UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Development of stabilized plasmid lipid particles as intracellular gene delivery vehicles Palmer, Lorne R. 2000

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2000-566005.pdf [ 11.65MB ]
Metadata
JSON: 831-1.0089815.json
JSON-LD: 831-1.0089815-ld.json
RDF/XML (Pretty): 831-1.0089815-rdf.xml
RDF/JSON: 831-1.0089815-rdf.json
Turtle: 831-1.0089815-turtle.txt
N-Triples: 831-1.0089815-rdf-ntriples.txt
Original Record: 831-1.0089815-source.json
Full Text
831-1.0089815-fulltext.txt
Citation
831-1.0089815.ris

Full Text

DEVELOPMENT OF STABILIZED PLASMID LIPID PARTICLES AS INTRACELLULAR GENE DELIVERY VEHICLES by L O R N E R. P A L M E R B.Sc. (Hons.), Chemistry, Acadia University, 1991 M . S c , Biochemistry, University of Guelph, 1994  A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Biochemistry and Molecular Biology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A September, 2000 © Lome R. Palmer, 2000  UBC  Special Collections - Thesis Authorisation Form  Page 1 of 1  I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t copying or p u b l i c a t i o n of t h i s t h e s i s for f i n a n c i a l gain s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  Department The U n i v e r s i t y o f B r i t i s h Columbia Vancouver, Canada Date  http://www.library.ubc.ca/spcoll/thesauth.html  9/2/99  ABSTRACT  This thesis focuses on the development of an efficient, non-viral system capable of delivering genetic information (i.e. plasmid DNA) to cells in vitro and eventually in vivo. The approach taken is to develop small liposomal systems containing plasmid that can deliver intact D N A to disease sites in vivo, and then to examine methods to improve the transfection potential of these systems. In Chapter 2, a detergent dialysis procedure is described which allows encapsulation of plasmid D N A within a lipid envelope, where the resulting particle is stabilized in aqueous media by the presence of a poly(ethyleneglycol) (PEG) coating. These "stabilized plasmid-lipid particles" (SPLP) exhibit an average size of 70 nm in diameter, contain one plasmid molecule per particle and fully protect the encapsulated plasmid from digestion by serum nucleases. cationic  lipid  content,  with  maximum  Encapsulation is a sensitive function of entrapment  observed  at  dioleoyl-  dimethylammonium chloride (DODAC) contents of 5 to 10 mol%. The formulation process results in plasmid-trapping efficiencies of up to 70% and permits inclusion of "fusogenic" lipids such as dioleoylphosphatidylethanolamine (DOPE).  The in vitro  transfection capabilities of SPLP are demonstrated to be strongly dependent on the length of the acyl chain contained in the ceramide group used to anchor the P E G polymer to the surface of the SPLP.  Shorter acyl chain lengths result in a P E G coating which can  dissociate from the SPLP surface, transforming the SPLP from a stable particle to a transfection-competent entity. In Chapter 3, the ligand employed is a cationic poly (ethylene glycol) (PEG) lipid (CPL) consisting of a lipid anchor and a  PEG3400  spacer chain with 1, 2, 4, or 8 positive  ii  charges at the end of the P E G (CPLj, C P L , C P L , and C P L ) . These C P L are introduced 2  4  8  into preformed L U V s by a post-insertion method. The uptake of L U V - C P L by B H K cells is assessed both quantitatively and qualitatively.  The LUV-CPL4 system  (containing 4 positive charges) exhibits dramatically improved uptake compared to L U V in the absence of CPL. Chapter 4 examines the influence of CPL4 on the transfection potency of SPLP. It is shown that up to 4 mol% CPL4 can be inserted into preformed SPLP, resulting in up to 50-fold enhancements in uptake into baby hamster kidney (BHK) cells. In the presence of 2_|_  Ca  g  this results in up to 10 -fold enhancements in transgene expression as compared to  SPLP in the absence of CPL4 or Ca . These transfection levels are comparable to those observed for plasmid DNA-cationic lipid complexes (lipoplexes) but without the toxic effects noted for lipoplex systems. It is concluded that in the presence of Ca  and  appropriate ligands to stimulate uptake, SPLP are highly transfection potent, supporting their potential as in vivo gene therapy vectors.  iii  T A B L E OF CONTENTS  ABSTRACT  ii  T A B L E OF CONTENTS  .  iv  LIST OF T A B L E S  viii  LIST OF FIGURES  ix  ABBREVIATIONS  xii  ACKNOWLEDGEMENTS  xvi  DEDICATION  xvii  C H A P T E R 1: I N T R O D U C T I O N  1  1.1 D N A D E L I V E R Y VECTORS  4  1.1.1 Viral Vectors  4  1.1.2 Non-Viral Vectors  7  1.1.2.1 DNA-liposome complexes  8  1.1.3 Liposomes as cancer drug carrier systems  9  1.1.3.1 Targeting of L U V to disease tumour sites  11  1.2 LIPIDS  15  1.2.1 Chemistry and physics of lipid  15  1.2.1.1 Phospholipids  17  1.2.1.2 Sphingolipids  17  1.2.1.3 PEG-lipids  17  1.2.1.4 Cationic lipids  20  1.2.2 Structural behavior of lipids  21  1.2.2.1 Gel-liquid crystalline phase transition  23  1.2.2.2 Lipid polymorphism  23  iv  1.3 LIPOSOMES  28  1.3.1 Classification of liposomes  28  1.3.1.1 Multilamellar vesicles (MLVs)  28  1.3.1.2 Large unilamellar vesicles (LUVs)  28  1.3.1.3 Small unilamellar vesicles (SUVs)  30  1.3.2 Preparation of L U V s  30  1.3.2.1 Extrusion technique  30  1.3.2.2 Detergent dialysis  31  1.3.3 Membrane fusion  33  1.4 D E O X Y R I B O N U C L E I C ACID (DNA) 1.3.1 Composition of D N A 1.3.2 Plasmid D N A  36 36  :  1.3.3 Structure of Plasmid D N A 1.5 THESIS OBJECTIVES  37 37 40  C H A P T E R 2: S T A B I L I Z E D PLASMID-LIPID P A R T I C L E S : C O N S T R U C T I O N AND CHARACTERIZATION 41  2.1 INTRODUCTION  41  2.2 M A T E R I A L S & METHODS  43  2.2.1 Materials  43  2.2.2 Encapsulation of plasmid D N A  43  2.2.3 Assay for plasmid D N A encapsulation efficiency  44  2.2.4 Removal of unencapsulated D N A by anion-exchange chromatography  44  2.2.5 Isolation of encapsulated plasmid by sucrose density gradient centrifugation  44  2.2.6 Freeze-fracture electron microscopy  45  2.2.7 Serum stability assay  46  2.2.8 In vitro transfection  46  2.3 RESULTS  47  2.3.1 Entrapment of plasmid D N A within lipid particles by employing detergent dialysis  47  2.3.2 Plasmid D N A in stabilized plasmid-lipid particles is protected from serum nuclease  51  2.3.3 Stabilized plasmid-lipid particles can be isolated by density centrifugation ....53 2.3.4 Stabilized plasmid-lipid particles exhibit a narrow size distribution  55  2.3.5 In vitro transfection properties of stabilized plasmid-lipid particles  .55  2.4 DISCUSSION  59  CHAPTER 3: CATIONIC PEG-LIPIDS  68  3.1 INTRODUCTION  68  3.2 M A T E R I A L S & METHODS  71  3.2.1 Materials  71  3.2.2 Vesicle preparation  71  3.2.3 Insertion of CPL into pre-formed vesicles  72  3.2.4 Uptake of liposomes containing CPL by B H K cells  73  3.3 RESULTS  :  74  3.3.1 Insertion of CPL into pre-formed liposomes  74  3.3.2 Uptake of CPL-liposomes by B H K cells  77  3.4 DISCUSSION  82  CHAPTER 4: INCORPORATION OF CPL INTO SPLP FOR INCREASED IN VITRO UPTAKE AND TRANSFECTION  85  4.1 INTRODUCTION  85  4.2 M A T E R I A L S & METHODS  87  4.2.1 Materials  87  4.2.2 Preparation of SPLP-CPL  87  4  4.2.3 Uptake and transfection studies  89  4.3 RESULTS  93  4.3.1 Cationic PEG lipids can be inserted into preformed SPL  93  4.3.2 SPLP-CPL aggregate following insertion of C P L and de-aggregate 4  4  following addition of divalent cations 4.3.3 PEG-CerC  20  content and stability of SPLP-CPL4  95 99  4.3.4 SPLP-CPL4 exhibit enhanced uptake into B H K cells and dramatically enhanced transfection potency 4.3.5 C a  2+  is required for transfection activity of SPLP-CPL4  100 103  4.3.6 SPLP-CPL4 exhibit transfection potencies in vitro that are comparable to or greater than achieved using lipoplexes 4.3.7 SPLP-CPL4 are non-toxic and efficient transfection agents 4.4 DISCUSSION  107 110 114  C H A P T E R 5: S U M M A R Y A N D F U T U R E D I R E C T I O N S  118  5.1 S U M M A R Y OF RESULTS  118  5.2 F U T U R E DIRECTIONS  120  REFERENCES  122  LIST OF T A B L E S  Table 1.1 Transition temperatures of various phospholipids  25  Table 2.1 Procedures for encapsulating plasmid in lipid-based systems  61  LIST OF FIGURES  Figure 1.1  Methods of D N A delivery using gene therapy  Figure 1.2  A . Effect of PEG-lipids on the binding of proteins, which target the  5  liposomes to the RES. B. Chemical structures of PEG-DSPE and PEGCerC  20  12  Figure 1.3  Passive and active targeting of liposomes to cells at disease sites  13  Figure 1.4  Amphipathic lipids in membranes  16  Figure 1.5  Structures of common phospholipids  18  Figure 1.6  Structure of the sphingolipid and specifically the ceramides (Cg, C H , and C20) used in this thesis  19  Figure 1.7  Structures of commonly used cationic lipids  22  Figure 1.8  Gel to liquid-crystalline transition  24  Figure 1.9  Lipid polymorphism  26  Figure 1.10 Classification of liposomes  29  Figure 1.11 Schematic for the formation of liposomes using detergent dialysis  32  Figure 1.12 Schematic of the intracellular delivery of plasmid D N A following endocytosis of a liposomal system  34  Figure 1.13 Mechanisms of membrane fusion and intermediate structures  35  Figure 1.14 Map of plasmid D N A (pCMVLuc) used in this thesis  38  Figure 1.15 Conformations of plasmid D N A  39  Figure 2.1  Figure 2.2  Effect of D O D A C concentration on the encapsulation efficiency of plasmid D N A (pCMVCAT) in SPLP  49  Plasmid in SPLP is protected from serum nuclease cleavage  52  Figure 2.3  Separation of SPLP from empty vesicles by discontinuous sucrose density gradient centrifugation  Figure 2.4  54  QELS analysis of (a) empty liposomes from the top band of the sucrose gradient and (b) purified SPLP from the bottom band of the sucrose gradient.. 5 6  Figure 2.5  Freeze-fracture electron microscopy of purified SPLP and empty vesicles  57  Figure 2.6  Effect of PEG-Cer coating of SPLP on transfection activity in vitro  58  Figure 2.7  Model for the formation and possible structure of SPLP  64  Figure 3.1  Structures of the cationic P E G lipids (CPL)  70  Figure 3.2  Insertion of CPL into empty liposomes  75  Figure 3.3  CPL insertion into L U V s characterized by size exclusion chromatography  76  Figure 3.4  Influence of incubation time and temperature on CPL insertion into L U V s  78  Figure 3.5  Uptake of L U V - C P L with different C P L by B H K cells  80  Figure 3.6  Insertion of CPL4 into L U V results in uptake of the particles by B H K cells  81  Figure 4.1  Production of SPLP-CPL4  94  Figure 4.2  Time course for the insertion of CPL4 into SPLP at 60°C  96  Figure 4.3  Effect of cation concentration on the deaggregation of SPLP following insertion of CPL4  Figure 4.4  Freeze-fracture electron micrographs of (A) SPLP, (B) SPLP-CPL4, and (C) SPLP-CPL4 in the presence of 40 m M C a C l  Figure 4.5  97  98  2  Serum stability of SPLP-CPL4 as assayed by Southern analysis of encapsulated plasmid  Figure 4.6  101  A . Influence of the amount of CPL4 incorporated into SPLP on the uptake of SPLP- CPL4 into B H K cells  102  B. Fluorescence micrographs of B H K cells following uptake of SPLP and SPLP containing 4 mol% CPL4 following a 4 hour incubation  104  x  Figure 4.7  Luciferase expression in B H K cells following transfection by SPLP containing various amounts of CPL4  105  Figure 4.8  Influence of C a  106  Figure 4.9  Effect of C a  2+  2+  and M g  and M g  2 +  2 +  on the transfection potency of SPLP-CPL4  on the uptake of SPLP-CPL4 by B H K cells  108  Figure 4.10 Luciferase expression in B H K cells as a function of transfection time for SPLP, SPLP-CPL , and lipoplexes  109  4  Figure 4.11  A . The transfection potency of SPLP-CPL4 containing 4 mol% CPL4 and Lipofectin lipoplexes. B . The toxicity of SPLP-CPL and Lipofectin 4  lipoplexes as a function of transfection time  Ill  Figure 4.12 Fluorescence and phase contrast micrographs of B H K cells transfected with SPLP-CPL4 and lipoplexes containing a plasmid coding for GFP  113  xi  ABBREVIATIONS  BHK  baby hamster kidney  BSA  bovine serum albumin  CAT  chloramphenicol acetyl transferase  CFTR  cystic fibrosis transmembrane regulator  14  C-CHE  14  C-labeled cholesteryl hexadecyl ether  Choi  cholesterol  cmc  critical micellar concentration  CPL  cationic PEG lipid conjugate  CPLi  CPL possessing 1 positive charge  CPL  2  CPL possessing 2 positive charges  CPL4  CPL possessing 4 positive charges  CPLg  CPL possessing 8 positive charges  CPRG  chlorophenolred galactopyranoside  DC-CHOL  3-5-[N-(N',N'-dimethylaminoethyl)carbamoyl] cholesterol  DDAB  N',N'-distearyl-N',N'-dimethylammonium bromide  DEAE  diethylaminoethyl  DMEM  Dulbecco's Modified Eagle Medium  DMRIE  N-[2,3-dimyristyloxy)propyl]-N,N-dimethyl_N-hydroxyethylammonium bromide  DNA  deoxyribonucleic acid  DNase  deoxyribonuclease I  DODAC  N,N-dioleoyl-N,N-dimethylammonium chloride  DODAB  N,N-dioleoyl-N,N-dimethylammoniurn bromide  DOPE  1,2-dioleoyl-3-phosphatidylethanolamine  DOTAP  1,2-dioleoyloxy-3 -(trimethylamino)propane  DOTMA  N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium chloride  DSPE  distearoylphosphatidylethanolamine  DSPC  distearoylphosphatidylcholine  FBS  fetal bovine serum  H '  tritium labeled  Hn  inverted hexagonal phase  HBS  HEPES buffered saline  HEPES  N-(2-hydroxyethyl)piperazine-N"-2-ethanesulfonic acid  HSV-tk  tyrosine kinase gene of the Herpes simplex virus  ICAM  intracellular adhesion molecule  i.v.  intravenous  LUV  large unilamellar vesicles  LUV-CPL  L U V s containing CPL  MLV  multilamellar vesicles  MPS  mononuclear phagocytic system  OGP  n-octyl-B-D-glucopyranoside  PA  phosphatidic acid  PBS  phosphate buffered saline  PC  phosphatidylcholine  pCMVB  plasmid containing cytomegalovirus promoter and the P-gal gene  xiii  pCMVCAT  plasmid containing cytomegalovirus promoter and the C A T gene  pCMVGFP  plasmid containing cytomegalovirus promoter and the green fluorescence protein gene  pCMVLuc PE  plasmid containing cytomegalovirus promoter and the luciferase gene phosphatidylethanolamine  PEG  poly(ethylene glycol)  PEG3400  poly (ethylene glycol) with a molecular weight of-3400  PEG-CerCi4  l-0-(2'-(co-methoxypolyethyleneglycol(2ooo))succinoyl)-2-Nmyristoylsphingosine  PEG-CerC2o  1 -0-(2' -(co-methoxypolyethyleneglycol(2ooo))succinoyl)-2-Narachidoylsphingosine  PEG-DSPE  poly(ethylene glycol) conjugated to distearoylphosphatidylethanolamine  PG  phosphatidylglycerol  PI  phosphatidylinositol  POPC  1 -palmitoyl-2-oleoyl-3 -phosphatidylcholine  PS  phosphatidylserine  QELS  quasi-elastic light scattering  RES  reticuloendothelial system  Rh-PE  N-(lissamine rhodamine B sulonyl)-l,2-dioleoy-3-phosphatidyl ethanolamine  RNA  ribonucleic acid  SPLP  stabilized plasmid lipid particles  SPLP-CPL  SPLP containing CPL  SUV  small unilamellar vesicles  xiv  T  m  gel to liquid crystalline phase transition temperature  TMC  trans-monolayer contact  Triton X-100  /-octylphenoxypolyethoxyethanol  XV  ACKNOWLEDGEMENTS I would like to thank all the members of the Cullis lab for their help and friendship. I would especially like to thank my supervisor Dr. Pieter Cullis for providing me with a good project, much encouragement and a good learning environment. Also, I must thank the senior scientists in the lab: Dr. K i m Wong, Dr. Dave Fenske, Dr. Norbert Maurer, and Dr. Peter Scherrer for allowing me to discuss ideas for my thesis and not letting me get too stressed out about any one thing. Especially, Dr. Wong for looking after the running the lab and for demonstrating/performing freeze fracture E M ; Dr. Fenske for helping with some of the experiments for the insertion of CPL into preformed L U V s ; and Dr. Scherrer for the critical reading of this thesis.  The friendship of my  fellow graduate students in the lab: Angel Lam, Ismail Hafez, John Finn, Ammen Sandhu, and Lenore Louie, as well as our technician Tabitha Wong was very much appreciated. Angel and Tabitha were also helpful with experiments, Angel for helping with the Southern blot analysis and Tabitha with technical help in the lab. Finally, I would like to thank the members of my committee: Dr. Marcel Bally and Dr. Bob Molday for their suggestions for my thesis.  xvi  my loving wife Dora, whose love and encouragement helped me through the tough times  CHAPTER 1 INTRODUCTION  Advances in molecular biology, such as detection and replacement of mutant codons, and the sequencing of the human genome have greatly increased the knowledge of the molecular mechanisms and genetic defects causing disease. This has led to the development of a new class of pharmaceuticals, the gene-based drugs. The goal of these drugs is to manage disease at the molecular level by controlling gene function. This may include the replacement of mutant genes with their wild-type counterparts for genetic diseases or introduction of a new gene to produce an enzyme to manipulate the physiology of diseased cells. Genetic disorders requiring a gene therapy approach (i.e. replacing the defective gene) include cystic fibrosis, Huntington's disease, hemophilia, and some cancers (Knoell and Y i u , 1998; Constantini et al., 2000; van den Driessche et al., 1999; Roth and Cristiano, 1997). In most cases, conventional drugs can only treat the symptoms of the disease but do not restore normal function. For example, in the case of cystic fibrosis, the only treatment available treats symptoms, facilitating the removal of mucus and controlling lung infection with drugs such as antibiotics. Gene-based drugs offer the possibility of replacing the mutated CFTR gene with the normal gene resulting in the expression of the wild-type CFTR and the restoration of function (Conrad et al., 1996). Cancers may also be treated using genetic therapies.  For example, in many  cancers, tumour growth arises from a mutated p53 gene (Takahashi et al., 1992). The p53 gene is involved in the regulation of apoptosis, such that the expression of the p53 protein signals the cell to terminate growth and die. However, i f a mutated p53 gene is  1  present the expression of the mutant protein may not cause cell death, resulting in continued cell growth and division. The mutated p53 gene is passed on to daughter cells, which pass it on to their daughter cells. The result is the presence of a large number of cells with uncontrolled growth (i.e. a tumour). Replacement of the mutated p53 gene in these cells with the wild-type p53 results in tumour cell apoptosis (Cai et al., 1993) and suppresses cell transformation and neoplastic cell proliferation. In another cancer gene therapy known as "suicide" gene therapy, tumour cells are destroyed following the selective delivery and expression of a gene within the tumour cells that produces an enzyme that converts a prodrug to an active drug (e.g. by phosphorylation or catalytic cleavage). For example, the prodrug ganciclovir can be phosphorylated to the active drug ganciclovir-phosphate by a thymidine kinase, whose corresponding gene is introduced to tumour cells by transfection employing a vector based on the Herpes simplex virus (HSV-tk) (Kwong et al., 1996). Advantages of this type of therapy include the lack of activity and toxicity of the prodrug, the selective killing of the tumour cells which express the gene, and that transfection of only a small number of tumour cells is required for effective killing. This is known as the "bystander effect", and involves the activated drug crossing gap junctions and killing the cells surrounding the originally transfected cells (Kruse et al., 2000). Another strategy using gene-based drugs involves the down-regulation of specific proteins within the cell. This type of drug includes antisense and ribozymes, which bind to messenger R N A . Down-regulation occurs due to the inability of the cell to translate the messenger R N A into the corresponding proteins due to blocking of the protein translation complex or R N A degradation (Putnam, 1996). This type of therapy has many  2  potential applications. One example is for gene therapy of inflammatory diseases, since the down-regulation of some proteins (e.g. the intracellular adhesion molecule (ICAM)) within cells results in the inhibition of the inflammation cascade process (Klimuk et al., 2000). Most gene-based drugs have limited stability in vivo due to the susceptibility of D N A to degradation by nucleases and have limited ability to cross cellular membranes due to the large size and highly charged nature of the D N A . Thus, a system that protects the D N A from degradation i.v. and delivers it across cytoplasmic membranes is a major requirement for the successful development of treatments for diseases using DNA-based drugs. A n exception is D N A vaccination, which has been performed by direct injection of naked D N A into muscle. Two basic requirements for many gene delivery systems are a therapeutic gene and a delivery system to deliver the protected D N A across the cellular membrane. A number of steps must be met to obtain a therapeutic protein within the cell. In most cases, the therapeutic gene must first be encapsulated within a carrier system (viral or non-viral). Secondly, the therapeutic gene must be delivered to the cytoplasm of the cell, which is, in most cases, achieved by fusion between the delivery system and the endosomal membrane following receptor-mediated endocytosis. Thirdly, the gene must enter the nucleus and in some cases be integrated into the host genome.  Finally,  transcription and translation will result in the production of the therapeutic protein. This thesis will focus on the development of i) a plasmid delivery system that protects the plasmid from degradation and is capable of ii) intracellular delivery.  3  1.1 D N A D E L I V E R Y V E C T O R S  1.1.1  V i r a l Vectors Viral vectors are the most commonly used carriers for delivery of therapeutic  genes into target diseased cells (Figure 1.1 A). Viruses interact with target cells in such a way that the gene is inserted into the cytoplasm. For a large number of viruses, the viral D N A enters the cytoplasm following receptor-mediated endocytosis and viral fusion with the endosomal membrane (Hoekstra and Kok, 1989). Once in the cytoplasm, the viral D N A is transported into the nucleus where the virus takes over the cell's expression system and replicates itself many times over. In order to use viral vectors for gene therapy, the viruses must be made replication-incompetent.  A viral vector for gene  therapy also requires replacing a portion of the viral D N A with a therapeutic gene, which results in a construct that retains the infection properties of the original virus and results in the expression of the therapeutic gene product, under control of the viral promoter (Berkner, 1988). Some common viral vectors are adenovirus (Berkner, 1988; StratfordPerricaudet and Perricaudet, 1994), adeno-associated virus (McLaughlin et al., 1988; Muzyczka, 1992), herpes simplex virus (Andersen et al., 1992; Glorioso et al., 1994), and the retroviruses (Miller, 1990; Dunckley and Dickson, 1994). Viral vectors are very efficient gene transfer agents. However, a number of major limitations exist with viral systems.  A major safety concern is the possibility that  recombinant events occur rendering the viral particles pathogenic. Viral particles may also cause an immune response, which leads to the production of antibodies (Yang et al., 1994a).  This makes repeat treatments ineffective since viral particles would be  4  A.  B.  DNA Cationic  LUV  C.  DNA /  //  LUV  Figure 1.1. Methods of D N A delivery using gene therapy. They can be divided into viral (A) and non-viral liposomal techniques: (B) lipoplexes and (C) D N A encapsulated within liposomes.  5  eliminated rapidly on subsequent (or repeat) injections. Also, viral systems are rapidly cleared from the circulation by cells of the MPS following intravenous (i.v.) injection, resulting in transfection primarily in first pass organs, such as the lung and liver. In addition, the size of the gene that may be inserted into the viral genome is limited. The viral genome of adenoviruses, the viral vector which gives the best overall results, can accommodate  a therapeutic  gene of up to 14 kb (Evans and Robbins, 2000).  