UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

DNA-triggered release liposomes : a pharmaceutical strategy for improving the tumor exposure of chemotherapeutic… Wong, Hayes Ga-Hei 2003

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

Item Metadata

Download

Media
831-ubc_2003-0672.pdf [ 7.11MB ]
Metadata
JSON: 831-1.0091404.json
JSON-LD: 831-1.0091404-ld.json
RDF/XML (Pretty): 831-1.0091404-rdf.xml
RDF/JSON: 831-1.0091404-rdf.json
Turtle: 831-1.0091404-turtle.txt
N-Triples: 831-1.0091404-rdf-ntriples.txt
Original Record: 831-1.0091404-source.json
Full Text
831-1.0091404-fulltext.txt
Citation
831-1.0091404.ris

Full Text

DNA-triggered release liposomes: A pharmaceutical strategy for improving the tumor exposure of chemotherapeutic agents by Hayes Ga-Hei Wong B . S c , The University of British Columbia, 1999 A thesis submitted in partial fulfillment of the requirement for the degree of MASTER OF SCIENCE in THE FACULTY OF G R A D U A T E STUDIES (Division of Pharmaceutics, Faculty of Pharmaceutical Sciences) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 2003 © Hayes Ga-Hei Wong, 2003 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada 11 ABSTRACT Long circulating liposomes have been demonstrated to be an attractive delivery system for anticancer agents with favorable tumor drug accumulation. However, the slow rate of drug release from these liposomes may limit the level of drug exposure in the tumor available for cell uptake and one strategy to address this problem is to develop long circulating liposomes with selective drug release at the tumor. With the well-known propensity of fusogenic cationic liposomes to associate with DNA and strong evidences indicating selective localization of extracellular DNA in solid tumors, it is hypothesized that a fusogenic cationic liposome formulation containing DOTAP and D O P E would exhibit selective drug release in the tumor. This thesis presents a stepwise approach to the design of such liposomes. In order to develop a triggered release liposome that selectively targets the extracellular DNA in the tumor, the characteristic features of these DNA are first studied. In vivo results in Chapter 2 demonstrate an elevated level of extracellular DNA in the tumor in comparison with other organs and that they are in the size range of 200-3000 bp. Secondly, it is essential for an optimum triggered release liposome to remain stable in the circulation before reaching the tumor. Results from Chapter 3 demonstrate that the incorporation of D S P E - P E G 350 into DOTAP/DOPE liposomes prevents their characteristic salt-induced aggregations in physiological solutions while retaining the capacity to be triggered by extracellular DNA to release its entrapped contents. The in vivo results from Chapter 4 demonstrate the prolonged circulation time of DOTAP/DOPE/DSPE-350 (50:35:15) liposomes in H460 tumor bearing mice. These liposomes were stable in the circulation with selective tumor release of the entrapped contents. The results from these studies provide the basis for developing DNA triggered release liposomes that can selectively release their entrapped therapeutic agents in the tumor. iii TABLE OF CONTENTS A B S T R A C T ii TABLE OF C O N T E N T S iii LIST OF T A B L E S vi LIST OF F IGURES vii ABBREVIATIONS ix A C K N O W L E D G M E N T S x DEDICATIONS xi CHAPTER 1 Introduction 1.1. Project Overview 1 1.2. Liposome formulations: physiological properties 2 1.2.1. Phospholipids 3 1.2.2. Lipid polymorphism 5 1.2.3. The molecular basis of lipid polymorphism 8 1.2.4. Cationic lipids 12 1.2.5. D O P E 13 1.3. Liposomes as drug delivery vehicles 15 1.4. D S P E - P E G 17 1.5. Applications of lipid polymorphic behaviour in the delivery of 21 therapeutic agents 1.5.1. Membrane fusion 21 1.5.2. pH-sensitive liposomes 23 1.5.3. Lipid based gene delivery 24 1.6. Potential of fusogenic cationic liposomes as triggered release 24 delivery vehicles 1.7. Extracellular DNA in the solid tumors as potential tumor specific 25 target in selective anticancer drug delivery 1.7.1. Necrotic regions in solid tumors 25 1.7.2. Localization of extracellular DNA in solid tumors 26 1.8. Research rationale and hypothesis 29 1.9. Summary of research objectives 31 CHAPTER 2 Characterization of extracellular DNA in solid tumors 2.1. INTRODUCTION 33 2.2. MATERIALS AND METHODS 35 2.2.1. Materials 35 2.2.2. Tumor models and tissue staining with haematoxylin and eosin 35 2.2.3. Extracellular DNA extraction 36 2.2.4. DNA spectrophotometric quantitation 36 2.2.5. DNA agarose gel electrophoresis 37 IV 2.2.6. Statistical analysis 37 2.3. R E S U L T S 2.3.1. Visualization of tumor necrotic area in H & E slides 38 2.3.2. Tumor extracellular DNA size determination on agarose gel 38 electrophoresis 40 2.3.3. Extracellular DNA extraction from solid tumors in the presence of standard Dnase inhibitors 40 2.3.4. Comparison of extracellular DNA levels between tumor and healthy organs 43 2.4. C H A P T E R DISCUSSION 45 CHAPTER 3 In vitro characterization of DOTAP/DOPE/DSPE-PEG liposomes 3.1. INTRODUCTION 49 3.2. MATERIALS AND METHODS 51 3.2.1. Materials 51 3.2.2. Preparation of cationic liposomes 51 3.2.3. Q E L S particle sizing 52 3.2.4. 1 4 C-lactose release assays 52 3.2.5. Statistical analysis 53 3.3. R E S U L T S 54 3.3.1. Comparison between conventional and fusogenic cationic 54 liposomes 3.3.2. Effect of D S P E - P E G 2000 on 1 4C-lactose retention properties of 57 DOTAP/DOPE liposomes 3.3.3. Effect of D S P E - P E G 2000 on the interaction between DNA and 58 DOTAP/DOPE liposomes and the DNA triggered release of entrapped contents 3.3.4. Effect of varying the molecular weights of D S P E - P E G on 63 DOTAP/DOPE liposome stability in an ionic medium 3.3.5. Effect of varying the molecular weights of D S P E - P E G on DNA 68 induced triggered release properties of DOTAP/DOPE liposomes 3.3.6. DNA triggered release following interaction with tumor 70 extracellular DNA 3.4. C H A P T E R DISCUSSION 73 V CHAPTER 4 In vivo characterization of DOTAP/DOPE/DSPE-PEG liposomes 4.1. INTRODUCTION 77 4.2. MATERIALS AND METHODS 79 4.2.1. Materials 79 4.2.2. Preparation of various liposomes 79 4.2.3. Plasma elimination and tissue distribution of various formulations 80 of cationic liposomes in non-tumor bearing mice 4.2.4. Comparison of plasma elimination, tissue distribution and 81 encapsulated lactose release between DNA-triggered release cationic liposomes and DSPC/Chol liposomes in tumor bearing mice 4.2.5. In vivo recovery and analysis of injected liposomes 81 4.2.6. Statistical analysis 82 4.3. R E S U L T S 83 4.3.1. Effect of PEG-lipids on the plasma removal and the tissue 83 distribution of D O T A P / D O P E / D S P E - P E G 350 liposomes in non-tumor bearing mice 4.3.1.1. Plasma removal 83 4.3.1.2. Tissue distribution 85 4.3.2. Analysis of in vivo distribution and tumor triggered release of 87 D O T A P / D O P E / D S P E - P E G 350 liposomes in tumor bearing mice 4.3.2.1. Plasma removal 87 4.3.2.2. Liposome accumulation in tumor and various other organs 87 4.3.2.3. Lactose retention and DNA triggered release capacity of 94 D O T A P / D O P E / D S P E - P E G 350 (50:35:15) liposomes recovered from the plasma post injection 4.3.2.4. In vivo DNA triggered release of encapsulated contents from 96 D O T A P / D O P E / D S P E - P E G 350 liposomes in H460 solid tumors 4.4. C H A P T E R DISCUSSION 101 CHAPTER 5 Overall discussion 107 CHAPTER 6 Conclusions and future studies 112 6.1 Conclusions 112 6.2 Future Studies 113 REFERNCES 115 VI LIST OF TABLES Table 3.1. Comparison of liposome size stability between DSPC/Cho l 57 and DOTAP/DOPE liposomes Table 3.2. Size stability of DOTAP/DOPE liposomes with varying 59 amount of D S P E - P E G 2000 incorporated Table 3.3. Size stability of DOTAP/DOPE liposomes with varying 66 molecular weights of D S P E - P E G incorporated Table 4.1. Tissue distribution of D O T A P / D O P E / D S P E - P E G 350 and 86 DSPC/Cho l liposomes with varying amounts of D S P E - P E G 350 incorporated 24 hours post injection in H460 tumor bearing mice Table 4.2 Plasma area under the curve values over 48 hours post 91 injection of cationic D O T A P / D O P E / D S P E - P E G 350 and conventional DSPC/Chol liposomes in H460 tumor bearing mice Table 4.3 Tumor area under the curve values over 48 hours post 92 injection of cationic D O T A P / D O P E / D S P E - P E G 350 and conventional DSPC/Cho l liposomes in H460 tumor bearing mice Table 4.4. Tissue distribution of D O T A P / D O P E / D S P E - P E G 350 and 93 DSPC/Cho l liposomes 24 hours post injection in H460 tumor bearing mice V l l LIST OF FIGURES Figure 1.1. Structures of lipids commonly used in liposomes 4 Figure 1.2. The three major polymorphic structures adopted by lipids on 6 hydration. Figure 1.3. Freeze-fracture electron micrograph of an Hn phase (H) 7 inclusion embedded a bilayer (L) membrane segment Figure 1.4. Polymorphic structures and the corresponding molecular 10 shapes of lipids Figure 1.5. Shape features exhibited by membrane lipids 11 Figure 1.6. Complementarity effects arising from the shape properties of 11 lipids Figure 1.7. Chemical structures of various phospholipids. A. Cationic 14 lipids. B. Non-bilayer lipid, DOPE Figure 1.8. Chemical structure of D S P E - P E G lipid 19 Figure 1.9. The mushroom versus brush conformation of P E G 2000 20 Figure 1.10. Proposed intermediates of membrane fusion 22 Figure 1.11. Illustration of DNA triggered drug release from cationic 30 liposomes Figure 2.1. Visualization of tumor necrotic areas in H460 human non- 39 small cell lung solid tumors collected from SCID/Rag2 mice Figure 2.2. Agarose gel electrophoresis of H460 tumor extracellular 41 DNA Figure 2.3. Agarose gel electrophoresis of H460 tumor extracellular 42 DNA extracted with or without the addition of Dnase inhibitors during the extraction process Figure 2.4. Extracellular DNA levels in H460 tumors and other healthy 44 tissues in female SCID/Rag2 tumor-bearing mice Figure 3.1. Comparison of DNA triggered release (A) and entrapped 55 lactose retention properties (B) between DSPC/Cho l (55:45) and DOTAP/DOPE (50:50) liposomes Figure 3.2. Percentage of encapsulated 1 4 C lactose retention in 60 DOTAP/DOPE liposomes with varying amounts of D S P E -P E G 2000 incorporated Figure 3.3. Percentage of DNA triggered encapsulated lactose release 61 from D O T A P / D O P E liposomes incubated with varying salmon testes DNA Figure 3.4. Percentage of DNA triggered encapsulated lactose release 64 from DOTAP/DOPE liposomes with varying amounts D S P E -P E G 2000 vi i i Figure 3.5. Percentage of encapsulated C lactose retention in 67 DOTAP/DOPE liposomes with varying molecular weights of D S P E - P E G Figure 3.6. Percentage of DNA triggered encapsulated lactose release 69 from DOTAP/DOPE liposomes with varying molecular weights of D S P E - P E G Figure 3.7. Comparison of DNA triggered lactose release between 72 D O T A P / D O P E / D S P E - P E G 350 (50:45:5) liposomes incubated with salmon testes DNA or H460 tumor extracted extracellular DNA Figure 4.1. Plasma removal profiles for DOTAP/DOPE liposomes with 84 varying amounts of D S P E - P E G 350 Figure 4.2. Plasma removal profiles for D O T A P / D O P E / D S P E - P E G 350 88 and DSPC/Cho l liposomes Figure 4.3. Tumor Accumulation of D O T A P / D O P E / D S P E - P E G 350 and 90 DSPC/Cho l liposomes in H460 tumor bearing SCID/Rag2 mice over a 48-hour period Figure 4.4. The lactose retention (A) and DNA-induced triggered release 95 capacity (B) of D O T A P / D O P E / D S P E - P E G 350 and DSPC/Cho l liposomes recovered from plasma of H460 tumor bearing mice one hour post injection Figure 4.5. The lactose-to-lipid ratios in the tumor and other tissues of 99 H460 tumor bearing mice following a single 100 mg/kg bolus injection of 1 4 C-lactose encapsulated DSPC/Cho l (55:45) liposomes labeled with 3 H - C H E lipids over a 48-hour period Figure 4.6. The lactose-to-lipid ratios in the tumor and other tissues of 100 H460 tumor bearing mice following a single 100 mg/kg bolus injection of 1 4 C-lactose encapsulated D O T A P / D O P E / D S P E P E G 350 (50:35:15) liposomes labeled with 3 H - C H E lipids over a 48-hour period Figure 4.7. An illustration of the proposed model of DNA-triggered 104 release liposomes post injection into tumor bearing mice ABBREVIATIONS A U C area under the plasm concentration-time curve C H E cholesterylhexadecyl ether CHOL cholesterol DNA deoxyribonucleic acid DODAC N,N-dioleoyl-N,N-dimethylammonium chloride DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOTAP 1,2-dioleoyl-3-trimethylammonium propane DOTMA 1,2-dioleoyl-3,3,3-trimethylaminopropane chloride D S P C 1,2-distearoyl-sn-glycero-3-phosphocholine D S P E - P E G 350 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 350] D S P E - P E G 750 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 750] D S P E - P E G 2000 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2000] EDTA ethylenediaminetetraacetic acid G M I monosialoganglioside Hn inverted hexagonal phase H & E hematoxylin and eosin HBS H E P E S buffered saline L a liquid crystalline bilayer M P S mononuclear phagocytic system P C phosphatidylcholine PE phosphatidylethanolamine P E G poly(ethylene glycol) PEG-lipids poly(ethylene glycol)-conjugated lipids PS phosphatidylserine TBE Tris-Borate-EDTA ACKNOWLEDGMENTS This thesis could not be completed without the contributions of many people. Foremost, I would like to sincerely thank my supervisor, Lawrence, for his guidance, insights and patience throughout the years. Thank you for accepting me as a graduate student and for being a great supervisor. Also, I would like to acknowledge my committee members Drs. Kishor Wasan, my co-supervisor, Thomas Chang, Ron Reid and Katherine MacLeod for their input and assistance. I would like to thank each and every member of Advanced Therapeutics for their help and support during my stay in the lab: Gigi (for answering and tolerating my many questions), Maggie, Spencer, Vincent, Ludger, Sheela, Jennifer, Nancy, Catherine, Yan Ping, Corinna, Euan, Margaret, Ghania, Chris, Lincoln, Dawn, Ellen, Jason, Jehan, Malathi, Josephine and especially Marcel, for the wonderful atmosphere and camaraderie in the lab. Special thanks to Corinna and Euan for teaching me how to extract DNA from tissues and run gels. I am also grateful to Dana, Rebecca, Hong and Sophia for their invaluable assistance in the animal experiments. DEDICATIONS To my parents 1 Chapter 1 Introduction 1.1. Project Overview In cancer chemotherapy, there is often high toxicity associated with treatment. This is due to the fact that cancerous cells arise from the abnormal growth of the body's own cells. The differences between healthy and diseased tissues are therefore quite subtle and many conventional anticancer drugs developed to combat this disease have significant toxicity at therapeutic levels. Liposomal drug delivery has been one of the most extensively researched approaches to enhance anticancer drug selectivity, with the aim of increasing drug concentrations specifically at tumor site, while decreasing exposure to other tissues typically associated with deleterious side effects. Encapsulating toxic anticancer drugs inside liposomes has been shown to increase circulation lifetimes and tumor accumulation in preclinical and clinical trials (1, 2). These improvements arise from the fact that liposomes remain stable in the circulation and are not taken up by most normal healthy tissues associated with drug toxicity. However, they preferentially accumulate in sites of tumor growth as a result of the increased permeability of tumor blood vessels (3, 4). Within the tumor, anticancer drug released from the liposomes can diffuse through the tumor and has direct access to tumor cells, similar to unencapsulated agents. There the rate of drug release from the liposomes can influence the level of anticancer drug in the tumor available for cell uptake. 2 A contradiction therefore arises: while liposomes displaying high level of drug retention will promote selective accumulation of liposomal encapsulated drug in the tumor, the rate of drug release in the disease sites will be compromised. This illustrates a long standing problem in the development of an ideal liposomal delivery system: in favoring long circulating liposomes with stable drug retention properties that minimize systemic drug exposure, the level of drug in the tumors available for cell uptake may be severely limited as a result (5, 6). One approach to address this problem is to develop a liposomal formulation that would selectively release its encapsulated agent at the tumor site, while maintaining stable drug retention in normal physiological conditions, thus increasing the levels of bioavailable anticancer drug at the tumors without compromising drug selectivity. The objective of this thesis is to characterize a novel deoxyribonucleic acid (DNA) - triggered release liposome formulation that would satisfy the abovementioned criteria. 1.2. Liposome formulations: physicochemical properties Liposomes are lipid vesicles composed of an aqueous core encapsulated by a lipid bilayer. Most typically, liposomes are made up of phospholipids. Advances in liposome technology have included the use of surface modifying hydrophilic polymers and cationic lipids. These components will be discussed within the context of this thesis. 3 1.2.1. Phospholipids Phospholipids are one of the essential components of life, present in every type of bacterial, plant and animal cells. The ability of phospholipids to adopt the bilayer organization forms the basis of the biological membrane structure. The bilayer arrangement maintains a permeability barrier between the internal and the external environment (7). Phospholipids consist of two hydrophobic acyl chains bound to a glycerol backbone. The third carbon atom of the glycerol backbone is linked to a polar hydrophilic head group via a phosphate group (Figure 1.1). The size and charge of the head group, the length and degree of saturation of the acyl chains can vary in different phospholipid species. Bilayer formation is a self-assembly process and is driven by the amphipathic character of the phospholipids, where the polar sections tend to orient towards the aqueous phase, while the hydrophobic sections are sequestered from water, such that the resulting structure has achieved the most thermodynamically favorable organization. 4 Choline (Phosphatidylcholine) head group O / glycerol backbone o= common head groups vo-) Ethanolamine (Phosphatidylethanolamine) -o-) Serine (Phosphatidylserine) xo-) Glycerol (Phosphatidylglycerol) OH OH-OH T~OH Myo-inositol (Phosphatidylinositol) OH -o-) acyl chains Figure 1.1: Structures of lipids commonly used in liposomes. The structure of the phospholipid molecule distearoyl-phosphatidylcholine is represented schematically. Head groups for other classes of phospholipids are also shown (Adapted from reference # 8). 5 1.2.2. Lipid polymorphism Besides the bilayer structure, lipids can also adopt other distinct structures (or phases) on hydration. This is commonly referred to as lipid polymorphism. The three major structures assumed are illustrated on Figure 1.2. The bilayer and the inverted hexagonal organizations are the predominant structures adopted by biological membrane lipids, while lipids forming micellar phase are commonly minor components (8). In the bilayer phase (L a), lipids are arranged in extended bimolecular layer sheets, whereas in the inverted hexagonal phase (Hn) phase, lipids are arranged in a hydrocarbon matrix penetrated by hexagonally packed aqueous cylinders with diameters of about 2 nm. In Figure 1.3, a freeze-fracture electron microscopic visualization of an Hn phase inclusion embedded within a bilayer membrane is presented. In addition, contrary to the lipid bilayer, the Hn phase is not capable of maintaining a permeability barrier between the external and internal aqueous compartments (8). A large proportion of membrane lipids prefers the H M phase on hydration in isolated pure lipid systems. The most notable example of a H N phase lipid is phosphatidylethanolamine (PE), which commonly makes up to 35% of biological membrane lipids. In mixed lipid systems, non-bilayer lipids such as PE can be stabilized in a bilayer structure by the presence of 10-50 mol% of bilayer forming lipids such as phosphatidylcholine (PC) and phosphatidylserine (PS) (9). Transitions between different lipid polymorphic phases can be modulated by a wide variety of factors, such as degree of 6 hydrocarbon chain saturation, temperature, head group size, ionization and hydration. The basis of these transitions will be addressed in the following section. Micellar Bilayer Hexagonal ( H||) Figure 1.2: The three major polymorphic structures adopted by lipids on hydration (Adapted from reference # 13). 7 Figure 1.3: Freeze-fracture electron micrograph of an Hn phase (H) inclusion embedded a bilayer (L) membrane segment (10). 