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Factors influencing the uptake and release of doxorubicin by liposomes Tai, Linda C.L. 1988

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FACTORS INFLUENCING THE UPTAKE AND RELEASE OF DOXORUBICIN BY LIPOSOMES BY LINDA C L . TAI B.Sc, University of B r i t i s h Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Biochemistry We accept t h i s thesis as conforming to the required standard THE U N I V E R S I T Y OF B R I T I S H C O L U M B I A A p r i l 1988 © L i n d a T a i , 1988 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 fobOteMl^'Tg^ The University of British Columbia Vancouver, Canada •ate afc,f<Wft DE-6 (2/88) ABSTRACT The use of liposomes exhibiting a transmembrane pH gradient (inside acidic) to accumulate doxorubicin into the i n t e r i o r aqueous compartment has been shown to achieve drug trapping e f f i c i e n c i e s i n excess of 98% i n a manner which i s independent of l i p i d composition. Doxorubicin entrapment appears to be most o e f f i c i e n t at 60 C with 100% drug accumulation occurring a f t e r 5 minutes. An increase i n internal buffering capacity and trap volume of the v e s i c l e s s i g n i f i c a n t l y enhances doxorubicin sequestration. I n i t i a l drug to l i p i d r a t i o s as high as 2:1 (wt:wt) have been used, although trapping e f f i c i e n c i e s f a l l below 95% at drug to l i p i d r a t i o s i n excess of 1:2 (wt:wt). As v e s i c l e s i z e i s decreased the i n i t i a l drug to l i p i d r a t i o must be reduced to 1:10 to maintain high trapping e f f i c i e n c i e s . In addition to e f f e c t i n g e f f i c i e n t doxorubicin entrapment, the transmembrane pH gradient also reduces the rate of doxorubicin leakage. For example, i n liposomes exhibiting a pH gradient greater than 2 units, release i s less than 5% of the o encapsulated doxorubicin over 24 hours at 37 C whereas release rates are s i g n i f i c a n t l y higher i n the absence of a pH gradient. F i n a l l y , a procedure for a rapid colorimetric t e s t for determining the amount of unencapsulated doxorubicin i s described. The t e s t i s based on a spectral s h i f t of doxorubicin peak absorption from 480nm to approximately 600nm upon addition of a l k a l i to the liposomal doxorubicin. The r e s u l t i n g color change of untrapped drug from orange to purple can be quantitated spectrophotometrically or v i s u a l l y . i i TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v i LIST OF FIGURES v i i ABBREVIATIONS ix ACKNOWLEDGEMENTS X 1 INTRODUCTION 1.1 Model Membrane Systems 1 1.2 Preparation and Properties of Liposomes 2 1.3 Rationale for Drug Encapsulation i n Liposomes 3 1.4 Factors A f f e c t i n g Drug Encapsulation i n Liposomes.. 8 1.5 Doxorubicin 12 1.5.1 Structure 12 1.5.2 Mechanism of Action 12 1.5.3 Therapeutic Uses and C l i n i c a l Toxocity 14 1.5.4 D i s t r i b u t i o n and Metabolism 15 1.6 Rationale for Doxorubicin Encapsulation i n Liposomes 16 2 Uptake of Doxorubicin into Large Unilamellar V e s i c l e s i n Response to a pH Gradient 2 .1 Introduction 17 2.2 Materials and Methods 2.2.1 Materials 18 2.2.2 Preparation of large unilamellar v e s i c l e s .... 18 2.2.3 Generation of the pH gradient 19 i i i 2.2.4 Doxorubicin uptake i n response to pH gradients 2 0 2.2.5 Passive entrapment of doxorubicin 21 2.2.6 Trap volume determinations 21 2.3 Results 2.3.1 Temperature dependence of doxorubicin uptake . i- 22 2.3.2 E f f e c t of cholesterol on doxorubicin uptake . 24 2.3.3 E f f e c t of lyso PC on doxorubicin uptake 27 2.3.4 Doxorubicin concentration and v e s i c l e permeability and s t a b i l i t y 28 2.3.5 E f f e c t of buffering capacity on doxorubicin uptake 31 2.3.6 E f f e c t of varying i n i t i a l drug to l i p i d r a t i o s on uptake 34 2.3.7 Vesicle s i z e and drug to l i p i d r a t i o s on doxorubicin uptake 38 2.4 Discussion 43 3 Factors Influencing Release of Doxorubicin from Large Unilamellar Vesicles 3.1 Introduction 48 3.2 Materials and Methods 49 3.2.1 Materials 49 3.2.2 Methods 3.2.2.1 Release Assay 49 3.2.2.2 Colorimetric Assay 50 3.3 Results 3.3.1 E f f e c t of l i p i d composition on doxorubicin release 51 3.3.2 Ef f e c t of buffering capacity on doxorubicin release 54 iv 3.3.3 E f f e c t of varying i n i t i a l drug to l i p i d r a t i o s on doxorubicin release 57 3.3.4 Colorimetric assay 61 3.4 Discussion 66 4 SUMMARIZING DISCUSSION 68 BIBLIOGRAPHY 72 v LIST OF TABLES 1. Uptake o f d o x o r u b i c i n a t v a r i o u s c h o l e s t e r o l c o n c e n t r a t i o n s 2 6 2. Uptake o f d o x o r u b i c i n i n v e s i c l e s w i t h v a r y i n g l y s o PC c o n c e n t r a t i o n s 26 3. E x t e r n a l d o x o r u b