Furthermore, viral vectors, with the possible exception of adeno-associated viruses, may insert heterologous D N A randomly into the host genome, which could result in the activation of proto-oncogenes. Many clinical trials using viral-based gene therapy have been conducted (Knoell and Yiu, 1998; Dubinett et al., 1999; Tait et al., 1999; Wagner et al., 1999; Losordo et al., 1998). These include trials for the treatment of cystic fibrosis, cancers and coronary diseases. However, due to rapid clearance by the MPS and the immunogenicity of viral systems, these trials do not involve systemic delivery of the therapeutic genes. Rather, direct injection at the tumour site and inhalation in the case of cystic fibrosis are the methods used for administering these genetic drugs. The production of viral vectors is not a trivial process, requiring manipulation of the viral particle system and questions remain regarding the safety and efficiency of this therapeutic modality (Berkner, 1988). Viral  vectors are also not suited for systemic  applications due in part to their rapid clearance to first pass organs. The development of non-viral alternatives that can deliver gene-based drugs systemically is particularly needed.  6  1 . 1 . 2 Non-Viral Vectors Non-viral gene delivery methods have been developed initially for in vitro transfection.  Early methods using DEAE-dextran (Vaheri and Pagano, 1965) and  calcium phosphate co-precipitation (Graham and van der Eb, 1973) rely on the absorption of plasmid D N A to the surface of dextran or on precipitation in the presence of calcium phosphate, with subsequent uptake by the cells after the particles fall to the surface of the cells.  These methods result in inconsistent and low transient transfections.  development  of physical techniques  The  such as microinjection (Capecchi, 1980),  electroporation (Wong and Neumann, 1982; Neumann et al., 1982), and biolistic particle bombardment (Ye et al., 1990; Jiao et al., 1993) followed. In vitro uptake using these methods results in higher transfection efficiencies in some cell lines, but none of these techniques can be widely used for all cell types.  Systems such as cationic liposomes  (Feigner et al., 1987) and poly-L-lysine (Alio et al., 2000) neutralize the negative charges on D N A and condense it into particles possessing an overall positive charge.  These  systems are able to transfect, to high levels, many different cell types in vitro than the former non-viral methods. In vivo, the most widely used non-viral gene delivery methods include direct injection of naked D N A (Choate and Khavari, 1997), particle bombardment (Yang et al., 1994b), and DNA-cationic liposome complexes (Zhu et al., 1993). The direct injection method is a straightforward procedure that provides reasonable transfection efficiency in muscle and skin (Ghazizadeh et al., 1999). D N A can also be introduced to the cell by particle bombardment using a "gene-gun" which forces microscopic gold or tungsten particles coated with D N A into tissues (Nanney et al., 2000). In both of these cases, the  7  application is limited to the introduction of D N A directly into muscle and skin tissue. However, these systems cannot be used for systemic applications for the treatment of diseases such as cancer, inflammation, and infections, since D N A directly injected into the circulation will be rapidly degraded due to serum nucleases.  1.1.2.1 DNA-liposome complexes The most common non-viral liposome-based delivery systems for both in vitro and in vivo applications are DNA-liposome complexes (Figure L I B ) . These complexes or "lipoplexes" were first introduced in 1987 by Feigner who synthesized N-[2,3(dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), a cationic lipid with two oleoyl chains and a quaternary amine head group (Feigner et al., 1987). Since that time a large number of other cationic lipids have been synthesized including 3-i?-[N(N',N'-dimethylaminoethyl)carbamoyl] cholesterol (DC-CHOL; Gao and Huang, 1991), N',N'-distearyl-N',N'-dimethylammonium bromide (DDAB; Rose et al., 1991), N-[2,3dimyristyloxy)propyl] -N,N-dimethyl_N-hydroxyethylammonium  bromide  (DMRIE;  Feigner et a l , 1994), and N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC; Wong et al., 1996).  Homogeneously-sized small liposomes composed of equimolar  amounts of the cationic lipid and DOPE, a fusogenic lipid, can be prepared by the extrusion method of Mayer et al. (1986), and form complexes with plasmid D N A . These complexes have been shown to be capable of transfecting cells in vitro with great efficiency (Feigner et al., 1987; Feigner et al., 1994). However, since the complexes grow to very large sizes (on the order of microns) and possess a large amount of positive charge, systemic administration primarily results in rapid accumulation of these particles  8  only in "first pass" organs, such as the lung, liver, and spleen with transfection primarily occurring in these organs (Huang and L i , 1997). Thus, complexes are not useful for systemic administration to distal tumors.  In many cases, the D N A is not completely  protected by association with the liposomes, and is therefore susceptible to degradation from serum nucleases (Li et al., 1999). The high concentration of cationic lipid in these complexes causes toxicity to cells both in vivo and in vitro (Li and Huang, 1997; Feigner et al., 1994). Therefore, the development of small long circulating lipid-based D N A carriers, with D N A encapsulated within these particles, for systemic in vivo applications is a major requirement for gene therapy. A n ideal gene therapy delivery system would possess D N A encapsulated within liposomes of-100 nm (Figure 1.1C). Following systemic (i.v.) administration of such systems, the D N A would be protected from serum degradation and the small long circulating liposomes would accumulate at solid tumour sites as observed for liposomes containing conventional cancer drugs, as described in the following section. And finally, once at the disease site, an ideal delivery system will mediate efficient transfection.  1.1.3 Liposomes as cancer drug carrier systems The most widely used clinical application of liposomes is for the delivery of anticancer drugs to solid tumour sites.  Typically, drugs are encapsulated in large  unilamellar vesicles (LUV) with diameters in the range of 50-150 nm. Drugs can be loaded into L U V by passive or active techniques. Passive loading of hydrophobic drugs, such as Amphotericin B , occurs by preferential partitioning into the hydrophobic lipid bilayer (Madden et al., 1990a). Water soluble drugs can be entrapped inside the aqueous  9  core of the liposomes. However, only a small proportion of water soluble drugs can be passively trapped.  Many water soluble drugs can be encapsulated using active  encapsulation techniques; examples of such drugs include doxorubicin, vincristine and mitoxantrone (Boman et al., 1993; Mayer et al., 1990). Lipophilic drugs possessing an ionizable amino function can be loaded using a pH gradient (inside acidic) across the bilayer of preformed liposomes (Madden et al., 1990b). In the relatively high pH outside the liposomes, the neutral form of the ionizable drug moves across the lipid bilayer into the aqueous core where the drug is protonated due to the lower pH. This positively charged drug cannot escape, resulting in accumulation at high concentrations inside the liposome, with close to 100% of the drug being encapsulated by this technique. Following i.v. injection, liposomes are cleared from circulation by the macrophages of the mononuclear phagocytic system (MPS; Senior, 1987).  In the  circulation, various proteins, including plasma proteins and lipoproteins, can interact with the liposomes carrying the drug.  The interaction of lipoproteins with liposomes can  cause leakage of encapsulated drug due to lipid exchange, compromising the integrity of the liposomal membrane (Kirby et al., 1980; Silverman et al., 1984). The binding of plasma proteins marks the liposomes for uptake by the resident macrophages of the liver, spleen, lung, and bone marrow (Coleman, 1986; Moghimi and Patel, 1989). The size of the liposomes is also an important property affecting the in vivo clearance of liposomal systems.  Very large systems (200-2000 nm dia.), such as  multilamellar vesicles (MLVs), are cleared very rapidly from circulation primarily by macrophages of the liver (Juliano and Stamp, 1975). Large unilamellar vesicles (LUVs, 50-200 nm dia.), on the other hand, can circulate for longer periods of time. The lipid  10  composition also strongly influences the clearance behavior of liposomes. Under most circumstances, liposomes possessing charged lipids are cleared very rapidly from solution compared to liposomes possessing neutral lipids (Chonn et al., 1991; Chonn et al., 1992; Senior et al., 1991) due to increased binding of serum proteins to the former. Also, the more fluid (i.e. unsaturated) the bilayer, the greater the binding of serum proteins which results in clearance by the MPS. Methods for enhancing the circulation lifetimes of liposomes have been developed.  This has involved the incorporation of MPS-avoiding lipids into the  liposomal bilayer.  MPS-avoiding lipids include the ganglioside G M i (a naturally-  occurring lipid; Allen and Chonn, 1987) and P E G [poly(ethylene glycol)] - lipids (synthetic lipids; Allen et al., 1991b).  The hydrophillic coating on L U V s results in  decreased binding of serum proteins and thus decreased the recognition and clearance by cells of the MPS (Figure 1.2A) (Lasic, 1994). The most widely used PEG-lipid is PEGDSPE (Figure 1.2B).  1.3.1.1 Targeting of L U V to disease tumour sites In most cases, liposomal drugs (i.e. conventional and polynucleic acid drugs) must accumulate at disease sites to exert their therapeutic effect. Liposomes with long circulation lifetimes have been shown to accumulate at some disease sites (Figure 1.3 A), including sites of infection, inflammation, and solid tumour sites (Gabizon et al., 1990; Gabizon, 1992) due to the increased permeability of the vasculature in these regions. This is known as passive targeting. This type of targeting can lead to -10 times more cancer drug accumulation at a tumour site compared to the same dose of free drug.  11  A.  B.  H,C,  *0  —(X)—N H  c.  v  ^0 I 0 = P - 0 "  I  0  I CH,—CH — C H ,  I I  f o =  PEG-DSPE  c  ? c=o  PEG-Ceramide C,  0  Figure 1.2. A . Effect of PEG-lipids on the binding of proteins, which target the liposomes to the MPS. B. Chemical structures of PEG-DSPE and PEG-CerC 20  12  A.  r  Target Cell  I ^nucleus^ (nucleus^  B. '  >s  Target Cell  I Tumor Fenestrations Figure 1.3. Passive (A) and active (B) targeting of liposomes to cells at disease sites.  13  In the case of conventional drugs encapsulated within long circulating liposomes, the liposomes do not need to be internalized in order for the drug to act on tumour cells. Instead, after accumulation in the tumour interstitial space, the drugs can leak from these liposomes and be taken up by the cells by normal free drug uptake mechanisms (Lim et al., 2000). For larger gene-based drugs (i.e. DNA), intracellular delivery into target cells is required. Liposomes can be delivered to particular cells using 'active' (i.e., ligandmediated) targeting techniques following accumulation by passive targeting (Figure 1.3B). For active targeting, targeting moieties are included on the liposomal exterior. These active targeting moieties can take the form of an antibody to a protein on the cell surface (Hansen et al., 1995; Kao et al., 1996; Meyer et al., 1998), a protein with a specific receptor on the cell surface (Zalipski et al., 1995; Zalipiski et al., 1997), or a small molecule (e.g. folate or glucose) which binds to a specific receptor molecule on the surface of the target cell (Gabizon et al., 1999; Holladay et al., 1999). In some cases, following interaction of the ligands of these systems with the corresponding cellular protein, the liposomes can be endocytosed (if the receptor undergoes endocytosis) and the drug released into the cytoplasm. These systems have shown much increased uptake by cells in vitro; however, actively targeted liposomes have proved less useful in vivo (Harding et al., 1997).  This is due to their more rapid clearance from circulation,  reducing passive targeting to the disease site.  From the above discussion, it should follow that gene delivery systems possessing D N A encapsulated within long circulating L U V s should result in greater amounts of  14  D N A accumulation at diseased sites due to increased capillary permeabilities.  The  following sections describe the properties of lipids and liposomes which are important for the design and characterization of plasmid delivery systems such as stabilized plasmid lipid particles (SPLP). The final section describes the structure and properties of D N A used in this thesis.  1.2 L I P I D S  Lipids are very important molecules in nature. Some lipids store energy in the form of fats while others are structural, assembling into lipid bilayer membranes.  These  lipid membranes separate cellular compartments from each other and from the external environment. Membrane lipids are amphipathic, possessing hydrophobic and hydrophilic regions (Figure 1.4). Within the bilayer, the hydrophilic lipid headgroups are oriented toward the aqueous environment while the hydrophobic tails are facing each other. Proteins spanning the cytoplasmic membrane are required for cellular uptake of metabolites and the supply of energy to the cell by transporting H ions across the +  permeability barrier in mitochondria.  1.2.1 Chemistry and physics of lipids The lipids relevant to this thesis can be classified into four different categories based on their structure and function. These include phospholipids, sphingolipids, PEGlipids, and cationic lipids.  15  Hydrophilic i  Hydrophobic  Biological Membrane  Figure 1.4. Amphipathic lipids in membranes.  16  1.2.1.1 Phospholipids Phospholipids are the most abundant class of naturally occurring lipids, and contain a glycerol backbone and a phosphate-containing head group. The phospholipids include phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylserines (PS), phosphatidic acids (PA), phosphatidylglycerols (PG), phosphatidylinositols (PI), and cardiolipins (CL). The structures of these phospholipids are shown in Figure 1.5. The head groups of these lipids are important in determining their various properties. Some of the phospholipids (PS, P G , PI, and CL) are anionic while others are zwitterionic (PC and PE). Phospholipid diversity also results from the length of the acyl chains and their degree of unsaturation. Typically acyl chain lengths range from 16 to 24 hydrocarbons. The most common saturated fatty acids found in phospholipids are palmitic and stearic acid, while the most common unsaturated fatty acids are oleic and linoleic acids. Some of these lipids possess bilayer-forming properties while others prefer the hexagonal phase (see section 1.2.2.2).  1.2.1.2 Sphingolipids This class of membrane lipids is based on the long chain amino alcohol sphingosine.  The linkage of a fatty acid via an amide bond to the amino group of  sphingosine yields a class of sphingolipid referred to as a ceramide (Figure 1.6).  1.2.1.3 PEG-lipids PEG-lipids are designed primarily to allow the liposomal system to avoid the mononuclear phagocytic system (MPS), as described earlier, resulting in increased  17  CH  Neutral Phospholipids  y  3  C H — C H — N\—CH, CH  Choline Phosphatidylcholine (PC)  2  Ethanolamine  2  •CH —CH —NH 2  2  3  3  Phosphatidylethanolamine (PE)  o  Headgroup  I  Negative Phospholipids Phosphatidic Acid (PA)  0=P—O"  -H  I  CH  2  o CH—CH  2  Glycerol Backbone  O  0=C  CH—CH — C - O  Phosphatidylserine (PS)  I  c=o / H,C H,C \ CH, CH, / H,C H,C \ CH, CH, H,C H,C CH, CH, H,C H,C CH, CH, H,C H,C CH, CH, H,C H,C CH, CH, H,C H,C CH, CH, H,C CH,  Serine  + NH,  Glycerol  CH —CH — C H  I  2  2  OH  2  Phosphatidylglycerol (PG)  2  •  I  OH  OH  2  2  OH  Inositol Phosphatidylinositol (PI) OH  - A c y l Chain  Saturated Fatty Acids Laurie (12:0)  CH (CH ) COOH  Myristic (14:0)  CH (CH ) COOH  Palmitic (16:0)  CH (CH ) COOH  Stearic (18:0)  CH (CH ) COOH  3  2  3  10  2  3  12  2  3  14  2  16  Unsaturated Fatty Acids Palmitoleic (16:1 A )  CH (CH ) CH=CH(CH ) COOH  Oleic (18:1 A')  CH (CH ) CH= CH(CH ) COOH  9  3  3  2  5  2  2  7  7  2 7  Linoleic (18:2A' ) JI2  CH (CH ) CH = 3  2 4  C H C H C H = CH(CH ) COOH 2  2 7  Figure 1.5. Structures of common phospholipids.  18  Sphingolipid  Sphingosine OR  -  R = C O R " ; R' = H  Ceramide (Cer)  R = C O R " * ; R' = Phosphocholine  Sphingomyelin (SM)  (R" = hydrocarbon chain)  Ceramides  Figure 1.6. Structure of the sphingolipids and specifically the ceramides (Cs, C M , and C20) used in this thesis.  19  circulation lifetimes.  These PEG-lipids possess a polyfethylene glycol) molecule  attached to the headgroup of a lipid molecule (Figure 1.2 B and C). The most widely used PEG-lipid is PEG-DSPE. This molecule is prepared by the conjugation of a P E G molecule through the amino group in the headgroup of DSPE (Figure 1.2B). The lipid possesses a net negative charge. Although incorporation of PEG-DSPE into liposomes decreases the elimination from circulation, it also may inhibit interactions required for liposomal uptake by target cells. For conventional drugs, this is not a problem as leakage of the drug from the liposomes is all that is required for drug uptake by the tumour cells. However, large drugs (e.g. D N A ) require intracellular delivery. One approach is to design the P E G coating so that it only remains in the lipid bilayer long enough for the liposomes to accumulate at the diseased site. To achieve this, PEG-ceramides have been developed (Webb et al., 1998). These PEG-ceramides possess dissociation rates dependent on their acyl chain lengths (Wheeler et al., 1999), with those possessing shorter acyl chains dissociating from the bilayer at earlier time points. Three ceramides, with different acyl chain lengths, have been used in the production of these PEG-ceramides (Figure 1.6) with the PEG moiety conjugated to the head group of the ceramide at the hydroxyl of the CI carbon (Figure 1.2C).  1.2.1.4 Cationic lipids Biologically, cationic lipids are extremely rare, appearing in nature only as sphingosine and stearylamine. Cationic lipids possess a positive charge in the headgroup. This positive charge allows for electrostatic interactions with polynucleic acids (e.g.  20  D N A , antisense), important in the formation of plasmid DNA-cationic liposome complexes (lipoplexes) used for D N A delivery. The first synthetic cationic lipids, prepared in the late 1970's, were N , N didodecyl-N,N-dimethylammonium bromide (Kunitake and Okhata, 1977) and 1,2dimyristoyloxy-3-(trimethylammino)propane (DMTAP)  (Eibl  and  Wooley, 1979).  However, it was not until 1987 that Feigner and colleagues reported the first use of DNA/cationic  liposome  complexes  composed  of  DOPE  and  N-[l-(2,3-  dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) for mediating in vitro D N A transfection (Feigner et al., 1987). Since then, a large variety of different synthetic cationic lipids have been synthesized (Figure 1.7).  The different structures  include those with multiple charges, quaternary amines, protonatable headgroups, ester linkages between the alcohol of glycerol and the carboxyl of the fatty acid, or an ester linkage between the tails and the glycerol (Figure 1.7).  1.2.2 Structural behavior of lipids Both the chemical structure of the lipid and its surrounding environment govern its molecular behavior. Lipids in an aqueous environment adopt particular organizations or phases. The different phases that lipids can adopt include the lipid bilayer, the most common phase, and a number of non-bilayer phases, such as the hexagonal (Hn) or isotropic phases. The ability of lipids to adopt different phases is referred to as lipid polymorphism. The presence of ions, temperature, or pH all influence the type of phase adopted by a given lipid. Also, depending on the temperature, the phospholipids in the bilayer phase may be in one of two different states: the gel or liquid crystalline state.  21  CI  DODAC (N,N-dioleoyl-N,N-dimethylarnmonium chloride)  DOTAP (N-[2,3-(dioleoyloxy)propyl]-N,N,N-trimethylamrnonium chloride)  DOTMA (N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylarnrnonium chloride) Br  DDAB (N,N-distearyl-N,N-dimethylammonium bromide)  DMRIE (N-[2,3-(dimyristyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide) H I  DC-CHOL (S-^-fN-CN'^'-dimethylaminoethyOcarbamoyl] cholesterol)  NH  3  DOSPA (2,3-(dioleyloxy)-N-[2-(spermine carboxamido)ethyl]N,N-dimethyl-l-propanaminium trifluoroacetate)  Figure 1.7. Structures of commonly used cationic lipids.  22  1.2.2.1 Gel - liquid crystalline phase transition Phospholipids in a bilayer are characterized by the T , the temperature below m  which the bilayer is in its gel state (Lp). Phospholipids can exist in either the "frozen" gel state (Lp) or a "fluid" liquid crystalline state ( L ) (Figure 1.8). The temperature of the gel a  to liquid crystalline phase transition (T ) is mainly dependent upon the length and degree m  of saturation of the acyl chains. At temperatures below the T , the acyl chains are in a m  rigid or ordered conformation, known as the gel state (Lp). Above the T , however, the m  molecular motion of the acyl chains is increased, and the lipids are able to diffuse laterally in the plane of the bilayer (Cullis and Hope, 1991; Fenske et al., 1995). The T values of a number of different lipids are given in Table 1.1. Clearly, an m  increase in the acyl chain length results in an increase in the T (Papahadjopoulos et al., m  1973).  Saturation of the acyl chain is also an important factor; increased acyl chain  unsaturation decreases the T of the lipid (Papahadjopoulos et al., 1973). The charge on m  the head group also affects the T . m  The repulsion of charged head groups increases  packing distance between lipids, resulting in an increase in mobility of the lipids in the bilayer, and favoring the liquid crystalline state. Thus the charged lipids tend to have lower T (Table 1.1). m  1.2.2.2 Lipid polymorphism Lipids in the liquid crystalline state can adopt a number of different lipid polymorphic phases, including the bilayer (lamellar) phase, the inverted hexagonal (Hn) phase, and other non-bilayer phases known as cubic phases. The molecular shape of the lipid determines the polymorphic phase it adopts (Israelachvilli et al., 1980; Cullis et al.,  23  Gel State (L ) p  Liquid-Crystalline State (L ) a  Figure 1.8. Gel to liquid-crystalline transition. Transition between the gel and liquid crystalline states in a bilayer. The acyl chains of lipids in the gel state are tilted and ordered while in the liquid crystalline state they are in motion and unordered.  24  Table 1.1. Transition temperatures of various phospholipids composed of different acyl chain length, degree of saturation, and head group moiety. Values taken from Marsh (1990).  L i p i d Species  Transition Temperature (°C)  dilauroyl PC (12:0, 12:0)  -1  dimyristoyl PC (14:0, 14:0)  24  dipalmitoyl PC (16:0, 16:0)  41  distearoyl PC (18:0, 18:0)  55  stearoyl, oleoyl PC (18:0, 18:1)  6  stearoyl, linoleoyl PC (18:0, 18:2)  -13  dipalmitoyl P A (16:0, 16:0)  67  dipalmitoyl PE (16:0, 16:0)  63  dipalmitoyl PS (16:0, 16:0)  55  dipalmitoyl P G (16:0, 16:0)  41  25  Shape  Structure  Inverted Micellar  Figure 1.9. Lipid polymorphism.  Common shapes and resulting lipid shapes. A .  micellar, B. lamellar, and C. inverted micellar. Taken from Cullis and deKruijff (1979).  