8 1.2.3. The Molecular Basis of Lipid Polymorphism The molecular shape concept A generalized molecular shape concept developed by Cullis and de Kruijff (11) and expanded by Israelachvili et al. (12) has been used to rationalize the polymorphic behaviour of lipids. This concept is illustrated in Figure 1.4 where bilayer phase lipids are proposed to exhibit cylindrical geometry, while Hn phase lipids have a cone shape where the acyl chains occupy a larger cross-sectional area than the polar headgroup region. Detergent-type lipids that form micellar structures are proposed to have a reversed geometry corresponding to an inverted cone shape. The geometrical packing constraints associated with each lipid shape and the thermodynamic driving force, which minimizes water-hydrocarbon contact, determine the lipid macromolecular aggregate adopted. A simple index termed the critical packing parameter (S) has been used to describe the molecular shape of a lipid molecule, which is based on the ratio of acyl chains and head group areas: S = v h / A 0 * L L Here Ao represents the surface area per lipid molecule at the hydrocarbon/water interface (m2), v h is the volume per molecule (m3) and L L is the length of the fully extended acyl chain (m). Figure 1.5 shows how the packing parameter relates to the molecular shape, where an additional parameter A H , the cross-sectional area of the hydrophobic end of a molecule, is defined (13, 14). Applying basic geometry, it can be shown that lipids with an S value less than 0.5 have an inverted cone shape (micellar lipids). S values between 0.5 and 1.0 correspond to cylindrical shapes (bilayer lipids) and S values more than 1.0 corresponds to a cone shape (Hn lipids). It is important to note that molecular lipid shape is a general concept inclusive of a large variety of complex intermolecular forces. For example, Ao would be expected to reflect the steric size of the head group, electrostatic repulsion, hydration and hydrogen bonding. Alternatively, A H would be sensitive to factors influencing the spread of the hydrocarbon chains, such as acyl chain unsaturation, chain length and temperature. The molecular shape concept has been successful in explaining many experimental observations. For example, the concept would predict that appropriate mixtures of inverted cone shaped (micellar) lipids and cone shaped Hn lipids would complement each other and assume a bilayer organization (Figure 1.6). Such lipid behaviour has been demonstrated in detergent (micellar lipid) and PE (HM lipids) mixed lipid systems (15). 10 LIPIDS Lysophospholipids Detergents PHASE Micellar MOLECULAR SHAPE Inverted cone Phosphatidylcholine Sphingomyelin Phosphatidylserine Phosphatidylinositol Phosphatidylglycerol Phosphatidic Acid Cardiolipin Digalactosyldiglyceride mm Bilayer Cylinder Phosphatidylethanolamine Cardiolipin - C a 2 + Phosphatidic Acid - C a 2 + Phosphatidic Acid (pHO.O) Phosphatidylserine (pH<4.0) Monogalactosyldiglyceride Hexagonal (H||) Cone Figure 1.4: Polymorphic structures and the corresponding molecular shapes of lipids (Adapted from reference # 8). P H A S E L IP IO S H A P E M I C E L L A R B I L A Y E R I N V E R T E D M I C E L L A R H II L L Figure 1.5: Shape features exhibited by membrane lipids (Adapted from reference # 13). Micelle Hexagonal H„ m A A A A V V V V Figure 1.6: Complementarity effects arising from the shape properties of lipids. 12 1.2.4. Cationic Lipids The use of cationic lipids as liposomal components has long been investigated. However, because of their rapid clearance and toxic nature in vivo, cationic liposomes were not shown much interest initially when they were first introduced in the early seventies (16, 17). However, interest was revived when the potential of cationic lipids as gene delivery vehicles was first introduced by Feigner (18). Cationic lipids commonly consist of a cationic head group with one or more amine groups and hydrophobic tails of various chain lengths. Various novel cationic lipids have been synthesized with variations in the polar head group, degree of alkyl chain saturation, number of hydrocarbon moieties on the amine group and the linkage between head group and alkyl chain (19, 20). A few examples are illustrated in Figure 1.7. Cationic lipids associate with DNA by electrostatic interactions to form complexes favorable for gene transfection. The mechanism of lipid-based gene transfection is further discussed in Section 1.5.3. In this thesis, the cationic lipid, DOTAP, is included as the cationic component in our DNA-triggered release formulation. DOTAP was one of the first cationic lipids to be synthesized (21) and its structure is shown in Figure 1.7. DOTAP consists of a permanent quartenary amine group and its positive charge is not affected by variations in pH in the external environment (22). Cationic liposomes have the tendency to self-aggregate in physiological salt 13 buffers (23-26). The stability of DOTAP containing liposomes is further investigated in Chapter 3 of this thesis. 1.2.5. DOPE A major component of the DNA-triggered release liposome formulation investigated in these studies is the phospholipid, DOPE. This synthetic lipid has been used extensively in liposomal gene delivery systems (27, 28). DOPE is composed of a PE head group linked to hydrophobic tails of 18-carbon acyl chains via a glycerol backbone. It is a non-bilayer forming lipid, preferring the hexagonal phase structure on hydration. As described earlier, it can also be stabilized into a bilayer vesicle formation by the inclusion of 10-50 mol% of a bilayer forming charged lipid, such as DOTAP. D O P E has often been included as a "helper" lipid with cationic lipids to form liposomal transfection vehicles for the purpose of gene delivery. Incorporation of DOPE has been shown to enhance transfection success rate, by its ability to promote liposomal membrane fusion with endosomal membrane (29). The mechanism of D O P E assisted membrane fusion will be discussed in Section 1.5. o 1,2-dioleoyl-34rimethylammonium propane (DOTAP) B. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) Figure 1.7: Chemical structures of various phospholipids. A. Cationic lipids. B. Non-bilayer lipid, D O P E . 15 1.3. Liposomes as Drug Delivery Vehicles After the first descriptions of self-assembled enclosed spherical liposomes by Bangham in the 1960's (30), the potential utility of liposomes as drug delivery vehicles was recognized quickly and effective liposomal carriers of a wide range of therapeutic agents such as anticancer drugs, antimicrobial agents, genes and anti-sense oligonucleotides have been developed (31). Encapsulating conventional anticancer agents into liposomal carriers have been shown to reduce the toxicity associated with these cytotoxic agents. This is due to the ability of liposomes to alter the tissue distribution of these agents, resulting in reduced drug exposure to healthy tissues and selective drug accumulation in the tumors. Lipid carriers also protected the encapsulated drugs from degradation and elimination from the circulation after injection, promoting extended plasma circulation time and increased drug exposure to the disease sites (32-35). For example, delivery of liposomal doxorubicin to tumor sites has been found to achieve tumor drug exposure five times greater than observed with free drug, along with significant decreases in heart, kidney and Gl toxicity (36, 37). Reduced toxicity to healthy organs also allows higher doses to be administered, increasing the effectiveness of the therapeutic treatment. The ability of liposomes to selectively localize in solid tumors has been attributed to "passive tumor targeting", which is a result of the unique physiological characteristics of the tumor vasculature (3, 4). In rapidly growing tumors, vascular endothelium is often immature with adnormal fenestrations 16 devoid of the basement membrane layer. Large intercellular openings are observed with sizes ranging from 380-780 nm, and a maximum up to 2 urn (38). This abnormal tumor vascular architecture gives rise to increased vascular permeability to macromolecules (39). In conjunction with a lack of a developed lymphatic drainage system, it contributes to the passive accumulation of liposomes in the sites of tumor growth (40). Liposomes designed for drug delivery to solid tumors typically involved a combination of a long-chained P C (e.g. DSPC) and cholesterol (30-50 mol%), forming neutral unilamellar liposomes of approximately 100 nm in size, with stable drug retention and long plasma circulation longevity after injection. These liposomes, relatively simple in their design, are often referred to as first generation or conventional liposomes. They typically possessed circulation half-life of approximately 5 hours and were primarily removed from circulation by the mononuclear phagocytic system (MPS) (41). The M P S consists of monocytes and macrophages, and is a part of the host defense system with a role in recognizing and removing dead, foreign or altered cells. It is also involved in other cellular functions including immune and inflammatory reactions (42, 43). After recognition by the M P S , liposomes are primarily redistributed to the liver and spleen for elimination. Increasing the circulation longevity of liposomal anticancer drugs does not necessarily ensure enhanced drug exposure in the tumors. Lim et al. (1997) demonstrated no increase in drug efficacy with long circulating mitoxantrone encapsulated in conventional DSPC/Chol liposomes than that of 17 the free drug (44). Although an increase in plasma circulation time improves selective accumulation of liposomal agents in the tumor sites via passive tumor targeting, it does not necessarily translate to an increase in drug availability. This lack of correlation between enhanced tumor delivery and treatment efficacy could be due to the slow release of encapsulated therapeutic agents from the liposome carrier. In the process of developing long circulating liposomes with stable drug retention, the release of encapsulated drugs in the disease sites is compromised. In this thesis, we seek to find a solution to this problem by developing a novel liposome formulation with long circulation properties, accompanied by a selective triggered release mechanism in the tumor sites. 1.4. DSPE-PEG Many strategies have been undertaken to develop liposomes exhibiting enhanced circulation lifetimes. Initially, inclusion of monosialoganglioside GMI into conventional liposomes demonstrated prolonged circulation and reduced M P S uptake in mice (45). However, one of the major drawbacks to the use of G M i was its cost and difficulty in production. Therefore, alternatives to GMI were sought and the synthetic hydrophilic polymer, poly(ethylene glycol) (PEG), attached to a lipid anchor (usually PE) was found to be most the effective in providing extended circulation longevity (46). P E G is a neutral polyether available in a variety of molecular weights (commonly 350 to 5000) and is soluble in water and most organic solvents. 18 The structure of the poly(ethylene glycol)-conjugated lipid (PEG-lipid) is shown in Figure 1.8. P E G has been shown to enhance circulation times when attached to therapeutic proteins (47). Covalent attachment of P E G to therapeutic surfaces has also been demonstrated to reduce protein absorption (48). These observations led to the use of P E G polymers in liposome technology with the aim of enhancing circulation longevity. P E G can be attached to a liposome surface by a number of methods. However, incorporation of PEG-lipids in the original lipid mixture before liposome formation has been most commonly preferred because of the ease and reproducibility of this method. PEG-lipid incorporated into liposome surfaces can adopt two different conformations depending on the density of the polymer content (49). At low polymer densities, P E G polymer resembles a "mushroom" model while at high polymer densities, a "brush"-like model is preferred (Figure 1.9). The length of P E G 2000 polymer extension from liposome surface has been reported to be 5 nm and 6.5 nm for the "mushroom" and "brush" models, respectively (23, 50). Liposomes containing PEG-lipids in the bilayer have often been referred to as sterically stabilized liposomes (SSL). Several mechanisms have been proposed to explain the prolonged circulation longevity of pegylated SSL . Increases in surface hydrophilicity and surface hydration have been proposed as reasons for the improved liposome stability and reduction in M P S uptake (51, 52). The most widely accepted mechanism involves the 19 ability of P E G to generate a steric repulsive barrier on the surface of the liposomes, preventing protein binding, membrane fusion, liposome aggregation and cellular uptake by the M P S organs (23, 50, 53). In this thesis, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) (DSPE-PEG) polymers are incorporated into our liposome formulation to enhance the circulation time as well as the steric stability of the cationic liposomes, which have been demonstrated in previous studies to self-aggregate in physiological salt solutions. The impact of D S P E -P E G on DNA triggered release liposomes will be addressed in Chapter 3. 0 J linker D S P E (C 1 f i ) polv(ethvlene oxide) Figure 1.8: Chemical structure of a D S P E - P E G lipid with carbamate linkage 20 5 nm Mushroom A 6.5 nm Brush Figure 1.9: The mushroom versus brush conformation of P E G 2000. The extensions of P E G 2000 from the liposome surfaces for the two conformations are also illustrated (Adapted from reference # 20). 21 1.5. Applications of lipid polymorphic behaviour in the delivery of therapeutic agents 1.5.1. Membrane fusion Increasing interest has been shown in employing the polymorphic behaviour of phospholipids in lipid-based drug delivery systems, such as pH-sensitive liposomes and gene delivery vehicles. These lipid systems exploit the functional role of Hn phase preferring lipids in lipid membrane fusion. Membrane fusion is a process involving the union of two opposing bilayers in order to complete biological processes such as exocytosis. In order for the two bilayers to merge into one, there must be local transient deviations from the bilayer organization. Although the exact nature of these membrane transient structures has not yet been elucidated, studies have implicated key involvement of the Hn phase lipids (54). Currently, there are two proposed models on the structures of these fusion membrane intermediates: the inverted micelle intermediate (IMI) and the transmembrane contact (TMC) intermediate (55). As illustrated in Figure 1.10, both of these intermediate structures are favored by the incorporation of Hn phase adopting lipids. Studies have demonstrated that Hn-phase adopting lipids, such as D O P E , promote membrane fusion of lipid vesicles, while in contrast, micellar lysolipids have been shown to inhibit lipid membrane fusion (56, 57). 22 ?8W8?8IW8?88888f8? IMI mm mm mm mmmmmmm mmmmmmmmmm mnmmmmm i mm mm mmm Si "mm& mmm mm fus ion mm m pore T M mm yMff K( « « « « Figure 1.10: Proposed intermediates of membrane fusion. Two opposed bilayers are schematically represented to undergo fusion through either an inverted micelle intermediate (IMI) or the transmembrane contact (TMC) intermediate (Adapted from reference # 59). 23 1.5.2. pH-sensitive liposomes The ability of Hn-adopting lipids such as D O P E to promote membrane fusion has generated increasing interest in the development of "fusogenic" triggered-release liposomal systems. In these liposomes, Hn "fusogenic" lipids such as D O P E are incorporated into bilayer vesicles by a species of ionizable lipids that prefers bilayer arrangement at neutral physiological pH. As the pH decreases, the ionizable lipids become protonated, leading to a reduction in charge and the effective head group size, which results in the transition from the bilayer phase to the Hn phase. Thus at acidic pH, these liposomes become structurally unstable and fusogenic. The first fusogenic pH-sensitive liposomes were composed of phosphatidylserine (PS) and D O P E (58). These liposomes were stable at neutral pH but underwent membrane fusion at an acidic pH (<4). pH-sensitive liposomes have been investigated as potential delivery vehicles for the intracellular delivery of various macromolecules, including nucleic acids, protein toxins and antibiotics (59). The exact mechanism(s) of these liposomes in intracellular delivery has not been well established. It is proposed that following endocytosis into the target cell, pH-sensitive liposomes are introduced into acidic endosomes. There the acidic pH triggers liposomal membrane fusion with the endosomal membrane, resulting in the release of the encapsulated materials into the cytoplasm. Thus these lipid systems would be useful in the protection of hydrolytically sensitive materials from degradation in the lysosomal pathway. 24 1.5.3. Lipid based gene delivery Bilayer-Hn polymorphic transition has also been employed for the delivery of genetic materials. In lipid-based gene delivery systems, nucleic acids such as DNA are associated with positively charged, cationic lipids to form lipid-DNA particles called lipoplexes (8). It is proposed that after endocytosis into the cell, lipoplexes are stored in endosomes. There the cationic lipid would interact with the negatively charged, anionic lipids in the endosomal membrane via electrostatic forces, leading to the displacement of genetic materials from association with the cationic lipids. The cationic-anionic lipid aggregates would have a reduction in the effective head group size. The resulting induction of Hn phase structures would lead to membrane fusion and consequently, the release of unassociated genetic materials into the cytoplasm, preventing degradation via the lysosomal pathway. Incorporation of Hn preferring lipids such as D O P E to form fusogenic cationic liposome formulations have been shown to enhance the degree of gene transfection (27) highlighting their functional role in lipid membrane fusion. 1.6. Potential of fusogenic cationic liposomes as triggered release delivery vehicles As described above, "fusogenic" cationic liposomes have been studied as vehicles for delivery of DNA oligomers and genes. They are composed of a non-bilayer forming neutral lipid (e.g. DOPE) stabilized in a bilayer organization by a bilayer forming cationic lipid (e.g. DOTAP), and associate with DNA by electrostatic interactions, forming DNA/liposome complexes (60). This association was found to disrupt membrane stability resulting in the 25 transition from a liposomal bilayer phase to non-bilayer polymorphic structures, such as hexagonal (Hn) structures, sheets or tubules in which DNA is embedded (61, 62). In these structures, DNA provides a cross-link between liposomes, leading to lipid fusion, aggregation and membrane destabilization. Previous studies have shown that after the addition of plasmid DNA to cationic liposomes composed of DOTAP and DOPE (50/50 mole %), release of encapsulated aqueous markers is induced, accompanied by vesicle aggregation and fusion (63). It has also been observed that reducing the membrane stability of DOPE-containing liposomes increases encapsulated drug release (64). These findings highlight the potential of using fusogenic cationic liposomes as a specific trigger release formulation for targeted drug delivery. 1.7. Extracellular DNA in the solid tumors as potential tumor specific target in selective anticancer drug delivery 1.7.1. Necrotic regions in solid tumors The characteristic presence of a high percentage of necrotic cells in a tumor has long been recognized. Using tritiated thymidine as labeling index in in vitro and in vivo experiments, Steel (1967) estimated that the cell loss in a wide variety of human tumor types often exceeds 50% of the growing tumor populations (65). Another investigation in tumor growth estimated that necrotic tissue occupies a mean of 60-70% of the tumor by volume (66). In another study, a 90% rate of cell loss was found in 3 squamous cell carcinomas of the skin (67). Tumor cell death is often attributed to poor vascularization, where the rate of tumor blood vessels development cannot 26 sufficiently provide for the rapid growth rate of tumors. Consequently necrotic foci develop in the generally poorly perfused central tumor region, resulting in cells dying from a lack of nutrients (e.g. glucose and oxygen) and the injurious effects of high concentrations of toxic metabolites (e.g. lactic acid) (66). 