pLUDNA complexes, measured by !turn of EtBr fluorescence, and found, as predicted, mt complexes formed using small pLL are more easily isrupted by physiological salts. Small complexes such ;; these are attractive for size-restricted gene delivery pplications, for example, where vectors must extrav;;ate through small interendothelial cell fenestrations , gain access to target tissues. However, to apply 1ese small complexes in biological environments it ill be necessary to stabilize them against disruption ~ salts. Several techniques are available to achieve 1is, including surface modification with multivalent HPMA, as described above. The use of such complexes . particularly enticing following arrival within the ~11. since removal of the stabilizing polymer should 1able salt-assisted dissociation of the complex, proiding the DNA in a form suitable for efficient tran:ription. In addition, such systems would be ideally 1ited for delivery of mRNA, where it is known that the Licleic acid must be released within the cytoplasm in a !latively free form, to undergo efficient ribosomal anslation (12). :KNOWLEDGMENTS We are grateful to the Cancer Research Campaign and the Biochnological and Biological Sciences Research Council for support.

EFERENCES . Hwang, S. ]., and Davies, M. E. (2001) Cationic polymers for gene delivery: Designs for overcoming barriers to systemic administration. Curr. Opin. Mol. Ther. 3, 183-191.

2. Wolfert, M.A., Dash, P. R., Nazarova, 0., Oupicky, D .. Seymour. L. W., Smart, S., Strohalm. J ., and Ulbrich, K. (1999) Polyelectrolyte vectors for gene delivery: Influence of cationic polymer on biophysical properties of complexes fanned with DNA. Bioconjug. Chem. 10, 993-1004. 3. Kircheis, R., Wightman, L, Schreiber. A., Robiza, B.. Rossler, V .. Kursa, M., and Wagner, E. (2001) Polyethylenimine/DNA complexes shielded by transferrin target gene expression to tumours after systemic application. Gene Ther. 8, 28-40. 4. Kabanov, A., Szoka, F., and Seymour, L. W. (1998) Interpolyelectrolyte complexes for gene delivery Polymer aspects of transfection activity, in Self-Assembling Vectors for Gene Therapy (Kabanov, A_, Feigner, P., and Seymour, L. W., Eds.), Wiley, New York_ 5. Lane, D., Prentki, P., and Chandler, M. (1992) Use of gel retardation to analyze protein-nucleic acid interactions. Microbial. Rev. 56, 509-528. 6. Xu, Y.. and Szoka, F. C., Jr. (1996) Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry 35, 5616-5623. 7. Konak, C., Mrkvickova, L., Nazarova, 0., Ulbrich, K., and Seymour, L. W. (1998) Formation of DNA complexes with diblock copolymers of poly-N(2-hydrox:ypropyl)methacrylamide and polycations. Supramol. Sci. 5, 67-74. 8. Tstgimoto, Y. (1989) Overexpression of the human BCL-2 gene product results in growth enhancement of Epstein-Barr virusimmortalized B cells. Proc. Natl. Acad. Sci. USA 86, 1958-1962. 9. Bronich, T. K., Nguyen, H. K., Eisenberg. A., and Kabanov, A V. (2000) Recognition of DNA topology in reactions between plasmid DNA and cationic copolymers. J. Am. Chem. Soc. 122, 83398343. 10. Dash, P.R., Read, M. L., Fisher, K. D_, Howard, K. A., Wolfert, M., Oupicky, D .. Subr, V., Strohalm, J., Ulbrich, K., and Seymour, L. W. (2000) Decreased binding to proteins and cells of polymeric gene delivery vectors surface modified with a multivalent hydrophilic polymer and retargeting through attachment of transferrin. J. Bioi. Chem. 275, 3793-3802. 11. Wolfert, M. A., and Seymour, L. W. (1996) Atomic force microscopic analysis of the influence of the molecular weight of poly(L-lysine) on the size of polyelectrolyte complexes formed with DNA. Gene Ther. 3, 269-273. 12. Bettinger. T., Carlisle, R. C., Read, M. L., Ogris, M., and Seymour, L. W. (2001) Peptide-mediated RNA delivery: A novel approach for enhanced transfection of primary and post-mitotic cells. Nucleic Acids Res. 29, 3882-3891.

JACS COMMUNICATIONS Published on Web 1211112001

Laterally Stabiliz~d Complexes of DNA with Linear Reducible Polycations: Strategy for Tnggered Intracellular Activation of DNA Delivery Vectors David Oupicky,* Alan L. Parker, and leonard W. Seymour CRC Institute for Cancer Studies, University of Birmingham, Birmingham BJ5 21T. United Kingdom Received June 18, 2001

Polyelectrolyte complexes of DNA represent an important alternative to viruses in the development of efficient vectors for gene therapy. Our research focuses on polycation-based vectors surface stabilized with hydrophilic polymers to decrease nonspecifi~ interactions with biological components and ensure suitability for intravenous delivery. We have developed a method to surfacemodify the polycation/DNA vectors using multivalent hydrophilic copolymers of N-(2-hydroxypropyl) methacrylamide with methacryloylglycylglycine 4-nitrophenyl ester (PHPMA). In contrast to coating with monovalent polymers, the multivalent coating introduces lateral stability into the complexes-a factor that promotes extended circulation in the bloodstream. This is a prerequisite for targeting dissen_rinated disease targets (e.g. metastatic cancers). 1 The versatile nature of this vector allows receptor-mediated targeting of genes to selected cells and manipulation of intracellular trafficking. The vector is stable enough to allow efficient in vivo delivery and prevent DNA degradation, however relatively low levels of gene expression suggest that the DNA is not available for transcription due to the retention of the polymer coating, restricting enzymatic access, and transcription efficiency.2 This indicates that poor release of DNA from this vector can be an important limiting step and highlights the need for a specific intracellular activation mechanism. Strategies of specific intracellular activation of drug and gene delivery vectors already have been established for several types of carriers.3 - 7 Here we describe an innovative approach to the design ofthe PHPMA-stabilized vectors, which makes use of the significant intracellular reducing capacity8·9 to reverse the lateral stabilization introduced by surface coating with PHPMA, facilitating release of the delivered DNA. The design of the vector can thus satisfy the contradictory requirements for high stability in the extracellular environment and easy intracellular availability of DNA. This is achieved by a unique combination of steric and reversible lateral stabilization by PHPMA of vectors based on plasmid DNA complexed with a reducible linear polycation. Intracellular reductive degradation of the polycation to lower molecular weight species removes the lateral stabilizing effect of the PHPMA coating, enabling efficient transcription of the plasmid DNA (Scheme 1). Oxidative JX->lycondensation ofCys(Lys) 10Cys peptide (prepared by standard Fmoc/tBoc chemistry) with dimethyl sulfoxide 10 provided the reductively cleavable linear polycations (RPC) by oxidation of the terminal cysteinyl thiol groups (Scheme I). The reaction kinetic shows typical features of step-growth polymerization, i.e., molecular weight of the resulting polymer increases with time, and high conversion degrees of functional thiol groups must be reached to obtain high molecular weight products (Figure 1). 11 Molecular weight of the RPC can be easily controlled by introducing compounds containing single thiol functionality into the polycon*To whom correspondence should be addressed.

8 VOL 124, NO.1, 2002 • J. AM. CHEM. SOC.

Exuaoellutu enviromlent: resistaJI stniC:IUral inrepity 10 CJ

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