26  1986). Some lipids, such as detergents, possess head groups that occupy large areas in comparison to their tails (Figure 1.9A). These lipids can be thought of as having a "cone" shape, and tend to form micelles. Lipids that adopt the bilayer phase possess head groups which have cross-sectional areas similar to that of their tails, for example the PCs. These lipids exhibit a cylindrical shape (Figure 1.9B). Finally, lipids such as the unsaturated species of PE exhibiting an inverted cone shape due to their relatively small neutral head groups and more flexible acyl chains adopt an inverted hexagonal (Hn) phase structure (Figure 1.9C). The polymorphic properties of the different lipids have been reviewed elsewhere (Cullis and deKruijff, 1979, Gruner et al., 1985; Lindblom and Rilfors, 1989; Seddon, 1990). Lipid polymorphism can also be modulated by a number of other factors (Cullis et a l , 1986). With increasing temperature, the mobility of the acyl chains increases, thus increasing the relative cross-sectional area of the hydrophobic section, thus favoring Hn phase formation. The pH and ionic strength can alter the charge of the head group, with charged head groups effectively increasing the cross-sectional area occupied by the hydrophilic section. Therefore, the relative changes in the size of the head group and tail determine the polymorphic phase of the molecule.  For example, the structure of  unsaturated PS changes from the bilayer phase to the inverted hexagonal phase when the pH is decreased below 4 (Cullis et al., 1986).  This results in the protonation (i.e.  neutralization) of the carboxyl group of the PS. As a neutral head group occupies a smaller volume, formation of the Hn phase structure is promoted.  27  1.3 L I P O S O M E S  Liposomes form spontaneously on dispersion of bilayer-forming lipids in aqueous media. Liposomes have been used as model membrane systems for the study of lipids and membrane proteins, and also as delivery vehicles for conventional and genetic drugs. The next section describes the different classifications of liposomes and methods of preparing large unilamellar vesicles (LUVs).  1.3.1 Classification of liposomes  1.3.1.1 Multilamellar vesicles (MLVs) Multilamellar vesicles (MLVs) are formed by dispersion of bilayer forming lipids in aqueous media and were first observed by Bangham et al. (1965) as an onion skin arrangement of concentric lamellae.  These  concentric bilayer structures have  heterogeneous diameters in the range of 1 to 10 um (Figure 1.1 OA) and low trapping volumes (0.5 uL/umol). M L V s are useful for studying the structural and motional properties of lipids.  However, M L V s are not widely used as drug delivery vehicles  because of their large size, leading to rapid elimination from circulation.  1.3.1.2 Large unilamellar vesicles (LUVs) Large unilamellar vesicles (LUVs) possess a single bilayer and range in diameter from 50 to 200 nm (Figure 1.10B). L U V s can be prepared by a number of different techniques,  28  MLV  LUV  C. 20-50 nm  SUV Figure 1.10. Classifications of liposomes.  Schematic representation of A . M L V , B .  L U V , and C. SUV.  29  including reverse phase evaporation (Szoka and Papahadjopoulos,  1978), detergent  dialysis (Szoka and Papahadjopoulos, 1980), and extrusion (Mayer et a l , 1986). The most convenient method for producing L U V s is extrusion through polycarbonate filters with well-defined pore size (Mayer et al., 1986).  The techniques of extrusion and  detergent dialysis will be discussed in Section 1.3.2. For drug delivery applications, L U V s are most commonly used.  1.3.1.3 Small unilamellar vesicles (SUVs) Small unilamellar vesicles (SUVs) are single bilayer vesicles with diameters of about 20 to 50 nm (Figure 1.10C). These vesicles are formed by subjecting M L V s to sonication (Huang, 1969) or passage through a French press (Barenholtz et al., 1979). As drug delivery vehicles, the SUVs' trapped volume is small and the liposomes are unstable due to their radius of curvature.  1.3.2 Preparation of L U V s  1.3.2.1 Extrusion technique The most efficient technique for the formation of homogeneous L U V s is extrusion (Olson et al., 1979). In this technique, a lipid dispersion is repeatedly forced through polycarbonate filters under 200-400 psi pressure. The size of the vesicles is dependent on the pore size of the filters, which for L U V s range from 50 to 200 nm. For most liposomal drugs, the drugs are encapsulated within 100 nm vesicles prepared by  extrusion (Mayer et al., 1986). Homogeneous populations of 100 nm L U V s can be prepared by multiple passes through 100 nm filters.  1.3.2.2 Detergent dialysis Detergent dialysis techniques also result in production of L U V .  Detergents,  possessing a hydrophilic head and a hydrophobic tail, have been used in the preparation of L U V s by a number of investigators (Szoka and Papahadjopoulos, 1980; Lasch et al., 1983; Freytag, 1985; Liu and Huang, 1989). At low detergent concentrations, detergents exist as a monomeric dispersion in solution. At a specific detergent concentration known as the critical micelle concentration (cmc), micelles (clusters of detergent monomers) are formed. Micellar solutions contain a mixture of both micelles and monomers in solution. The monomers present in one micelle can constantly exchange with other monomers in solution or in other micelles (Lindman and Wennerstom, 1980). The formation of liposomes by detergent dialysis requires a mixture of lipids with a detergent in aqueous solution. Mixed micelles containing both detergent and lipid molecules coexist with detergent monomers in solution.  Dialysis, using dialysis  membranes of low molecular weight cut off, removes detergent monomers from the mixed micellar solution. As the concentration of monomers decreases inside the dialysis bag, detergent monomers exchange from the micelles into the buffer solution. As the detergent concentration decreases macromolecular lipid intermediates are formed. These include cylindrical micelles, lamellar sheets or leaky vesicles (Ollivon et al., 1988; Vinson et al., 1989). As the rest of the detergent is removed as monomers, L U V s form (Figure 1.11). The L U V size can be controlled by the rate of dialysis. Detergents most  31  ........  £  i  Lipids  Detergent  ^^^^^  Mixed Micelles  Dialysis  Liposomes Figure 1.11. Schematic for the formation of liposomes using detergent dialysis.  32  suitable for dialysis to form L U V s are those with high cmc values (> 1 mM) and small micelle size (Dencher and Heyn, 1978; Furth, 1980).  1.3.3 Membrane fusion In order for liposomes to be useful drug delivery vehicles their contents must access the interior of target cells. Conventional drugs released from liposomes following accumulation at solid tumors are able to permeate into nearby tumour cells. However, large molecules such as D N A require intracellular delivery (Figure 1.12). Following endocytosis of the liposomes, fusion between the liposomal and endosomal membranes or between the liposomal membrane and the plasma membrane can result in the release of the D N A into the cytoplasm. This fusion can be facilitated by a number of liposomal components. These include liposomal membrane-associated fusogenic lipids, fusogenic peptides or proteins, and fusogenic polymers.  Fusogenic lipids are lipids that  preferentially adopt non-bilayer (Hn phase) structures in isolation. In this thesis, the fusogenic phospholipid dioleoyl phosphatidylethanolamine (DOPE) will be used. Three models for membrane fusion exist. In the first, inverted micellar structures form between the two closely apposed bilayers (e.g. endosomal and liposomal membranes) involved in the fusion event (Figure 1.13A). These are known as inverted micellar intermediates (Cullis and deKruijff, 1979; Cullis et a l , 1986). Non-bilayer lipids, such as PE, favor the formation of these intermediate structures. In the second model, a "stalk" intermediate (Markin et al., 1984; Chernomordik et al., 1985) is formed by coalescence of the outer monolayers of the apposing membranes (Figure 1.13B). This structure is then expanded radially becoming a trans-monolayer contact (TMC).  The  33  1. Liposome Interaction With Cytoplasmic Membrane  2. Endocytosis of the Liposome  4. Formation of a Fusion Pore Through Which the Plasmid D N A Escapes  3. Fusion of Liposomal Membrane With Endosomal Membrane  Figure 1.12. Schematic of the intracellular delivery of plasmid DNA following endocytosis of a liposomal system.  Following endocytosis, the endosomal membrane  fuses with the liposomal membrane resulting in a fusion pore through which the DNA is capable of escaping to the cytoplasm at which point it can proceed to the nucleus (Hafez and Cullis, 2000). Modification of a figure from Xu and Szoka (1996).  34  JP  n  o E o  Q.  s  •—5*  o «J O  E  ro IL.  c  8»  si < J3  f i i  Jut  III  Figure 1.13. Mechanisms of membrane fusion and intermediate structures. Modification of a figure from Siegel and Epand (1997).  35  T M C continues to radially expand, forming a single bilayer diaphragm between the two apposed membranes ("hemifusion intermediate"), which then rapidly ruptures to yield a fusion pore (Chernomordik and Zimmerberg, 1995). This is sometimes called the "stalkpore" model. The final model is an extension of the "stalk-pore" model, known as the "modified stalk model". It also involves the formation of stalk and T M C intermediates; however, the TMCs do not radially expand to form a "hemifusion intermediate". Instead, membrane fusion is mediated as the diaphragm ruptures locally in the T M C (Figure 1.13C) (Siegel, 1993; Siegel and Epand, 1997).  1.4 D E O X Y R I B O N U C L E I C A C I D (DNA)  1.4.1 Composition of D N A D N A is composed of a sequence of nucleotides joined via phosphodiester linkages between the 5' hydroxyl group of deoxyribose on one nucleotide and the 3' hydroxyl group of the next nucleotide.  Nucleotides are composed of a purine or a  pyrimidine (nitrogenous) base attached to a deoxyribose sugar via a (5-glycosidic bond, and a phosphate group. D N A contains four different bases: adenine (A), guanine (G), cytosine (C), and thymine (T). In vivo, a D N A molecule is usually found as a double helix possessing two nucleotide strands running anti-parallel. The strands are linked together internally via hydrogen bonding between the purine and pyrimidine bases such that the A bonds with the T (two hydrogen bonds) and the G with the C (three hydrogen bonds). Double-stranded D N A is stabilized by hydrophobic interactions resulting from  36  base stacking within the double helix. D N A used in this thesis is plasmid D N A and is described in the next section.  1.4.2 Plasmid D N A Plasmids  are  small, circular, double  stranded  pieces  of D N A found  extrachromosomally in many bacteria and other species. When introduced into a cell, plasmids can migrate to the nucleus, where genes contained in the plasmid can be expressed.  Plasmid D N A can possess either a reporter gene (to determine the extent of  transfection) or a therapeutic gene (to correct for a dysfunction in a cell). In this thesis, the plasmid pCMVLuc, possessing the reporter gene encoding for luciferase, is used (Figure  1.14).  This plasmid possessses the gene for luciferase, the C M V  (cytomegalovirus) promoter, the C o l E l origin of replication, and an ampicilin resistance gene, which allows for the selection of tranformants.  Plasmid can be overexpressed in  bacteria, and can be easily isolated and purified due to their unique properties (Sambrook et al., 1989).  Plasmid D N A obtained in this manner can be composed of different  conformations. These conformations are described in the following section.  1.4.3 Structure of Plasmid D N A Depending on the degree of torsional constraint, plasmid D N A can adapt different conformations including linear D N A , open circular D N A , and supercoiled D N A (Figure 1.15). Open circular D N A is produced from supercoiled D N A when a nick is made in only one strand of the supercoiled D N A . The linear form can be produced from either  ColEI Ori  Figure 1.14. Map of plasmid D N A (pCMVLuc) used in this thesis.  38  Supercoiled DNA  DNase  Nick in D N A single strand  Restriction Endonuclea  DNase  WW Degraded DNA Restriction Endonuclease (cleavage of both strands)  Linear DNA  DNase  Figure 1.15. Conformations of plasmid DNA. Effect of DNase (deoxyribonuclease) and restriction endonucleases on plasmid D N A conformation.  39  supercoiled D N A or open circular D N A by cutting both strands of the double stranded D N A . In most cases this is achieved in vitro using restriction endonucleases which cut at specific sites in the D N A . D N A in any form can be degraded in the presence of DNase which randomly cuts D N A into many small particles.  1.5 T H E S I S O B J E C T I V E S  The major objective of this thesis was to develop a non-viral liposomal gene delivery system that is small in size (<100 nm diameter), is stable in serum, possesses a long circulation half life, and shows high transfection potencies. This was achieved by first developing a procedure for the encapsulation of plasmid D N A within fusogenic, cationic liposomes. Due to the PEG coating of these particles, their transfection potency is low due to decreased cellular interactions. To increase the liposomal uptake by cells and thus the transfection potency of these particles, a non-specific targeting ligand was developed and incorporated within these particles. This resulted in increased transfection due to increased intracellular delivery.  40  CHAPTER 2 STABILIZED PLASMID-LIPID PARTICLES: CONSTRUCTION AND CHARACTERIZATION  2.1 INTRODUCTION  Currently available gene delivery systems for gene therapy protocols have limited utility for systemic applications. Viral systems, for example, are rapidly cleared from the circulation, limiting potential transfection sites to "first-pass" organs such as the lungs, liver and spleen. In addition, these systems induce immune responses which compromise transfection resulting from subsequent injections. In the case of non-viral systems such as plasmid DNA-cationic lipid complexes (lipoplexes), the large size and positively charged character of these aggregates also result in rapid clearance, and the highest expression levels are again observed in first-pass organs, particularly the lungs (Thierry et al., 1995; Hofland et al., 1997; Huang and L i , 1997; Templeton et a l , 1997). Plasmid DNA-cationic lipid complexes can also result in toxic side effects both in vitro (Feigner et al., 1994) and in vivo (Li and Huang, 1997). The need for a gene delivery system for treatment of systemic disease is obvious. For example, for cancer gene therapy there is a vital need to access metastatic disease sites as well as primary tumours.  Similar considerations apply to other systemic  disorders, such as inflammatory diseases.  The design features for lipid-based delivery  systems that preferentially access such disease sites are increasingly clear. It is now generally recognized that preferential delivery of anticancer drugs to tumour sites following intravenous injection can be achieved by encapsulation of these drugs in large unilamellar vesicles (LUVs) exhibiting a small size (< 100 nm diameter) and extended  41  circulation lifetimes (circulation half-life in mice > 5 h) (Profitt et al., 1983; Gabizon and Papahadjopoulos, 1988; Chonn and Cullis, 1995).  The accumulation of these drug  delivery systems at some disease sites, which include sites of infection and inflammation as well as tumours, has been attributed to enhanced permeability of the local vasculature in diseased tissue (Kohn et al., 1992). A gene delivery system containing an encapsulated plasmid for systemic applications should therefore be small (< 150 nm diameter) and must exhibit extended circulation lifetimes to achieve enhanced delivery to disease sites. This requires a highly stable, serum-resistant plasmid-containing particle that does not interact with cells and other components of the vascular compartment. In order to maximize transfection after arrival at a disease site, however, the particle should interact readily with cells at the site, and should have the ability to destabilize cell membranes to promote intracellular delivery of the plasmid. In this work we show that a straightforward detergent dialysis procedure can produce stabilized plasmid-lipid particles (SPLP) which satisfy the demands of plasmid encapsulation, small size and serum stability. Furthermore, it is shown that the transfection properties of these systems can be modulated by employing poly(ethylene glycol) (PEG) coatings which can dissociate from the SPLP, transforming the particle from a stable particle to a transfection-competent entity.  42  2.2 M A T E R I A L S & M E T H O D S  2.2.1 Materials 1,2-Dioleoyl-phosphatidylethanolamine  (DOPE)  and  l-palmitoyl-2-oleoyl-  phosphatidylcholine (POPC) were obtained from Northern Lipids (Vancouver, B C , Canada). The lipids PEG-CerCn and PEG-CerC2o were kindly provided by Z. Wang of Inex Pharmaceuticals and N,N-dioleoyl-N,N-dimethyl ammonium chloride (DODAC) was supplied by S. Ansell of Inex. Octyl-P-D-galactoside (OGP), HEPES and NaCl were obtained from Sigma  (St. Louis, MO).  The plasmids pCMVLuc (5.6 kb) and  p C M V C A T (4.5 kb) were supplied by Inex Pharmaceuticals.  14  C-cholesteryl hexadecyl  ether ( C-CHE) was obtained from Mandel Scientific (Guelph, ON). H-plasmid D N A 14  ( P C M V C A T ) was prepared at Inex Pharmaceuticals.  3  Mouse serum was obtained from  CedarLane (Mississauga, ON). Dialysis tubing (SpectraPor 12 000 to 14 000 mwco) was obtained from Fisher Scientific (Ottawa, ON), DEAE-Sepharose CL-6B and Sepharose CL-4B were from Sigma, and the luciferase assay kit was obtained from Promega (Madison, WI).  2.2.2 Encapsulation of plasmid D N A DOPE:DODAC:PEG-Cer or POPC:DODAC:PEG-Cer, at 10 mg/mL total lipid, with varying DODAC:DOPE (or POPC) ratios and a fixed PEG-Cer content of 10 mol% were prepared, as follows. Plasmid D N A (50-400 ug) spiked with a small amount of H 3  labeled D N A and D O D A C that had been dried down from a chloroform stock was incubated for 30 min at room temperature in 500 uL of 0.2 M OGP and 150 m M NaCl,  43  5mM HEPES pH 7.4. The plasmid-DODAC mixture was then added to the co-lipid (DOPE or POPC) and one of the PEG ceramides ( P E G - C e r d , or C ) dissolved in 500 4  20  uL of 0.2 M OGP and 150 m M NaCl, 5mM HEPES pH 7.4. As a lipid marker, a small amount of C - C H E was added to the lipid mixture. Each of the plasmid-lipid mixtures 14  was dialyzed against 5 m M HEPES, 150 m M NaCl pH 7.4 (HBS) for 36 to 48 hours with two buffer changes.  2.2.3 Assay for plasmid DNA encapsulation efficiency Following dialysis, the encapsulation efficiency of each sample was measured by loading 50 uL aliquots on to a DEAE-Sepharose CL-6B column (lmL) equilibrated with HBS. The column was eluted with HBS and the fractions were assayed for plasmid D N A and lipid by dual-label scintillation counting using H and C , respectively. 3  14  2.2.4 Removal of unencapsulated DNA by anion-exchange chromatography Unencapsulated D N A was removed by loading the dialyzed plasmid/lipid mixture onto a DEAE-Sepharose CL-6B column equilibrated with 5 m M HEPES, 150 m M NaCl pH 7.4.  Plasmid-containing liposomes were eluted in the void volume while  unencapsulated D N A is retained on the column matrix.  2.2.5 Isolation of encapsulated plasmid by sucrose density gradient centrifugation Liposomes containing D N A were isolated from empty liposomes using density gradient centrifugation. Fractions from the D E A E column that contained both lipid and D N A were combined (~1 mL) and applied to a discontinuous sucrose gradient in a 12.5  44  mL ultracentrifuge tube. The gradient was formed by the consecutive layering from bottom to top of 3.8 mL each of 10%, 2.5%, and 1% sucrose.  The gradients were  centrifuged for 2 hours at 36 000 rpm in a SW41Ti rotor in a Beckman ultracentrifuge. For the initial analysis of the gradient separation, 250 uL fractions were removed from top to bottom and each fraction was assayed for H-plamid and C-CHE by dual label 3  14  counting. To directly isolate the SPLP band following centrifugation, the centrifuge tube may be punctured with a syringe and the liposomes drawn out with a syringe. This is possible because following centrifugation, the lipid-encapsulated plasmid DNA is banded between 2.5% and 10% sucrose, whereas the unassociated lipid appears as a smear between the top of the gradient to the interface between 1% and 2.5% sucrose. In many cases the isolated SPLP had to be concentrated using Aquacide II with the sample in 12000-14000 mwco dialysis tubing.  When the desired volume was achieved, the  formulation was transferred into a new dialysis bag and dialyzed overnight against HBS to adjust the NaCl concentration to 150 mM and remove sucrose.  2.2.6 Freeze-fracture electron microscopy  Freeze-fracture was performed on a Balzers Freeze-Etching system, BAF (Balzers, Lichenstein).  Samples were cryofixed in the presence of 25% glycerol by  plunging them into liquid freon 22. The fractured surface was shadowed unidirectionally with platinum/carbon (45°) and coated with carbon (90°) immediately after fracturing. Replicas were analyzed using a Jeol model JEM 1200 EX electron microscope (Jeol, Montreal, PQ).  45  2.2.7 Serum stability assay SPLP formulations were assayed for serum stability in the presence of 90% serum in vitro. A 50 uL aliquot of the formulations was added to 450 uL of mouse serum and incubated at 37°C for 30 min. The mixture was then loaded onto a Sepharose CL-4B column and eluted with HBS. The fractions were analyzed by dual label scintillation counting to determine the amount of original plasmid D N A associated with the liposomes, (i.e. the D N A that is protected from degradation by DNase).  2.2.8 In vitro transfection COS-7 cells were grown in an incubator at 37°C and 5% CO2 in 24-well plates with Dulbecco's modified Eagle's medium ( D M E M ; Life Technologies) and 10% fetal bovine serum (FBS; Intergen Co., Purchase, M A ) . Cells were plated at 2 x 10 /well. 4  Cells were incubated with the transfection media for up to 24 hours at 37°C.  The  transfections were carried out using liposome-encapsulated plasmid pCMVLuc, which contains the gene for the enzyme luciferase. Liposome-encapsulated plasmid had been cleared of unencapsulated D N A prior to addition of the media. Upon completion of the incubation, the cells were washed with PBS and then lysed with lysing buffer (0.5% TritonX-100 in PBS, pH8). The lysate was then assayed for luciferase activity using the Promega Luciferase Assay System reagent kit (Promega El501) according to the manufacturer's instructions. The activity was measured using a Dynex Technologies ML3000 microplate luminometer (Dynex Technologies, Ghentilly, VT). Luminescence readings were calibrated according to a standard curve obtained using Photinunus  pyralis  luciferase standard (Boehringer Mannheim, Laval, PQ).  46  2.3 R E S U L T S  2.3.1 Entrapment of plasmid DNA within lipid particles by employing detergent dialysis. Previous work has shown that incubation of plasmid D N A with cationic lipids can result in a hydrophobic particle that is soluble in organic solvent (Reimer et al., 1995). It is of interest to determine whether this hydrophobic particle can be surrounded by an outer monolayer of lipid, which would then result in small, plasmid-containing particles stabilized in an aqueous medium. Detergent dialysis is a logical technique for achieving this, as the detergent may be expected to solubilize the hydrophobic plasmid D N A cationic lipid particles.  The addition of phospholipid and subsequent removal of  detergent by dialysis could then result in the exchange of the solubilizing detergent with phospholipid, leaving particles that are stable in aqueous suspension. Initial experiments employed the cationic lipid D O D A C , the plasmid p C M V C A T , the  non-ionic  detergent  OGP  and  the  bilayer-forming  lipid  palmitoyl-  oleoylphosphatidylcholine (POPC). When D O D A C was added to plasmid in distilled water, the formation of large (> 1000 nm diameter) precipitates was observed. However, the subsequent addition of OGP (200 mM) resulted in solubilization of the precipitate, forming an optically clear suspension consistent with entrapment of hydrophobic plasmid DNA-cationic lipid particles within detergent micelles. This optically clear quality was maintained when POPC solubilized in OGP was added.  