1.7.2. Localization of extracellular DNA in solid tumors Previous preclinical and clinical investigations have demonstrated the specific localization of extracellular DNA fragments in the solid tumors, which are released from the high percentage of degenerating cells in the necrotic tumor regions. In histochemical studies, free DNA and histones have been found to deposit on the vascular walls of small blood vessels in various types of human tumors including small cell anaplastic lung carcinoma, retinoblastoma, oat-cell carcinoma, medulloblastoma, malignant choroidal melanoma, anaplastic seminoma of the mediastinum and embryonal rhabdomyosarcoma of the buttock (68). The occurrence of extracellular DNA coating of blood vessels was found to be independent of size and spread of the tumor, as well as the type of anticancer treatment, while detection was most frequent in small blood vessels in necrotic regions of the tumors (69). Free extracellular DNA levels in the serum of patients with various types of cancer have been found to be significantly higher than levels in healthy individuals (70-74). Using radioimmunoassay and P C R detection techniques, the mean free DNA levels in cancer patients was determined to be more than ten times higher than observed in healthy volunteers, with values highest in patients with fast-growing and advanced metastatic 27 carcinomas. Investigations on the characteristics of the serum DNA from cancer patients showed that an important part of these DNA originates from the necrotic cells of the tumor region, with cancerous gene mutations detected in these DNA. These results indicated a notable amount of DNA fragments are released from the necrotic tissues of solid tumor growth. This phenomenon of increased extracellular localization of normally intracellular antigens (including DNA) released from necrotic tumor cells with degenerating membranes was further confirmed in immunohistological studies in which a variety of serum proteins (e.g. albumin, immunoglobulins, transferrin) was found intracellularly in a proportion of malignant tumor cells of various types of human tumors (102). Such normally extracellular proteins are believed to have entered the cell cytoplasm due to the increased permeability of the membrane of degenerating tumor cells in different stages of necrosis. Based on these unique characteristics of cancer pathophysiology, the increased exposure of intracellular substances such as DNA and histones in the extracellular space of solid tumors has been investigated as potential targeting ligands to distinguish between cancerous growths and normal healthy tissues (75-79). In studies by Epstein et al. (1988), radiolabeled monoclonal antibodies with specificity towards DNA and histones were injected intravenously into mice bearing tumors of various human tumor cell lines. Antibody level was found to be significantly higher in the tumors, where tumor-to-blood ratios of 4.9-6.9 to 1 and tumor-to-organ ratios in the range of 3.8 to 29.1:1 were obtained with no signs of specific binding in the major 28 organs. The degree of antibody accumulation corresponds directly to the degree of necrosis in the tumor. In one study, tumor-to-blood ratio as high as 131:1 was observed (75). Furthermore, in vivo imaging studies on tumor bearing mice also showed preferential accumulation of these antibodies in the tumor growth while microscopic morphological studies confirmed selective antibody binding to the nuclei of degenerating cells, with no labeling in the viable proliferating tumor cells. Subsequently, these DNA-directed antibodies have been investigated as potential targeting ligands to increase the selectivity of radio- and immuno-therapeutic anticancer treatments. The above observations provided evidence in support of a characteristic localization of DNA fragments in the extracellular space of solid tumors, including the tumor vasculature and interstitial space. This unique pathophysiological feature of cancer growth has been demonstrated to have potential therapeutic value where it can be employed as a specific target with the aim of increasing the selectivity of existing anticancer treatments. 29 1.8. Research Rationale and Hypothesis In the past, researchers have investigated various aspects of tumor physiology as potential targets for liposomal targeted drug delivery. As our knowledge on the differences between healthy normal cells and neoplastic cells increases, more specific approaches for the treatment of cancer have emerged. Past histological and clinical research have shown an elevated level of deoxyribonucleic acid (DNA) fragments in the extracellular space of solid tumors as compared to healthy organs, due to the high percentage of cells undergoing necrosis and cell lysis in solid tumor. This unique feature of tumor physiology has the potential to be utilized as a specific target for anticancer drug delivery, allowing tumors to be readily distinguished from normal tissues. In this regard, we hypothesize that fusogenic cationic liposomes, composed of a bilayer forming cationic lipid (DOTAP) and a non-bilayer forming neutral lipid (DOPE), would have greater drug release at solid tumors compared to healthy tissues by their ability to have electrostatic interactions with the increased level of extracellular DNA in the solid tumors. After intravenous injection, these liposomes will remain stable in the circulation, retaining the encapsulated drugs. When they reach the extracellular space of the tumor, they will interact with the increased level of extracellular DNA, inducing the formation of non-bilayer DNA/liposome complexes, disrupting membrane stability and the specific release of the encapsulated therapeutic agents in the solid tumor (See Figure 1 for illustration). 30 A. Cationic liposomes stable in circulation Drug • • • DNA Figure 1.11: Illustration of DNA triggered drug release from cationic liposomes 31 1.9. Summary of research objectives The overall objective of this thesis is to develop a novel DNA triggered release liposomal formulation with selective release of the encapsulated contents in solid tumors. The focus of this thesis is to engineer the components of fusogenic cationic liposomes to achieve functional DNA-triggered release properties with respect to the characteristics of the extracellular DNA found in the solid tumors. The specific objectives are as follows: (1) To characterize the properties of the extracellular DNA in the necrotic tumor regions as compared to healthy tissues using a human tumor xenograft mouse model. This is described in Chapter 2, where the molecular size range and quantitative levels of the extracellular DNA were evaluated in comparison with other major organs. (2) To develop an optimal salt-stable DNA-triggered release liposomal formulation from different compositions of cationic lipid (DOTAP), non-bilayer forming neutral lipid (DOPE) and DSPE-PEG lipids. This is described in Chapter 3, where in vitro studies were conducted to determine the ability of different formulations of D O T A P / D O P E / D S P E -P E G to maintain stability and DNA triggered release properties in physiological salt buffers. 32 (3) To determine the elimination profile and tumor accumulation behaviour after a single dose of the optimal DNA-triggered release liposomes in non-tumor bearing and tumor bearing mice. This is described in Chapter 4, where a series of in vivo studies were performed to determine the circulation longevity and tumor accumulation levels of these liposomes. (4) To demonstrate the selective release of encapsulated contents from these optimal DNA-triggered release liposomes in the solid tumors of tumor bearing mice. This is described in Chapter 4, where in vivo studies were conducted to evaluate the specific triggered release of the encapsulated contents from these liposomes in the solid tumors in a human xenograft mouse model after a single intravenous injection. In Chapter 5, the therapeutic implications of a tumor specific DNA triggered release liposomal formulation, as well as future directions in research will be discussed. 33 Chapter 2 Characterization of extracellular DNA in solid tumors 2.1. Introduction The presence of a large degenerating cell population in fast-growing tumors has long been recognized. It is not surprising that in the process of cell necrosis and cell lysis, free DNA and other intracellular markers are released and become readily exposed to the extracellular space of solid tumors. As described in Chapter 1, the accumulation of extracellular DNA in the walls of tumor blood vessels has been observed in histological studies on isolated human tumors (68, 69). Extracellular DNA in the tumor has also been investigated as a potential specific marker to differentiate tumor cells from healthy tissue (75-79). In these studies, monoclonal antibodies targeting DNA or DNA associated histones were found to selectively accumulate in the solid tumors in human xenograft mouse models. Although previous clinical and animal studies have provided strong evidence for the existence of an elevated level of extracellular DNA in the solid tumors, a detailed characterization has not been performed previously. In our effort to develop a DNA-triggered release liposome formulation targeting these extracellular DNA for selective drug release in the tumor, it was important to first complete a characterization of the content and nature of extracellular DNA. In this chapter, extracellular DNA in the solid tumor is evaluated: in particular, quantitative evaluation with comparison to other healthy tissues, and determination of the physical size range of the DNA was performed. First, an extraction method was developed to isolate extracellular DNA from the tumors and other healthy organs in a human xenograft mouse 34 model. We demonstrated here that solid tumors have elevated levels of extracellular DNA compared to healthy tissues and the extracellular DNA was found to be degraded fragments in the size range of 200-3000 base pairs. 35 2.2. Materials and Methods 2.2.1. Materials Cholesterol was purchased from Fisher Scientific (Nepean, Ontario). Hank's Balanced Salt Solution was obtained from Stemcell Technologies Inc. (Vancouver, BC). Salmon testes DNA was purchased from Worthington's Biochemical Corp. (Lakewood, NJ). Buffered-saturated phenol, 1 Kb Plus Ladder and agarose were obtained from Invitrogen Corp. (Carlsbad, CA). Collagenase Type II, ethylenediaminetetraacetic acid (EDTA) and Proteinase K were purchased from Sigma (St. Louis, MO). H460 human non-small cell lung carcinoma cell line was obtained from the National Cancer Institute. 2.2.2. Tumor models and tissue staining with haematoxylin and eosin All of the in vivo studies were completed following protocols approved by the University of British Columbia's Animal Care Committee. These studies met the current guidelines of the Canadian Council of Animal Care. Female severe combined immune deficient mice (SCID) with a mutation in the Rag2 gene, resulting in incomplete lymphocyte development and minimal production of immunoglobulins or T-cell receptors, were used in establishing the H460 and L C C 6 tumor models. Female SCID/Rag2 mice were inoculated unilaterally with 1 x 10 6 H460 cells subcutaneously in the back. When the weight of the tumors reached ~ 0.5 g, the mice were sacrificed and the tumors were removed, fixed in 10% buffered formalin. The tumors were then embedded in paraffin and 5 urn sections stained with hemotoxylin and eosin (H & E) on glass slides were prepared by Wax-It (Aldergrove, BC). 3 6 2.2.3. Extracellular DNA extraction Female SCID/Rag2 mice inoculated with H460 tumors were prepared as described in section 2.2.1. When the weight of the tumors reached ~ 0.5 g, the mice were sacrificed, and the tumors and organs (liver, kidney, lung, muscle from the left thigh) were removed and their weights determined using an analytical balance. Inside a sterile fume hood, the tissues were transferred to a culture dish and cut into small cubes (~ 1mm wide) with a surgical scalpel. The tissues were then incubated on ice in 2ml Hank's Balanced Salt Solution with 1.18 mg/ml of collagenase type II. After 2 hours on ice with frequent gentle shaking, the tissue mixtures were centrifuged for 10 min at 1500 rpm at 4°C to separate the tissues from the supernatant. DNA in the supernatant was separated from plasma proteins using phenol/chloroform extraction and then precipitated in 100% ethanol for >2 hrs at -20°C. Ethanol was removed from the precipitated DNA pellet after centrifugation at 14000 rpm at 4°C. The DNA pellets were then dissolved in 100 ul distilled water, pre-warmed to ~37°C to aid dissolution. 2.2.3. DNA spectrophotometric quantitation DNA levels in samples are determined using spectrophotometric quantitation by measuring its ultraviolet absorbance at 260nm (A26o) with the Hewlett Packard 8453 UV-Visible Spectroscopy System. A standard curve consisting of known amount of salmon testes DNA was performed, which was linear in the range of 0 - 1200 ug/ml (r2 = 0.9944). Sample readings were determined within this concentration range and DNA concentrations were 37 calculated with measured A26o absorbance of 1 corresponding to 50 ug/ml of double-stranded DNA. The ratio between readings at 260 and 280 nm (A260/A280) were obtained to estimate the purity of the nucleic acid in the sample. Pure preparations of DNA and RNA have A260/A280 values of 1.8 and 2.0, respectively (80). If there were contamination with protein or phenol, A260/A280 values will be significantly less than values given above. 2.2.4. DNA agarose gel electrophoresis The size distributions of the extracted DNA from solid tumors were determined using agarose gel electrophoresis with ethidium bromide staining. 100 ug of the extracted tumor DNA mixed with loading dye were run on a 0.8% agarose gel in 0.5X Tris-Borate-EDTA (TBE) buffer at a voltage of 100 V with the electrodes set at 20 cm apart. Samples were run alongside a 1 Kb DNA size ladder and 100 ug of salmon testes DNA with a known size range of 200 - 2000 bp for comparison. After the dye had run to the end of the gel, the gel was removed from the gel tray and stained with ethidium bromide by incubating in 0.5X TBE with 0.5 ug/ml ethidium bromide for 30 min. The gel was subsequently visualized using the Stratagene Eagle Eye II still video system. 2.2.5. Statistical analysis A N O V A (analysis of variance) was performed on the results obtained in Section 2.2.4. to compare the levels of extracellular DNA between tumor and other healthy organs. A post hoc Scheffe test was used to compare the different groups pairwise. Differences were considered significant at p < 0.05. 38 2.3. Results 2.3.1. Visualization of tumor necrotic area on H & E slides In order to access the characteristics of tumor extracellular DNA in the necrotic area of a solid tumor, the first step was to identify a tumor model exhibiting strong cell necrosis for in vivo evaluation. A number of tumor models which are well established in our laboratory have shown large necrotic centres and one of these models, H460 human non-small cell lung carcinoma, was stained with H & E for visualization of the necrotic regions. The results are shown in Figure 2.1. High levels of cell necrosis were observed, indicated by three characteristic features of cell necrosis on H & E stained tissues (104). First, the tumor sample exhibited a bright pink colour, indicating an intense staining of the dye, eosin, with denatured cytoplasmic proteins produced during cell necrosis. Secondly, dissolution of the cell membrane was demonstrated with the observable loss of definable cell outlines (cytolysis). Thirdly, the loss of nucleus (karyolysis) and the condensation of nuclear chromatin into small densely purple staining mass (pyknosis) were also seen, which are both characteristic nuclear changes during cell necrosis. These observations demonstrated large areas of necrotic regions in the H460 tumor model. 39 Figure 2.1. Visualization of tumor necrotic areas in H460 human non-small cell lung solid tumors collected from SCID/Rag2 mice. 0.5 tumor sections were prepared tumors weighted ~0.5g and stained with haematoxylin and eosin by Wax-It (Aldergrove, BC). Tumor necrosis is identified by intense staining with eosin (bright pink colour), the loss of definable cell outlines (cytolysis), the loss of nucleus (karyolysis) and the condensation of nuclear chromatin (pyknosis). (Magnification: 10x) 40 2.3.2. Tumor extracellular DNA size determination on agarose gel electrophoresis The sizes of the extracellular DNA fragments extracted from H460 solid tumors were determined using agarose gel electrophoresis. Tumors were extracted and analyzed as described in Sections 2.2.3. and 2.2.4. A sample of tumor extracted DNA was run alongside a 1 kb DNA size ladder and a sample of salmon testes DNA, which was randomly sheared to a size range of 200-2000 bp, for comparison. The results are shown in Figure 2.2. The tumor extracted extracellular DNA was observed to be degraded fragments with a size range estimated to be 200-3000 bp, comparable to the size range of the salmon testes DNA. The size of the extracellular DNA fragments observed was significantly smaller then a size of intracellular genomic DNA, which is commonly > 50 kbp (80). 2.3.3. Extracellular DNA extraction from solid tumors in the presence of standard DNase inhibitors Previous results demonstrated that extracellular DNA extracted from H460 solid tumors has a relatively small size range of 200-3000 bp when compared to intracellular genomic DNA, which is commonly > 50 kbp in size. However, extracted DNA using the method described above may have been degraded by endogenous DNase present in the tissue sample during the isolating procedures. Therefore, in order to determine whether the small DNA size was a result of such endogenous degradation, standard DNase inhibitors: EDTA (80-82) or proteinase K (83, 84) were included in the incubation buffer during the extraction process. 41 12,000 bp 2000 bp 1000 bp 500 bp — 200 bp 1 2 3 Figure 2.2. Agarose gel electrophoresis of H460 tumor extracellular DNA. Lane 1: DNA size ladder, Lane 2: sheared salmon testes DNA, Lane 3: H460 tumor extracellular DNA. DNA size (bp) is shown by the scale on the left. Extracellular DNA was isolated from H460 solid tumor as described in Section 2.2.3. 42 2 0 0 b p 1 2 3 4 Figure 2.3. Agarose gel electrophoresis of H460 tumor extracellular DNA extracted with or without the addition of DNase inhibitors during the extraction process. Lane 1: DNA size ladder, Lane 2: with no DNase inhibitors added, Lane 3: with 10 mM EDTA, pH 8, Lane 4: with 0.1 mg/ml proteinase K. DNA size (bp) is shown by the scale on the left. Extracellular DNA was isolated from H460 solid tumor as described in Section 2.2.3. 43 The size range of subsequently extracted DNA was determined with agarose gel electrophoresis and compared to a sample of H460 tumor DNA extracted without the addition of DNase inhibitors (Figure 2.3). The results do not show any significant differences in the DNA size ranges between sample with or without the addition of DNase inhibitors. This provided evidences supporting that the small size of the DNA extracted were not a consequence of degradation by endogenous DNase during the extraction process. 2.3.4. Comparison of extracellular DNA levels between tumor and healthy organs The levels of extracellular DNA in the tumors and other healthy organs from SCID/Rag2 tumor-bearing mice inoculated with H460 (n=4) tumor models were determined by UV spectrophotometric analysis as described in Section 2.2.4. A significantly higher level of tumor extracellular DNA was found in comparison to the healthy organs (lungs, liver, kidney and muscle from the left thigh) (p < 0.05). The extracellular DNA levels in the healthy organs were not statistically different from one another (p < 0.05). An average level of 899 ± 163 ug DNA/g tissue was found in the tumor, while a total combined value 220 ± 133 ug DNA/g tissue was found in the healthy organs. This corresponds to a ratio of approximately 4:1 between tumor/healthy tissue extracellular DNA. The combined average A260/A280 ratio from the tumor and healthy organ samples was determined to be 1.77 + 0.11, close to the value of 1.8, which is the standard A260/A280 ratio of pure DNA. This demonstrates that 44 there was no significant contamination with proteins, RNA (A260/A280 ratio of 2.0) or phenol during the extraction process. 1200 -, 1000 A o 3 800 </> (0 -S* 600 < D) 3 400-J 200 4 if-.-''1 •' ' tumor lung liver kidney muscle Figure 2.4. Extracellular DNA levels in H460 tumors and other healthy tissues in female SCID/Rag2 tumor-bearing mice (n=4). Extracellular DNA was isolated as described in Section 2.2.3. and quantitative levels were determined spectrophotometrically by measuring UV absorbance at 260 nm. The error bars represent S.D. * denotes that the value is statistically different from other groups, where the values are not statistically different from each other, as analyzed using one way ANOVA, post hoc Scheffe test, with p < 0.05. 4 5 2.4. Chapter 2 Discussion The specific localization of extracellular DNA in solid tumors, due to the high rate of tumor cell necrosis, has been implicated by previous histological and in vivo animal studies as described in Chapter 1. The ubiquitous nature of this tumor physiology has the potential to be utilized as a specific marker to distinguish between cancerous growths and other healthy tissues with the aim of improving anticancer drug selectivity. However, direct characterization on this tumor extracellular DNA has not been performed previously. In this chapter, the physical size range and quantitative levels of tumor extracellular DNA in solid tumors were investigated using a human xenograft mouse model. An in vivo extracellular DNA extraction method was developed using a human xenograft mouse model with a cancer cell line, H460 lung carcinoma, which has been demonstrated to contain a high degree of cell necrosis. In order to isolate extracellular DNA fragments for analysis without significant contamination from intracellular genomic DNA, a least invasive procedure was taken where the various tissue samples were rinsed with physiological buffers to isolate the extracellular components in the vascular and interstitial compartments. Using this method, extracellular DNA was then extracted from the isolated mixture for detailed analysis. Extracellular DNA in the tumor was determined to be degraded fragments in the size range of 200-3000 bp. Selective localization of this DNA in the tumor has also been confirmed where the amount of extracellular DNA was found to be approximately four times higher in the tumor than other healthy tissues. These results provided 46 evidence supporting tumor extracellular DNA as a target for selective drug delivery. However, the purity of the extracellular DNA extracted may be questioned with possible contamination by intracellular genomic DNA or RNA released from cells damaged during the extraction process. Several evaluations were performed to ensure the purity of the extracellular DNA and the following observations were obtained. First, standard A260 /A280 measurements were performed and the results demonstrated the purity of the DNA extracted, without contamination from intracellular RNA or plasma proteins. Secondly, the size of the DNA fragments extracted from the tumors was determined to be 200-3000 bp, significantly smaller than the large size of intracellular genomic DNA, typically > 50 kbp in size. However, the presence of intracellular DNA may not have been detected due to the fact that during extraction, endogenous DNase in the tissue sample might have contributed to degradation of the extracted DNA, resulting in the small DNA size observed. Therefore, studies were performed with the addition of standard DNase inhibitors, EDTA and proteinase K, to the incubation buffer during the extraction procedures with the purpose of inhibiting the activity of endogenous DNase in the samples. The size range of the extracted extracellular tumor DNA was subsequently determined. The results, as described in Section 2.3.3., demonstrated that the size of extracellular tumor DNA remained consistent at 200-3000 bp, ensuring that the DNA extracted was not degraded by endogenous DNase during the extraction process and there was no significant presence of intracellular genomic DNA. 47 The quantitative level of extracellular DNA in the tumor sites was significantly higher than the level obtained from the other healthy tissues, including muscle, lungs, kidneys and liver. These results were consistent with earlier histological studies where extracellular DNA was found to selectively accumulate in tumor vasculature (68, 69). In in vivo animal studies performed by Epstein er al., antibodies with specificity toward DNA were also found to accumulate in higher levels at the tumor sites than other healthy organs (75). The elevated level of DNA fragments in tumor extracellular space has been attributed to the high rate of cell necrosis in rapidly growing solid tumors. These observations corroborate that we have successfully isolated extracellular DNA fragments from solid tumors in a human xenograft mouse model without significant contamination from intracellular genomic DNA. As described in Section 2.3.2., the extracellular DNA fragments extracted from solid tumors has a size range of 200-3000 bp, which is comparable to the size range of a commercial preparation of mechanically sheared salmon testes with a size range of 200-2000 bp. Consequently, such sheared salmon testes DNA can be used as an appropriate model of tumor extracellular DNA during the in vitro development of an optimal DNA triggered-release liposome formulation, which will be described in the next chapter. In this chapter, extracellular DNA fragments from the solid tumors were isolated and were found at significantly higher levels in the tumor than other healthy organs. These results were consistent with previous observations and 48 in support of tumor extracellular DNA as a specific target for improving anticancer drug selectivity. In the next chapter, fusogenic cationic liposomes were investigated with the aim of developing a DNA triggered release liposome formulation, targeting this elevated level of extracellular DNA in the tumor. 4 9 Chapter 3 In vitro characterization of DOTAP/DOPE/DSPE-PEG liposomes 3.1. Introduction Results from the in vivo studies of Chapter 2 demonstrated an elevated level of extracellular DNA in H460 solid tumors in comparison to other healthy tissues. In light of these results, it was postulated that a liposome formulation with the ability to triggered-release its entrapped agents after interaction with tumor extracellular DNA fragments would show selective drug release in the area of tumor growth. In this chapter, we investigated the potential of developing such a DNA-triggered release liposome formulation, utilizing the unique properties of "fusogenic" cationic liposomes. As described in Chapter 1, "fusogenic" cationic liposomes, composed of a non-bilayer lipid stabilized in a bilayer vesicle structure by a bilayer-forming cationic lipid, has been well established to associate with DNA via electrostatic interactions to form non-bilayer structures where DNA is embedded (8, 61). The formation of these structures is accompanied by liposome fusion and aggregation, and the subsequent release of the encapsulated agents. However, previous research demonstrated the instability of these liposome formulations in physiological salt buffers where salt-induced aggregation was observed (23-26). This would be an obstacle to be overcome since stable drug retention in the circulation would be required to ensure selective drug delivery to the tumors. 50 Incorporation of hydrophilic P E G polymers has been shown to reduce salt-induced aggregation in cationic liposomes and other polycationic DNA carriers (23-26). In this chapter, we investigated the potential of fusogenic cationic liposomes, composed of the non-bilayer lipid, D O P E , and the bilayer cationic lipid, DOTAP, in developing a DNA-triggered release liposome formulation targeting the extracellular DNA in the tumors. In particular, we evaluated the effect of P E G polymers on the stability and the DNA triggered release properties of these liposomes in physiological salt buffers. It is demonstrated here that the liposome composition of D O T A P / D O P E / D S P E -P E G 350 exhibits the required formulation features of an optimum DNA triggered release liposome delivery system. 51 3. 2. Materials and Methods 3.2.1. Materials All lipids were obtained from Avanti Polar Lipids (Aladaster, AL). [3H] -C H E (cholesterylhexadecyl ether) was purchased from NEN/Dupont (Mississauga, ON). [14C] - Lactose was purchased from Amersham Biosciences (UK). Salmon testes DNA was purchased from Worthington's Biochemical Corp. (Lakewood, NJ). Sephadex G-50 size exclusion gel was purchased from Sigma (St. Louis, MO) and Microcon centrifugal filters (YM-100) were obtained from Millipore (Bedford, MA). 3.2.2. Preparation of cationic liposomes Large unilamellar liposomes (DOTAP/DOPE/DSPE-PEG) of different compositions and different molecular weights of D S P E - P E G (350, 750, 2000) were prepared by the extrusion method (85). Lipids were dissolved in chloroform and dried down under a stream of N 2 gas, followed by drying under vacuum for a minimum of 4 hours. The dried lipid film is hydrated on ice with filtered H E P E S buffer saline (HBS): 25mM H E P E S and 150 mM NaCI, pH 7.5, with vigorous vortexing. The resulting liposomes were then passed 10 times at approximately 400 psi through an extrusion apparatus (Lipex Biomembranes, Vancouver, BC) containing two stacked polycarbonate filters (Whatman, Clifton, NJ) of 0.08 urn and 0.10 um pore sizes, respectively, at room temperature. Lipid concentration was determined with the use of the non-exchangeable radioactive lipid tracer 3 H - C H E . 52 3.2.3. QELS particle sizing Quasi-elastic light, scattering (QELS) was employed to assess the mean diameters of liposomes and DNA/liposome complexes, using a Nicomp Model 270 Submicron Particle Sizer (Pacific Scientific, Santa Barbara, CA) with an argon laser at a wavelength of 632.8 nm. The resulting data were fitted to a normal distribution, generating the mean, standard deviation, coefficient of variation, and chi square values. 3.2.4.14C-lactose release assays The encapsulated content release properties of the cationic liposomes were assessed using 1 4 C-lactose as an aqueous marker where 1 4C-lactose was quantified with scintillation counting. 1 4 C-lactose has been employed in previous studies to evaluate the internal trapping volume of various liposome formulations (63). 1 4 C-lactose encapsulated liposomes was prepared by passive loading. Lipids were dissolved and dried to a lipid film as described previously. The lipid film was hydrated on ice with HBS pH 7.5 containing a trace amount of 1 4 C-lactose, followed by vigorous vortexing and extrusion as described above in 3.2.2. The resulting liposomes were passed through a Sephadex G-50 column to remove the unencapsulated lactose. A non-exchangeable radioactive lipid tracer 3 H - C H E was incorporated and dual-labeling ( 3H and 1 4 C ) scintillation counting was performed to determine lipid concentration and 1 4 C-lactose concentration. DNA-triggered release was assessed after addition of DNA to liposomes at 1 mM lipid concentration in HBS, pH 7.5. Released 1 4 C-lactose was separated from the liposomes using Microcon centrifugal filters (YM-100), with molecular weight cut-off limit of 53 100,000 Daltons, centrifuged at 7800xg for 5 min and subsequently quantified by scintillation counting to determine the percentage of encapsulated 1 4 C -lactose triggered released. The limits for quantitating the amount of liposomes (labeled with 3 H-CHE) and 1 4 C-lactose by liquid scintillation counting were 0.001 mg and 0.005 nmol, respectively, which were determined by ensuring the subsequent sample readings will be > 500 counts, the established radiolabel reliability limit in our laboratory to ensure readings can be differentiated from background levels (-100 counts). 3.2.5. Statistical analysis A N O V A (analysis of variance) was performed on the results obtained in this chapter to detect differences in the data from different liposome formulations in Q E L S particle sizing and 1 4 C-lactose release studies. A post hoc Scheffe test was used to compare the different groups pairwise. Differences were considered significant at p < 0.05. 54 3.3. Results 3.3.1. Comparison between conventional and fusogenic cationic liposomes Fusogenic cationic liposomes composed of cationic lipid, DOTAP, and the Hn lipid, D O P E , have been previously reported to associate with DNA via electrostatic interaction (61). This association results in the formation of non-bilayer DNA/liposome complexes and the subsequent triggered release of the encapsulated contents. Here, we first compared the release behaviour of DOTAP/DOPE liposomes with conventional liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (Choi), a formulation previously established to have stable drug retention properties and which have no known electrostatic associations with DNA (1, 2). D O T A P / D O P E (50:50) liposomes and DSPC/Cho l (55:45) were passively encapsulated with 1 4 C-lactose as an aqueous marker, as described in Section 3.2.4. 1 4 C-lactose release assays were performed where the liposomes were incubated with 25 ug/ml salmon testes DNA in H E P E S buffered saline solution. The percentage of encapsulated lactose triggered release was measured and the results are shown in Figure 3.1 A. Only 2.0 ± 0.1 % of the entrapped lactose was released from conventional liposomes after exposure to DNA, in contrast to a significantly higher release level of 49.4 ± 5.9 % seen with the fusogenic cationic formulation (p < 0.05). The results demonstrated the unique ability of fusogenic cationic liposomes to be triggered to release their entrapped contents by DNA. 55 10 TO V O V) o o re •c V « O) 60 50 - 40 2> 30 ^ 20 O) re •«—i c <D O u. 0) 0 . 1 0 4 D S P C / C h o l D O T A P / D O P E B. 100 80 -I 0) 0) i/> o u o <u O) re c a) u a. 60 40 -J 20 D S P C / C h o l D O T A P / D O P E Figure 3.1: Comparison of DNA triggered release (A) and entrapped lactose retention properties (B) between DSPC/Chol (55:45) and DOTAP/DOPE (50:50) liposomes. Liposomes were prepared and 1 4 C-lactose assays were performed as described in Sections 3.2.2. and 3.2.4., respectively. (A) Liposomes were incubated with 25 ug/ml salmon testes DNA for 30 min at room temperature. (B) Lactose retention was evaluated 24 hours after extrusion. Experiments were performed in triplicates and the error bars represent S.D. * denotes that the value is statistically different from the other group as analyzed using one way ANOVA, post hoc Scheffe test, with p < 0.05. 56 The physical size stability of these two liposome formulations in physiological salt solution was next evaluated. After extrusion through 80 and 100 nm polycarbonate filters as described section 3.2.2., liposome sizes were assessed by particle sizing and results are presented in Table 3.1. A significantly larger particle size was observed with the DOTAP/DOPE formulation (163 ± 22 nm) over the conventional formulation (104 + 5 nm) (p < 0.05). This is consistent with the long recognized salt-induced aggregation phenomenon of cationic liposomes. Next, liposome size was evaluated in the presence of DNA (Table 3.1). After incubation with 25 ug/ml salmon testes DNA, significant increases in liposome mean diameter were observed, from 163 + 22 nm to 210 ± 4 nm. On the other hand, no significant increase was seen with the conventional formulation, confirming the unique ability of cationic liposomes to interact with DNA, inducing liposome fusion and aggregation (p < 0.05). Finally, internal aqueous maker retention was evaluated. The percentage of encapsulated 1 4 C-lactose remaining in the liposomes were determined 24 hours after extrusion. DSPC/Chol liposomes maintained stable retention with 97.6 ± 0.7 % of the encapsulated lactose remaining. However, only 53.1 ± 2.2 % was retained in the DOTAP/DOPE formulation (Figure 3.1B). The leakiness of the cationic liposomes may be related to its structural instability in salt buffers where the salt-induced aggregation of the cationic liposomes has been long recognized. In order to develop a successful DNA triggered release liposome formulation with cationic lipids, the instability issue of cationic liposomes will have to be addressed. 57 Formulation Mol % Average Mean diameter (nm)a Average mean diameter after incubation with DNA (nm) a b DSPC/Chol 55:45 104 ± 5 105 + 4 DOTAP/DOPE 50:50 163 ± 2 2 * 2 1 0 ± 4 # Table 3.1: Comparison of liposome size stability between DSPC/Cho l and DOTAP/DOPE liposomes (n=3). Liposomes were prepared and Q E L S particle sizing was performed as described in Sections 3.2.2. and 3.2.3., respectively. a Liposomes were sized with QELS 24 hours after extrusion. b Liposomes were incubated with 25 uej/ml salmon testes DNA at 1mM lipid concentration at room temperature for 30min. * denotes that the value is statistically different from the corresponding value from the other formulation in the same column as analyzed by one way ANOVA, posr hoc Scheffe test, with p < 0.05. "denotes that the value is statistically different from the average mean diameter of the same liposome formulation before incubation with DNA presented in the same row, as analyzed by one way ANOVA, post hoc Scheffe test, with p < 0.05. 3.3.2. Effect of DSPE-PEG 2000 on 14C-lactose retention properties of DOTAP/DOPE liposomes As mentioned in Chapter 1, the inclusion of PEG-lipids into cationic liposomes has been demonstrated to increase liposome structural stability in physiological salt solutions by generating a steric barrier on the liposome surfaces. Here, the effect of the incorporation of D S P E - P E G 2000 on the size stability of DOTAP/DOPE liposomes in a salt solution (HEPES buffer saline, pH 7.5) was investigated. Liposome formulations with various inclusion ratios of D S P E - P E G 2000 were prepared as described in Section 3.2.2. QELS particle sizing was performed for size determination 24 hours after extrusion (Table 3.2.). The DOTAP content was maintained at 50 mol%. Liposome salt-58 induced aggregation and fusion were reduced following the incorporation of D S P E - P E G 2000. For instance, DOTAP/DOPE liposomes without P E G had a size population of 163 ± 22 nm, whereas the incorporation of 5 mol % of D S P E - P E G 2000 led to a decrease of liposome size to 125 ± 4 nm. In addition to Q E L S size determination, 14C-lactose release assays were also performed to assess the effect of D S P E - P E G 2000 on entrapped lactose retention (Figure 3.2). In the non-pegylated formulation, only 53.1 ± 2.2 % of C14-lactose remained entrapped 24 hours after extrusion, as mentioned in the previous section. The incorporation of P E G 2000 at 5 mol% and 10 mol% substantially improved entrapped lactose retention to approximately 86.3 ± 1.6 % and 98.4 ± 0.4 %, respectively. These results demonstrated the ability of D S P E - P E G 2000 to protect DOTAP/DOPE liposomes from self-aggregation and fusion in the presence of salt and the subsequent leakage of entrapped contents. 3.3.3. Effect of DSPE-PEG 2000 on the interaction between DNA and DOTAP/DOPE liposomes and the DNA triggered release of entrapped contents Next, the effect of D S P E - P E G 2000 incorporation on DOTAP/DOPE liposome/DNA interactions was investigated. DOTAP/DOPE liposomes incorporated with various percentages of D S P E - P E G 2000 were incubated with 25 ug/ml of salmon testes DNA in H E P E S buffered saline for 30 min. Liposome size was determined subsequently with Q E L S particle sizing (Table 3.2). Increases in liposome mean diameter were observed indicating liposome/DNA aggregation and fusion. For instance, the mean diameter of 59 Formulation Mol % Average of mean diameter (nm)a Average of mean diameter after incubation with DNA (nm) a b DOTAP/DOPE 50:50 163 ± 2 2 210 + 4 # DOTAP/DOPE/ DSPE-PEG 2000 50:49:1 151 + 1 192 + 1 9 * DOTAP/DOPE/ DSPE-PEG 2000 50:48:2 133 ± 6 162 ± 1 3 # DOTAP/DOPE/ DSPE-PEG 2000 50:45:5 1 2 5 ± 4 * 130 ± 9 Table 3.2. Size stability of DOTAP/DOPE liposomes (n=3) with varying amount of D S P E - P E G 2000 incorporated. Liposomes were prepared and Q E L S particle sizing was performed as described in Sections 3.2.2. and 3.2.3., respectively. a Liposomes were sized with QELS 24 hours after extrusion. b Liposomes were incubated with 25 ug/ml salmon testes DNA at 1mM lipid concentration at room temperature for 30min. * denotes that the value is statistically different from the corresponding value from the non-pegylated DOTAP/DOPE formulation in the same column as analyzed by one way ANOVA, post hoc Scheffe test, with p < 0.05. "denotes that the value is statistically different from the average mean diameter of the same liposome formulation before incubation with DNA presented in the same row, while value not denoted in the same column is not statistically different from the corresponding value, as analyzed by one way ANOVA, post hoc Scheffe test, with p < 0.05. 60 100 -, c o c 2 80-^ a> w o u JO a> « (/) a ra o c a> O re c a u a> Q. D O T A P / D O P E 50:50 D O T A P / D O P E / D S P E - P E G 2 0 0 0 50:48:2 14, D O T A P / D O P E / D S P E - P E G 2 0 0 0 50:45:5 D O T A P / D O P E / D S P E - P E G 2 0 0 0 50:40:10 Figure 3.2. Percentage of encapsulated "*C lactose retention in DOTAP/DOPE liposomes (n=3) with varying amounts of D S P E - P E G 2000 incorporated. Liposomes were prepared and 14C-lactose assays were performed as described in Section 3.2.2. and 3.2.4., respectively. The percentages of encapsulated lactose retention were determined 24 hours after extrusion in HBS, pH 7.5. Error bars represent S.D. * denotes that the value is statistically different from other groups, as analyzed using one way ANOVA, post hoc Scheffe test, with p < 0.05. 61 non-pegylated DOTAP/DOPE liposomes increased from 163 ± 22 nm to 210 ± 4 nm following incubation with DNA, as mentioned in Section 3.3.1. However, incorporation of D S P E - P E G 2000 inhibited this DNA-induced liposome aggregation. The mean diameter of DOTAP/DOPE liposomes with 5 mol % P E G content was 130 ± 9 nm, not significantly different from 125 ± 4 nm before the introduction of DNA (p < 0.05). DNA triggered release assays were performed to determine the effect of D S P E - P E G 2000 on DNA triggered release of entrapped contents from DOTAP/DOPE liposomes, as described in Section 3.2.4. Non-pegylated DOTAP/DOPE liposomes and DOTAP/DOPE liposomes with 5 mol % of D S P E - P E G 2000 were incubated with varying amounts of salmon testes DNA in H E P E S buffered saline. The results are shown in Figure 3.3. The level of DNA triggered release from the liposomes is directly proportional to the amount of DNA present. In the presence of 25 ug/ml DNA, significantly less entrapped lactose was triggered to release from DOTAP/DOPE liposomes with 5 mol% of D S P E - P E G 2000 included than the non-pegylated formulation. For instance, 49.4 ± 5.9 % of entrapped lactose was released from non-pegylated DOTAP/DOPE liposomes, whereas the incorporation of 5 mol% P E G 2000 led to a decrease in triggered lactose release to 21.3 ± 1.2 %. Subsequently, 25 ug/ml DNA was used to compare the DNA triggered release capability between different liposome formulations. 