However, during dialysis to  substitute the detergent associated with the particles for POPC, extensive precipitation of  47  the suspension was observed.  A method to stabilize the plasmid-containing particles  against aggregation and precipitation during the dialysis process was therefore required. Previous studies have shown that a PEG coating can prevent aggregation of L U V s induced by covalent coupling of protein to the surface of the L U V s (Harasym et al., 1995), and can inhibit fusion between L U V s (Holland et al., 1996a). It was therefore of interest to determine whether the stabilizing properties of a P E G coating could prevent aggregation  during  dialysis.  However,  the  use  of  the  standard  PEG-  phosphatidylethanolamine (PEG-PE) was contraindicated because the PEG-PE molecule bears a net negative charge and could displace the cationic lipid from the plasmid, as has been noted for other negatively charged lipids (Xu and Szoka, 1996).  Therefore, a  neutral molecule was produced by linking PEG2000 to ceramide, the hydrophobic anchor. Two ceramide anchors were synthesized which differed in the length of the ceramide acyl chain (CerCi and CerC2o). 4  When 10 mol% PEG-CerC o was incorporated into the 2  detergent mixture with POPC, D O D A C and plasmid D N A , precipitation was not observed during detergent dialysis.  Furthermore, a proportion of the plasmid was  encapsulated, as measured by co-elution of D N A with liposomes from a D E A E Sepharose CL-6B anion exchange column. As shown in Figure 2.1 A , the achieved encapsulation is a sensitive function of the D O D A C content, with encapsulation levels of 30% or higher at about 9 mol% to 12 mol% D O D A C . It should be noted that addition of plasmid to preformed vesicles with the same lipid composition, followed by D E A E chromatography, resulted in complete plasmid retention on the column. These results suggest that "stabilized plasmid-lipid particles" (SPLP) can be produced by detergent dialysis employing a POPC/DODAC/PEG-CerC  20  (79:11:10;  48  80  D O D A C (mol %) Figure 2.1. Effect of D O D A C concentration on the encapsulation efficiency of plasmid D N A (pCMVCAT) in SPLP. A . Lipid composition POPC, D O D A C and 10 mol% P E G CerC  20  B . Lipid composition DOPE, D O D A C and 10mol% PEG-CerC . Lipid (10 20  mg/ml total), dissolved in octylglucoside (0.2 M ) , was mixed with plasmid D N A (50 u.g/ml) in a total volume of lmL.  49  mol%) lipid mixture. However, it has been shown that when POPC is employed as a "helper" lipid in plasmid DNA-cationic lipid complexes, very low transfection rates are observed, whereas when dioleoylphosphatidylethanolamine (DOPE) is present, much higher transfection rates are achieved (Farhood et a l , 1995).  The encapsulation  properties of DOPE/DODAC/PEG-CerC o lipid mixtures were therefore investigated. 2  Figure 2.IB shows that encapsulation of D N A was dependent on D O D A C content in DOPE-containing systems, similar to the POPC-containing systems.  Significant  differences are that maximum encapsulation was greater (-70%) for the DOPEcontaining system and that optimum encapsulation was observed at about 6 mol% D O D A C , compared with approximately 9 mol% D O D A C for the POPC-containing particles. When PEG-CerCn was substituted for PEG-CerC o, very similar plasmid 2  encapsulation behaviour was observed. In  subsequent  experiments,  DOPE/DODAC/PEG-Cer  formulations  were  employed containing 6 mol% D O D A C . The effect of D N A concentration and D N A size were examined. By varying the plasmid (pCMVCAT) concentration over the range of 25 to 400 pg/ml in the DOPE/DODAC/PEG-CerC  20  (84:6:10; mol%) lipid mixture (10  mg/ml total lipid) it was found that encapsulation efficiencies of more than 50 % were achieved over this range (data not shown). In addition, at a plasmid concentration of 400 p,g/ml, similar levels of entrapment were observed for plasmids of 4.49 and 10 kbp in length (data not shown).  50  2.3.2 Plasmid DNA in stabilized plasmid-lipid particles is protected from serum nucleases It is important to demonstrate that the "encapsulated" plasmid in the particles obtained by the detergent dialysis process is, in fact, fully protected from the external environment. A rigorous test of SPLP stability and protection of encapsulated plasmid involves incubation in serum. Serum contains a variety of nucleases, and serum proteins can rapidly associate with lipid systems (Chonn et al., 1992), resulting in enhanced leakage and rapid clearance of liposomal systems. The ability of serum nucleases to degrade plasmid is illustrated in Figure 2.2A. Intact p C M V C A T elutes in the void volume of the Sepharose CL-4B column, whereas after incubation with mouse serum (90%) at 37°C for 30 min the plasmid is degraded into fragments which elute in the included volume. The behaviour of the DOPE/DODAC/PEG-CerC  20  (84:6:10; mol%)  SPLP system where non-encapsulated plasmid has not been removed is shown in Figure 2.2B. In this particular preparation, 53% of the plasmid D N A elutes with the lipid in the void volume, and 47% of the D N A , which represents degraded plasmid, elutes in the included volume. This indicates that 53% of the plasmid is encapsulated and protected from the external environment, in good agreement with a 55% trapping efficiency of this sample as determined by D E A E ion exchange chromatography. A final test of the stability of the SPLP formulation is given in Figure 2.2C, which shows the elution profile of the DOPE/DODAC/PEG-CerC  20  (84:6:10; mol:mol:mol)  SPLP system following removal of the external plasmid by D E A E chromatography and incubation in 90% mouse serum (30 min at 37°C).  In this case more than 95% of  plasmid applied to the column eluted in the void volume, demonstrating the stability and  51  0 0  10  20  30  40  50  Fraction Figure 2.2. Plasmid in SPLP is protected from serum nuclease cleavage. The SPLP (DOPE/DODAC/PEG-CerC ; 84:6:10; mol:mol:mol) were prepared containing Relabeled C H E as a lipid marker and H-labeled plasmid D N A . Samples were incubated in the presence of 90% mouse serum for 30 min at 37°C and eluted on a Sepharose CL-4B column equilibrated in HBS. (a) Elution of undegraded (•) and degraded (o) D N A (b) Elution profile of D N A (•) and lipid (o) following incubation of SPLP in 90% mouse serum, (c) Elution profile of D N A (•) and lipid (o) following incubation of SPLP with mouse serum where unencapsulated plasmid was removed by anion exchange chromatography prior to the serum treatment. 20  3  52  the plasmid protection properties of the SPLP formulation. It should also be noted that SPLP containing PEG-CerCi  4  in place of PEG-CerC o exhibited similar plasmid 2  protection properties.  2.3.3 Stabilized plasmid-lipid particles can be isolated by density centrifugation The detergent dialysis process clearly results in plasmid-containing particles where the plasmid is protected from the external environment. However, it is likely that empty vesicles are also produced, as detergent dialysis of lipids (in the absence of plasmid) is well known to result in the formation of small lipid vesicles (Mimms et a l , 1981). These empty vesicles may be expected to be less dense than SPLP. The density gradient profile of a DOPE/DODAC/PEG-CerC  (84:6:10; mol%) SPLP preparation  20  (plasmid-to-lipid ratio of 200 pg D N A to 10 mg lipid) was therefore examined employing sucrose density step gradient centrifugation. As shown in Figure 2.3, after centrifugation at 160,000 x g for 2 h, the encapsulated D N A is present as a band which was localized at the 2.5% sucrose-10% sucrose interface in the step gradient. It is interesting to note that less than 10% of the total lipid (as assayed by the H - C H E lipid marker) is associated 3  with the plasmid D N A , which corresponds to 55% of the total D N A . The plasmid-tolipid ratio in these purified SPLP was determined (as indicated in Materials and Methods) to be 62.5 u.g plasmid per umol lipid. SPLP generated by detergent dialysis and purified by density gradient centrifugation may be concentrated using carboxymethyl cellulose to achieve plasmid concentrations of 1 mg/ml or higher.  53  Fraction  Figure 2.3. Separation of SPLP from empty vesicles by discontinuous sucrose density gradient centrifugation. The solid circles indicate the behaviour of the H-labeled plasmid (pCMVLuc), whereas the open circles indicate the distribution of lipid as reported by the 14  C-labeled C H E lipid marker.  54  2.3.4 Stabilized plasmid-lipid particles exhibit a narrow size distribution The sizes of the empty lipid vesicles in the upper band and SPLP in the lower band of the sucrose density gradient were examined by QELS and freeze-fracture E M techniques. QELS analysis (Figure 2.4) indicated that the mean diameter of the empty liposomes was -44 nm, whereas the isolated SPLP were larger having a mean diameter of-75 nm. Freeze-fracture E M studies gave similar results (Figure 2.5). A size analysis of the particles in these micrographs indicated a mean diameter of 36 ± 15 nm for the empty vesicles and 64 ± 9 nm for the isolated SPLP.  2.3.5 In vitro transfection properties of stabilized plasmid-lipid particles SPLP consisting of DOPE/DODAC/PEG-CerC  20  (84:6:10) containing p C M V L u c  coding for the luciferase reporter gene were prepared for transfection studies. As shown in Figure 2.6, after incubation of these SPLP with COS-7 cells for 24 h, little i f any transfection activity was observed. It is probable that the presence of the P E G coating on the SPLP inhibits the association and fusion of the SPLP with cells in the same manner that P E G coatings inhibit fusion between lipid vesicles (Holland et al., 1996a), and thus inhibits intracellular delivery of the encapsulated plasmid.  In this regard, previous  studies (Holland et al., 1996a) on L U V s with P E G coatings attached to phosphatidylethanolamine (PE) anchors have demonstrated that, for PE anchors containing short acyl chains, the PEG-PE can rapidly exchange out of the L U V , rendering the L U V s increasingly able to interact and fuse with each other.  The transfection properties of  SPLP containing PEG-Cerdo were therefore compared to SPLP containing PEG-CerCn,  100-  a •'o  >  >  tf  a  806040200 10  20  50  100  200  500  100" 0)  I  80-  >  60-  >  40-  o  tf  20-  Lk  0 10  20  50  100  -200  500  Diameter (nm)  Figure 2.4. QELS of (a) empty liposomes from the top band of the sucrose gradient and (b) purified SPLP from the bottom band of the sucrose gradient.  56  Figure 2.5. Freeze-fracture electron microscopy of purified SPLP and empty vesicles. SPLP containing pCMVLuc were prepared as indicated in the legend to Figure 2.1 and separated into (a) empty vesicles and (b) SPLP employing discontinuous sucrose density gradient centrifugation. The bar indicates 100 nm.  57  1 5 0  Time (h)  Figure  2.6. Effect of PEG-Cer coating of SPLP on transfection activity in vitro. Plasmid  (pCMVLuc)  was  encapsulated  in  SPLP  (DOPE/DODAC/PEG-Cer;  84:6:10;  mol/mol/mol) containing PEG-CerC2o (O) or PEG-CerCn («).The SPLP preparation (1 [ig plasmid) was then added to COS-7 cells at a density of 2 x 10 per 24-well plate. 4  The cells were incubated with the SPLP for the times indicated, and luciferase activity was measured. This work was done in collaboration with Dr. J. Wheeler.  58  which has a shorter acyl chain. As shown in Figure 2.6, after incubation with COS-7 cells for 24 h, the SPLP containing PEG-CerCi exhibits substantially higher levels of 4  transfection compared with the system containing PEG-Cerdo- This is consistent with the ability of the PEG-CerCi coating to diffuse away from the SPLP surface. The SPLP 4  containing either PEG-CerCi or 4  PEG-CerC2o  exerted no apparent toxic effects on the  cells as evaluated by monitoring protein content in the cell extract.  59  2.4  DISCUSSION  In this chapter a new method for the encapsulation of plasmid D N A in small, stable particulate systems that may find utility as gene delivery vehicles has been presented. O f particular interest are the relationship between the properties of SPLP and those of other lipid-based systems containing plasmids, the structure of SPLP, and the potential utility of SPLP with exchangeable P E G coatings. These areas are discussed in turn. The SPLP protocol for plasmid entrapment allows trapping efficiencies of up to 70% and results in stable particles containing low levels of cationic lipids and high levels of fusogenic lipids such as DOPE. These particles are small (< 100 nm diameter), are resistant to external nucleases, exhibit high DNA-to-lipid ratios (62.5 p,g/pmol) and can be concentrated to achieve high plasmid D N A concentrations (1 mg/ml). Furthermore, the detergent dialysis procedure is a gentle procedure that results in little, if any, plasmid degradation.  These features of SPLP contrast favourably with previous plasmid  encapsulation procedures. Plasmid D N A has been encapsulated by a variety of methods, including reverse phase evaporation (Fraley et al., 1980; Soriano et al., 1983; Nakanishi et al., 1985), ether injection (Fraley et al., 1979; Nicolau and Rottem, 1982), detergent dialysis in the absence of PEG stabilization (Stravridis et al., 1986; Wang and Huang, 1987), lipid hydration and dehydration-rehydration techniques (Lurquin, 1979; Alino et al., 1993; Baru et al., 1995), and sonication (Jay and Gilbert, 1987; Puyal et al., 1995; Ibanez et al., 1997), among others. The characteristics of these protocols are summarized in Table 2.1. None of these procedures yields small, serum-stable particles at high  60  00  •a •a  73  73  *-'  3  CJ  S  o  E  E 2 i  Q Z  O  w  cu cO O Q Q  tu  ¥¥  00  B  <  8  IT ¥  C  o o OO CT\  —' CN  <3 E  II  o o  B  e e  "So "Sb  z  A  o  A  A  A  A  A  "oo "So  OO ON  o  CN  ox c >. uI CL  B  "o "o B B  eg  VI O ON ON  <  Z  Q  o  -  0  m o  CJ  < a o  o o o 2  r> o '  CL  s  O  o CJ •a  o  a?  oo O CJ  o CU  2  CJ  J3  PH  0.  CJ CJ  OH  OH OH  CJ ^  ^s  O o  CJ  Q  K)  CJ  cj  0H I o- tU i  o  "o 75 '  O o  CJ CJ  J3 —'  o  O  f  OH  U  6  C o  OH NO tU O OH T.  a  tu ,-.  x: . CJ i CJ I OH I tU I  o o •* •a Q S2-  as  <u C3 X>  «3  r  T3  B  c  73 I-  Oi  o  Q u  o u  2  a o e u <U  < H Q +  "ed  g  'S o g. <  CL)  -o  Z  U Q  o  61  plasmid concentrations and plasmid-to-lipid ratios in combination with high plasmidencapsulation efficiencies. Trapping efficiencies comparable with the SPLP procedure can be achieved employing methods relying on sonication. However, sonication is a harsh technique which can shear nucleic acids (Ausubel et a l , 1995). Size ranges of 100 nm diameter or less can be achieved by reverse phase techniques; however, this requires an extrusion step through filters with 100 nm or smaller pore size which can often lead to significant loss of plasmid. Finally, it may be noted that the plasmid DNA-to-lipid ratios that can be achieved for SPLP are significantly higher than those achievable by any other encapsulation procedure. With regard to the structure of SPLP, any model must take into account two important observations.  First, SPLP form only at a critical cationic lipid content of  approximately 6 mol%.  At higher cationic lipid contents, aggregation is observed,  whereas lower cationic lipid contents lead to little or no plasmid encapsulation. Second, purified SPLP exhibit a plasmid DNA-to-lipid ratio of 62.5 ug/umol. For a 4.49 kbp (pCMVCAT) plasmid, this corresponds to a plasmid-to-particle ratio of 0.97 for an SPLP diameter of 70 nm (the average of the freezerfracture electron microscopy and QELS results), assuming a lipid molecular area (King et al., 1985) of 0.67 nm and an average 2  nucleotide molecular weight of 330 g/mol. It may therefore be concluded that SPLP contain one plasmid per particle. The model that guided the construction of SPLP relied on the hypothesis that the plasmid combines with the cationic lipid to form a hydrophobic "inverted micellar" structure that is stabilized in aqueous media by the detergent. In this model the addition of DOPE and PEG-Cer and subsequent dialysis results in deposition of a monolayer of  62  DOPE and PEG-Cer around the hydrophobic intermediate, resulting in a stabilized plasmid-lipid particle.  It is instructive to perform some simple calculations to see  whether this model is consistent with experimental observations. In particular, i f each negative charge on the plasmid has a cationic lipid associated with it, the total volume of each hydrophobic plasmid-cationic lipid particle can be calculated to be -1.35 x 10 nm 4  3  for a 4.49 kbp plasmid. This calculation assumes that plasmid D N A has a density of 1.7 g/ml, the molecular weight of each base is 330 g/mol, and that, as an upper limit, the volume per molecule of the cationic lipid is 1.5 nm , which is the volume of a liquid 3  crystalline bilayer-forming lipid such as dioleoylphosphatidylcholine (lipid length 2.2 nm and area per molecule 0.67 nm ) (King et al.,1985). Thus, i f each SPLP contained one 2  p C M V C A T plasmid completely neutralized by associated cationic lipid and arranged in a spherical conformation, the predicted diameter would be -30 nm. The freeze-fracture electron microscopy results presented here indicate that SPLP containing the p C M V C A T plasmid exhibit a diameter of approximately 70 nm, and are therefore too large to be composed solely of a plasmid-lipid particle with no interior aqueous volume. A n alternative working model for SPLP formation and structure is shown in Figure 2.7. It is unlikely that plasmid associates directly with the micelles, as the presence of high levels of detergent may be expected to dilute the positive surface charge due to the cationic lipid to the extent that the electrostatic association is reduced. A probable first step of the dialysis process is the formation of macromolecular lipid intermediates, which may include cylindrical micelles, lamellar sheets or leaky vesicles that form as detergent is removed. These structures have been observed as intermediates in the micelle to vesicle transition undergone by dispersions of egg phosphatidylcholine  63  as detergent (OGP) is removed by dialysis (Ollivon et al., 1988; Vinson et al., 1989). As shown in the lowest panel of Figure 2.7, low concentrations of cationic lipid would result in little association of plasmid with these intermediate structures, which is consistent with little or no plasmid entrapment following detergent dialysis. At high concentrations of cationic lipid, intermediate structures may be expected to associate with the plasmid and, if the cationic lipid content is too high, plasmid-lipid-plasmid association could dominate as dialysis proceeds, leading to formation of aggregates (Figure 2.7, top panel). If the cationic lipid content is at a critical level (Figure 2.7, central panel), the positive surface charge on the plasmid-associated intermediates will be reduced below that needed to associate with other plasmids, due to charge neutralization. This would mitigate against further aggregation. Further dialysis will result in fusion between intermediates to eventually produce empty vesicles or in fusion between intermediates and the plasmid-lipid particle. Fusion with the particle will result in the deposition of excess bilayer lipid, leading to the formation of an associated vesicle in the final SPLP. In the structure presented, the plasmid is associated with the inner monolayer of the vesicle that is produced as more lipid is deposited in the particle. It should be noted that the forces driving a partial removal of the plasmid lipid coat are not clear, and it is possible that the plasmid resides in a hydrophobic domain inside the particle. The final area of discussion concerns the potential utility of SPLP with exchangeable P E G coatings. As previously indicated the SPLP system has been designed for systemic (intravenous) gene therapy applications.  This places two potentially  conflicting demands on the delivery system. First, the carrier must circulate long enough to achieve accumulation at disease sites, such as solid tumours, by taking advantage of  65  the increased vascular permeability in these regions. Second, the carrier must be able to bind to target cells and to destabilize the plasma or endosomal membrane after arrival at the disease site in order to facilitate intracellular delivery of the enclosed plasmid. The first requirement implies a very stable carrier that does not interact with cells, whereas the second requirement necessitates a particle that can bind to cells and exhibit a membrane-destabilizing "fusogenic" character. P E G coatings that can dissociate from a carrier provide a potential solution to these demands. First, the presence of a P E G coating allows SPLP to be formed with a large proportion of DOPE in the outer monolayer. Previous work has shown that DOPE prefers the (non-bilayer) hexagonal Hn phase at temperatures above 10°C (Cullis and deKruijff, 1978), and that P E G lipids can stabilize DOPE in the bilayer organization (Holland et al., 1996b). Thus in the absence of the PEG-Cer the SPLP would be expected to be highly unstable and fusogenic. The detergent dialysis procedure therefore allows an intrinsically fusogenic plasmid-containing particle to be formed, where the stability of the particle is dependent on the presence of the P E G coating. As demonstrated here, these particles are stable in the presence of serum nucleases, consistent with an ability to protect encapsulated D N A in the circulation. In addition, the small size and presence of the P E G coating would be expected to promote the extended circulation lifetimes required to achieve preferential accumulation at disease sites such as tumours, following intravenous administration. The stability of the SPLP would, however, be expected to mitigate against uptake and intracellular delivery of the plasmid. The use of P E G coatings that dissociate from the SPLP after arrival at a disease site provides a potential solution to this problem. This  66  is supported by the in vitro results presented here, which show that a PEG-CerC o 2  coating, which has a long residence time in lipid bilayers, exhibits poor transfection properties, whereas improved transfection is observed for the SPLP containing a PEGCerCi4 coating, which can dissociate from lipid bilayers more rapidly (~1.1 hr for the C14 system compared to greater than 13 days for the C20 system) (Wheeler et a l , 1999). In summary, in this chapter a method has been presented for the encapsulation of plasmid D N A in particulate systems that have the properties of small size, high plasmidto-lipid ratio and high content of fusogenic lipid, and that can be concentrated to achieve high plasmid concentrations.  These SPLP are stabilized by the presence of a P E G  coating that can be designed to dissociate, thus increasing the transfection potency of the SPLP. It is expected that these systems will find utility as delivery systems for systemic gene therapy.  