62 Figure 3.3. Percentage of DNA triggered encapsulated lactose release from DOTAP/DOPE liposomes (n=3) incubated with varying salmon testes DNA. Liposomes were prepared and 14C-lactose assays were performed as described in Section 3.2.2. and 3.2.4., respectively. The percentages of DNA triggered encapsulated lactose release were determined after incubation with salmon testes DNA for 30 min at RT in HBS, pH 7.5. Error bars represent S.D. * denotes that the value is statistically different from the corresponding value from the other group, as analyzed using two way ANOVA, post hoc Scheffe test, with p < 0.05. 63 The effect of D S P E - P E G 2000 on DNA triggered release from DOTAP/DOPE liposomes was further compared between formulations with different mol % of D S P E - P E G 2000 (Fig. 3.4). Consistent with the above results, incorporation of D S P E - P E G 2000 significantly decreased entrapped lactose release in the presence of DNA. The decrease in DNA-induced aggregation and DNA-triggered release by D S P E - P E G 2000 is consistent with D S P E - P E G generating a steric barrier over the charged surface of liposomes. This steric barrier reduces interaction between DNA and cationic liposomes, leading to inhibition in DNA triggered bilayer destabilization into non-bilayer structures, and the subsequent release of entrapped markers. 3.3.4. Effect of varying the molecular weights of DSPE-PEG on DOTAP/DOPE liposome stability in an ionic medium The results thus far have demonstrated the ability of D S P E - P E G 2000 to effectively reduce DOTAP/DOPE liposome salt-induced aggregation and leakage of encapsulated markers. However, liposome/DNA interaction and DNA-induced triggered release is also inhibited. A successful triggered release formulation would require stability in physiological buffers while also maintaining substantial effectiveness to interact with DNA fragments. Therefore, we investigated the effect of varying the molecular weights of the polymer moiety of D S P E - P E G in the formulation, on size stability and DNA triggered release properties. 64 D O T A P / D O P E 50:50 D O T A P / D O P E / D S P E - P E G 2 0 0 0 50:48:2 D O T A P / D O P E / D S P E - P E G 2 0 0 0 50:45:5 D O T A P / D O P E / D S P E - P E G 2 0 0 0 50:40:10 Figure 3.4. Percentage of DNA triggered encapsulated lactose release from DOTAP/DOPE liposomes (n=3) with varying amounts D S P E - P E G 2000. Liposomes were prepared and 14C-lactose assays were performed as described in Section 3.2.2. and 3.2.4., respectively. The percentages of encapsulated lactose triggered release were determined after incubation with 25 ug/ml salmon testes DNA for 30 min at RT in HBS, pH 7.5. Error bars represent S.D. Percentages of lactose release from the controls not treated with DNA were deducted from the results. * denotes that the value is statistically different from other groups, where the values are not statistically different from each other, as analyzed using one way ANOVA, post hoc Scheffe test, with p < 0.05. 65 Here, DOTAP/DOPE liposomes incorporated with D S P E - P E G of different molecular weights at 5 mol % were prepared as described in Section 3.2.2. Liposome size was determined 24 hours after extrusion (Table 3.3). Incorporation of D S P E - P E G with smaller molecular weights (350,750) maintained liposome size stability comparable to D S P E - P E G 2000, where inclusion of D S P E - P E G 2000 resulted in liposome mean diameter of 125 ± 4 nm, and D S P E - P E G 350 and 750 led to mean diameters of 127 ± 4 nm and 142 ± 12 nm, respectively. Next, the effect of varying the molecular weight of D S P E - P E G on liposome retention was investigated. DOTAP/DOPE liposomes were encapsulated with 1 4 C-lactose and 1 4 C-lactose release assays were performed as described in Section 3.2.4. Incorporation of smaller molecular weight D S P E - P E G (polymer weights of 350 and 750) maintained lactose retention at 86.8 ± 3.2 % and 84.3 ± 3.8 %, respectively, while incorporation of D S P E - P E G 2000 resulted in lactose retention level of a comparable 86.3 ± 1.6 % (p < 0.05). Increasing the D S P E - P E G 350 content to 15% further increased lactose retention to 96.5 ± 0.8 %, confirming the ability of smaller molecular weights of D S P E - P E G to maintain size stability and reduce encapsulated content leakage in a salt solution. 66 Formulation Mol % Average of mean diameter (nm)a Average of mean diameter after incubation with DNA (nm) a b DOTAP/DOPE 50:50 163 + 22* 2 1 0 ± 4 # DOTAP/DOPE/ DSPE-PEG 2000 50:45:5 125 ± 4 1 3 0 ± 9 DOTAP/DOPE/ DSPE-PEG 750 50:45:5 142 ± 1 2 241 ± 33 # DOTAP/DOPE/ DSPE-PEG 350 50:45:5 1 2 7 ± 4 2 0 6 ± 1 5 # DOTAP/DOPE/ DSPE-PEG 350 50:35:15 115 ± 7 208 ± 2 4 # Table 3.3. Size stability of DOTAP/DOPE liposomes (n=3) with varying molecular weights of D S P E - P E G incorporated. Liposomes were prepared and Q E L S particle sizing was performed as described in Sections 3.2.2. and 3.2.3., respectively. a Liposomes were sized with QELS 24 hours after extrusion. b Liposomes were incubated with 25 ug/ml salmon testes DNA at 1mM lipid concentration at room temperature for 30min. * denotes that the value is statistically different from the corresponding values from the other formulations in the same column as analyzed by one way ANOVA, post hoc Scheffe test, with p < 0.05. * denotes that the value is statistically different from the average mean diameter of the same liposome formulation before incubation with DNA presented in the same row, while value not denoted in the same column is not statistically different from the corresponding value, as analyzed by one way ANOVA, post hoc Scheffe test, with p < 0.05. 67 100 - , £ 80 -J 60 4 40 -\ 20 A DOTAP/DOPE DOTAP/DOPE/ DOTAP/DOPE/ DOTAP/DOPE/ DOTAP/DOPE/ 50:50 DSPE-PEG 2000 DSPE-PEG 750 DSPE-PEG 350 DSPE-PEG 350 50:45:5 50:45:5 50:45:5 50:35:15 Figure 3.5. Percentage of encapsulated 1 4 C lactose retention in DOTAP/DOPE liposomes (n=3) with varying molecular weights of D S P E -P E G . Liposomes were prepared and 14C-lactose assays were performed as described in Section 3.2.2. and 3.2.4., respectively. The percentages of encapsulated lactose retention were determined 24 hours after extrusion in HBS, pH 7.5. Error bars represent S.D. * denotes that the value is statistically different from other groups, where the values are not statistically different from each other, as analyzed using one way ANOVA, post hoc Scheffe test, with p < 0.05. 68 3.3.5. Effect of varying the molecular weights of DSPE-PEG on DNA induced triggered release properties of DOTAP/DOPE liposomes The effect of varying D S P E - P E G molecular weight on liposome/DNA interactions and DNA triggered release was investigated. Here, liposomes with varying molecular weights of D S P E - P E G (at 5 mol % inclusion) were incubated with salmon testes DNA in H E P E S buffered saline. Liposome size was determined using Q E L S particle sizing to evaluate DNA/liposome aggregation (Table 3.3). Liposomes with shorter polymer lengths of D S P E -P E G (350, 750) exhibited DNA-induced size aggregation from 127 ± 4 nm to 206 ± 15 nm, and 142 ± 12 nm to 241 ± 33, respectively, comparable to the levels observed with non-pegylated DOTAP/DOPE liposomes (p < 0.05). This is in contrast to the inhibition observed with D S P E - P E G 2000, as described earlier. This demonstrated that inclusion of PEG-lipids with smaller molecular weights do not reduce liposome interaction with DNA fragments. Next, 1 4 C-lactose release assays were performed to study the DNA triggered release properties of these liposome formulations. Liposomes containing 5 mol % of D S P E - P E G with varying molecular weights were incubated with 25 ug/ml of salmon testes DNA in H E P E S buffer saline at pH 7.5 (Figure 3.6). Incorporation of D S P E - P E G 2000 reduced the percentage of triggered lactose release to 21.3 ± 1.2 %, compared to 49.4 ± 5 . 9 % in the non-pegylated formulation. On the other hand, incorporation of D S P E - P E G of 69 D O T A P / D O P E D O T A P / D O P E / DOTAP/DOPE/ D O T A P / D O P E / D O T A P / D O P E / 50:50 D S P E - P E G 2000 D S P E - P E G 750 D S P E - P E G 350 D S P E - P E G 350 50:45:5 50:45:5 50:45:5 50:35:15 Figure 3.6. Percentage of DNA triggered encapsulated lactose release from DOTAP/DOPE liposomes (n=3) with varying molecular weights of D S P E -P E G . Liposomes were prepared and 14C-lactose assays were performed as described in Section 3.2.2. and 3.2.4., respectively. The percentages of encapsulated lactose triggered release were determined after incubation with 25 ug/ml salmon testes DNA for 30 min at RT in HBS, pH 7.5. Error bars represent S.D. Percentages of lactose release from the controls not treated with DNA were deducted from the results. Values denoted by * statistically different from other non-denoted groups and not statistically different from each other, as analyzed using one way ANOVA, post hoc Scheffe test, with p <0.05. 70 shorter polymer lengths (350 and 750) resulted in higher levels of DNA triggered release. In the case of P E G 350, 56.0 ± 5.7 % of encapsulated lactose was released, which compares favorably with the non-pegylated formulation (p < 0.05). Furthermore, DNA triggered release level consistently remained high at a level of 40.0 ± 1.0 %, even when the D S P E - P E G 350 content was raised to 15 mol %. These results reveal that the incorporation of D S P E - P E G 350 satisfies the requirements for an optimum DNA triggered release formulation; where the liposome stability in physiological salt solution is maintained without inhibiting DNA-induced triggered release of the entrapped agents. 3.3.6. DNA triggered release following interaction with tumor extracellular DNA In the previous experiments, salmon testes DNA was used as an experimental model for tumor extracellular DNA to evaluate the DNA-triggered release capability of various liposomes formulations. Although this commercial preparation of salmon testes DNA has been shown to possess comparable size range as tumor extracted DNA fragments, as mentioned in Chapter 2, other differences may exist which may influence its association with liposomes. To ensure that salmon testes DNA is an appropriate experimental model for tumor extracellular DNA and that the behaviour observed so far with our fusogenic cationic liposome formulations is an appropriate estimation of in vivo behaviour in the tumors, our optimum formulation composed of D O T A P / D O P E / D S P E - P E G 350, was incubated with 71 extracted tumor extracellular DNA isolated from H460 tumor bearing mice. The results are compared with the DNA triggered release levels obtained earlier with salmon testes DNA. Tumor extracellular DNA was extracted as described in Section 2.2.2. 1 4 C-lactose encapsulated in D O T A P / D O P E / D S P E - P E G liposomes (50:45:5) was incubated with 25 ug/ml salmon testes DNA or tumor extracted DNA. The subsequent DNA-triggered lactose release was evaluated. The results are presented in Figure 3.7. The levels of DNA triggered lactose release was not statistically different between the two groups with 46.2 ± 5.7 % with salmon testes DNA and 39.6 ± 5.4 % with tumor extracted extracellular DNA (p < 0.05). The results provide evidence that salmon testes DNA is an appropriate model for tumor extracellular DNA, and that D O T A P / D O P E / D S P E - P E G 350 liposomes exhibit favorable DNA-triggered release of encapsulated contents following interaction with tumor extracellular DNA. 72 50 A ~ 40 -J 30 4 20 -J « 10 J Salmon testes DNA Tumor extracted extracellular DNA Figure 3.7. Comparison of DNA triggered lactose release between D O T A P / D O P E / D S P E - P E G 350 (50:45:5) liposomes incubated with salmon testes DNA or H460 tumor extracted extracellular DNA (n=3). Liposomes were incubated in H E S P E S buffered saline, pH 7.5, with 25 ug/ml of commercially available salmon testes DNA or 25 ug/ml of tumor extracellular DNA extracted from H460 tumor bearing mice, as described in Section 2.2.2. Error bars represent S.D. The two levels of triggered lactose release was found to be not statistically different using one-way ANOVA, post hoc Scheffe test, p < 0.05. 73 3.4. Chapter 3 Discussion Fusogenic cationic liposomes, composed of a bilayer cationic lipid and a non-bilayer "helper" lipid, have been extensively investigated for the purpose of gene delivery, as described in Chapter 1. Bilayer cationic liposomes electrostatically associate with negatively charged DNA fragments to form non-bilayer lipid/DNA complexes favorable for gene transfection (18, 28, 60). From previous observations in our laboratory, release of internally entrapped lactose from fusogenic cationic liposomes was observed following the introduction of DNA and the formation of DNA/liposome complexes (63). In conjunction with the observations that there is a higher level of extracellular DNA in solid tumors, compared to other healthy tissues, we recognized the potential of fusogenic cationic liposomes as delivery vehicles for anticancer agents with selective triggered release at the tumor sites. An optimum formulation of this triggered release delivery system would be required to exhibit stable drug retention properties in the general circulation as well as the ability to interact with DNA fragments. However, cationic liposomes have been shown to be unstable with salt-induced aggregations in salt solutions including physiological buffers. It has been reported that the inclusion of the hydrophilic polymers, P E G , on the surface of liposomes can reduce these aggregations (23-26). In this chapter, we tested different formulations of DOTAP/DOPE liposomes with different molecular weights of D S P E - P E G incorporated with the aim of developing an optimum DNA triggered-release formulation. 74 We first investigated the effect of D S P E - P E G 2000, the most commonly employed PEG-lipid, on the behaviour of D O T A P / D O P E liposomes in in vitro studies. The physical size stability of these liposomes was determined. Incorporation of D S P E - P E G 2000 reduced salt-induced aggregations and improved the level of entrapped lactose retention in physiological salt buffers. This is consistent with previous results where P E G -polymers have been shown to prevent salt-induced aggregations in polycation/DNA complexes as well as cationic liposomes containing various cationic lipids, such as DODAC and DOTMA (61, 86). However, our results also demonstrated substantial inhibition of this liposome/DNA interaction and the subsequent DNA-induced release of the encapsulated markers. This is not surprising as long-chained P E G polymers have been reported to reduce cationic lipid/DNA association as well as the rate of gene transfection with DNA/polycation complexes (86). This effect is generally attributed to the steric hindrance generated by long-chained P E G polymers, such as P E G 2000. In light of these results, we investigated the effect of varying the polymer length of P E G , using D S P E - P E G lipids with smaller molecular weights (PEG 350 and P E G 750). Here, we found that both D S P E - P E G 350 and D S P E - P E G 750 exhibited similar effectiveness as D S P E - P E G 2000 in preventing salt-induced aggregation and improving high retention properties, while also showing significantly less inhibition of DNA/liposome interactions. In the case of D S P E - P E G 350, the level of DNA-triggered release was maintained at a comparable level as non-pegylated DOTAP/DOPE liposomes. Similar results have been previously reported where the surface 75 charge and transfection efficiency of polycationic gene carriers were found to be inversely related to the molecular weights of P E G polymers incorporated (23). These results can be explained by the difference in the repulsive barriers generated by these PEG-lipids of different polymer lengths. Physical characterization on D S P E - P E G lipids revealed distinct structural behaviour between P E G 350 and P E G 2000 polymers. D S P E - P E G 350 with the shorter side chain form bilayer when hydrated in aqueous medium, while D S P E - P E G 2000 forms micelles (23). In terms of the molecular shape theory as described in Chapter 1, these observations correspond to D S P E - P E G 350 exhibiting a relatively "cylindrical" shape and D S P E - P E G 2000 a relatively "inverted cone" shape where the effective head group including the P E G side chain is substantially larger than the corresponding section in D S P E - P E G 350. It has also been demonstrated that the range and magnitude of the steric repulsive force generated by D S P E - P E G lipids on the liposome surface are strong functions of the molecular weight of the P E G side chain (50). In the case of D S P E - P E G 2000 incorporated into DOTAP/DOPE liposomes, the P E G side chains drastically reduce association with DNA fragments by "shielding" a large area of the positively charged liposome surface. In combination with their long range of extension, P E G 2000 inhibits DNA-induced release of encapsulated contents while improving liposome stability in physiological buffers. In the case of D S P E - P E G 350, the short range repulsive force is effective in preventing salt-induced aggregation with 76 the extension of the P E G polymers preventing apposing liposome surfaces from being "brought together" to close proximity, hence decreasing the occurrence of subsequent salt-induced membrane fusion and aggregation. However, as mentioned earlier with the molecular shape theory, D S P E - P E G 350 has a substantially smaller or "less bulky" P E G side chain, therefore, the liposome surface area being "shielded" is significantly less than P E G 2000. This leads to an increase in the accessibility of DNA fragments to associate with the liposome surface where DNA can act as a link between neighboring liposomes to promote liposome fusion, the formation of non-bilayer DNA/liposome structures and the subsequent release of encapsulated markers. In this chapter, we successfully developed an optimum DNA triggered release formulation, composed of the cationic lipid, DOTAP, the fusogenic lipid, D O P E and the PEG-lipid, D S P E - P E G 350. This fusogenic cationic formulation exhibited optimal properties for a tumor selective triggered release delivery system, namely, stability in physiological buffers combined with the ability to be triggered release of the entrapped markers in the presence of tumor extracellular DNA fragments, the tumor specific target in our studies. In the following chapter, the in vivo distribution, tumor targeting effectiveness and the specific tumor release properties of this DNA-triggered release liposome formulation will be investigated in a human xenograft mouse model. 77 Chapter 4 In vivo characterization of DOTAP/DOPE/DSPE-PEG liposomes 4.1. Introduction In this chapter, we evaluated the in vivo distribution as well as the tumor accumulation behaviour of the optimum cationic DNA triggered release liposome formulation developed previously in Chapter 3. As described in Chapter 1, liposomal delivery of anticancer agents is favorable due to the ability of liposomes to passively accumulate in the solid tumors via the abnormal fenestrations in the tumor vasculature. Because of this, long circulating liposome formulations are preferred to increase liposomal drug exposure to the disease sites. However, one of the major disadvantages of employing cationic liposomes in drug and gene delivery has been its rapid elimination from the circulation via the M P S (87, 88). Inclusion of hydrophilic P E G polymers, usually at 5 - 8 mol % of D S P E -P E G 2000 on neutral liposome surface, has been well established to reduce M P S recognition. The P E G polymers act as a steric barrier to reduce plasma protein binding and cellular uptake. Here, we investigated the effect of D S P E -P E G 350 on cationic liposome circulation longevity and passive tumor targeting in a human xenograft mouse model. We compared the results with the in vivo behaviour of the conventional liposome formulation, DSPC/Chol (55:45), which has been well established in preclinical and clinical studies to exhibit favorable prolonged circulation time and effective tumor selective targeting. 78 Finally, we evaluated the in vivo selective triggered release behaviour of our liposome formulation in the tumor. The results here demonstrated a prolonged circulation lifetime as well as effective tumor targeting for the liposome formulation, D O T A P / D O P E / D S P E - P E G 350 (50:35:15), in conjunction with selective encapsulated marker release in the tumor, supporting further research of this DNA triggered release formulation as a carrier of anticancer therapeutic agents. 79 4.2. Materials and methods 4.2.1. Materials All lipids were purchased from Avanti Polar Lipids (Alabaster, AL). The 3 H - C H E was obtained from NEN Life Sciences Products (Oakville, ON). 1 4 C -Lactose was purchased from. Sephadex G-50 and Sepharose CL-4B size exclusion gel were purchased from Sigma (St. Louis, MO). 4.2.2. Preparation of various liposomes The preparation of large unilamellar cationic liposomes with different compositions of DOTAP, DOPE, and D S P E - P E G 350 followed the procedures described in Section 2.2.2. 1 4 C-lactose encapsulated cationic liposomes were prepared as described in Section 3.2.3. DSPC/Cho l (55:45) liposomes were prepared according to the following procedure. Lipids were dissolved in chloroform and dried under a stream of nitrogen gas. The resulting film was placed under vacuum for > 4 hours. The lipid film was hydrated in HBS pH 7.5 at 65°C with vigorous vortexing. The resulting preparation was frozen and thawed 5 times prior to extrusion 10 times through 2 stacked polycarbonate filters of 0.08 um and 0.10 um, respectively, with an extrusion apparatus (Lipex Biomembranes, Vancouver, BC) at 65°C. A trace amount of the non-exchangeable lipid 3H-CHE was included in the formulation as a radioactive marker. 1 4 C-lactose encapsulated DSPC/Cho l liposomes were prepared similarly, with the hydrating buffer including a trace amount of 1 4 C-lactose for passive loading. The extruded preparation was subsequently passed through a 20 cm Sephadex G-50 column to remove the unencapsulated 1 4 C-lactose. 80 4.2.3. Plasma elimination and tissue distribution of various formulations of cationic liposomes in non-tumor bearing mice All of the in vivo studies were completed following protocols approved by the University of British Columbia's Animal Care Committee. These studies met or exceeded the current guidelines of the Canadian Council of Animal Care. Female Balb-C mice (BC Cancer Agency, Vancouver, BC) of 20 - 22 g in weight were injected with 100 mg/kg of 3 H - C H E labelled cationic liposomes via the lateral tail vein with a single bolus injection volume of 200 uL. At various times after liposome injection (t = 1, 2, 4, 8, 24 h), mice were terminated by CO2 asphyxiation. Blood was collected by cardiac puncture, and was placed into EDTA-coated microtainer tubes (Becton-Dickinson). After centrifuging the blood samples at 4°C for 15 min at 3000 rpm, plasma was isolated and 100 uL aliquots were obtained and counted in 5 mL of scintillation fluid. The limit of lipid quantitation in this case was 1 ug per ml plasma, which was determined by ensuring the subsequent sample readings will be >500 counts, the established radiolabel reliability limit in our laboratory to ensure readings can be differentiated from background levels (-100 counts). Spleen, liver and lung were also harvested from each time group to determine liposome distribution in these tissues. Briefly, 0.5 mL Solvable (Packard Bioscience Co.) was added to whole organs (spleen and lungs) and liver homogenate (50% w/v), and the mixture was incubated overnight at 50°C. After cooling to room temperature, 50 uL of 200 mM EDTA, 200 uL of 30% hydrogen peroxide and 25 uL of 10 N HCI were added, and the mixture was incubated at room temperature for -1 hour. 0.5 mL of scintillation fluid 81 was added and the mixture was counted 24 h later. The limit of lipid quantitation in tissues was 1 ug. 4.2.4. Comparison of plasma elimination, tissue distribution and encapsulated lactose release between DNA-triggered release cationic liposomes and DSPC/Chol liposomes in tumor bearing mice Female SCID/Rag2 mice (BC Cancer Agency, Vancouver, BC) of 20 -22 g in weight were inoculated unilaterally with 1 x 10 6 H460 cells subcutaneously in the back. When the weight of the tumors reached ~ 0.5 g, the mice were injected with 100 mg/kg of 3 H - C H E labeled and 1 4C-lactose encapsulated D O T A P / D O P E / D S P E - P E G 350 (50/35/15) liposomes or DSPC/Chol (55/45) liposomes via the lateral tail vein with a single bolus injection volume of 200 uL. At various times after liposome injection (t = 1, 2, 4, 8, 24, 48 h), mice were terminated by C 0 2 asphyxiation. Blood and other tissues (tumors, lungs, liver, spleen) were collected and processed as described in Section 4.2.3. Samples were counted by 3 H / 1 4 C dual scintillation counting to determine 3 H / 1 4 C ratios. 4.2.5. In vivo recovery and analysis of injected liposomes Female SCID/Rag2 mice with H460 tumors were injected with 3 H - C H E labeled and 1 4 C-lactose encapsulated liposomes ( D O T A P / D O P E / D S P E - P E G 350 or DSCP/Chol) as described in Section 4.2.4. One hour after injection, blood was collected into EDTA-coated microtainers. Plasma was isolated as described in Section 4.2.4. and was applied to a 1.0 cm x 50 cm Sepharose CL-4B gel column, eluted with HBS pH 7.5 buffer. The use of column chromatography to isolate injected liposomes from plasma components has 82 been previously employed in various studies (103). Column fractions were analyzed for radioactivity to determine the fractions containing the liposomes. The two fractions with the highest radioactivity were pooled and DNA triggered release was assessed after addition of 25 ug/ml salmon testes DNA. The released 1 4 C-lactose was separated from the liposomes using Microcon centrifugal filters (YM-100) centrifuged at 7800xg for 5 min and quantitated by scintillation counting. 4.2.6. Statistical analysis A N O V A (analysis of variance) was performed on the results obtained after administration of the various liposomal formulations. Common time points were compared using the post hoc comparison of means, Scheffe test. Differences were considered significant at p < 0.05. In Section 4.3.2.2., plasma area under the curve (AUC) analysis from plasma-time curves was performed using non-compartmental analysis WinNonlin software (version 1.5; Pharsight Corp., Mountain View, CA). First order elimination was assumed, and three to four time points were used in the estimation of the terminal end phase. Mean tumor A U C values from tumor accumulation-time curves were determined using the trapezoidal rule. 83 4.3. Results 4.3.1. Effect of PEG-lipids on the plasma removal and the tissue distribution of DOTAP/DOPE/DSPE-PEG 350 liposomes in non-tumor bearing mice 4.3.1.1. Plasma removal Cationic liposomes are rapidly eliminated from the circulation, a phenomenon believed to be due to the extensive uptake of positively charged lipids by the M P S . Here, DOTAP/DOPE cationic liposomes were injected intravenously into Balb-c non-tumor bearing mice for investigation. As presented in Figure 4.1, the rapid plasma removal of DOTAP/DOPE liposomes was consistent with this phenomenon, where only 0.13 ± 0.03 mg/ml -of liposomes remained in circulation one hour after injection, corresponding to ~ 4.8 % of the injected dose. As described earlier in Chapter 1, it is widely accepted that the incorporation of 5 - 8 mol % of D S P E - P E G 2000 into conventional liposomes can extend circulation longevity by reducing liposome interaction with plasma proteins. However, although the inclusion of 5 mol% of D S P E - P E G 2000 was able to promote liposome stability, it also resulted in inhibition of liposome/DNA interaction and subsequent DNA triggered release. Therefore D S P E - P E G 2000 was not ideal in a DNA-triggered release liposome formulation. On the other hand, as described in the previous chapter, incorporation of D S P E - P E G 350 was shown to promote stability as well as maintaining significant DNA triggered release in physiological buffer, and thus the plasma removal of DOTAP/DOPE liposomes with different amounts of D S P E - P E G 350 included was investigated. 84 * - • - D O T A P / D O P E 50:50 - • - • D O T A P / D O P E / D S P E - P E G 350 50:45:5 -- • - - D O T A P / D O P E / D S P E - P E G 350 50:35:15 1 1 1 ' I 1 I 10 15 20 25 time after injection (hours) Figure 4.1: Plasma removal profiles for DOTAP/DOPE liposomes with varying amounts of D S P E - P E G 350. Liposomes were injected as a single 100 mg/kg IV dose into Balb-c mice via the lateral tail vein. Three mice were used for each time point with the error bars representing S.D. * denotes that the value is statistically different from the corresponding value from the non-pegylated formulation, as analyzed using two way ANOVA, post hoc Scheffe test, with p < 0.05. 85 As presented in Figure 3.1, the inclusion of 5 mol% of D S P E - P E G 350 was not effective in prolonging liposome circulation longevity. This is reflected by the low plasma lipid concentration of 0.27 ± 0.06 mg/ml at one hour after injection. Increasing the D S P E - P E G 350 content to 15 mol%, however, was able to increase the circulation longevity of the DOTAP/DOPE liposomes, with a lipid concentration of 0.94 ± 0.09 mg/ml one hour after injection, representing ~ 36 % of the injected dose, which was significantly higher than the corresponding levels exhibited by the non-pegylated formulation or with 5 mol % P E G 350 included (p < 0.05). 4.3.1.2. Tissue distribution The lipid concentrations of different liposome formulations in the liver and spleen 24 hours post injection are shown in Table 4.1. The corresponding lipid levels in the these organs were found to be similar among the three liposome formulations with varying amount of D S P E - P E G 350 included, with concentrations of approximately 1.2 and 1.5 mg per g tissue in the liver and spleen, respectively (p < 0.05). A high level of lipid accumulation was observed in these major M P S organs, accounting for a combined total of approximately 67% of the injected dose. 8 6 Liposomes mg lipid per g tissue +/- S.D. liver spleen DOTAP/DOPE (50:50) 1.1+0.1 1.5 ±0.2 (51%) (7%) DOTAP/DOPE/DSPE-PEG350 (50:45:5) 1.1 ±0 .2 1.3 ±0.6 (57%) (6%) DOTAP/DOPE/DSPE-PEG350 (50:35:15) 1.27 ±0.03 1.60 ±0.05 (71%) (8%) Table 4.1: Tissue distribution of D O T A P / D O P E / D S P E - P E G 350 and liposomes with varying amounts of D S P E - P E G 350 incorporated 24 hours post injection in H460 tumor bearing mice. Data represent the averages ± S.D. of three mice. The injected lipid dose was 100 mg/ml as a single IV bolus. The amount of lipid per gram tissue was determined as described in Section 4.2.4. Shown in parentheses are the percent injected doses recovered in each organ. Corresponding values from the different liposomes formulations were found to be not statistically different from each other, as analyzed by one-way ANOVA, post hoc Scheffe test, p < 0.05. 87 4.3.2. Analysis of in vivo distribution and tumor triggered release of DOTAP/DOPE/DSPE-PEG 350 liposomes in tumor bearing mice 4.3.2.1. Plasma removal The circulation longevity of DOTAP/DOPE liposomes containing 15 mol % of D S P E - P E G 350 was studied in SCID/Rag2 mice bearing H460 tumors. The results were compared with the plasma removal profile of conventional DSPC/Cho l (55:45) liposomes, which have been shown to possess favorable prolonged circulation lifetime and tumor targeting in previous preclinical and clinical studies. As presented in Figure 4.2, the plasma removal of D O T A P / D O P E / D S P E - P E G 350 liposomes follows a similar trend as the conventional DSPC/Chol liposomes, where the two trends were found to be not statistically different from one another (p < 0.05). One hour after injection, 0.64 ± 0.03 mg/ml of the cationic formulation remained in circulation, compared to 1.3 ± 0.3 mg/ml for conventional liposomes, corresponding to 24 ± 1 % and 52 ± 10 % of the respective injected doses. The corresponding plasma area under the curve values over 48 hours post injection (AUCo-48h) are presented in Table 4.2. The plasma AUCo-48h value of the D O T A P / D O P E / D S P E - P E G 350 liposomes was estimated to be 8.95 mg«h/ml, which is approximately 2.6X lower than 23.5 mg»h/ml, the corresponding value from the conventional liposomes. 4.3.2.2. Liposome accumulation in tumor and various other organs The tumor accumulation of D O T A P / D O P E / D S P E - P E G 350 (50:35:15) liposomes was determined over a 48-hour time period post injection and the results are shown in Figure 4.3. The amount of D O T A P / D O P E / D S P E - P E G 88 Figure 4.2: Plasma removal profiles for D O T A P / D O P E / D S P E - P E G 350 and DSPC/Cho l liposomes. Liposomes were injected as a single 100 mg/kg IV dose into H460 tumor bearing SCID/Rag2 mice via lateral tail vein. Three mice (20 - 22 g) were used for each time point with the error bars representing S.D. Corresponding values from the different liposome formulations were found to be not statistically different from each other, as analyzed by two-way ANOVA, post hoc Scheffe test, p < 0.05. 89 DSPC/Chol DOTAP/DOPE/DSPE-PEG 350 (55:45) (50:35:15) AUC 0 - 4 8 h a ' D (mg*h/ml) 23.5 8.95 R2 0.9167 0.9944 Corr (x:y) -0.9574 -0.9972 Table 4.2: plasma area under the curve values over 48 hours post injection (AUC0-48h) of cationic D O T A P / D O P E / D S P E - P E G 350 and conventional DSPC/Chol liposomes after i.v. injection into H460 tumor bearing mice. a AUCo-48h was calculated from values presented on Figure 4.3 using non-compartmental analysis and linear trapezoidal rule from software WinNonlin version 1.5. Three to four time points were used for the estimation of the terminal elimination phase. In the calculations, the dose was entered as an IV bolus with no lag time and first order elimination was assumed. Uniform weighting was used. b AUCo-48h, area under the curve from time of dosing (t = 0 h) up to the last measured concentration (t = 48 h); R2, goodness of fit statistic for the terminal elimination phase; Corr (x:y) correlation between time (X) and log concentration (Y) for the points used in the estimation of the first order rate constant associated with the terminal portion of the plasma concentration-time curve via linear regression. 90 0 . 1 4 - , 0 . 1 2 4 0.10 -1 o E ~ 0.08-1 S 0 0 6 - | E 0 .04 4 0 .02 4 0 . 0 0 DOTAP/DOPE/DSPE-PEg 350 55:35:15 DSPC/Chol 55:45 (1.2%) I (1.2%) (0.5%) (0.6%) 1 0 2 0 3 0 times after injection (hours) 4 0 -1 5 0 Figure 4.3: Tumor Accumulation of D O T A P / D O P E / D S P E - P E G 350 and DSPC/Cho l liposomes in H460 tumor bearing SCID/Rag2 mice over a 48-hour period. The lipid dose injected as single IV bolus of 100 mg/kg, and the mice weighed 20 - 22 g. Each data point represents the mean from three mice, with the error bars representing S.D. Shown in parentheses are the corresponding percentages of the injected doses. Corresponding values from the different liposome formulations were found to be not statistically different from each other, as analyzed by two-way ANOVA, post hoc Scheffe test, p < 0.05. 91 350 liposomes accumulated in the tumors 48 hours post injection was determined to be 0.040 ± 0.05 mg/g tumor, compared to 0.10 ± 0.01 mg/g tumor for the conventional liposomes (0.5% and 1.2% of total injected dose, respectively). The corresponding tumor AUC 0 -48h values are presented in Table 4.3. The tumor AUC 0 - 4 8h value of the D O T A P / D O P E / D S P E - P E G 350 formulation was found to be 1.58 mg»h/ml, approximately 2.5X lower than 3.96 mg»h/ml, the AUC 0 -48h value of the DSPC/Chol formulation. The tumor targeting efficiency (TE) factor, which is defined as the area under the plasma concentration-time curve from 0 to 48 hours (AUC 0-48h) in the tumor divided by AUCo-48h in the plasma, was calculated to evaluate the passive tumor targeting ability of this liposome formulation. This factor has been used previously to evaluate the tumor uptake properties of conventional and sterically stabilized liposomes (34). The estimated T E value for D O T A P / D O P E / D S P E - P E G 350 liposomes was 0.177, comparable to the value of 0.168 obtained with the conventional formulation, which has been well established in previous literature to show favorable tumor targeting capacity. In Table 4.4, the level of lipids distributed in the M P S tissues 24 hour after injection is presented. The concentration of D O T A P / D O P E / D S P E - P E G 350 liposomes in the liver and spleen was found to be 1.42 ± 0.07 and 1.4 ± 0.5 mg/g tissue, corresponding to ~ 61 and ~ 4 % of the total injected dose, respectively. The corresponding levels with the conventional DSPC/Chol liposomes, were 0.9 ± 0.1 mg/g and 0.8 ± 0.2 mg/g tissue (~ 36 % and 1 % of the injected dose) in the liver and spleen, respectively. 92 DSPC/Chol DOTAP/DOPE/DSPE-PEG 350 (55:45) (50:35:15) AUCrj-48h 3 3.96 1.58 (mg«h/g) Table 4.3: tumor area under the curve values over 48 hours post injection (AUCo-48h) of cationic D O T A P / D O P E / D S P E - P E G 350 and conventional DSPC/Chol liposomes after i.v. injection into H460 tumor bearing mice. a Tumor AUC0-48h, area under curve from time of dosing (t = 0 h) up to the last measured concentration (t = 48 h), was calculated from the mean values presented on Figure 4.4 using the trapezoidal rule. 93 Liposomes mg lipid per g tissue +/- S.D. liver spleen DSPC/Chol 0.9 ±0.1 0.8 ±0.2 (55:45) (36%) (1%) DOTAP/DOPE/DSPE-PEG350 1.42 ± 0 . 0 7 * 1.4 ±0.5 (50:35:15) (61%) (4%) Table 4.4: Tissue distribution of D O T A P / D O P E / D S P E - P E G 350 and DSPC/Chol liposomes 24 hours post injection in H460 tumor bearing mice. Data represent the averages ± S.D. of three mice. The injected lipid dose was 100 mg/ml as a singe IV bolus. The amount of lipid per gram tissue was determined as described in Section 4.2.4. * denotes that the value is statistically different from the corresponding value from the other liposome formulation, as analyzed by one-way ANOVA, post hoc Scheffe test, p < 0.05. 9 4 4.3.2.3 Lactose retention and DNA triggered release capacity of DOTAP/DOPE/DSPE-Peg 350 (50:35:15) liposomes recovered from the plasma post injection After liposomes are injected into the bloodstream, plasma components begin to interact with the liposome surface. Therefore it is important to determine whether DOTAP/DOPE liposomes maintain stable retention of the entrapped contents as well as functional DNA triggered release. Here, D O T A P / D O P E / D S P E - P E G 350 (50:35:15) and DSPC/Cho l (55:45) liposomes were passively loaded with 1 4 C-lactose as described in Section 3.2.2. Liposomes were recovered one hour after injection from SCID/Rag2 mice bearing H460 tumors. The percentage of entrapped lactose retention was determined as described in Section 3.2.2. and the results were presented in Figure 4.4A. One-hour post injection, 85.9 % of the entrapped lactose remained in the DOTAP/DOPE liposomes, while 97.6 % remained in the conventional liposomes, demonstrating stable retention of both formulations in the circulation. DNA triggered release assays were subsequently performed on the recovered liposomes where the percentage of entrapped lactose release was determined after incubation with 25 ug/ml of H460 tumor extracted DNA, as outlined in section 3.2.3. Results are shown in Figure 4.4B. 20.1 % of DNA-induced lactose release was observed with the recovered D O T A P / D O P E / D S P E - P E G 350 liposomes, while no significant release was observed for the conventional formulation, consistent with previous results where no DNA triggered release capacity was observed. The results demonstrated that after injection into the bloodstream, D O T A P / D O P E / D S P E -P E G 350 liposomes were able to maintain stable entrapped agent retention as well as functional DNA-triggered release capacity. 95 100 c .2 80 c 0) •*-. 0) o in o u re 0) o> re c Q) U L_ a> a . 60 40 20 DSPC/Chol (55/45) B. DOTAP/DOPE/ DSPE-PEG 350 (50/35/15) 30 -, 20 4 o in re a> o u> o *-> o re T3 a « O) ° 10 o 0) re c V u «) DSPC/Chol (55/45) DOTAP/DOPE/ DSPE-PEG 350 (50/35/15) Figure 4.4: The lactose retention (A) and DNA-induced triggered release capacity (B) of D O T A P / D O P E / D S P E - P E G 350 and DSPC/Cho l liposomes recovered from plasma of H460 tumor bearing mice one hour post injection. The injected lipid dose was 100 mg/kg as a single IV bolus. Liposomes were recovered from plasma nd assessed for lactose retention and DNA-triggered release as described in Section 4.2.5. Data represent the averages of duplicates. 