67  CHAPTER 3 CATIONIC PEG-LIPIDS  3.1 I N T R O D U C T I O N  The stabilized plasmid-lipid particles (SPLP) described in Chapter 2 meet some of the requirements for a plasmid delivery system with systemic administration. They are small in size (-70 nm diameter) and contain a single plasmid molecule, which is fully protected from degradation by serum nucleases. The poly(ethylene glycol) (PEG) surface coating provides extended circulation lifetimes, resulting in significant (-6% of the injected dose) accumulation at a distal tumour site (Tarn et al., 2000). However, only low levels of transfection were observed with SPLP in vivo in tumour tissue (Tarn et al., 2000) and with cells in vitro (Mok et al., 1999; Wheeler et al., 1999). A reason for the low transfection potency of SPLP is that the SPLP are not readily taken up by the cells. This limited cell interaction results from the P E G coating of SPLP which provides a steric barrier that prevents rapid clearance by the MPS but also minimizes interactions with cells. In this regard, inclusion of either negative (Allen et al., 1991a; Lee et al., 1992; Lee et a l , 1993; Miller et al., 1998) or positive (Miller et al., 1998) charges in liposomes can lead to enhanced cellular uptake. For example, plasmid DNA-cationic lipid complexes (lipoplexes), which are efficient in vitro transfection agents (Feigner et al., 1987; Jarnagin et a l , 1992), associate with cells due to their net positive charge (Miller et a l , 1998). In this chapter, a new class of cationic lipid, known as cationic P E G lipids (CPL), described by Chen et al. (2000) to enhance non-specific targeting by increasing the  68  electrostatic attraction between liposomes and cells have been used to increase the cellular uptake of preformed LUVs.  The CPL are inserted into the preformed L U V s  using a procedure developed by Fenske et al. (2000), which was modified from the work of Uster et al. (1996) and Ishida et al. (1999). The CPL, which contain 1-8 positive charges at the termini of a PEG lipid, consist of four moieties conjugated in the following order: (i) a phospholipid anchor (DSPE), (ii) a lysine spacer containing a dansyl label on the s-amino group, (iii) a PEG3400 chain, at the distal end of which is covalently attached (iv) a positively charged headgroup made of linked lysine residues (Figure 3.1). The nomenclature used simply specifies the charge of the headgroup as given in Figure 3.1 (e.g., CPL4 possesses 4 positive charges).  Using optimal conditions determined by  Fenske et al. (2000) for the post-insertion, L U V - C P L are prepared and the in vitro cellular uptake in the presence of various CPL is studied.  69  Figure 3.1. Structures of the cationic-PEG-lipids.  70  3.2 M A T E R I A L S & M E T H O D S  3.2.1 Materials 1,2-dioleoylphosphatidylethanolamine phosphoethanolamine-N-(Lissamine  (DOPE), and l,2-dioleoyl-sn-glycero-3-  Rhodamine B Sulfonyl) (Rhodamine-PE) were  obtained from Avanti Polar Lipids. Cholesterol, octyl-p-D-glucopyranoside (OGP), and HEPES were obtained from Sigma Chemical Co.  D O D A C and PEG-CerC , P E G 20  CerCn, and PEG-CerCg were generous gifts from Dr. Z. Wang at Inex Pharmaceuticals. The cationic P E G lipids (CPL) possessing 1, 2, 4, and 8 positive charges (Fig. 3.1) were synthesized by Drs. K . Wong and T. Chen , as described by Chen et al. (2000). B H K cells were obtained from Dr. R. MacGillivray of the Department of Biochemistry and Molecular Biology, U B C .  3.2.2 Vesicle Preparation Liposomes composed of the same lipid composition as the SPLP were formed using the detergent dialysis procedure outlined in Chapter 2. The lipids, dissolved in chloroform, were mixed to give 10 umol of DOPE:DODAC:PEG-CerC  20  (84:6:10,  mol/mol/mol), and the solvent removed by evaporation to obtain a dry lipid film.  The  lipid film was dissolved by the addition of 200 uL of the non-ionic detergent octyl-p-Dglucopyranoside (OGP) (1 M in water), and 800 uL of 20 m M HEPES, 150 m M NaCl pH 7.5 (HBS) added to give a final detergent concentration of 200 m M . This lipiddetergent solution was then transferred to a dialysis bag (12,000-14,000 M W C O , Fisher Scientific) and dialyzed at room temperature against HBS (2 L) over a period of 48 h  71  with 3-4 buffer changes. The concentration of the liposomes was determined using the Fiske-Subbarow (1925) phosphorus assay.  3.2.3 Insertion of C P L into pre-formed vesicles The CPL were stored at 4°C as micellar solutions in 20 m M Hepes, 150 m M NaCl pH 7.5.  Post-insertion was performed as previously described (Fenske et al., 2000).  Briefly, CPL micelles (0.04-0.08 umol) were added to vesicles (1 pmol of total lipid) to give the desired initial molar ratio (between 4 and 8 mol% CPL of total lipid), followed by incubation for a given time at the desired temperature. Typically, the insertions were carried out at 60°C with a 2-3 h incubation in the presence of 1 umol of liposomes. Following incubation, the L U V s containing CPL (LUV-CPL) were separated from excess free C P L micelles by Sepharose CL-4B column (1.5 cm x 15 cm) chromatography. The column was equilibrated with HBS, and the fractions analyzed for both L U V and CPL, as described below. To determine the levels of CPL insertion, the vesicles were labeled with 0.50 mol%  rhodamine-PE, and the C P L contained a dansyl group.  The rhodamine  fluorescence was measured using excitation and emission wavelengths of 560 nm and 590 nm, respectively while dansyl fluorescence was measured using excitation and emission wavelengths of 340 nm and 510 nm, respectively. In general, the excitation and emission slit widths were 10 and 20 nm, respectively. The amount of CPL inserted into the vesicles was calculated based on the initial dansyl/rhodamine ( D / R ) fluorescence ratio, and the D / R ratio of the isolated L U V - C P L using the following equation. %-insertion = ([D/R] PL-LUV)*100/[D/R], IAL C  NIT  72  The assays were performed as follows.  L U V - C P L before (2-3 uL) and after column  chromatography (20-40 uL) were dissolved in 20 uL of 10% Triton X-100 and dansyl and rhodamine fluorescence were measured following addition of 2 mL of HBS.  3.2.4 Uptake of liposomes containing C P L by B H K cells Approximately 1x10 B H K cells were seeded on 12-well plates in 2 mL of s  complete media ( D M E M + 10% FBS) and incubated overnight at 37°C in 5% C 0 . The 2  cells were washed twice with 1 mL of PBS and then incubated with 1 mL of P B S / C M G containing 20 nmol of DOPE:DODAC:PEG-CerC  20  (84:6:10, mol%) L U V s with either  no inserted CPL, 8 mol% C P L , 7 mol% C P L , or 4 mol% C P L for 1, 2, 4, and 6 h at 4 2  3  4  °C and 37 °C. At each time point, the cells were washed with PBS and lysed with 600 uL of 0.1%) Triton X-100 in PBS (pH 8.0).  Rhodamine fluorescence, using the  parameters given above, and protein concentration were measured in the lysate. The lipid uptake was calculated based on a rhodamine standard curve and normalized to the amount of protein in the lysate determined using the B C A assay kit (Pierce, Rockford, IL). At 4°C, internalization (if any) would be inhibited and therefore binding of L U V CPL to the cell surface can be determined. Cellular uptake of L U V - C P L was also monitored by fluorescence microscopy. Fluorescence micrographs were taken on an Axiovert 100 Zeiss fluorescence microscope (Carl Zeiss Jena GmbH) using a rhodamine filter from Omega Opticals (Brattleboro, V T ) with the following specifications: excitation at 560 ± 20 nm, long pass emission at 600 nm, and a dichroic filter at 590 nm.  3.3 R E S U L T S  3.3.1 Insertion of C P L into pre-formed liposomes The approach to be described here requires the insertion of novel C P L into preformed liposomes, which leads to a cationic vesicle in which the positive charge involved in cell interaction is located some distance from the liposomal surface.  The insertion  process is illustrated in Figure 3.2 for the insertion of CPL4 into sterically-stabilized L U V s composed of DOPE, the cationic lipid D O D A C , and PEG-CerC . 20  This lipid  composition was chosen as it was used in Chapter 2 for SPLP preparation (Wheeler et al., 1999). CPL with 1, 2, 4, and 8 positive charges were synthesized by Drs. Wong and Chen using methods described by Chen et al. (2000). In each case the C P L was added, in micellar form, to vesicles of a lipid composition identical to SPLP (DOPE/DODAC/PEGCerC o [84:6:10, mol/mol/mol]). Following incubation at 60°C, C P L insertion was 2  detected by size exclusion chromatography on a Sepharose CL-4B column. Figure 3.3 shows the elution profile for the insertion of CPL4 into the preformed L U V s . The C P L micelles detected by dansyl fluorescence elute in the included volume while the vesicles, identified by rhodamine fluorescence, elute in the void volume (Figure 3.3A). Following incubation with vesicles, CPL elutes in two peaks. The first CPL peak co-elutes (peak at 9 mL) with the vesicles while the second peak (broad peak centered at 16 mL) elutes in the same fractions as free CPL micelles. The two peaks are well separated allowing for easy isolation of the L U V - C P L . Association of C P L with the vesicles was calculated to be approximately 70% based on the fluorescence distribution, corresponding to -4.5  74  Figure 3.2. Insertion of C P L into empty liposomes.  75  Figure 3.3. C P L insertion into L U V characterized by size exclusion chromatography. L U V s (5 umol lipid), composed of DOPE/DODAC/PEG-CerC (84/6/10), were incubated with 0.355 pmol C P L in a total volume of 519 uL at 60 °C for 3 hours. Following incubation, the sample was applied to a Sepharose CL-4B column equilibrated with HEPES-buffered saline. Fractions of 1 mL were collected and assayed for dansylCPL (•) and rhodamine-PE (•) fluorescence, as described in Materials and Methods. The elution profile is shown in A for lipid (•) and CPL (•). Open triangles (A) show the elution profile of CPL micelles only. In B the elution profile of the main L U V fraction obtained in A rechromatographed on the same column. Fractions of 1 mL each were collected and assayed for dansyl-CPL (•) and rhodamine-PE (•). This work was done in collaboration with Dr. D. Fenske. 20  4  76  mol% C P L  4  insertion.  When the isolated L U V - C P L were rechromatographed on a  second Sepharose CL-4B column, all the C P L co-eluted in the L U V peak (Figure 3.3B). This indicates that the inserted C P L does not exchange out of the vesicles, which would indicate a very loose association instead of the tight one that is seen here. The work described here was done in collaboration with Dr. D. Fenske. The C P L insertion is time- and temperature- dependent as shown in Figure 3.4. CPL  4  micelles  (0.54  mM)  DOPE:DODAC:PEG-CerC  20  were  incubated  with  LUVs  (12.5  mM)  of  (84:6:10, mol/mol/mol) at room temperature (25 °C), 40  °C, and 60 °C. Aliquots were withdrawn at 1, 3, and 6 h and analyzed for CPL insertion by Sepharose CL-4B column chromatography.  At a CPL to lipid molar ratio of 0.0433,  CPL insertion at room temperature and 40°C were similar reading -30% after 1 h and increased only a little at 40°C over the 6 h period to 40%. However, increasing the incubation temperature to 60°C resulted in approximately 70% C P L insertion within 1 h and increased further to 80-85% after 6 h. At all three temperatures, the majority of insertion occurred within 1 h. Based on these data, a 2-3 hour incubation at 60°C was chosen as standard insertion conditions for C P L .  This work was also done in  collaboration with Dr. D. Fenske.  3.3.2 Uptake of CPL-liposomes by B H K cells Vesicles  composed  of DOPE:DODAC:PEG-CerC2o  containing  differently  charged C P L in the outer monolayer and labeled with Rh-PE were added to B H K cells in P B S / C M G and incubated over 6 h. At different time intervals, cells were washed and lysed.  The uptake of L U V - C P L was determined by measuring the  rhodamine  77  Time (hr) Figure 3.4. Influence of incubation time and temperature on C P L insertion into L U V s . DOPE/DODAC/PEGCerC L U V s (3 pmol) were incubated with 0.13 umol C P L (total 20  4  volume 240 uL) for 1, 3, and 6 hours at various temperatures. Aliquots containing 1 umol of lipid were removed and C P L insertion determined following Sepharose CL-4B column chromatography, as described in Materials and Methods. Percent C P L insertion into L U V s following incubation at 25 °C (•), 40 °C (•), and 60 °C (A) is plotted as a function of time. This work was done in collaboration with Dr. D. Fenske.  78  fluorescence in the lysate. The incubations were performed at both 4 and 37°C and the rhodamine assayed for each. The fluorescence obtained at 4°C is subtracted from that at 37°C, as the measurement at 4°C is assumed to be give an approximation of the particles bound to the surface of the cells. L U V s containing 8 mol% CPL2 were taken up by the cells to a similar extent as L U V s without CPL2 (Figure 3.5). The uptake of L U V s with 7 mol% CPL3 was increased by a factor of two. However, a significant increase in uptake of L U V s with 4.1 mol% CPL4 was observed. After a 6 hour incubation, the lipid uptake with LUV-CPL4 was approximately ten times higher than for L U V s without CPL. The difference in binding and uptake of L U V - C P L by B H K cells as compared to L U V s can clearly be visualized using fluorescence microscopy (Figure 3.6).  In the  absence of C P L , vesicles composed of DOPE:DODAC:PEG-CerC o exhibit little 4  2  binding to cells (Figure 3.6 A ) , as indicated by the lack of rhodamine fluorescence. Incorporation of 3 mol% CPL4 leads to high levels of vesicle binding and uptake (Figure 3.6 B). Although most of the lipid appears to be bound to the cell surface, small punctate structures can be seen, indicating uptake of L U V s by endocytosis. A n important point to note is that the cells incubated with L U V - C P L appear healthy, in contrast to cells incubated with L U V s containing high concentrations of quaternary cationic lipids (e.g. DNA-cationic lipid complexes) where toxic effects are usually clearly visible.  79  Incubation Period (hr) Figure 3.5. Uptake of L U V - C P L with different CPL by B H K cells. Rhodamine-labeled L U V - C P L were prepared by post-insertion of  CPL2, CPL3,  and CPL4 by incubation at  various CPL/lipid ratios for 2 hours at 60°C. The L U V - C P L (20 mM) were incubated in P B S / C M G on ~10 B H K cells at 37°C for various incubation periods. 5  The bound  particles were measured by incubating at 4°C. Following the incubations, the cells were washed and lysed, and assayed as described in Materials and Methods to determine the uptake of DOPE:DODAC:PEG-CerC  20  (84/6/10) L U V s containing no CPL (•), 8 mol%  C P L ( • ) , 7 mol% C P L ( • ) , and 4 mol% C P L (•). To determine the lipid uptake, the 2  3  4  intensities at 4°C were subtracted from those at 37°C. The lipid uptake is plotted as a function of incubation period for L U V s possessing various CPL.  80  Figure 3.6. Insertion of CPL4 into L U V results in uptake of the particles by B H K cells. B H K cells (1 x 10 ) were incubated for 6 hour with 20 m M D O P E / D O D A C / P E G - C e r C vesicles labeled with 0.5 mol% rhodamine-PE with and without 3 mol% CPL4 inserted. Fluorescence microscopy was performed as described in Materials and Methods after a 6 hour incubation. Panel A shows a fluorescence micrograph of B H K cells following incubation with LUVs. Panel B shows a fluorescence micrograph of B H K cells following incubation with L U V - C P L . Panels C and D show phase contrast micrographs of the cells in A and B, respectively. 5  20  81  3.4 D I S C U S S I O N  In this chapter, a non-specific targeting approach is described that increases the association between preformed L U V and cells. Insertion of CPL leads to localization of positive charge above the PEG surface layer, thereby facilitating electrostatic interactions between the L U V and the cell surface, leading to uptake of the particles by endocytosis. This results in dramatic increases in the binding and uptake of L U V by B H K cells in vitro. The insertion procedure is simple and relatively gentle, involving an incubation of CPL and L U V for 2-3 h at 60°C and results in insertions of approximately 60-80% of the available CPL. Maximum insertions of ~6 mol% CPL in the L U V could be achieved. There are two major advantages to the post-insertion process. First, the procedure allows the incorporation of surface information into liposomal systems that may not be possible to incorporate during vesicle formation. For example, inclusion of C P L into SPLP during the formulation process would be complicated by electrostatic interactions between the C P L and plasmid. Alternatively, the post-insertion process should allow the incorporation of targeting information in the form of peptides and proteins coupled to the PEG-PE that are insoluble in the organic solvents used to mix the L U V components prior to L U V formation (Ishida et al., 1999). A second advantage is that the procedure is general, and allows liposomal systems of varying complexity to be built in a modular fashion, building on a well-defined basic unit such as the SPLP system or a drug-loaded liposome (Ishida et al., 1999). CPL and L U V containing C P L have not been described previously. A nonfluorescent monovalent cationic amino-PEG-DSPE lipid, similar to C P L i has been shown  82  to induce prolonged circulation lifetimes (Zalipski et al., 1994).  Other workers have  described the synthesis of a non-phospholipid-anchored P E G with a distal divalent cationic headgroup (Schulze et al., 1999) and a dansyl-labeled PEG (Pendri et al., 1995). Advantages of CPL include the phospholipid anchor that allows incorporation of CPL into preformed liposomal systems.  Secondly, the dansyl label permits accurate and  convenient quantification of the C P L in the bilayer using fluorescence  techniques.  Finally, the number of positive charges on the cationic headgroup in the C P L can easily be modified. Previous workers have shown that the presence of PEG on the liposome surface inhibits interactions with cells (Miller et al., 1998). This work shows that inclusion of CPL4 at 4 mol% gives rise to a ten-fold increase in uptake into B H K cells following a 6 h incubation as compared to vesicles in the absence of CPL.  This demonstrates that the  presence of positively charged groups at some distance from the L U V surface can lead to significant increases in cellular uptake. However, it is not total charge alone that plays a role in enhanced cell uptake. For example, the presence of 7 mol% CPL2 leads to little uptake whereas 4 mol% CPL4 leads to efficient uptake. It would appear that localization of a sufficient positive charge density at the distal end of the C P L molecule is an important parameter in ensuring interaction with cells. At least four charges seem to be required for efficient cell binding to occur. A recent study by Zalipsky and co-workers (Gabizon et al., 1999) reported on the incorporation of folic acid-PEG-DSPE conjugates into vesicles, and demonstrated enhanced binding to cells expressing the folate receptor. The best binding was observed for vesicles containing only the folate-PEG-DSPE conjugate (i.e., in the absence of  83  MePEG-DSPE).  The presence of additional MePEG-DSPE greatly reduced binding,  particularly when the length of the P E G in the two molecules was identical. Enhanced binding was only observed for vesicles containing folate-PEG335o-DSPE and  MePEG2ooo-  DSPE. This mirrors our system, where the M W s of the P E G in the C P L and PEG-Cer are 3400 and 2000, respectively, and suggests we should be able to modulate cell binding by varying the lengths of the two P E G spacers. In Chapter 4, the results from this chapter will be incorporated to increase the cellular uptake and expression of a reporter gene within cells in vitro. In particular, the insertion protocol developed in this chapter and the result that the presence of C P L in 4  pre-formed L U V s gives optimal uptake in vitro will be used in the development of SPLPC P L in Chapter 4.  84  CHAPTER 4 INCORPORATION OF CPL INTO SPLP FOR INCREASED IN VITRO UPTAKE AND TRANSFECTION  4.1 INTRODUCTION  In Chapter 2, techniques were developed that allow the efficient encapsulation of plasmid D N A in well-defined "stabilized plasmid-lipid particles" (SPLP) of -70 nm diameter containing one plasmid per vesicle (Wheeler et al., 1999). It has also been shown that SPLP exhibit extended circulation lifetimes and preferential accumulation of intact plasmid at distal tumour sites following intravenous injection (Tarn et al., 2000). The levels of transgene expression observed at the tumour site following i.v. injection of SPLP containing the luciferase marker gene are superior to the levels that can be achieved employing lipoplexes or naked D N A but are low compared to the values that may be required for therapeutic benefit (Tarn et al., 2000). Previous work showing that SPLP are less readily taken up by cells in comparison to lipoplexes suggests that the low transfection potencies of SPLP may be related to low levels of binding and uptake (Mok et a l , 1999). In Chapter 3 it was shown that CPL, specifically C P L , when incorporated 4  into L U V results in increased cellular uptake.  This chapter examines whether the  insertion of C P L into SPLP leads to enhanced cell association, thus leading to enhanced 4  in vitro transfection properties. It is shown that C P L can be inserted into preformed SPLP, using the post4  insertion process, and that the resulting SPLP-CPL  4  exhibit improved uptake and  markedly improved in vitro transfection potency in B H K cells in vitro. These results  85  establish that the SPLP system is intrinsically a highly potent transfection vector that requires only the addition of factors to stimulate uptake into target cells.  86  4.2 M A T E R I A L S A N D M E T H O D S 4.2.1 Materials DOPE was obtained from Northern Lipids Inc. (Vancouver, BC). Rh-PE, and PicoGreen were obtained from Molecular Probes (Eugene, OR). D O D A C  was  synthesized and supplied by Dr. S. Ansell of Inex Pharmaceuticals (Vancouver, B C ) . PEG-CerC2o  was synthesized as indicated elsewhere (Webb et al., 1998) and was  supplied by Dr. Z. Wang of Inex Pharmaceuticals (Vancouver, BC). The p C M V L u c plasmid (~6 kb) encodes for the Photinus pyralis luciferase gene under the control of the human C M V early promoter and was supplied by Dr. P. Tarn of Inex Pharmaceuticals (Vancouver, BC). The p C M V G F P plasmid (~5 kb) contains the gene for the green fluorescent protein from Aequorea victoria and was supplied by Dr. P. Tarn of Inex Pharmaceuticals (Vancouver, BC). DEAE-Sepharose CL-6B, Sepharose CL-4B, octyl-pD-galactoside, and HEPES were obtained from Sigma-Aldrich (Oakville, ON). Lipofectin was obtained from Gibco B R L (Burlington, ON). B H K cells were obtained from Dr. R. MacGillivray of the Department of Biochemistry and Molecular Biology, UBC.  4.2.2 Preparation of S P L P - C P L  4  SPLP composed of DOPE:DODAC:PEG-CerC  20  (84:6:10) and containing the  plasmid pCMVLuc (or pCMVGFP) were prepared according to the method of Wheeler et al.  (1999)  using  purification by  anion  exchange  (DEAE-Sepharose CL-6B)  chromatography and sucrose density gradient centrifugation to remove unencapsulated plasmid and empty vesicles, respectively. SPLP containing Rh-PE were prepared by  87  dissolving Rh-PE with other component lipids in CHC1 at a molar ratio of 83.5:10:6:0.5 3  (DOPE:DODAC:PEG-CerC :Rh-PE) prior to forming the lipid film. 20  C P L was inserted into preformed SPLP by incubating SPLP (500 nmol lipid) 4  with C P L (12.5, 19, and 30 nmol) at 60°C for 2 to 3 h in Hepes buffered saline (HBS), 4  pH 7.5, unless otherwise indicated. Unincorporated C P L was removed by gel filtration 4  chromatography on a Sepharose CL-4B column equilibrated in HBS.  Fractions (1 ml)  were collected and assayed for CPL4, phospholipid and D N A content.  Fractions  containing all three components were pooled and concentrated. CPL4 content was determined by the fluorescence of the dansyl-labeled C P L at A, =510 nm following em  excitation at A.  ex  = 340 nm employing a Perkin Elmer LS52  Luminescence  spectrophotometer with excitation and emission slit widths of 10 and 20 nm, respectively. A standard curve was derived from a stock solution of dansylated CPL in HBS. For SPLP containing Rh-PE the phospholipid content was determined from the fluorescence of the Rh label measured at A, 590 nm following excitation at A,ex 560 nm, using excitation =  =  em  and emission slit widths of 10 and 20 nm, respectively. For SPLP that did not contain the Rh label, phospholipid was determined using the method of Fiske-Subbarow (Fiske and Subarrow, 1925), following lipid extraction according to Bligh and Dyer (1959). Plasmid DNA  was determined using the PicoGreen Assay kit (Molecular Probes, Eugene,  Oregon) as previously described (Mok et al., 1999). For the Rh-PE-containing systems, the incorporation of CPL4 was determined by dividing the dansyl to rhodamine ratio before the Sepharose column by that after the column multiplied by 100%. For the other systems, incorporation was determined by dividing the CPL4 content by the total lipid content and multiplying by 100%.  