96 4.3.2.4. In vivo DNA triggered release of encapsulated contents from DOTAP/DOPE/DSPE-PEG 350 liposomes in H460 solid tumors In order to investigate the selective triggered release properties of the D O T A P / D O P E / D S P E - P E G 350 (50:35:15) formulation in the tumors, 3 H - C H E labeled liposomes were encapsulated with 1 4 C-lactose as an internal aqueous marker and injected intravenously into H460 tumor bearing mice. The H460 non-small cell lung cancer solid tumor model has been demonstrated in Chapter 2 to have an elevated level of extracellular DNA fragments in the tumor when compared to other healthy tissues. The ratio of 1 4 C-lactose to 3 H -lipid was determined in the plasma, tumors and other organs by 1 4 C - and 3 H -dual labeled scintillation counting. This allowed the amount of encapsulated lactose released from the injected liposomes to be determined. For example, if the encapsulated lactose is triggered to release from the liposomes, the observed lactose-to-lipid ratio will change, as the number of 1 4C-lactose molecules associated with each liposome will vary accordingly. The results were subsequently evaluated in comparison with the lactose-to-lipid ratios of the conventional DSPC/Cho l (55:45) formulation, which has been shown to have no DNA-triggered release properties. The results over a 48-hour time period post injection for the conventional liposome formulation is shown in Figure 4.5. The initial lactose-to-lipid ratio before injection was 0.25. In the plasma and spleen, the ratio consistently maintained at approximately 0.25 over 48 hours post injection, while the ratio in the tumor, lung and liver gradually decreased. At 48 hours post injection, the lactose-to-lipid ratio in the liver was ~ 0.045, corresponding 97 to a ~ 98% reduction from the initial ratio, indicating a gradual release and elimination of the entrapped lactose. At the tumor, a more moderate decrease was observed, where the ratio was ~ 0.13 at 48 hours post injection, corresponding to a ~ 48 % lactose release from the liposomes in the tumor. In Figure 4.6, the lactose-to-lipid ratio of the D O T A P / D O P E / D S P E -P E G 350 (50:35:15) liposome formulation is presented. The pattern of lactose-to-lipid ratios in the plasma, tumor and other organs 48 hours after injection were drastically different from the results of the convention formulation. Here, the initial lactose-to-lipid ratio before injection was 0.29. In the plasma, the ratio was maintained consistently at ~ 1.25 post injection, which corresponds to ~ 43 % of the initial injection ratio. These results showed that approximately 43 % of the initially entrapped lactose remained within the liposomes in the circulation after injection. In the M P S organs, the ratios reduced rapidly after injection, where at one hour post injection, value of ~ 0.11 was observed in the liver and spleen. The results showed rapid release and elimination of the entrapped lactose in these organs. However, a different result was observed in the tumors, where the lactose-to-lipid ratios were determined to be 0.57 ± 0.07, 0.42 ± 0.03 and 0.39 ± 0.07 at 1, 2, 4 hours after injection, respectively, which correspond to ~ 2.OX, 1.4X, 1.3X of the initial injected ratio. The elevated values of lactose-to-lipid ratios showed that there was an increase in the number of lactose molecules in relation to the lipids as compared to the initial 1 4 C-lactose encapsulation into the liposomes. The only way this could occur is if 98 entrapped lactose is released from the liposomes selectively in tumor blood vessels during transient passage through the tumor vasculature, since no significant lipid accumulation in the tumor was observed in the corresponding early time points post injection. From 4 hours after injection onwards, the lactose-to-lipid ratio gradually decreased, eventually reaching 0.07 ± 0.01 (~ 76 % lactose release) at 48 hour post injection, corresponding to a gradual removal of lactose from the tumors. The results here differ significantly from the behaviour of non-triggered release conventional liposomes and provided evidence in support of selective lactose release from the D O T A P / D O P E / D S P E - P E G 350 liposomes in the tumor. 9 9 0.7- , 0.6 A 0.5 A — • — plasma - # - liver -A - spleen tumor 0.4 A 0.3 A 0.2 A 0.1 -4 5-0.0 — r ~ 20 10 30 40 50 time after injection (hours) Figure 4.5: The lactose-to-lipid ratios in the tumor and other tissues of H460 tumor bearing mice following a single 100 mg/kg bolus injection of 1 4 C-lactose encapsulated DSPC/Cho l (55:45) liposomes labeled with 3 H - C H E lipids over a 48-hour period. Three mice were used for each time point with the error bars representing S.D. The initial lactose-to-lipid ratio before injection was 0.25. Data were determined as described in Section 4.2.4. 100 0.7 _ 0.6-\ 0.5 A 5 0.4 J 0.3 A — • — plasma - #- liver -A - spleen — t u m o r u JS 0.2 A 0.1 -H 0.0 10 - r -20 30 40 50 time after injection (hours) Figure 4.6: The lactose-to-lipid ratios in the tumor and other tissues of H460 tumor bearing mice following a single 100 mg/kg bolus injection of 1 4 C-lactose encapsulated D O T A P / D O P E / D S P E - P E G 350 (50:35:15) liposomes labeled with H-CHE lipids over a 48-hour period. Three mice were used for each time point with the error bars representing S.D. The initial lactose-to-lipid ratio before injection was 0.29. Data were determined as described in Section 4.2.4. 101 4.4. Chapter 4 Discussion The in vivo distribution behaviour of liposomes can be influenced by a number of physiochemical properties of the lipids, including its surface charge, hydrophilicity and the physical structure of the liposomes (51, 52). Previous studies have shown that positively charged liposomes composed of cationic lipids such as DOTAP, DODAC and DOTMA are rapidly eliminated from the circulation, with circulation lifetimes in the order of minutes (87, 88). Protein binding values of 500-800 g protein/mole lipid have been reported for cationic liposome recovered in vivo, indicating extensive interaction with plasma proteins after injection. The inclusion of PEG-lipid polymers has been demonstrated to prolong circulation longevity of cationic liposomes (23-26). There are several proposed mechanisms for the ability of P E G to reduce plasma elimination, as described in Chapter 1. The most widely accepted concept involves the generation of a repulsive steric barrier on the surface of the liposomes, reducing the extent of protein binding, liposome aggregation and subsequent cellular uptake by the M P S (23, 50, 53). In this chapter, we investigated the plasma longevity and tissue distribution behaviour of our DNA triggered release liposome formulation, composed of DOTAP/DOPE and D S P E - P E G 350. As described in the previous chapter, this composition of fusogenic cationic liposomes has been shown to exhibit structural stability and stable retention properties in physiological buffers, as well as maintaining effective triggered release capability after incubation with extracellular DNA fragments extracted from solid tumors in in vitro studies. 102 In in vivo studies with non-tumor bearing mice, DOTAP/DOPE liposomes (50/50) were rapidly eliminated from the circulation, consistent with previous results in the literature. Incorporation of 15 mol % of D S P E - P E G 350 significantly increased the circulation time, as described in Section 4.3.1.1. Therefore, the in vivo distribution and tumor accumulation properties of this D O T A P / D O P E / D S P E - P E G formulation were further investigated in H460 tumor bearing mice. Conventional DSPC/Chol liposomes have been extensively characterized and shown to exhibit favourable extended circulation longevity and selective tumor targeting in preclinical and clinical studies (32-34, 37, 41). Our results showed that compared to the conventional liposomes, our D O T A P / D O P E / D S P E - P E G 350 (50:35:15) DNA-triggered release liposomes, revealed a comparable prolonged circulation time, plasma A U C values and tumor accumulation levels over a 48-hour time period following injection. The efficiency in passive tumor targeting were similar, indicating that the DNA-triggered release formulation maintains the ability to selectively accumulate in the tumor via fenestrations in the tumor vasculature. Finally, tumor-selective DNA triggered release of our fusogenic cationic formulation, D O T A P / D O P E / D S P E - P E G , was evaluated. The in vivo entrapped lactose to lipid ratio in the tumor, plasma and other tissues were monitored over 48 hours after injection and compared to the corresponding values from the conventional DSPC/Chol liposomes, which has been shown to have no DNA triggered release properties in Chapter 3. A different pattern 103 of lactose-to-lipid ratio was observed for the DNA triggered release formulation when compared to the conventional formulation. For the cationic liposomes, a rapid rise of the lactose-to-lipid ratio in the tumor was accompanied by a decrease in the plasma and other organs. In contrast, for the conventional liposomes the lactose-to-lipid ratio was relatively constant in the plasma and a gradual decrease of the lactose-to-lipid ratio was observed in the tumor and other organs over the 48 hours time period after injection. To explain the observed data, we propose the following model, as illustrated in Figure 4.7: 1.) Initially following injection, our DNA triggered release liposomes remain stable in the circulation, retaining lactose while maintaining the ability to be triggered to release their contents upon exposure to DNA fragments, as demonstrated in section 4.3.2.3. 2.) Upon passage through the tumor vasculature, the liposomes interact with the elevated level of extracellular DNA present in the vasculature and the interstitial space of the tumor immediately surrounding the vasculature. 3.) Formation of DNA/liposome complexes via electrostatic interaction result in the selective release of entrapped lactose. 4.) The released lactose is taken up by the tumor cells and endothelial cells in the periphery of the tumor vasculature while the DNA/liposome complexes return to the circulation, resulting in the increased lactose-to-lipid ratios observed in the tumor at the early time points and the significantly reduced ratios in the plasma and other tissues. After the initial transient passages through the tumor vasculature, the 104 S lactose encapsulated liposomes tumor extracellular DNA fenestrated tumor vasculature released lactose DNA/liposome complex Figure 4.7: An illustration of the proposed model of DNA-triggered release liposomes post injection into tumor bearing mice. 1. Liposome in circulation after injection. 2. Liposomes enter tumor vasculature and interact with tumor extracellular DNA. 3. Liposomes associate with tumor extracellular DNA to form non-bilayer liposome/DNA complexes leading to the release of encapsulated lactose. 4. Liposome/DNA complexes return to the circulation. 105 remaining liposomes and liposome/DNA complexes passively accumulate in the tumor via the abnormal fenestrations in the tumor vascular architecture, as observed in Section 4.3.2.2. These extravasations of liposomes between the tumor interstitial space and tumor vasculature (influx/efflux) have been described in several studies investigating liposome kinetics in the tumor. Additional research will be required to further elucidate the behaviour of this DNA-triggered release liposome formulation. However, we believe the results provided proof-of-principle evidences in support of tumor selective triggered release of the entrapped lactose in solid tumors. We recognized the limitations of the current DNA-triggered release formulation as a result of its tissue distribution behaviour. Specifically, a high level of accumulation of lipids in the M P S organs (e.g. liver and spleen) was observed, accompanied by low lactose-to-lipid ratios. This may be due to the rapid uptake of the liposomes by the M P S in the circulation, which led to the subsequent redistribution of the liposomes from the plasma to the M P S organs, liver (-60%) and spleen (-10%), shortly after injection. Subsequently, the liposomes may be broken down during the elimination process and release of the entrapped contents may ensue. This characteristic may influence which anticancer therapeutic agents would be best suited for delivery by these liposomal carriers. For example, agents that are highly cytotoxic to the liver and spleen may not be desirable for this method of delivery. However, this liposomal formulation may be beneficial for treatments where specific delivery to the liver or spleen is desirable, especially when the 106 selective targeting of small undetected tumors is necessary, in which the ubiquitous nature of this tumor targeting mechanism will be highly attractive. In this chapter, we have demonstrated that the fusogenic cationic formulation, D O T A P / D O P E / D S P E - P E G 350 (50:35:15), exhibited prolonged plasma longevity and selective targeting to the solid tumors in in vivo studies. We have also demonstrated stable entrapped content retention in the circulation as well as specific triggered release in the tumor sites, fulfilling the necessary requirements of a successful DNA-triggered release liposome formulation for the delivery of anticancer therapeutic agents. 107 Chapter 5 Overall Discussion The overall objective of this thesis was to develop a DNA triggered release liposome formulation targeted to the elevated level of extracellular DNA in the solid tumors. This research has followed a stepwise approach: 1) characterization of the properties of the extracellular DNA in the solid tumors; 2) development of a DNA triggered release liposome formulation with optimum retention and triggered release properties; 3) characterization of its in vivo distribution and tumor accumulation behaviour; and 4) evaluation of the specific triggered release of encapsulated markers in the solid tumors. At each step, various methodological and formulation issues have been discussed. In order to develop a tumor selective DNA triggered release liposome formulation with clinical relevance, extracellular DNA in the solid tumors were isolated from a human xenograft mouse model. These extracellular DNA fragments were determined to be in a small size range of 200-3000 bp and were localized in higher levels in the tumors than other healthy organs, in agreement with previous research in the literature. The existence of an elevated level of tumoral extracellular DNA was essential in the development of a DNA triggered release liposome delivery system with specific targeting to the tumor sites. The validity of the extracellular DNA extraction method developed was discussed in Chapter 2. In an ideal DNA-triggered release liposome formulation, the following features would be necessary: 1.) stable drug retention in the circulation and 108 2.) selective triggered release of the encapsulated drugs following interaction with extracellular DNA fragments. Although fusogenic cationic liposomes, composed of DOTAP and DOPE have been demonstrated previously to exhibit the latter feature, their retention properties have been limited by the tendency to self-aggregate in a salt solution such as physiological buffer. The inclusion of PEG-lipids was investigated as a solution to this formulation obstacle. Consistent with previous reports, the addition of D S P E - P E G 2000 resulted in the significant increase of cationic liposome stability and encapsulated marker retention in physiological buffer, however, liposome/DNA interaction and subsequent DNA-triggered release was severely compromised. The results reported in Chapter 3 showed that by using a lower molecular weight PEG-lipid, D S P E - P E G 350, with a shorter P E G polymer length, stable retention in physiological salt buffer can be achieved without compromising DNA triggered release properties. Hence, the liposome formulation, D O T A P / D O P E / D S P E - P E G 350, was found to satisfy the formulation requirements for an optimum DNA-triggered release liposome formulation. Cationic liposomes have been shown to be rapidly eliminated from circulation after injection and this issue was addressed in Chapter 4. Although the inclusion of long-chained D S P E - P E G , such as D S P E - P E G 2000, has been well established to significantly increase the circulation time of conventional liposomes, the effect of short-chained D S P E - P E G 350 on cationic liposomes has not been well characterized previously. Here, we evaluated the in vivo circulation longevity, tissue distribution and tumor 109 targeting effectiveness of D O T A P / D O P E / D S P E - P E G 350 liposomes in a human xenograft mouse model. The results demonstrated that the composition, D O T A P / D O P E / D S P E - P E G 350 (50:35:15), significantly improved plasma circulation time over non-pegylated DOTAP/DOPE liposomes. Tumor targeting effectiveness comparable with conventional liposomes, which have been well established to provide favorable tumor accumulation of anticancer agents, was also observed. Finally, the selective DNA triggered release function of D O T A P / D O P E / D S P E - P E G 350 (50:35:15) in solid tumors was evaluated. By using the entrapped-lactose-to-lipid ratio as an indicator of entrapped marker release, P E G 350 pegylated fusogenic cationic liposomes were successfully demonstrated to show selective triggered release in the tumors, when compared to conventional liposomes without DNA triggered release properties. These results provided evidence in support of a DNA triggered release liposome formulation, which exploits the unique physiological features of the solid tumors as well as the characteristic properties of fusogenic cationic liposomes. The need to develop a tumor selective triggered release liposome formulation was discussed previously in Chapter 1. In order to take advantage of the passive tumor targeting ability of liposomal carriers, long circulating liposomes with stable drug retention properties were desired. However, a resulting shortcoming of this approach is the ensuing slow release of the encapsulated therapeutic agents in the disease sites, as a result limiting drug 110 exposure in the tumor. In order to ensure high drug availability in the tumor, a liposome formulation possessing selective drug release in the tumor, accompanied by long circulation time and effective tumor targeting, is therefore desired. Different approaches, targeting various physiological features of cancerous growths, have been taken toward the development of such a liposome formulation. They include: 1.) thermosensitive liposomes, which target local hyperthermia in solid tumors (90-94); 2.) pH sensitive liposomes, which target the local acidic environment in hypoxic tumors (95, 96); and 3.) immunoliposomes that target tumor-associated cell surface antigens to enhance liposome uptake by tumor cells (97). However, these targeting approaches are strongly dependent on various biological factors, such as the size, location, degree of hypoxia and type of the tumor, which are highly variable among different patients. In this research, we have taken an alternative approach of targeting the elevated level of extracellular DNA fragments in the tumor region, which is a result of the high rate of tumor cell necrosis in rapidly growing tumors. As mentioned in Section 1.7.2., the occurrence of extracellular DNA localization in the tumor is an ubiquitous feature of solid tumors. The ubiquitous nature of such extracellular DNA also offers a unique advantage over other targeting strategies. Specifically, an amplifying "snowball" effect can occur where drug selectivity is expanded after each treatment. As the selective delivery of anticancer agents into the tumor sites continues with each treatment, the resulting treatment induced tumor cell death would lead to the release of more intracellular DNA into the I l l extracellular space, thereby expanding the level of extracellular DNA in the tumor available for targeting by subsequent DNA-triggered release liposome treatments. This unique feature enables progressive improvement in the effectiveness of continued liposomal drug delivery. In summary, the work presented in this thesis described a stepwise approach to the development of a DNA triggered release liposome formulation that may potentially be applied to improve the effectiveness of anticancer therapeutic treatments, providing enhanced tumor drug exposure accompanied by selective delivery to the solid tumors. 112 Chapter 6 Conclusions and future studies 6.1 Conclusions In Chapter 1, it is hypothesized that fusogenic cationic liposomes composed of the cationic lipid, DOTAP, and non-bilayer fusogenic lipid, DOPE, will have greater release of entrapped contents in the tumor than other organs by their capacity to associate with extracellular DNA in the solid tumor. The results presented in this thesis support this hypothesis. Essentially, four areas of investigation were explored in this thesis, as stated in the research objectives in Chapter 1. First, In vivo studies were performed, characterizing the size range of the extracellular DNA in the tumor and confirming the elevated presence of DNA in the tumor in comparison to other organs. Secondly, DOTAP/DOPE liposomes with the inclusion of D S P E - P E G 350 were found to satisfy the requirements of an optimum DNA-triggered release formulation, with stable retention properties in physiological salt solutions and the capacity to be triggered to release its encapsulated contents upon exposure to extracellular DNA. Thirdly, in vivo studies demonstrated the prolonged circulation time of D O T A P / D O P E / D S P E - P E G 350 (50:35:15) in tumor bearing mice, accompanied by selective accumulation in the tumor. Finally, selective release of entrapped contents in the solid tumors were demonstrated in in vivo studies following a single intravenous injection into tumor bearing mice. In conclusion, the results from this study demonstrated the potential of D O T A P / D O P E / D S P E - P E G 350 liposomes as an anticancer drug delivery system with selective triggered release capacity in the solid tumor. 