88  Lipoplexes were prepared at a charge ratio of 1.5:1 (positive-to-negative) by adding 25 uL of 88 ug/mL plasmid D N A (pCMVLuc or pCMVGFP) with 25 uL of DOPE:DODAC (0.8 mM) while vortexing followed by incubation at room temperature for 30 min prior to addition to cells. Lipofectin lipoplexes were similarly prepared. Quasi-elastic light scattering (QELS) studies were conducted employing a Nicomp Model 270 Submicron Particle Sizer operating in the vesicle mode. Freezefracture electron microscopy studies were performed as described by Wheeler et al. (1999). D N A for Southern analysis was extracted using a phenol:chloroform extraction following incubation of SPLP systems with 50% mouse serum. The resulting D N A was then subjected to electrophoresis through a 1% agarose gel, denatured to give singlestranded D N A , transferred to a nylon membrane (Amersham) and subjected to Southern analysis. The membrane was exposed to random-primed P-labelled PvuII restriction 32  fragment from the luciferase gene according to current protocols. Hybridization intensities were quantified using a Phosphorimager™ SI from Molecular Dynamics. The data were converted to give amounts of intact D N A relative to undigested D N A . Levels of PEG-CerC2o and DOPE were determined by H P L C analyses performed by Nothern Lipids, Inc, Vancouver, B.C.  4.2.3 Uptake and transfection studies A B H K cell line (tk") transformed by polyoma virus was used for all uptake and transfection studies. To determine the cellular uptake of SPLP, 1x10 B H K cells were s  89  seeded in each well of a 12-well plate and incubated overnight in 2 ml of complete media ( D M E M containing 10% FBS) at 37°C in 5% C 0 . SPLP, S P L P - C P L 2  4  in media  containing 40 m M CaCl , or DOPE:DODAC lipoplexes in a volume of 200 uL were 2  mixed with 800 uL of complete media at a final lipid dose of 20 u M and was added to the cells. Plasmid D N A concentrations corresponded to 1.4 ug/mL and 2.2 p.g/mL for the SPLP systems and the lipoplexes, respectively. Cells were incubated at 37°C for indicated periods, washed twice with PBS and lysed with 600 uL of lysis buffer (0.1% Triton X 100 in PBS). Pvhodamine fluorescence was determined using a X of 560 nm and a X ex  em  of 600 nm with slit widths of 10 and 20 nm, respectively. A n emission filter of 530 nm was also used. Lipid uptake was determined by comparison of the fluorescence in the lysate to that of a lipid standard and normalized to the cell number as determined by the B C A protein assay (Pierce, Rockford, IL). Where indicated, fluorescence micrographs were obtained using an Axiovert 100 Zeiss Fluorescent microscope (Carl Zeiss Jena GmbH) using a rhodamine filter from Omega Opticals (Brattleboro, V T ) with the following specifications: excitation 560±20/dichroic filter 590/long pass emission 600. The effect of C a  2+  and M g  2 +  on lipid uptake was determined as described above  with the following exceptions. B H K cells (5x10 per well) were seeded in a 24-well plate 4  in 1 mL of complete media and incubated overnight at 37°C. SPLP-CPL4 (40 nmol) were mixed with C a C l or M g C l in a total volume of 100 uL. Complete media (400 uL) was 2  2  added to the SPLP-CPL4 resulting in final cation concentrations of 4 to 14 m M . This mixture was then added to the cells and incubated for 4 h at 37°C. Cells were then washed twice with PBS and lysed in 600 uL of lysis buffer (0.1% Triton X-100 in PBS).  90  Unless otherwise indicated, transfection studies were performed employing l x l 0  4  B H K cells plated in each well of a 96-well plate in 150 uL complete media prior to overnight incubation at 37°C in 5% C 0 . SPLP and SPLP-CPL corresponding to 0.5 pg 2  4  of p C M V L u c in 20 uL HBS (SPLP), or HBS containing 40 m M C a C l (SPLP-CPL ) 2  4  were added to 80 uL of complete media for a plasmid concentration of 5.0 p,g/mL. A transfection time of 4 h with a total incubation time of 24 h was used routinely. The transfection time is defined as the time the cells are incubated with the plasmidcontaining particles whereas the total incubation time is the transfection time (after which the transfection media is replaced) plus the subsequent time the cells are incubated for prior to assaying for transgene expression. After 24 h, the cells were lysed with 100 u.L of lysis buffer, and 40 uL of the lysate was transferred to a 96-well luminescence plate. Luciferase activity was determined using a Luciferase reaction kit (Promega, Madison, WI), a luciferase standard (Boehringer-Mannheim), and a ML3200 microtiter plate luminometer from Molecular Dynamics (Chantilly, V A ) . Activity was normalized to the number of cells as determined by the B C A protein assay (Pierce, Rockford, IL). The transfection time-course study included SPLP, SPLP-CPL, and Lipofectin (Gibco B R L , Burlington, ON) and DOPE/DODAC lipoplexes containing pCMVLuc. The lipoplexes were prepared as described earlier. After transfection times of 4, 8, and 24 h the transfection media was removed and in the case of the 4 and 8 h transfections, was replaced with complete media for a total incubation time of 24 h. At 24 h, all cells were lysed and assayed for luciferase activity and protein content (BCA assay), as described above.  SPLP-CPL4, DOPE:DODAC lipoplexes and Lipofectin lipoplexes containing p C M V G F P were prepared as described for pCMVLuc. The transfections were performed as described above at a plasmid D N A dose of 5.0 ug/mL. Following incubation of the samples for 24 and 48 h, the transfection media was removed, the cells were washed, and fresh media was added to the cells.  The cells were then viewed under the Zeiss  fluorescence microscope. The number of cells expressing GFP were counted using a fluorescein filter (Omega Opticals) with the following  specifications: excitation  475±20/dichroic filter 500/emission 535±22.5. The transfection efficiency was expressed as percentage of cells expressing GFP.  92  4.3 R E S U L T S  4.3.1 Cationic P E G lipids can be inserted into preformed SPLP  Previous work has shown that SPLP exhibit lower uptake into cells and much lower transfection potencies than lipoplexes (Mok et al., 1998). It was shown in Chapter 3 that surface-associated cationic P E G lipids (CPL), particularly those containing four charges at the end of the P E G molecule (CPL ; for structure see Figure 4.1 A), can 4  dramatically enhance the uptake of L U V into cells (Fenske et a l , 2000). Furthermore, CPL were shown in Chapter 3 to insert into pre-formed L U V with lipid compositions similar to SPLP employing a straightforward incubation protocol (Fenske et al., 2000). A similar procedure to that used in Chapter 3 was developed for the insertion of C P L into 4  SPLP. SPLP containing pCMVLuc were prepared by the detergent dialysis procedure, described in Chapter 2 from a lipid mixture containing 6 mol% of the cationic lipid N , N dioleoyl-N,N-dimethyl ammonium chloride (DODAC), 84 mol% of the "fusogenic" helper lipid dioleoylphosphatidyethanolamine (DOPE) and 10 mol% of a stabilizing lipid consisting of PEG2000 attached to a ceramide (Cer) anchor (PEG-Cer) (Wheeler et al., 1999). The ceramide anchor of the PEG-Cer contained a C20 acyl chain (PEG-CerC2o) which does not readily exchange out of the SPLP and leads to a highly stable SPLP system (Wheeler et al., 1999). The detergent dialysis procedure leads to a mixture of SPLP containing one plasmid per vesicle, free plasmid and empty vesicles. SPLP were purified by removing free plasmid and empty vesicles by D E A E column chromatography and density centrifugation, respectively, as described elsewhere (Wheeler et al., 1999).  93  B CPU micelles  SPLP Incubation at6CPC for3h  Uninserted CPU micelles SPLP-CPL4 Sepharose CL-4B column Uninserted CPL4 micelles  SPLP-CPL4  Figure 4.1. Production of SPLP-CPL4. A . Structure of dansylated CPL4. CPL4 possesses four positive charges at the end of a PEG3400 molecule attached to a lipid anchor, DSPE. B. Protocol for insertion of CPL4 into preformed SPLP. The SPLP and CPL4 are incubated together at 60°C for 3 hours, and unincorporated CPL4 is removed using Sepharose CL-4B column chromatography. For further details see Materials and Methods.  94  The procedure for post-insertion of C P L into the preformed SPLP is illustrated in 4  Figure 4.IB. Purified SPLP were incubated with C P L (~5 mol%) at 60°C for up to 3 h 4  and then separated from non-incorporated C P L by column chromatography. As shown 4  in Figure 4.2, this resulted in association of up to 80% of the available C P L with the 4  SPLP, which corresponds to 4 mol% of the total lipid in the S P L P - C P L system (i.e., 8 4  mol% in the outer monolayer).  4.3.2 SPLP-CPL4  aggregate following insertion of C P L 4 and de-aggregate following  addition of divalent cations Previous work has shown that L U V containing C P L tend to aggregate and that this aggregation can be inhibited by increasing the ionic strength of the medium (Fenske et al., 2000). It was found that SPLP-CPL were also susceptible to aggregation, and that 4  this aggregation could be reversed by adding NaCl, C a C l or M g C b to the S P L P - C P L 2  4  formulation. This effect is illustrated in Figure 4.3 which shows the effect of the addition of CaCl2 and MgCl2 on aggregation of SPLP-CPL4 as monitored by the change in the standard deviation of the mean diameter of the particles measured by quasi-elastic light scattering (QELS).  For both cations the standard deviation decreases with increasing  cation concentration with optimal de-aggregation occurring above 30 to 40 m M . This behaviour could also be visualized by freeze-fracture electron microscopy. As shown in Figure 4.4A, freeze-fracture micrographs of SPLP reveal small monodisperse particles, whereas SPLP-CPL prepared in the absence of C a C l are highly aggregated (Figure 4  2  4.4B). As shown in Figure 4.4C, the addition of 40 m M CaCb reverses this aggregation to produce monodisperse particles similar to the SPLP preparation.  95  100  0  1  2  3  4  Incubation Time (h)  Figure 4.2. Time course for the insertion of CPL4 into SPLP at 60°C. Dansylated CPL4 (0.3 umol) was added to SPLP composed of 6 umol DOPE:PEG-CerC :DODAC:Rh-PE 20  (83.5:10:6:0.5; mol%) containing 360 pg pCMVLuc in a total volume of 1.5 mL and incubated at 60°C. Aliquots (250 uL) of the mixture were taken at the times indicated and unincorporated  CPL4  was  removed  employing  Sepharose  CL-4B  column  chromatography. CPL4 incorporation was determined as described in Materials and Methods.  96  90  Figure 4.3. Effect of cation concentration on the de-aggregation of SPLP following insertion of CPL4. SPLP were prepared and CPL4 was inserted as described in Materials and Methods. The mean diameter and standard deviation of the mean diameter of the SPLP-CPL4 in the presence of increasing concentrations of C a  2+  (•) and M g  2 +  (•) was  determined by QELS. C a C l or M g C l from 500 m M stock solutions was added to SPLP2  2  CPL4 (180 nmol in 400 uL). The addition of C a  2+  or M g  2 +  results in a more monodisperse  preparation as indicated by a reduction in the standard deviation of the mean diameter at cation concentrations above 30 m M .  97  Figure 4.4. Freeze-fracture electron micrographs of (A) SPLP, (B) SPLP-CPL and (C) 4  SPLP-CPL in the presence of 40 mM CaCl . The SPLP-CPL4 were prepared as 4  2  described in Materials and Methods, and contained 4 mol% CPL4. The bar in plate A corresponds to 200 nm.  98  The sizes of SPLP and SPLP-CPL4 in the presence of CaCh were compared using QELS and freeze-fracture electron microscopy. QELS studies revealed the mean diameter of SPLP and SPLP-CPL4 to be 80 ± 19 nm and 76 ± 15 nm, respectively, whereas the freeze-fracture studies indicated diameters of 68 ± 11 nm and 64 ± 14 nm. These values for SPLP are in close agreement those determined in Chapter 2 (Wheeler et al., 1999).  4.3.3 PEG-CerC content and stability of SPLP-CPL 20  The observation that  CPL4  4  can be inserted to achieve levels as high as 4 mol% of  the total SPLP lipid indicates that the level of C P L 4 in the outer monolayer of the SPLPCPL4  is 8 mol%. Given that the initial concentration of PEG-CerC2o is 10 mol%, this  suggests that the total levels of PEG-lipids in the outer monolayer of the SPLP-CPL4 can approach 18 mol%. These levels are higher than the levels of PEG-lipids that can usually be incorporated into lipid vesicles (Woodle and Lasic, 1992) leading to the possibility that some of the PEG-CerC2o in the outer monolayer exchanged out as  CPL4  was  inserted. This was examined by measuring the ratio of PEG-CerC2o-to-DOPE for the SPLP before and after insertion of C P L 4 by H P L C .  CPL4  was incorporated within SPLP  as described in Materials and Methods. Analysis following removal of non-incorporated material determined that 4 mol% C P L  4  (corresponding to the total SPLP lipid) was  inserted into the SPLP. Prior to insertion of the C P L the PEG-CerC -to-DOPE ratio 4  20  was 0.091, corresponding to a PEG-CerC o content of 7.6 mol%, assuming that the 2  DOPE constituted 84 mol% of the lipid content. Following insertion of the  CPL4  the  PEG-CerC2o-to-DOPE ratio was found to be 0.072, indicating a PEG-CerC2o content of  6.0 mol%. Assuming that all of the PEG-CerC o lost from the SPLP during insertion of 2  99  the C P L 4 is lost from the outer monolayer, this indicates that the PEG-CerC2o content of the outer monolayer decreases from 7.6 mol% to 4.4 mol% during the insertion process. The total PEG-lipid content in the outer monolayer of the SPLP-CPL4 can then be estimated to be 12.4 mol% of the outer monolayer lipid. The stability of SPLP and SPLP-CPL4 following incubation in 50% mouse serum for up to 4 h is illustrated in Figure 4.5. In all cases, the encapsulated plasmid D N A was fully protected from serum degradation. In contrast, essentially complete degradation of the plasmid in lipoplexes was observed within 30 min of incubation in serum (data not shown).  4.3.4 SPLP-CPL4 exhibit enhanced uptake into B H K cells and dramatically  enhanced transfection potency The next set of experiments was aimed at determining the influence of incorporated CPL4 on the uptake of SPLP into B H K cells and the resulting transfection potency of the SPLPCPL4 system. SPLP containing up to 4 mol% CPL4 were prepared in the presence of 40  m M CaCb and were added to B H K cells (final CaCk concentration 8 mM) and incubated for varying times. The cells were then assayed for associated SPLP-CPL4 as indicated in Methods. As shown in Figure 4.6A, uptake of SPLP that contain no C P L is minimal 4  even after 8 h of incubation; however, uptake is dramatically improved for SPLP containing 3 mol% or higher levels of CPL4. For example, SPLP containing 4 mol% CPL  4  exhibit accumulation levels at 8 h that are approximately 50-fold higher than  achieved  for  SPLP  in  the  absence  of  CPL.  This  enhanced  uptake  is  100  Lane: 1  2  3  4  5 6 7  8 9  10  11  12  Figure 4.5. Serum stability of SPLP-CPL4 as assayed by Southern analysis of encapsulated plasmid. SPLP were prepared as indicated in the legend to Figure 2 and 4 mol% of CPL4 inserted using the post-insertion protocol. SPLP-CPL4 containing 5 ug p C M V L u c were incubated in the presence of 50% mouse serum at 37°C for the times indicated, an aliquot of the mixture corresponding to 1 pg of plasmid D N A was removed and plasmid D N A was extracted and subjected to Southern analysis, as described in the Materials and Methods. Lanes 1-4 indicate the behaviour of naked plasmid D N A following 0, 1,2, and 4 h incubation times respectively; Lanes 5-8 indicate the behaviour of plasmid extracted from SPLP following 0, 1,2, and 4 h incubation times; Lanes 9-12 show the behaviour of plasmid D N A extracted from SPLP containing 4 mol% C P L  4  following 0, 1,2, and 4 h incubation times.  101  0  2  4  6  8  Incubation Time (h) Figure 4.6A. Influence of the amount of CPL4 incorporated into SPLP on the uptake of SPLP-CPL4 into B H K cells. Uptake of SPLP containing 0 (•), 2 (•), 3 ( • ) , or 4 ( • ) mol% C P L was investigated; the uptake of DOPE:DODAC lipoplexes (O) is given for 4  comparison. The insertion of CPL4 into SPLP and the preparation of lipoplexes was performed as described in Materials and Methods. The S P L P - C P L media contained 40 4  m M CaCb to prevent aggregation, addition to the B H K cells resulted in dilution of the CaCb concentration to 8 m M . The uptake protocol involved incubation of SPLP-CPL4 (20 u M Total Lipid) with 10 B H K cells in D M E M containing 10% FBS. Following 5  incubation, the cells were lysed and uptake of rhodamine-PE was measured as described in Materials and Methods.  102  visually illustrated in Figure 4.6B, which shows fluorescence micrographs of B H K cells following incubation with rhodamine-labeled SPLP and SPLP-CPL for 4 h. 4  The  transfection  properties  of  SPLP,  SPLP-CPL4  and  lipoplexes  (DODAC/DOPE; 1:1) were examined using the lipoplex transfection protocol. A charge ratio (positive-to-negative) of 1.5 was employed to construct the lipoplexes. 0.5 pg of p C M V L u c was incubated on 10 B H K for 4 h, followed by incubation for a further 20 h. 4  As shown in Figure 4.7, the presence of various amounts of CPL4 resulted in dramatic increases in the transfection potency for the SPLP system.  SPLP-CPL4 containing 4  mol% C P L exhibited luciferase expression levels some 3x10 higher than achieved with 3  4  SPLP.  However, the liposomes, under these conditions, still did not achieve the same  level of expression as the DOPE:DODAC complexes.  4.3.5 C a is required for transfection activity of SPLP-CPL4 2+  Previous work has shown that the transfection potency of SPLP is highly sensitive to the presence of C a , where the presence of ~10 m M C a 2+  2+  enhances transfection  potency several hundred-fold (Lam et al., 2000). It was therefore of interest to determine the influence of C a  2+  on the transfection activity of SPLP-CPL4. SPLP containing 4  mol% CPL4 were incubated with B H K cells for 48 h in the presence of varying amounts of M g C l and CaCb and the luciferase activities then determined. As shown in Figure 2  2_|_  4.8, the transfection activity was almost completely dependent on the presence of Ca in the transfection medium. At the optimum CaCb concentration of 10 m M , SPLP-CPL4 exhibit transfection potencies that were more than 10 times higher than i f MgCL; was 5  present.  103  -  v  v v V  *  J .  ^ v ^  v  Figure 4.6B. Fluorescence micrographs of B H K cells following uptake of SPLP (Panel I) and SPLP containing 4 mol% CPL4 (Panel II) following a 4 hour incubation. The micrographs on the left were taken in the phase contrast mode and those on the right in the (rhodamine) fluorescence mode.  104  iooo H  Figure  4.7. Luciferase expression in B H K cells following transfection by SPLP  containing various amounts of CPL4. SPLP containing 2, 3 and 4 mol% CPL4 were prepared employing the post-insertion process. SPLP, SPLP-CPL4 and D O P E : D O D A C (1:1) lipoplexes containing 5.0 pg/mL pCMVLuc were incubated with 10 B H K cells for 4  a 4 h pulse followed by a 20 h chase (for a complete 24 hour transfection), as described in Materials and Methods. The CaCb concentration in the SPLP-CPL4-containing systems following addition to the B H K cells was 8 mM. After transfection the cells were lysed and the luciferase and B C A assays performed as described in Materials and Methods.  105  Figure 4.8. Influence of Ca  (•) and M g  (•) on the transfection potency of SPLP-  CLP4. SPLP-CPL4 containing 4 mol% C P L were prepared by the post-insertion process 4  as described in Materials and Methods. Increasing concentrations of CaCb or M g C b were added to the SPLP-CPL4 (5.0 pg pCMVLuc/mL), transferred to B H K cells and incubated for 48 h in D M E M containing 10% FBS. The cells were then lysed and the luciferase activity and protein content were measured as described in Materials and Methods.  106  A previous study has shown that Ca has no effect on the cellular uptake of SPLP (Lam et al., 2000). In order to determine whether the SPLP-CPL4 system exhibits similar behaviour, uptake of SPLP-CPL4 into B H K cells was monitored following a 4 h incubation in the presence of M g C l or CaCl . 2  uptake is unaffected by M g  2 +  2  and C a . 2+  As shown in Figure 4.9, SPLP-CPL4  It may be noted that SPLP-CPL4 uptake  decreases as the concentration of divalent cations increases, likely due to the shielding of the negatively charged CPL4 binding sites on the surface of B H K cells.  4.3.6 SPLP-CPL4 exhibit transfection potencies in vitro that are comparable to or  greater than achieved using lipoplexes Figure 4.7 shows that lipoplexes yield ~100-fold higher levels of gene expression than SPLP-CPL4 when applied to B H K cells for a period of 4 hours. Given that SPLP are stable systems, it was considered likely that uptake could continue over extended time periods. Therefore, the transfection levels achieved when SPLP-CPL4 or the lipoplexes were applied to B H K cells were examined using a pulse of 8 and 24 h, followed by chase times of 16 and 0 h, respectively.  Two types of lipoplexes were used, namely  DOPE:DODAC (1:1) lipoplexes (charge ratio 1.5) and lipoplexes obtained using the commercial transfection  reagent Lipofectin,  consisting of D O P E / D O T M A  (1:1)  lipoplexes at a charge ratio of 1.5. As shown in Figure 4.10, the potency of SPLP-CPL4 increases markedly with increased incubation times, suggesting that the rate of uptake of the SPLP-CPL system is still a limiting factor for transfection. After 24 h continuous 4  incubation, transfection levels are comparable to those achieved by Lipofectin or the DOPE/DODAC lipoplexes.  107  Figure 4.9. Effect of Ca  z+  (•) and Mg  I+  (•) on the uptake of S P L P - C P L by B H K cells. 4  S P L P - C P L were prepared with increasing cation concentration as indicated for Figure 8 4  and incubated with B H K cells (-80 u M lipid and -5.0 ug pCMVLuc/mL per well) for 4 h in D M E M containing 10% FBS. The cells were then lysed and the SPLP-CPL4 content (as indicated by the Rh-PE lipid label) and cellular protein measured as described in Materials and Methods.  108  3000  2500 H  Transfection Time (h)  Figure 4.10. Luciferase expression in BHK cells as a function of transfection time for SPLP, SPLP-CPL and lipoplexes. SPLP-CPL4 containing 4 mol% CPL were prepared 4  4  by the post-insertion process. BHK cells in DMEM and 10% FBS were incubated with SPLP, SPLP-CPL4 and lipoplexes (5.0 pg/mL pCMVLuc) employing transfection times of 4, 8 and 24 h and total incubation times of 24 h. The final CaCb concentration following addition of media was 8 mM. The cells were then assayed for luciferase activity and protein content. Luciferase activity following transfection with SPLP-CPL4 (•), SPLP (T), DOPE:DODAC lipoplexes (•), and Lipofectin lipoplexes (•) is plotted as a function of transfection time. Lipoplexes were prepared at a charge ratio of 1.5:1.  109  Further experiments were conducted to determine transfection levels after 24 and 48 h incubations with luciferase activities assayed immediately following the incubation period. As shown in Figure 4.11 A , the activity of Lipofectin (DOPE:DOTMA) lipoplexes leveled off at -2000 ng luciferase per mg of cell protein after 24 h. Similar results were obtained for the DOPE:DODAC lipoplexes (data not shown). In contrast, the activity of the SPLP-CPL formulation continued to increase as the incubation time was increased, 4  achieving luciferase expression levels corresponding to 4000 ng per mg of cell protein at 48 h. This activity is approximately 10 times higher than observed for SPLP (in the 6  absence of Ca ) and almost double the levels that can be achieved by Lipofectin 2+  lipoplexes.  