113 6.2 Future studies In this thesis, we have successfully demonstrated fusogenic cationic liposomes as potential effective carriers of anticancer therapeutic agents using radioactive 1 4 C-lactose as an aqueous internal marker. The loading of an appropriate anticancer agent would be the next step to further elucidate the drug retention properties and DNA triggered release capability of this liposome formulation. Numerous strategies have been employed to load anticancer agents into liposomal carriers. The most common approach involves drug encapsulation by means of a transmembrane proton gradient, where encapsulation efficiency may approach 100% for compounds such as doxorubicin (32, 98, 99). We recognized that an indirect approach has been taken in our studies to evaluate tumor selective triggered release of encapsulated contents, where entrapped-lactose-to-lipid-ratio was employed as a marker for lactose release. With the encapsulation of fluorescent compounds such as doxorubicin, a more direct approach can be applied. These compounds can provide visual evidence of triggered drug release in the tumor with the use of confocal fluorescence microscopy. This approach has been successfully demonstrated in previous studies on liposomal doxorubicin formulations (100, 101). Furthermore, successful anticancer drug loading would allow evaluation of the therapeutic efficacy of this DNA triggered release liposomal delivery system, providing the opportunity to establish the relationship between enhanced selective triggered drug release in the tumor and therapeutic effectiveness of the treatment. 114 The association of extracellular DNA fragments to liposomes to induce morphological transformation resulting in the formation of non-bilayer DNA/liposome complexes is essential to trigger the loss of lipid permeability barriers and the subsequent release of encapsulated agents in DNA-triggered release liposomal carriers. Previous studies have provided evidence in support of such DNA/liposome association and the resulting phase transition, as described in Chapter 1. In this thesis, as mentioned in Chapter 3, we have employed the use of Q E L S particle sizing and 1 4 C-lactose release assays as methods to access DNA/liposome association and the subsequent release of the encapsulated agents. However, we recognized that these studies do not provide detailed information on the above-mentioned DNA induced morphological changes. Therefore, for future studies, cryogenic electron microscopy would be an appropriate approach to elucidate the morphological behaviour of our DNA triggered release formulation in the presence of extracellular DNA fragments. Cryogenic electron microscopy has been employed in various studies in previous literature to evaluate the morphological structures of different liposome formulations, including conventional liposomes encapsulating various anticancer agents as well as different cationic lipid carriers for the purpose of gene delivery (61).' 115 References 1. Swenson, C. E., J . Freitag, and A. S. Janoff. 1998. The Liposome Company: Lipid-based pharmaceuticals in clinical development. In Medical Applications of Liposomes. D. D. Lasic, and D. Papahadjopoulos, eds. Elsevier, New York, p. 689. 2. Martin, F. 1998. Clinical pharmacology and antitumor efficacy of DOXIL (pegylated liposomal doxorubicin): Sequus Pharmaceuticals, Inc. In Medical Applications of Liposomes. D. D. Lasic, and D. Papahadjopoulos, eds. Elsevier, New York, p. 635. 3. Jain, R. K. 1987. Transport of molecules across tumor vasculature. Cancer Metastasis Rev. 6:559. 4. Yuan, F., M. Leunig, S. K. Huang, D. A. Berk, D. Papahadjopoulos, and R. K. Jain. 1994. Microvascular permeability and interstitial penetration of sterically stabilized (stealth) liposomes ina human tumor xenograft. Cancer Res 54:3352. 5. Mayer, L. D., P. R. Cullis, and M. B. Bally. 1994. The use of transmembrane pH gradient-driven drug encapsulation in the pharmacodynamic evaluation of liposomal doxorubicin. J Liposome Res 4:529. 6. Bally, M. B., H. Lim, P. R. Cullis, and L. D. Mayer. 1998. Controlling the drug delivery attributes of lipid-based drug formulations. J Liposome Res 8:299. 7. Singer, S. J . , and G. L. Nicholson. 1972. The fluid mosaic model of the structure of cell membranes. Science 175:720. 8. Cullis, P. R., D. B. Fenske, and J . Hope. 1996. Physical properties and functional roles of lipids in membranes. In Biochemistry of Lipids, Lipoproteins and Membranes. D. E. V. a. J . E. Vance, ed. Elsevier Science B.V. 9. Litzinger, D. C , and L. Huang. 1992. Phosphatidylethanolamine liposomes: drug delivery, gene transfer and jmmunodiagnostic applications. Biochimica et Biophsica Acta 1113:201. 10. Seddon, J . M. 1990. Strucuture of the inverted hexagonal (HII) phase, and non-lamellar phase transitions of lipids. Biochimica et Biophsica Acta 1031:1. 11. Cullis, P. R., and B. de Kruijff. 1978. The polymorphic phase behaviour of phosphatidylethanolamines of natural and synthetic origin: A 31P-NMR study. Biochimica et Biophsica Acta 513:31. 116 12. Israelachvili, J . N., S. Marcelja, and R. G. Horn. 1980. Physical principles of membrane organization. Q. Rev. Biophys. 13:121. 13. Cullis, P. R., M. J . Hope, and C. Tilcock. 1986. Lipid polymorphism and the roles of lipids in membranes. Chemistry and Physics of Lipids 40:127. 14. Janes, N. 1996. Curvature stress and polymorphism in membranes. Chemistry and Physics of Lipids 81:133. 15. Madden, T. D., and P. R. Cullis. 1982. Stabilization of bilayer structure for unsaturated phosphatidylethanolamines by detergents. Biochimica et Biophsica Acta 684:149. 16. Juliano, R. L., and D. Stamp. 1975. The effect of particle size and charge on the clearance rate of liposomes and liposome encapsulated drugs. Biochem Biophys Res Commun 63:651. 17. Adams, D. H., G. V. Joyce, J . Richardson, B. E. Ryman, and H. M. Wisniewski. 1977. Liposome toxicity in the mouse central nervous system. J Neurol Sci 31:173. 18. Feigner, P. L , T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J . P. Northrop, G. M. Ringold, and M. Danielson. 1987. Lipofection: a highly efficient, lipid-mediated DNA -transfection procedure. Proc Natl Acad Sci USA 84:7413. 19. Byk, G. , C. Dubertret, V. Escriou, M. Frederic, G. Jaslin, R. Rangara, B. Pitard, J . Crouzet, P. Wils, B. Schwartz, and D. Sherman. 1998. Synthesis, activity, and structure-activity relationship studies of novel cationic lipids for DNA transfer. J Med Chem 41:224. 20. Ansell, S. M., F. Feng, A. Lau, and L. Ahkong. 1997. The design and synthesis of simple quaternary ammonium salts for gene therapy formulations. In Artificial Self-Assembling Systems for Gene Delivery. Cambridge Healthtech Institute, Coronado, CA. 21. Feigner, J . H., R. Kumar, C. N. Sridnar, C. J . Wheeler, Y. J . Tsai, R. Border, P. Ramsay, M. Martin, and P. L. Feigner. 1994. Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol Chem 269:2550. 22. Stamatatos, L., R. Leventis, M. J . Zuckermann, and J . R. Silvius. 1988. Interactions of cationic lipid vesicles with negatively charged phospholipid vesicles and biological membranes. Biochemistry 27:3917. 117 23. Needham, D., T. J . Mcintosh, and D. D. Lasic. 1992. Repulsive interactions and mechanical stability of polymer-grafted lipid membranes. Biochim Biophys Acta 1108:40. 24. Ogris, M., S. Brunner, S. Schuller, R. Kircheis, and E. Wagner. 1999. PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther. 6:595. 25. Plank, C , K. Mechtler, S. F. C. Jr, and E. Wagner. 1996. Activation of the complement system by synthetic DNA complexes: a potential barrier for intravenous gene delivery. Hum Gene Ther. 7:1437. 26. Collard, W. T., Y. Yang, K. Y. Kwok, Y. Park, and K. G. Rice. 2000. Biodistribution, metabolism, and in vivo gene expression of low molecular weight glycopeptide polyethylene glycol peptide DNA co-condensates. J Pharm Sci. 89:499. 27. Farhood, H., N. Serbina, and L. Huang. 1995. The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. Biochim Biophys Acta 1235:289. 28. Hui, S. W., M. Z. Langner, Y-L., P. Ross, E. Hurley, and K. Chan. 1996. The role of helper lipids in cationic liposome-mediated gene transfer. Biophys J 71:590. 29. Xu, Y., and F. C. Szoka. 1996. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry 35:5616. 30. Bangham, A. D., M. M. Standish, and J . C. Watkins. 1965. Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol 13:238. 31. Lasic, D. D., and D. Papahadjopoulos. 1998. Medical Applications of Liposomes. Elsevier, New York. 32. Mayer, L. D., L. C. Tai, D. S. Ko, D. Masin, R. S. Ginsberg, P. R. Cullis, and M. B. Bally. 1989. Influence of vesicle size, lipid composition, and drug-to-lipid ratio on the biological activity of liposomal doxorubicin in mice. Cancer Res 49:5922. 33. Gabizon, A., A. Dagan, D. Goren, Y. Barenholz, and Z. Fuks. 1982. Liposomes as in vivo carriers of adriamycin: reduced cardiac uptake and preserved antitumor activity in mice. Cancer Res 42. 34. Mayer, L. D., R. Krishna, and M. B. Bally. 2001. In Polymeric biomaterials. S. Dumitriu, ed. Marcel Decker, New York, p. 823. 118 35. Mayer, L. D. 1998. Future developments in the selectivity of anticancer agents: drug delivery and molecular targer strategies. Cancer Metastasis Re v. 17:211. 36. Huang, S. K., E. Mayhew, S. Gilani, D. D. Lasic, F. J . Martin, and D. Papahadjopoulos. 1992. Pharmacokinetics and therapeutics of sterically stabilized liposomes in mice bearing C-26 colon carcinoma. Cancer Res 52:6774. 37. Mayer, L. D., G. Dougherty, T. O. Harasym, and M. B. Bally. 1997. The role of tumor-associated macrophages in the delivery of liposomal doxorubicin to solid murine fibrosarcoma tumors. J Pharmcol Exp Ther 280:1406. 38. Hobbs, S. K., W. L. Monsky, F. Yuan, W. G. Roberts, L. Griffith, V. P. Torchilin, and R. K. Jain. 1998. Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment. Proc Natl Acad Sci USA 95:4607. 39. Hashizume, H., P. Baluk, S. Morikawa, J . W. McLean, G. Thurston, S. Roberge, R. K. Jain, and D. M. McDonald. 2000. Openings between defective endothelial cells explain tumor vessel leakiness. Am J Pathol 156:1363. 40. Buocher, Y., and R. K. Jain. 1992. Microvascular pressure is the principle driving force for interstitial hypertension in solid tumors: Implications for vascular collapse. Cancer Res 52:5110. 41. Allen, T. M., and A. Chonn. 1987. Large unilamellar liposomes with low uptake into the reticuloendothelial system. FEBS Lett 223:42. 42. Senior, J . H. 1987. Fate and behavior of liposomes in vivo: a review of controlling factors. Crit Rev Ther Drug Carrier Syst 3:123. 43. Pratten, M. K., and J . B. Lloyd. 1986. Pinocytosis and phagocytosis: the effect of size of a particulate substrate on its mode of capture by rat peritoneal macrophages cultured in vitro. Biochim Biophys Acta 881:307. 44. Lim, H. J . , D. Masin, T. D. Madden, and M. B. Bally. 1997. Influence of drug release characteristics on the therapeutics activity of liposomal mitoxantrone. J Pharmacol Exp Ther 281:566. 45. Allen, T. M., and C. B. Hansen. 1991. Pharmacokinetics of stealth versus conventional liposomes: effect of dose. Biochim Biophys Acta 1068:133. 46. Woodle, M. C , and D. D. Lasic. 1992. Sterically stabilized liposomes. Biochim Biophys Acta 1113:171. 119 47. Abuchowski, A., J . R. McCoy, N. C. Palczuk, T. van Es, and F. F. Davis. 1977. Effects of coyalent attachment of polyethylene gylcol on immunogenicity and circulatin life of bovine liver catalase. J Biol Chem 252:3882. 48. Mori, Y., S. Nagaoka, H. Takiguchi, T. Kikuchi, N. Noguchi, H. Tanzawa, and Y. Noishiki. 1982. A new antithrombogenic material with long polyethyleneoxide chains. Trans Am Soc Artif Internal Organs 28:459. 49. de Gennes, P. G. 1988. In Physical basis of cell-cell adhesion, Vol. 39-60. P. Bongrand, ed. C R C Press, Florida. 50. Kenworthy, A. K., K. Hristova, D. Needham, and T. J . Mcintosh. 1995. Range and magnitude of the steric pressure between bilayers containing phospholipids witrh covalently attached polyethylene glycol. Biophys J 68:1921. 51. Senior, J . , C. Delgado, D. Fisher, C. Tilcock, and G. Gregoriadis. 1991. Influence of surface hydrophilicity of liposomes on their interaction with plasma protein and clearance from the circulation: studies with poly(ethylene glycol) -coated vesicles. Biochim Biophys Acta 1062:77. 52. Tirosh, O., Y. Barenholz, J . Katzhendler, and A. Priev. 1998. Hydration of polyethylene glycol-grafted liposomes. Biophys J 74:1371. 53. Kuhl, T. L , D. E. Leckband, D. D. Lasic, and J . N. Israelachvili. 1994. Modulation of interaction forces between biilayers exposing short-chained ethylene oxide headgroups. Biophys J 66:1479. 54. Cullis, P. R., and M. J . Hope. 1978. Effects of fusogenic agent on membrane structure of erthytocyte ghosts and the mechanism of membrane fusion. Nature 271:672. 55. Siegel, D. P. 1999. The modified stalk mechanism of lamellar/inverted phase transitions and its implications for membrane fusion. Biophys J 76:291. 56. Ellens, H., J . Bentz, and F. C. Szoka. 1985. H+- and Ca2+-induced fusion and destabilization of liposomes. Biochemistry 24:3099. 57. Yeagle, P. L , F. T. Smith, J . E. Young, and T. D. Flanagan. 1994. Inhibition of membrane fusion by lysophosphatidylcholine. Biochemistry 33:1820. 58. Hope, M. J . , D. C. Walker, and P. R. Cullis. 1983. Ca2+ and pH induced fusion of small unilamellar vesicles consisting of 120 phosphatidylethanolamine and negatively charged phospholipids: a freeze fracture study. Biochem Biophys Res Commun 110:15. 59. Hafez, I. M., and P. R. Cullis. 2001. Roles of lipid polymorphism in intracellular delivery. Advanced Drug Delivery Reviews 47:139. 60. Feigner, P. L , and G. M. Ringold. 1989. Cationic liposome-mediated transfection. Nature 337:387. 61. Wasan, E., P. Harvie, K. Edwards, G. Karlson, and M. B. Bally. 1999. A multi-step lipid mixing assay to model structural changes in cationic lipoplexes used for in vitro transfection. Biochimica et Biophysica Acta 1461:27. 62. Koltover, I., T. Salditt, O. Radler, and C. R. Safinya. 1998. An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery. Science 281:78. 63. Wasan, E. K., A. Fairchild, and M. B. Bally. 1998. Cationic l iposome-plasmid DNA complexes used for gene transfer retain a significant trapped volume. J Pharm Sci. 87:9. 64. Davis, S., and F. Szoka. 1998. Cholesterol phosphate derivatives: synthesis and incorporation into a phosphate and calcium-sensitive triggered release liposome. Bioconjugate Chem 9:783. 65. Steel, G. G. 1967. Cell loss as a factor in the growth rate of human tumors. Eur J Cancer 3:381. 66. Tannock, I. F. 1968. The relation between cell proliferation and the vascular system in a transplanted mouse mammary tumor. BrJ Cancer 22:258. 67. Frindel, E., E. Malaise, and M. Tubiana. 1968. Cell proliferation kinetics in five human solid tumors. Cancer 22:611. 68. Loefflerm, K. U., and P. G. McMenamin. 1987. An ultrastructural study of DNA precipitation in the anterior segments of eyes with retinoblastoma. Ophthalmology 94:1160. 69. Looser, P., and J . A. Laissue. 1980. Extrazellulare desoxyibonukleinsaure in kleinzelligen bronchuskarzinomen. Sheiz Med Wschr 110:1342. 70. Leon, S. A., B. Shapiro, D. M. Sklaroff, and M. J . Yaros. 1977. Free DNA in the serum of cancer patients and the effects of therapy. Cancer Res 37:646. 121 71. Nawroz, H., W. Koch, P. Anker, M. Stroun, and D. Sidransky. 1996. Microsatellite alterations in serum DNA of head and neck cancer patients. Nat Med 2:1035. 72. Chen, X. Q., M. Stroun, J.-L. Magnenat, L. P. Nicod, A . -M. Kurt, J . Lyautey, C. Lederrey, and P. Anker. 1996. Microsatellite alterations in plasma DNA of small cell lung cancer patients. Nat Med 2:1033. 73. Anker, P., H. Mulcahy, X. Q. Chen, and M. Stroun. 1999. Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients. Cancer Metastasis Rev. 18:65. 74. Jahr, S., H. Hentze, S. Englisch, D. Hardt, F. O. Fackelmayer, R.-D. Hesch, and R. Knippers. 2001. DNA Fragments in the blood plasma of cancer patients: quantitations and evidences for their origin from apoptotic and nectotic cells. Cancer Res 61:1659. 75. Epstein, A. L , F. Chen, and C. R. Taylor. 1988. A novel method for the detection of necrotic lesions in human cancers. Cancer Res 48:5842. 76. Miller, G. K., G. S. Naeve, S. A. Gaffar, and A. L. Epstein. 1993. Immunologic and biochemical analysis of TNT-1 and TNT-2 monoclonal antibody binding to histones. Hybridoma 12:689. 77. Chen, F., E. B. Hansen, C. R. Taylor, and A. L. Epstein. 1991. Diffusion and binding of monoclonal antibody TNT-1 in multicellular tumor spheroids. J Nat Can Int 83:200. 78. Chen, F., C. Liu, and A. L. Epstein. 1991. Effects of 131-labeled TNT-1 radioimmunotherapy on HT-29 human colon adenocarcinoma spheroids. Cancer Immunol. Immunother. 33:158. 79. Hornick, J . L , J . Sharifi, L. A. Khawli, P. Hu, B. H. Biela, M. M. Mizokami, A. Yun, C. R. Taylor, and A. L. Epstein. 1998. A new chemically modified chimeric TNT-3 monoclonal antibody directed against DNA for the radioimmunotherapy of solid tumors. Cancer Biother Radiopharm 13:255. 80. Sambrook, J . , D. W. Russell, and J . Sambrook. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York. 81. Junowicz, E., and J . H. Spencer. 1973. Studies on bovine pancreatic deoxyribonuclease A. II. The effect of different bivalent metals on the specificity of degradation of DNA. Biochim Biophys Acta 312:85. 82. Junowicz, E., and J . H. Spencer. 1973. Studies on bovine pancreatic deoxyribonuclease A. I. General properties and activation with different bivalent metals. Biochim Biophys Acta 1312:72. 122 83. Wiegers, U., and H. Hilz. 1971. A new method using 'proteinase K' to prevent mRNA degradation during isolation from HeLa cells. Biochem Biophys Res Commun 44:513. 84. Hilz, H., U. Wiegers, and P. Adamietz. 1975. Stimulation of proteinase K action by denaturing agents: application to the isolation of nucleic acids and the degradation of 'masked' proteins. Eur J Biochem 56:103. 85. Mayer, L. D., M. J . Hope, and P. R. Cullis. 1986. Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta 858:161. 86. Sung, S . -J . , S. H. Min, K. Y. Cho, S. Lee, Y. Min, I. Yeom, and J.-K. Park. 2003. Effect of polyethylene glycol on gene delivery of polyethylenimine. Biol Pharm Bull 26:492. 87. Cullis, P. R., A. Crohn, and S. C. Semple. 1998. Interactions of liposomes and lipid-based carrier systems with blood proteins: Relation to clearance behaviour in vivo. Adv Drug Deliv Rev 32:3. 88. Senior, J . H., K. R. Trimble, and R. Maskiewwicz. 1991. Interaction of positively-charged liposomes with blood: implications for their application in vivo. Biochim Biophys Acta 1070:173. 89. Fung, V., G. Chiu, and L. D. Mayer. 2003. Application of purging biotinylated liposomes from plasma to elucidate influx and efflux processes associated with accumulation of liposomes in solid tumors. Biochim Biophys Acta 1611:63. 90. Yatvin, M. B., J . M. Weinstein, W. H. Dennis, and R. Blumenthal. 1978. Design of liposomes for enhanced local release of drugs by hyperthermia. Science 202:1290. 91. Weinstein, J . N., R. L. Magin, M. B. Yatvin, and D. S. Zaharko. 1979. Liposomes and local hyperthermia: selective delivery of methotrexate to heated tumors. Science 204:188. 92. Anyarambhatla, G. R., and D. Needham. 1999. Enhancement of the phase transition permeability of D P P C liposomes by incorporation of M P P C : a new temperature-sensitive liposome for use with mild hyperthermia. J Liposome Res 9:491. 93. Kong, G. , G. Anyarambhatla, W. P. Petros, R. D. Braun, O. M. Colvin, D. Needham, and M. W. Dewhirst. 2000. Efficacy of liposomes and hyperthermia in a human tumor xenograft model: importance of triggered drug release. Cancer Res 60:6950. 123 94. Needham, D., and M. W. Dewhirst. 2001. The development and testing of a new temperature-sensitive drug delivery system for the treatment of solid tumors. Adv Drug Deliv Rev 53:285. 95. Connor, J . , and L. Huang. 1986. pH-sensitive immunoliposomes as an efficient and target-specific carrier for antitumor drugs. Cancer Res 46:3431. 96. Choi, M.-J., H.-S. Han, and H. Kim. 1992. pH-sensitive liposomes containing polymerized phosphatidylethanolamine and fatty acid. J Biochem 112:694. 97. Mastrobattista, E., G. A. Koning, and G. Storm. 1999. Immunoliposomes for the targeted delivery of antitumor drugs. Adv Drug Deliv Rev 40:103. 98. Mayer, L. D., M. B. Bally, and P. R. Cullis. 1986. Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient. Biochim Biophys Acta 857:123. 99. Mayer, L. D., M. B. Bally, M. J . Hope, and P. R. Cullis. 1985. Uptake of antineoplastic agents into large unilamellar vesicles in response to a membrane potential. Biochim Biophys Acta 816:294. 100. Krishna, R., M. St-Louis, and L. D. Mayer. 2000. Increased intracellular drug accumulation and complete chemosensitization achieved in multidrug-resistant solid tumors by co-administering valspodar (PSC 833) with sterically stabilized liposomal doxorubicin. IntJ Cancer 85:131. 101. Krishna, R., G . Chiu, and L. D. Mayer. 2001. Visualization of bioavailable liposomal doxorubicin using a non-perturbing confocal imaging technique. Histol Histopathol 16:693. 102. Mason, D.Y., and P. Biberfeld. 1980. Technical aspects of lymphomas immunohistology. J Histochem Cytochem 28:731-745. 103. , Chiu, G. , M.B. Bally, and L.D. Mayer. 2001. Selective protein interactions with phosphatidylserine containing liposomes alter the steric stabilization properties of poly(ethylene glycol). Biochim. Biophy. Acta 1510:56-69. 104. Burkitt, H.George, Barbara Young and John VV. Heath. 1993. Wheater's Functional Histology: A Text and Colour Atlas. Churchill Livingstone Inc., Edinburgh, UK. 

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.831.1-0091404/manifest

Comment

Related Items