4.3.7 SPLP-CPL4  a r e n o n - t o x i c a n d efficient t r a n s f e c t i o n agents  It is well known that lipoplexes can be toxic to cells. The SPLP-CPL4 contain low levels of cationic lipid and are potentially less toxic than lipoplexes. The toxicities of SPLP-CPL4 and lipoplexes were assayed by determining cell viability following a 24 h and 48 h exposure to levels of SPLP-CPL4 and lipoplexes corresponding to 5.0 ug/mL plasmid, corresponding to total lipid doses approximately 80 u M and 45 u M for SPLPCPL4 and lipoplexes, respectively. As shown in Figure 4.1 IB, SPLP-CPL4 exhibited little toxicity whereas lipoplexes were highly toxic. Cell survival was only 30% after a 48 h incubation with Lipofectin complexes whereas -95% of the cells were viable following a 48 h incubation with SPLP-CPL4. Studies were also conducted to determine the efficiency of transfection as indicated by the proportion of cells transfected by SPLP-CPL4.  The proportion of  110  5000  Transfection T i m e (h)  Figure 4 . 1 1 . A. The transfection potency of SPLP-CPL4 (•) containing 4 mol% C P L  4  and Lipofectin lipoplexes ( • ) following extended transfection times with B H K cells. SPLP-CPL4 and lipoplexes were generated as indicated for Figure 10. B H K cells were transfected in D M E M containing 10% FBS for 24 and 48 h with SPLP-CPL4 and Lipofectin lipoplexes (charge ratio of 1.5:1) containing 5.0 p.g/mL pCMVLuc. Following transfection the luciferase expression levels and cell protein levels were determined in the cell lysate. The luciferase activity was normalized for protein content in the lysate and plotted as a function of transfection time. B . The toxicity of SPLP-CPL4 (•) containing 4 mol% CPL4 and Lipofectin lipoplexes ( • ) as a function of transfection time, as assayed by cell survival based on the protein concentration in the cell lysate.  ill  transfected cells was determined by employing plasmid containing the green fluorescent protein (GFP) gene. GFP expression was detected by fluorescence microscopy.  As  shown in Figure 4.12A and 4.12B, approximately 35% of the cells at 24 h and 50% at 48 h were transfected by SPLP-CPL4, with no apparent cell death. In contrast, Lipofectin complexes exhibit maximum transfection efficiencies of less than 35% and only -50% cell survival after the 24 h transfection period (Figure 4.12C). Similar low transfection efficiencies and high toxicities were also seen with DOPE:DODAC complexes (data not shown).  112  Figure 4.12. Fluorescence and phase contrast micrographs of B H K cells transfected with SPLP-CPL4 and lipoplexes containing a plasmid coding for GFP. Cells were transfected with SPLP-CPL4 for 24 h ( A l , A2) and 48 h ( B l , B2) and with lipofectin for 24 h ( C l , C2). SPLP and lipoplexes were prepared with p C M V G F P as described in Materials and Methods. SPLP-CPL4 containing 4 mol% CPL was prepared by the post-insertion process and contained CaCh, resulting in an 8 m M CaCL. concentration in the transfection medium. B H K cells (10 ) were incubated with SPLP-CPL4 or Lipofectin (5.0 ug/mL) in D M E M containing 10% FBS for the 24 and 48 h transfection times and examined immediately after the transfection period. 5  113  4.4 D I S C U S S I O N  The results of this study demonstrate that the incorporation of C P L into SPLP 4  results in improved uptake into B H K cells and dramatically enhanced transfection potencies of SPLP when C a  2+  is present. There are three points of interest. The first  concerns the chemical composition and structure of the SPLP-CPL4 system and the generality of the post-insertion procedure for modifying the trophism and transfection potency of SPLP. The second concerns the relation between enhanced uptake of SPLP, the presence of C a  2+  and the transfection activities observed. Finally, it is of interest to  compare the properties of the SPLP-CPL4 system with lipoplexes.  These areas are  discussed in turn. The results presented here demonstrate that the cationic P E G lipid CPL4 can be inserted into preformed SPLP employing a simple process involving incubation at 60°C. The ability to insert C P L to levels corresponding to 8 mol% of the total lipid in the SPLP 4  outer monolayer is consistent with results of other workers demonstrating that PEG-PE can be inserted into preformed L U V employing a similar incubation protocol, resulting in systems exhibiting extended circulation lifetimes (Uster et al., 1996). It is also consistent with previous results from this laboratory showing that CPL4 can be inserted into preformed L U V with a lipid composition similar to the SPLP system (Fenske et al., 2000). The total levels of PEG-lipids achieved in the outer monolayer (12.4 mol%) are high given that maximum levels of incorporation of PEG-lipids into L U V are commonly 7-10 mol% (Woodle and Lasic, 1992). However, a number of authors have reported that PEG-lipids can be incorporated into L U V to levels as high as 15 mol% before lytic effects are observed (Hristova et al., 1995; Kenworthy et al., 1995; Edwards et al., 1997).  114  These include cryo-electron microscopy studies that indicate that structural changes (from spheres to discs) are only observed for distearoylphosphatidylcholine (DSPC) liposomes at PEG-PE levels above 12 mol%, with lytic effects observed above 15 mol% (Edwards et al., 1997).  Similarly, X-ray studies indicate that non-bilayer micellar  structures are only observed for PEG-lipid levels above 15 mol% (Kenworthy et al., 1995; Edwards etal., 1997). The tendency for the SPLP-CPL system to aggregate following insertion of the 4  C P L is consistent with previous observations that L U V containing CPL4 also aggregate 4  (Fenske et a l , 2000).  The reason for this aggregation is not currently understood;  however, two general points can be made. First, the interaction is likely due to electrostatic interactions between vesicles given the inhibition of aggregation at higher ionic strengths. Second, the aggregation is not a consequence of the post-insertion process itself as such aggregation is also observed for L U V containing CPL4 where the CPL4 was present in the lipid mixture from which the L U V were formed (Fenske et al., 2000). It is possible that the cationic headgroup interacts with apposed membranes at the level of the phospholipid phosphate group. Alternatively, the aggregation phenomenon may be related to the ability of PEG coatings to adopt a conformation that is able to bind proteins such as streptavidin (Sheth and Leckband, 1997). These and other possibilities are currently under investigation. The second point of discussion concerns the mechanism whereby CPL4 increases the transfection potency of the SPLP system. A number of studies have indicated that the cationic lipids contained in lipoplex systems play a direct role in stimulating uptake into cells (Miller et al, 1998) and that this uptake arises due to the positive charge on the  115  lipoplexes (Van der Woude et al., 1995). It has been suggested that heparin-sulfonated proteoglycans on the cell surface play a primary role in this process (Mislick and Baldeschwieler, 1996; Mounkes et al., 1998).  Enhanced uptake of SPLP following  addition of the CPL4 could be due to similar mechanisms; however, the increase in transfection potency is largely dependent on the additional presence of C a . Previous 2+  work has shown that the presence of C a  2+  results in a maximum increase in SPLP  transfection potency of -600 fold and that this increase results from an ability of C a  2 +  to  assist in destabilizing the endosomal membrane following uptake, rather than from an increase in uptake (Lam et al., 2000). It could therefore be suggested that the observed improvements in transfection potency for SPLP-CPL4 improvements in uptake mediated by the C P L  4  over SPLP  result  from  coupled with enhanced abilities to  destabilize the endosomal membrane due to the presence of C a . In this regard the 2+  transfection potency of SPLP-CPL4 (in the presence of Ca ) is increased by a factor of 2+  ~10 (Fig. 4.7) in comparison to the transfection potency of SPLP in the absence of C a . 4  2+  This could be accounted for by an increase in uptake of SPLP into B H K cells by approximately 50-fold due to the presence of 4 mol% C P L (Fig. 4.6A, 4 h incubation) 4  multiplied by a factor of ~ 600 due to the presence o f C a . 2+  The final area of discussion concerns the advantages of the SPLP-CPL4 system over other non-viral vectors, which include the well-defined modular nature of the SPLPCPL4 system as well as toxicity and potency issues. First, SPLP-CPL4 are small, homogeneous, stable systems containing one plasmid per particle (Wheeler et al., 1999), in contrast with other non-viral systems such as lipoplexes, which are large, heterogeneous, unstable systems containing ill-defined numbers of plasmids per particle.  116  A n important point is that SPLP are basic components of more sophisticated systems, such as SPLP-CPL4, that can be constructed in a modular fashion. For example, postinsertion of PEG-lipids containing specific targeting ligands in place of the cationic groups of C P L should result in SPLP that are specifically targeted to particular cells and tissues.  With regard to toxicity, SPLP-CPL4 are markedly less toxic to B H K cells in  tissue culture than are lipoplexes. This is presumably related to the low proportions of cationic lipid contained in SPLP as compared to lipoplexes. Finally, SPLP without C P L  4  2_|_  or Ca  exhibit transfection properties in vivo following systemic administration that are  already superior to the transfection properties of plasmid DNA-cationic lipid complexes or naked plasmid D N A (Tarn et al., 2000). The results presented here suggest that further gains can be expected through the use of ligands that encourage SPLP uptake into cells and methods leading to local increases in C a  2+  concentrations.  In summary the results presented here demonstrate that a cationic P E G lipid can be post-inserted into SPLP, resulting in well-defined SPLP-CPL4 systems that exhibit improved uptake into B H K cells in vitro. In the presence of C a  SPLP- CPL4 systems  2+  give rise to transfection potencies that are increased by up to 10 -fold as compared to 6  SPLP in the absence of C a . These results indicate that the SPLP system is a non-toxic, 2+  highly transfection potent entity following uptake into cells and suggests that SPLP targeted to cell-surface ligands that undergo endocytosis should lead to significant enhancement of transfection potency in vivo.  117  CHAPTER 5 SUMMARY AND FUTURE DIRECTION  5.1 SUMMARY OF RESULTS The results presented in this thesis detail the development of a potent, non-viral liposomal gene delivery system with the potential for systemic gene therapy. In Chapter 2, a method for producing stabilized plasmid lipid particles (SPLP), is developed. It is shown that SPLP are small, stable, well-defined systems containing one plasmid per vesicle that fully protect encapsulated plasmid from serum nucleases. Other workers have shown that SPLP exhibit long circulation lifetimes in vivo and accumulate at disease sites such as tumour sites (Zhang et al., 1999; Monck et al., 2000; Tarn et al., 2000). However, the transfection potency of these systems at distal tumour sites in vivo, while superior to other systemic gene delivery systems, is low. A reason for this is that SPLP have a P E G coating and are not highly charged, both of which are factors that reduce cell association and uptake. In order to demonstrate that SPLP can be potent transfection agents, methods to enhance cell uptake have been developed. Due to the demanding formulation procedure for producing SPLP, methods of enhancing cell uptake that can be used on preformed SPLP are required. In Chapter 3 it is shown that cationic P E G lipids (CPL) can be inserted into preformed L U V and that L U V containing CPL with four positive charges give rise to improved uptake into cells. These C P L possess positive charges at the terminus of a poly(ethylene glycol) (PEG) molecule attached to a lipid anchor, DSPE. The conditions for post-insertion into preformed L U V of C P L possessing different  118  numbers of positive charges at the distal end of the P E G moiety were determined and uptake into B H K cells characterized. It is shown that L U V containing 4 mol% C P L  4  exhibit particularly efficient uptake. In Chapter 4 it is shown that CPL4 can be post-inserted into preformed SPLP using the insertion procedure for L U V developed in Chapter 3. These particles were shown to be as serum stable as SPLP and of the same mean diameter, but exhibited markedly improved uptake into B H K cells, with uptake levels up to 50-fold higher than were achieved for SPLP in the absence of CPL4. In turn, this resulted in dramatically improved transfection potencies in vitro. In particular, in the presence of C a , it is shown 2+  that the transfection potency of SPLP-CPL4 can be up to 10 higher than SPLP. SPLP6  C P L particles were shown to be non-toxic compared to lipoplexes and highly efficient in 4  terms of the percentage of available cells transfected. These results demonstrate that SPLP can be highly potent transfection agents as long as factors giving rise to sufficient cell uptake and adequate C a  2+  levels are present.  119  5.2 F U T U R E D I R E C T I O N S The next step is to develop a version of SPLP that gives rise to improved levels of transfection in vivo following i.v. administration. The SPLP-CPL4 system developed in Chapter 4 has the advantages of high transfection potencies in vitro, but is not likely to give rise to improved transfection levels in vivo compared to the basic SPLP system. This is because the presence of the C P L 4 on the end of a PEG3450 tether extends beyond the PEG2000  cloud surrounding the SPLP, giving rise to interactions with serum proteins and  macrophages and associated rapid clearance from the circulation. There are a number of ways in which this problem could be circumvented. The first approach would be to develop a version of  CPL4  with a shorter PEG tether such as P E G 1000 and employ SPLP  containing PEG-Ceramides that have a shorter residence time on the SPLP than the P E G CerC2o-  This may allow the CPL4 to be "hidden" in the P E G cloud long enough for the  SPLP to accumulate at tumour sites and, after accumulation, dissociation of the P E G Ceramides would reveal the CPL4, giving rise to improved uptake into tumour cells. A second approach could involve the post-insertion of PEG-lipids containing specific targeting ligands on the end of the P E G (as opposed to positive charge) that bind to target cells, thus triggering accumulation. A remaining problem concerns the requirement for Ca , which exerts a remarkable effect on the transfection potency of both SPLP and SPLP-CPL4. It is clear that ways must be found to transport adequate amounts of Ca  along with the SPLP  system to give rise to the very significant benefits observed here. The studies of Lam et al. (2000) indicate that encapsulation of C a  2+  in SPLP does not give rise to improved  transfection, and thus the challenge is to develop ways to transport transfection-  120  competent amounts of Ca  associated with the exterior of the SPLP-CPL system. It is  possible that inclusion of Ca -chelating agents on the SPLP exterior may allow this 2+  problem to be approached.  121  REFERENCES  Alino S.F., Bobadilla, M . , Garcia-Sanz, M . , Lejarreta, M . , Unda, F., and Hilario, E. (1993) In vivo delivery of human alpha 1-antitrypsin gene to mouse hepatocytes by liposomes. Biochem. Biophys. Res. Comm. 192, 174-181. Allen,T.M., and Chonn, A . (1987) Large unilamellar liposomes with low uptake into the reticuloendothelial system. FEBS Lett. 223, 42-46. Allen, T . M . , Austin, G.A., Chonn, A . , Lin, L . , and Lee, K . C . (1991a) Uptake of liposomes by cultured mouse bone marrow macrophages: influence of liposome composition and size, Biochim. Biophys. Acta 1061 56-64. Allen, T . M . , Hansen, C , Martin, F., Redemann, C , and Yau-Young A . (1991b) Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim. Biophys. Acta 1066, 29-36. Alio, J.C., Midoux, P., Merten, M . , Souil, E., Lipecka, J., Figarella, C , Monsigny, M . , Briand, P., ans Fajac, I. (2000) Efficient gene transfer into human normal and cystic fibrosis tracheal gland serous cells with synthetic vectors. Amer. J. Resp. Cell Molec. Biol. 22, 166-175. Andersen, J.K., Garber, D.A., Meaney, C.A., and Breakefield, X.O. (1992) Gene transfer into mammalian central nervous system using Herpes virus vectors: extended expression of bacterial lacZ in neurons using the neuron-specific enolase promoter. Hum. Gene Ther.3, 487-499. Ausubel F. et al. (eds). (1995) Preparation and analysis of D N A . In: Short Protocols in Molecular Biology, 3 edn. John Wiley and Sons: New York,, pp A1-A58. rd  Bangham, A.D., Standish, M . M . , and Watkins, J.C. (1965) Diffusion of univalent ions across the lamellae of swollen phospholipids. J. Mol. Biol. 13, 238-252. Barenholtz, Y . , Amslem, S., and Lichtenberg, D. (1979) A new method for preparation of phospholipid vesicles (liposomes) by French press. FEBS Lett. 99, 210-214. Baru, M . , Axelrod, J.H., and Nur, I. (1995) Liposome-encapsulated DNA-mediated gene transfer and synthesis of human factor IX in mice. Gene 161, 143-150. Berkner, K . L . (1988) Development of adenovirus vectors for the expression of heterologous genes. Biotechniques 6, 616-629. Bligh, E.G., and Dyer, W.J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917.  122  Boman, N . L . , Mayer, L . D . , and Cullis, P.R. (1993) Optimization of the retention properties of vincristine in liposomal systems. Biochim. Biophys. Acta 1152, 253-258. Cai, D.W., Mukhopadhyay, T., Liu, Y . , Fujiwara, T., and Roth, J.A. (1993) Stable expression of the wild-type p53 gene in human lung cancer cells after retrovirus-mediated gene transfer. Human Gene Ther. 4, 617-624. Capecchi, M.R. (1980) High efficiency transformation by direct microinjection of D N A into cultured mammalian cells. Cell 22, 479-488. Chen, T., Wong, K.F., Fenske, D.B., Palmer, L.R., Cullis, P.R. (2000) Fluorescentlabeled poly(ethylene glycol) lipid conjugates with distal cationic headgroups. Bioconj. Chem. 11, 433-437. Chernomordik, L . V . , Kozlov, M . M . , Melikyan, G.B., Abidor, I.G., Markin, V.S., and Chizmadzhev, Y . A . (1985) The shape of lipid molecules and monolayer membrane fusion. Biochim. Biophys. Acta 812, 643-655. Chernomordik, L . V . , and Zimmerberg, J. (1995) Bending membranes to the task: structural intermediates in bilayer fusion. Curr. Opin. Struct. Biol. 5, 541-547. Choate, K . A . , and Khavari, P.A. (1997) Direct cutaneous gene delivery in a human genetic skin disease. Human Gene Ther. 8, 1659-1665. Chonn, A . , Semple, S . C , and Cullis, P.R. (1991) Separation of large unilamellar liposomes from blood components by a spin column procedure: towards identifying plasma proteins which mediate liposome clearance in vivo. Biochim. Biophys. Acta 1070, 215-22. Chonn, A . , Semple, S . C , Cullis, P.R. (1992) Association of blood proteins with large unilamellar liposomes in vivo. Relation to circulation lifetimes. J. Biol. Chem. 267, 18759-18765. Chonn, A., and Cullis, P.R. (1995) Recent advances in liposome drug delivery systems. Curr. Opin. Biotech. 6, 698-708. Coleman, D.L. (1986) Regulation of macrophage phagocytosis. Eur. J. Clin. Microbiol. 5, 1-5. Conrad, C.K., Allen, S.S., Afione, S.A., Reynolds, T . C , Beck, S.E., Feemaki, M . , Barrazzaortiz, X . , Adams, R., Askin, F.B., Carter, B.J., Guggino, W.B., and Flotte, T.R. (1996) Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung. Gene Ther. 3, 658-668. Constantini, L . C . , Bakowska, J . C , Breakefield, X . O . , and Isacson, O. (2000) Gene therapy in the CNS. Gene Ther. 7, 93-109.  123  Cudd, A . , and Nicolau, C. (1985) Intracellular fate of liposome-encapsulated D N A in mouse liver. Analysis using electron microscope autoradiography and subcellular fractionation. Biochim. Biophys. Acta 845, 477-491. Cullis, P.R., and deKruijff, B . (1978) The polymorphic phase behaviour of phosphatidylethanolamines of natural and synthetic origin. Biochim. Biophys. Acta 513, 31-42. Cullis, P.R., and deKruijff, B . (1979) Lipid polymorphism and the roles of lipids in membranes. Biochim. Biophys. Acta 559, 399-420. Cullis, P.R., Hope, M.J., and Tilcock, C.P.S. (1986) Lipid polymorphism and the roles of lipids in membranes. Chem. Phys. Lipids 40,127-144. Cullis, P.R., and Hope, M J . (1991) "Physical properties and functional roles ofof lipids in membranes.", in Biochemistry of Lipids, Lipoproteins, and Membranes (Vance, D.E. and Vance, J., Eds.), pp. 1-41, Elsevier Science Publishers B . V . , Amsterdam, The Netherlands. Dencher, N . A . , and Heyn, M.P. (1978) Formation and properties of bacteriorhodopsin monomers in the non-ionic detergents octyl-d-D-glucose and Triton X-100. FEBS Lett. 96, 322-326. Dubinett, S.M., Miller, P.W., Sharma, S., and Batra, R.K. (1999) Gene therapy for lung cancer. Hematol./ Oncol. Clin. North Am. 12, 569-594. Dunckley, M . G . , and Dickson, G. (1994) Retroviral-mediated gene transfer and Duchenne muscular dystrophy. In Gene Therapeutics: Methods and applications of direct gene transfer. J.A. Wolff, editor. Birkhauser Boston, Cambridge, M A , 391-410. Edwards, K . , Johnsson, M . , Karlsson, G , and Silvander, M . (1997) Effect of polyethyleneglycol-phospholipids on aggregate structure in preparations of small unilamellar liposomes. Biophys. J. 73, 258-266. Eibl, H . , and Wooley, P. (1979) Electrostatic interactions at charged membranes. Hydrogen bonds in lipid membrane surfaces. Biophys. Chem. 10, 261-271. Evans, C.H., and Robbins, P.D. (2000) Gene therapy for inflammatory diseases - basic concepts. In Gene therapy in inflammatory diseases. C H . Evans and P.D. Robbins, editors. Birkhauser Boston, Cambridge, M A , 1-11. Farhood, H . , Serbina, N . , and Huang, L . (1995) The role of phosphatidylethanolamine in cationic liposome mediated gene transfer. Biophys. Acta 1235, 289-295.  dioleoylBiochim.  Feigner, P.L., Gadek, T.R., Holm, M . , Roman, R., Chan, H.W., Wenz, M . , Northrop, J.P., Ringold, G . M . , and Danielsen, M . (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA 84, 7413-7417. 124  Feigner, J.H., Kumar, R., Sridhar, C.N., Wheeler, C.J., Tsai, Y.J., Border, R., Ramsey, P., Martin, M , and Feigner, P.L. (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. 269, 2550-2561. Fenske, D.B., Monck, M.A., Hope, M.J., and Cullis, P.R. (1995) The functional roles of lipids in biological membranes. Biomembranes 1, 1-28. Fenske, D.B., Palmer, L.R., Chen, T., and Cullis, P.R. (2000) Cationic PEG-lipids incorporated into pre-formed vesicles enhance binding and uptake to B H K cells. Biochim. Biophys. Acta (submitted). Fiske, C.H., and Subbarow, Y. (1925) The colorimetric determination of phosphorous. J. Biol. Chem. 66, 375-400. Freytag JW. (1985) Large unilamellar lipid vesicles for use in therapeutic and diagnostic medicine. J. Microencap. 2, 31-38. Fraley,R.T., Fornari, C.S., and Kaplan, S. (1979) Entrapment of a bacterial plasmid in phospholipid vesicles: Potential for gene therapy. Proc. Natl. Acad. Sci. USA 76, 33483352. Fraley, R., Subramani, S., Berg, P., and Papahadjopoulos, D. (1980) Introduction of liposome-encapsulated SV40 D N A into cells. J. Biol. Chem. 255, 10431-10435. Furth, A . J . (1980) Removing unbound detergent from hydrophobic proteins. Anal. Biochem. 109, 207-215. Gabizon, A . , and Papahadjopoulos D. (1988) Liposome formulations with prolonged circulation time in blood and enhanced uptake by tumors. Proc. Natl. Acad. Sci. USA 85, 6949-6953. Gabizon, A . , Price, D . C , Huberty, J., Brescalier, R.S., and Papahadjopoulos, D. (1990) Effect of liposome composition and other factors on the targeting of liposomes to experimental tumors: biodistribution and imaging studies. Cancer Res. 50, 6371-6378. Gabizon, A . A . (1992) Selective tumor localization and improved therapeutic index of anthracyclines encapsulated in long-circulating liposomes. Cancer Res. 52, 891-896. Gabizon, A . , Horowitz, A.T., Goren, D., Tzemach, D., Mandelbaum-Shavit, F., Qazen, M . M . , and Zalipsky, S. (1999) Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)-grafted liposomes: In vitro studies. Bioconj. Chem. 10, 289-298. Gao, X . , and Huang, L . (1991) A novel cationic liposome reagent for efficient transfection and mammalian cells. Biochem. Biophys. Res. Commun. 179, 280-285.  125  Ghazizadeh, S., Harrington, R., and Taichman, L.B. (1999) In vivo transduction of mouse epidermis with recombinant retroviral vectors: implications for cutaneous gene therapy. Gene Ther. 6, 1267-1275. Glorioso, J.C., DeLuca, N.A., Goins, W.F., and Fink, D.J. (1994) Development of Herpes simplex virus vectors for gene transfer to the central nervous system. In Gene Therapeutics: Methods and applications of direct gene transfer. J.A. Wolff, editor. Birkhauser Boston, Cambridge, M A , 281-302. Graham, F.L., and van der Eb, A.J. (1973) A new technique for the assay of infectivity of human adenovirus 5 D N A . Virology 52, 456-467. Gruner, S.M., Cullis, P.RR., Hope, M.J., and Tilcock, C.P.S. (1985) Lipid polymorphism: the molecular basis of nonbilayer phases. Ann. Rev. Biophys. Biophys. Chem. 14, 211238. Hafez, I.M., and Cullis, P.R. (2000) On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids. Biophys. J. (submitted). Hansen, C.B., Kao, G.Y., Moase, E.H., Zalipsky, S., and Allen, T . M . (1995) Attachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of coupling procedures Biochim. Biophys. Acta 1239, 133-144. Harasym, T.O., Tardi, P., Longman, S.A., Ansell, S.M., Bally, M . B . , Cullis, P.R., and Choi,L.S.L.(1995) Poly(ethyleneglycol)-modified phospholipids prevent aggregation during covalent conjugation of proteins to liposomes. Bioconj. Chem. 6, 187-194. Harding, J.A., Engbers, C M . , Newman, M.S., Goldstein, N.I., and Zalipsky S. (1997) Immunogenicity and pharmacokinetic attributes of poly(ethylene glycol)-grafted immunoliposomes. Biochim.Biophys. Acta 1327, 181-92, Hoekstra, D., and Kok J.W. (1989) Entry mechanisms of enveloped viruses. Implications for fusion of intracellular membranes. Biosci. Rep. 9, 273-305. Hofland, H.E., Nagy, D., Liu, J.J., Spratt, K . , Lee, Y . L . , Danos, O., and Sullivan S.M. (1997) In vivo gene transfer by intravenous administration of stable cationic lipid/DNA complex. Pharmaceutical Res. 14, 742-749. Holladay, S.R., Yang, Z., Kennedy, M.D.,. Leamon, C P , Lee, R.J., Jayamani, M . , Mason, Low P.S. (1999) Riboflavin-mediated delivery of a macromolecule into cultured human cells. Biochim. Biophys. Acta 1426, 195-204. Holland, J.W., Hui, C , Cullis, P.R., and Madden, T.D. (1996a) Poly(ethyleneglycol)lipid conjugates regulate the calcium-induced fusion of liposomes composed of phosphatidylethanolamine and phosphatidylserine. Biochemistry 35, 2618-2624.  126  Holland, J.W., Cullis, P.R., Madden, T.D. (1996b) Poly(ethyleneglycol)-lipid conjugates promote bilayer formation in mixtures of non-bilayer forming lipids. Biochemistry 35, 2610-2617. Hristova, K., Kenworthy, A., and Mcintosh, T.J. (1995) Effect of bilayer composition on the phase behavior of liposomal suspensions containing poly(ethylene glycol)-lipids. Macromolecules 28, 7693-7699. Huang, C.-H. (1969) Studies of phosphatidylcholine vesicles. Formation and.physical characteristics. Biochemistry 8, 344-352. Huang, L . , and L i , S. (1997) Liposomal gene delivery: a complex package. Nature Biotech. 15, 620-621. Ibanez, M . , Gariglio, P., Chavez, P., Santiago, R., Wong, C , and Baeza, I. (1997) Spermidine-condensed D N A and cone-shaped lipids improve delivery and expression of exogenous D N A transfer by liposomes. Biochem. Cell. Biol. 74, 633-643. Ishida, T., Iden, D . M . , and Allen, T.M. (1999) A combinatorial approach to producing sterically stabilized (Stealth) immunoliposome drugs. FEBS Lett. 460, 129-133. Israelachvili, J.N., Marcelja, S., and Horn, R.G. (1980) Physical principles of membrane organization. Q. Rev. Biophys. 13, 121-200. Jarnagin, W.R., Debs, R.J., Wang, S.S., and Bissell, D . M . (1992) Cationic lipid-mediated transfection of liver cells in primary culture. Nucl. Acids Res. 20, 4205-4211. Jay, D.G., and Gilbert, W. (1987) Basic protein enhances the incorporation of D N A into lipid vesicles: Model for the formation of primordial cells. Proc. Natl. Acad. Sci. USA 84, 1978-1980. Jiao, S., Cheng, L., Wolff, J.A., and Yang, N.-S. (1993) Particle bombardment-mediated gene transfer and expression in rat brain tissues. Bio/Technology 11, 497-502. Juliano, R.L., and Stamp, D. (1975) The effect of particle size and charge on the clearance rate of liposomes and liposome encapsulated drugs. Biochem. Biophys. Res. Commun. 63, 651-658. Kao, G.Y., Change, L.J., and Allen, T.M. (1996) Use of targeted cationic liposomes in enhanced D N A delivery to cancer cells. Cancer Gene Ther. 3, 250-256. Kenworthy, A . K . , Simon, S.A., and Mcintosh, T.J. (1995) Structure and phase behavior of lipid suspensions containing phospholipids with covalently attached poly(ethylene glycol). Biophys. J. 68, 1903-1920.  127  King, G.I., Jacobs, R.E., White, S.H. (1985) Hexane dissolved in dioleoyllecithin bilayers has a partial molar volume of approximately zero. Biochemistry 24, 4637-4645. Kirby, C , Clarke, J., and Gregoriadis, G. (1980) Effect of cholesterol content of small unilamellar liposomes on their stability in vivo and in vitro. Biochem J. 186, 591-598. Klimuk, S.K., Semple, S.C., Nahirney, P.N., Mullen, M . C , Bennett, C.F., Scherrer, P., and Hope, M.J. (2000) Enhanced anti-inflammatory activity of a liposomal intercellular adhesion molecule-1 antisense oligodeoxynucleotide in an acute model of contact hypersensitivity. J. Pharmacol. Exp. Ther. 292, 480-488. Knoell, D.L., and Yiu, I.M. (1998) Human gene therapy for hereditary diseases: a review of trials. Amer. J. Health-Sys. Pharm. 55, 899-904. Kohn, S., Nagy, J.A., Dvorak, H.F., and Dvorak, A . M . (1992) Pathways of macromolecular tracer transport across venules and small veins. Structural basis for the hyperpermeability of tumor blood vessels. Lab. Invet. 67, 596-607. Kruse, C A , Lamb, C , Hogan, S., Smiley, W.R., Kleinschmidt-DeMasters, B.K., and Burrows, F.J. (2000) Purified herpes simplex virus thymidine kinase retroviral particles. II. Influence of clinical parameters and bystander killing mechanisms. Cancer Gene Ther. 7, 118-127. Kunitake, K . , and Okahata, Y . (1977) A totally synthetic bilayer membrane. J. Amer. Chem. Soc. 99,3860-3861. Kwong, Y . L . , Chen, S.H., Kosai, K., Finegold, M.J., and Woo, S.L. (1996) Adenoviralmediated suicide gene therapy for hepatic metastases of breast cancer. Cancer Gene Ther. 3, 339-344. Lam, A.M.I., Palmer, L.R., and Cullis, P.R. (2000) Calcium dramatically enhances the transfection potency of stabilized plasmid-lipid particles. Gene Ther. (submitted). Lasch, J., Berdichevsky, V.R., Torchilin, V.P., Koelsch, R., and Kretschmer, K . (1983) A method to measure critical detergent parameters. Preparation of liposomes. Anal. Biochem. 133, 486-491. Lasic, D.D. (1994) Sterically stabilized vesicles. Agnew. Chem. Int. Ed. Engl. 33, 16851698. Lee, K.D., Hong, K., and Papahadjopoulos, D. (1992) Recognition of liposomes by cells: in vitro binding and endocytosis mediated by specific lipid headgroups and surface charge density. Biochim. Biophys. Acta 1103, 185-197.  128  Lee, K.D., Nir, S., and Papahadjopoulos D. (1993) Quantitative analysis of liposome-cell interactions in vitro: rate constants of binding and endocytosis with suspension and adherent J774 cells and human monocytes. Biochemistry 32, 889-899. L i , S., and Huang, L . (1997) In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes. Gene Ther. 4, 891-900. L i , S., Tseng, W.C., Stolz, D.B., Wu, S.P., Watkins, S . C , and Huang L. (1999) Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum: implications for intravenous lipofection. Gene Ther. 6, 585-594. Lim, H.J., Masin D., Mcintosh, N.L., Madden, T.D., and Bally, M . B . (2000) Role of drug release and liposome-mediated drug delivery in governing the therapeutic activity of liposomal mitoxantrone used to treat human A431 and LSI80 solid tumors. J. Pharm. Exp. Ther. 292, 337-345. Lindblom, G., and Rilfors, L . (1989) Cubic phases and isotropic structures formed by membrane lipids - possible biological relevance. Biochim. Biophys. Acta 988, 221-256. Lindman, B., and Wennerstrom, H . (1980) Micelles. Amphiphile aggregation in aqueous solution. Top. Curr. Chem. 87, 1-83. Liu, D., and Huang L . (1989) Role of cholesterol in the stability of pH-sensitive, large unilamellar liposomes prepared by the detergent-dialysis method. Biochim. Biophys. Acta 981, 254-260. Losordo, D.W., Vale, P.R., Symes, J.F., Dunnington, C.H., Esakof, D.D., Maysky, M . , Ashare, A . B . , Lathi, K., and Isner, J.M. (1998) Gene therapy for myocardial angiogenesis - Initial clinical results with direct myocardial injection of phVEGF(165) as sole therapy for myocardial ischemia. Circulation 98, 2800-2804. Lurquin, P.F. (1979) Entrapment of plasmid D N A by liposomes and their interactions with plant protoplasts. Nucl. Acids Res. 6, 3773-3784. Madden, T.D., Janoff, A.S., and Cullis, P.R. (1990a) Incorporation of Amphotericin B into large unilamellar vesicles composed of phosphatidylcholine and phosphatidyl glycerol. Chem. Phys. Lipids 52, 189-198. Madden, T.D., Harrigan, P.R., Tai, L . C , Bally, M.B., Mayer, L.D., Redelmeier, T.E., Loughrey, H . C , Tilcock, C P . , Reinish, L.W., Cullis, P.R. (1990b) The accumulation of drugs within large unilamellar vesicles exhibiting a proton gradient: a survey. Chem. Phys. Lipids 53, 37-46. Markin, V.S., Kozlov, M . M . , and Borovjagin, V . L . (1984) On the theory of membrane fusion. The stalk mechanism. Gen. Physiol. Biophys. 5, 361-377.  129  Marsh, D. (1990) In C R C Handbook of Lipid Bilayers, CRC Press Inc., Boca Raton, F L , pp.230-233. Mayer, L.D., Hope, M.J., and Cullis, P.R. (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim. Biophys. Acta 858, 161-168. Mayer, L.D., Tai, L.C., Bally, M . B . Mitilenes, G.N., Ginsberg, R.S., and Cullis, P.R. (1990) Characterization of liposomal systems containing doxorubicin entrapped in response to pH gradients. Biochim. Biophys. Acta 1025, 143-151. McLaughlin, S.K., Collis, P., Hermonat, P.L., and Muzyczka, N . (1988) Adenoassociated virus general transduction vectors:analysis of proviral structures. J. Virol. 62, 1963-1973. Meyer, O., Kirpotin, D., Hong, K . , Sternberg, B., Park, J.W., Woodle, M . C , and Papahadjopoulos, D. (1998) Cationic liposomes coated with polyethylene glycol as carriers for oligonucleotides. J. Biol. Chem. 273, 15621-15627. Milas, M . , Y u , D.H., Lang, A.Q., Ge,T., Feig, B., El-Naggar, A . K . , and Pollock, R.E. (2000) Adenovirus-mediated p53 gene therapy inhibits human sarcoma tumorigenicity. Cancer Gene Ther. 7, 422-429. Miller, A . D . (1990) Retrovirus packaging cells. Hum. Gene Ther. 1, 5-14. Miller, C.R., Bondurant, B., McLean, S.D., McGovern, K . A . , and O'Brien, D.F. (1998) Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes. Biochemistry 37, 1287512883. Mimms, L.T., Zampighi, G., Nozaki, Y . , Tanford, C , and Reynolds, J.A. (1981) Phospholipid vesicle formation and transmembrane protein incorporation using octylglycoside. Biochemistry 20, 833-840. Mislick, K . A . , and Baldeschwieler, J.D. (1996) Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc. Natl. Acad. Sci. USA 93, 12349-12354. Moghimi, S.M., and Patel, H . M . (1989) Serum opsonins and phagocytosis of saturated and unsaturated phospholipid liposomes. Biochim. Biophys. Acta 984, 384-387. Mok, K . W . C , Lam, A.M.I., and Cullis, P.R. (1999) Stabilized plasmid-lipid particles: factors influencing plasmid entrapment and transfection properties. Biochim. Biophys. ActaU\9, 137-150. Monck, M . A . , Mori, A . , Lee, D., Tarn, P., Wheeler, J.J., Cullis, P.R., and Scherrer, P. (2000) Stabilized plasmid-lipid particles: pharmokinetics and plasmid delivery to distal tumors following intravenous injection. J. Drug Targ. 7, 439-452.  130  Monnard, P.-A., Oberholzer, T., and Luisi, P. (1997) Entrapment of nucleic acids in liposomes. Biochim. Biophys. Acta 1329, 39-50. Mounkes, L . C . , Zhong, W., Cipres-Palacin, G., Heath, T.D., and Debs R.J. (1998) Proteoglycans mediate cationic liposome-DNA complex-based gene delivery in vitro and in vivo. J. Biol. Chem. 273, 26164-26170. Muzyczka, N . (1992) Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr. Top. Microbiol. Immunol. 158, 97-129. Nakanishi, M . , Uchida, T., Sugawa, H . , Ishiura, M . , and Okada, Y . (1985) Efficient introduction of contents of liposomes into cells using H V J (Sendai virus). Exper. Cell. Res. 159, 399-409. Nanney, L.B., Paulsen, S., Davidson, M.K., Cardwell, N.L., Whitsitt, J.S., and Davidson, J.M. (2000) Boosting epidermal growth factor receptor expression by gene gun transfection stimulatesepidermal growth in vivo. Wound Repair Regen. 8, 117-127. Neumann, E., Schaefer-Ridder, M . , Wang, Y . , and Hofschneider, P.H. (1982) Gene transfer into mouse glyoma cells by electroporation in high electric fields. EMBO J. 1, 841-845. Nicolau, C , and Rottem, S. (1982) Expression of a (3-lactamase activity in Mycoplasma carpicolum transfected with the liposome-encapsulated E. coli pBR32 plasmid. Biochem. Biophys. Res. Commun. 108, 982-986. Ollivon, M . , Eidelman, O., Blumenthal, R., and Walter, A . (1988) Micelle-vesicle transition of egg phosphatidylcholine and octylglucoside. Biochemistry; 27: 1695-1703. Olson F., Hunt C.A., Szoka F.C., Vail, W.J., and Papahadjopoulos D. (1979) Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochim. Biophys. Acta 557, 9-23. Papahadjopoulos, D., Jacobson, K . , Nir, S., and Isac, T. (1973) Phase transitions in phospholipid vesicles. Fluorescence polarization and permeability measurements concerning the effect of temperature and cholesterol. Biochim. Biophys. Acta 311, 330348. Pendri, A., Martinez, A., Xia, J., Shorr, R.G., and Greenwald, R.B. (1995) Poly(ethylene glycol) fluorescent linkers. Bioconj. Chem. 6, 596-598. Proffitt, R.T., Williams, L.E., Presant, C.A., Tin, G.W., Uliana, J.A., Gamble, R.C., and Baldeschwieler, J.D. (1983) Liposomal blockade of the reticuloendothelial system: Improved tumor imaging with small unilamellar vesicles. Science 220, 502-505.  131  Putnam, D . A . (1996) Antisense strategies and therapeutic applications. Am. J. HealthSyst. Pharm. 53, 151-160. Puyal, C , Milhaud, P., Bienvenue, A., and Philippot J.R. (1995) A new cationic liposome encapsulating genetic material. A potential delivery system for polynucleotides. Eur. J. Biochem. 228, 697-703. Reddy, J.A., and Low, P.S. (1998) Folate-mediated targeting of therapeutic and imaging agents to cancers. Crit. Rev. Ther. Drug Carrier Syst. 15, 587-627. Reimer, D.L., Zhang, Y.-P., Kong, S., Wheeler, J.J., Graham, R.W., and Bally, M . B . (1995)Formation of novel hydrophobic complexes between cationic lipids and plasmid D N A . Biochemistry; 34, 12877-12883. Rose, J.K., Buonocore, L . , and Whitt, M . A . (1991) A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. Biotechniques 10, 520-525. Roth, J.A., and Cristiano, R.J. (1997) Gene therapy for cancer: what have we done and where are we going. J. Natl. Cancer Inst. 89, 21-39. Sambrook, J., Fritsch, E.F., and Maniatis (1989) Extraction and purification of plasmid D N A . In Molecular Cloning: Alaboratory Manual, Vol. 1. C. Nolan, editor. Cold Spring Harbor Laboratory Press, Plainview, New York. pp. 1.21-1.52, 5.28-5.32, and 6.3-6.19. Schulze, U . , Schmidt, H.-W., and Safinya, C R . (1999) Synthesis of novel cationic poly(ethylene glycol) containing lipids. Bioconjugate Chem. 10, 548-552. Seddon, J.M. (1990) Structure of the inverted hexagonal (Hn) phase, and non-lamellar phase transitions of lipids. Biochim. Biophys. Acta 1031, 1-69. Senior, J.H. (1987) Fate and behavior of liposomes in vivo: a review of controlling factors. CRC Crit. Rev. Ther. Drug Carrier Syst. 3, 123-193. Senior, J.H., Trimble, K.R., and Maskiewicz. R. (1991) Interaction of positively-charged liposomes with blood: implications for their application in vivo. Biochim. Biophys. Acta 1070, 173-179. Sheth, S.R., and Leckband, D. (1997) Measurements of attractive forces between proteins and end-grafted polyethylene glycol) chains. Proc. Natl. Acad. Sci. USA 94, 8399-8404. Siegel, D.P. (1993) Energetics of intermediates in membrane fusion: comparison of stalk and inverted micellar intermediate mechanisms. Biophys. J. 65, 2124-2140. Siegel, D.P., and Epand, R . M . (1997) The mechanism of lamellar-to-inverted hexagonal phase transitions in phosphatidylethanolamine: implications for membrane fusion mechanisms. Biophys. J. 73, 3089-3111.  132  Silverman, B.A., Carney, D.F., Johnston, C.A., Vanguri, P., and Shin, M X . (1984) Isolation of membrane attack complex to complement from myelin membranes treated with serum complement. J. Neurochem. 42, 1024-1029. Soriano P., Dijkstra, J., Legrand, A . , Spanjer, H . , Londos-Gagliardi, D., Roerdink, F., Scherphof, G., and Nicolau, C. (1983) Targeted and nontargeted liposomes for in vivo transfer to rat liver cells of a plasmid containing the preproinsulin I gene. Proc. Natl. Acad. Sci. USA 80, 7128-7131. Stavridis J.C., Deliconstantinos, G , Psallidopoulos, M . C , Armenakas, N.A., Hadjiminas, D.J., and Hadjiminas, J. (1986) Construction of transferrin-coated liposomes for in vivo transport of exogenous D N A to bone marrow erythroblasts in rabbits. Exp. Cell. Res. 164, 568-572. Stratford-Perricaudet, L . D . , and Perricaudet, M . (1994) Gene therapy: the advent of adenovirus. In Gene Therapeutics: Methods and applications of direct gene transfer. J.A. Wolff, editor. Birkhauser Boston, Cambridge, M A , 344-362. Szelei, J., and Duda, E. (1989) Entrapment of high-molecular-mass D N A molecules in liposomes for the genetic transformation of animal cells. Biochem. J. 259, 549-553 Szoka, F., and Papahadjopoulos, D. (1978) Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc. Natl. Acad. Sci. USA 75, 4194-4198. Szoka, F., and Papahadjopoulos, D. (1980) Comparative properties and methods of preparation of lipid vesicles (liposomes). Ann. Rev. Biophys. Bioeng. 9, 467-508. Tait, D.L., Obermiller, P.S., Jensen, R.A., and Holt, J.T. (1999) Ovarian cancer gene therapy. Hematol./ Oncol. Clin. North Am. 12, 539-552. Tarn, P., Monck, M . , Lee, D., Ludkovski, O., Leng, E . C , Clow, K., Stark, H., Scherrer, P., Graham, R.W., and Cullis, P.R. (2000) Stabilized plasmid-lipid particles for systemic gene therapy. Gene Ther. (in press). Takahashi, T., Cabone, D., Takahashi, T., Nau, N . M . , Hida, T., Linnoila, I., Ueda, R., and Minna, J.D. (1992) Wild-type but not mutant p53 suppresses the growth of human lung cancer cells bearing multiple genetic lesions. Cancer Res. 52, 2340-2343. Templeton, N.S., Lasic, D.D., Frederik, P.M., Strey, H.H., Roberts, D.D., and Pavlakis, G.N. (1997) Improved DNA:liposome complexes for increased systemic delivery and gene expression. Nature Biotech. 15, 647-652.  133  Thierry, A.R., Lunardi-Iskandar, Y . , Bryant, J.L., Rabinovich, P., Gallo, R.C., and Mahan, L.C. (1995) Systemic gene therapy: biodistribution and long-term expression of a transgene in mice. Proc. Natl. Acad. Sci. USA 92, 9742-9746. Uster, P.S., Allen, T.M., Daniel, B.E., Mendez, C.J., Newman, M.S., and Zhu, G.Z. (1996) Insertion of poly(ethylene glycol) derivatized phospholipid into pre-formed liposomes results in prolonged in vivo circulation time. FEBS Lett. 386, 243-246. Vaheri, A . , and Pagano, J.S. (1965) Infectious poliovirus R N A : a sensitive method of assay. Virology 27, 434-436. van den Driessche, T., Vanslembrouck, V . , Goovaerts, I., Zwinnen, H . , Vanderhaeghen, M . L . , Collen, D., and Chuah, M . K . L . (1999) Long-term expression of human coagulation factor VIII and correction of hemophilia A after in vivo retroviral gene transfer in factor VHI-deficient mice. Proc. Natl. Acad. Sci. USA 96, 10379-10384. van der Woude, I., Visser, H.W. ter Beest, M.B., Wagenaar, A., Ruiters, M . H . , Engberts, J.B., and Hoekstra, D. (1995) Parameters influencing the introduction of plasmid D N A into cells by the use of synthetic amphiphiles as a carrier system. Biochim. Biophys. Acta 1240, 34-40. Vinson, P.K., Talmon, Y . , and Walter, A . (1989) Vesicle-micelle transition of phosphatidylcholine and octylglucoside elucidated by cryo-transmission electron microscopy. Biophys. J. 56, 669-681. Wagner, J.A., Reynolds, T., Moran, M.L., Moss, R.B., Wine, J.J., Flotte, T.R., and Gardner, P. (1998) Efficient and persistent gene transfer of A A V - C F T R in maxillary sinus. Lancet351, 1702-1703. Wagner, J.A., Nepomuceno, I.B., Shah, N . , Moran, M X . , Norbash, A . M . Moss, R.B., Wine, J.J., and Gardner, P. (1999) Maxillary sinusitis as a surrogate model for C F gene therapy clinical trials in patients with antrstomies. J. Gene Med. 1, 13-21. Wang C.-Y., and Huang L . (1987) pH-sensitive immunoliposomes mediate target-cellspecific delivery and controlled expression of a foreign gene in mouse. Proc. Natl. Acad. Sci. USA 84, 7851-7855. Webb, M.S., Saxon, D., Wong, F.M.P., Lim, H.J., Wang, Z., Bally, M.B., Choi, L.S.L., Cullis, P.R., and Mayer, L . D . (1998) Comparison of different hydrophobic anchors conjugated to poly(ethylene glycol): effects on the pharmacokinetics of vincristine. Biochim. Biophys. Acta 1372, 272-282. Wheeler, J.J., Palmer, L . , Ossanlou, M . , MacLachlan, I., Graham, R.W., Zhang, Y . P . , Hope, M . J . , Scherrer, P., and Cullis, P.R. (1999) Stabilized plasmid-lipid particles: construction and characterization, Gene Ther. 6, 271-281.  134  Wong, F . M . , Reimer, D.L., and Bally, M B . (1996) Cationic lipid binding to D N A : characterization of complex formation. Biochemistry 35, 5756-5763. Woodle, M . C , and Lasic, D.D. (1992) Sterically stabilized liposomes. Biochim. Biophys. Acta 1113, 171-199. Wong, T.-K., and Neumann, E. (1982) Electric field mediated gene transfer. Biochim. Biophys. Res. Commun. 107, 584-587. X u , Y., and Szoka, F.C. (1996) Mechanism of D N A release from cationic liposome/DNA complexes used in cell transfection. Biochemistry 35, 5616-5623. Yang, Y . , Nunes, F.A., Berenesi, K . , Furth, F.E., Gonczol, E., and Wilson, J.M.L. (1994a) Cellular immunity to viral antigens limits El-deleted adenoviruses of gene therapy. Proc. Natl. Acad. Sci. USA 91, 4407-4411. Yang, N.-S., Luna, C D . , and Cheng, L . (1994b) Gene transfer via particle bombardment: applications of the accell gene gun. In Gene Therapeutics: Methods and applications of direct gene transfer. J.A. Wolff, editor. Birkhauser Boston, Cambridge, M A , 193-209. Ye, G.-N., Daniell, FL, and Sanford, J . C (1990) Optimization of delivery of foreign D N A into higher-plant chloroplasts. Plant Molec. Biol. 15, 809-819. Zalipski, S, Brandeis, E., Newman, M.S., and Woodle, M.C. (1994) Long circulating, cationic liposomes containing amino-PEG-phosphatidylethanolamine. FEBS Lett. 353, 71-74. Zalipski, S., Puntambekar, B., Boulikas, P., Engbers, C M . , and Woodle, M . C . (1995) Peptide attachment to extremities of liposomal surface grafted PEG chains - preparation of the long-circulating form of laminin pentapeptide, YIGSR. Bioconjugate Chem. 6, 705-708. Zalipsky, S., Mullah, N . , Harding, J.A., Gittelman, J., Guo, L., and DeFrees, S.A. (1997) Poly(ethylene glycol)-grafted liposomes with oligopeptide or oligosaccharide ligands appended to the termini of the polymer chains. Bioconjugate Chem. 8, 111-118. Zhang, Y.P., Sekirov, L . , Saravolac, E.G., Wheeler, J.J., Tardi, P., Clow, K . , Leng, E., Sun, R., Cullis P.R., Scherrer, P. (1999) Stabilized plasmid-lipid particles for regional gene therapy: formulation and transfection properties. Gene Ther. 6, 1438-1447. Zhu, N . , Liggitt, D., L i u Y . , and Debs, R. (1993) Systemic gene expression after intravenous D N A delivery into adult mice. Science 261, 209-211.  135  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0089815/manifest

Comment

Related Items