i c i n c o n c e n t r a t i o n and v e s i c l e l e a k i n e s s 29 4. Trap volumes o f v e s i c l e s w i t h i n t e r n a l l y and e x t e r n a l l y i n c u b a t e d d o x o r u b i c i n 29 5. S i z e d i s t r i b u t i o n o f v e s i c l e s e x t r u d e d t h r o u g h f i l t e r s 40 6. C h a r a c t e r i s t i c s o f l i p o s o m e - e n c a p s u l a t e d d o x o r u b i c i n p r e p a r a t i o n s 46 v i L I S T OF F I G U R E S 1. F r e e z e - f r a c t u r e e l e c t r o n m i c r o g r a p h o f m u l t i l a m e l l a r v e s i c l e s 4 2. F r e e z e - f r a c t u r e e l e c t r o n m i c r o g r a p h o f s m a l l u n i l a m e l l a r v e s i c l e s 5 3. F r e e z e - f r a c t u r e e l e c t r o n m i c r o g r a p h o f l a r g e u n i l a m e l l a r v e s i c l e s 6 4. S t r u c t u r e o f d o x o r u b i c i n 13 5. Temperature dependence o f d o x o r u b i c i n u p t a k e 2 3 6. S t r u c t u r e o f c h o l e s t e r o l 25 7. The r e l a t i o n s h i p between e n t r a p p e d p r o t o n c o n c e n t r a t i o n and c i t r a t e c o n c e n t r a t i o n 3 2 8. The i n f l u e n c e o f t r a p volume and b u f f e r i n g c a p a c i t y on d o x o r u b i c i n uptake 33 9. The e f f e c t o f v a r y i n g i n i t i a l d r u g t o l i p i d r a t i o s on t h e t r a p p i n g e f f i c i e n c y o f d o x o r u b i c i n 36 10. Comparison between r e s i d u a l transmembrane pH g r a d i e n t s w i t h v a r y i n g i n t e r n a l c i t r a t e c o n c e n t r a t i o n s 37 11. E f f e c t o f drug t o l i p i d r a t i o on t h e t r a p p i n g e f f i c i e n c y o f d o x o r u b i c i n i n v a r i o u s s i z e d v e s i c l e s 41 12. The i n f l u e n c e o f drug t o l i p i d r a t i o s on t h e maintenance o f t h e pH g r a d i e n t i n s m a l l e r v e s i c l e systems 42 13. Proposed mechanism o f d o x o r u b i c i n uptake i n LUVETs e x h i b i t i n g a transmembrane pH g r a d i e n t 4 4 14. The i n f l u e n c e o f c h o l e s t e r o l c o n c e n t r a t i o n on d o x o r u b i c i n r e l e a s e i n LUVETs 52 15. The e f f e c t o f l y s o PC on t h e r e l e a s e o f d o x o r u b i c i n i n LUVETs 53 16. R e l e a s e o f d o x o r u b i c i n i n v e s i c l e s d e r i v e d from MLVs 55 17. R e l e a s e o f d o x o r u b i c i n i n v e s i c l e s d e r i v e d from FATMLVs 56 v i i 18. The e f f e c t o f v a r y i n g i n i t i a l d r u g t o l i p i d r a t i o s on t h e r e t e n t i o n o f d o x o r u b i c i n i n l i p o s o m e s 58 19. V a r y i n g i n i t i a l d rug t o l i p i d r a t i o s on d o x o r u b i c i n i n l i p o s o m a l systems c o n t a i n i n g 1M c i t r a t e 60 20. The absorbance s p e c t r a between 400 and 700nm f o r d o x o r u b i c i n s o l u t i o n s a t pH 7.5 and 10.5 64 21. Comparison o f f r e e / t o t a l d o x o r u b i c i n r a t i o s w i t h t h e absorbance r a t i o a t 600nm b e f o r e and a f t e r a d d i t i o n o f t r i t o n X-100 t o a l k a l i n i z e d sample 65 v i i i A B B R E V I A T I O N S CL C a r d i o l i p i n Choi,C Cholesterol DCP Dicetylphosphate Dox Doxorubicin DPPC Dipalmitoylphosphatidylcholine EPC Egg phosphatidylcholine HBS Hepes buffered saline Hepes [4-(2-Hydroxyethyl)]-piperazineethansulfonic acid LUVs Large unilamellar v e s i c l e s LUVETs Large unilamellar v e s i c l e s by extrusion techniques MLVs Multilamellar v e s i c l e s OD Optical density PG Phosphatidylglycerol ApH Transmembrane pH gradient A^ Transmembrane potential PL Phospholipid SA Stearylamine SUVs Small unilamellar v e s i c l e s TX-100 Tri t o n X-100 VET„ # Vesicles produced by extrusion of a s p e c i f i c s i z e ix ACKNOWLEDGEMENTS As previous acknowledgements from t h i s lab are a hard act to follow, I w i l l t r y to keep mine as simple as possible. F i r s t of a l l , I must thank Pieter C u l l i s and Mick Hope for fostering an excellent ( and fun ) s c i e n t i f i c working environment. Other individuals who I would also l i k e to thank include : Marcel Bally, Colin Tilcock, Tom Madden, Kim Wong, Larry Reinish, Tom Redelmeier , Helen Loughrey, Richard Harrigan, Simon Eastman, Michel Lafleur, J e f f r e y Veiro, N e i l Kitson, Archie Chonn and Diane Tanguay (whew!). Special thanks to Lawrence Mayer for having the patience to read t h i s thesis more than once (albeit while drinking scotch I) and introducing me to the pleasures of caring for s a l t water f i s h . Individuals aside, I would also l i k e to extend my thanks to Lipex Biomembranes Inc. (biotechnology for the future) for organizing the best s k i t r i p s possible, providing me with pocket money and most importantly for inducing my elusive alcohol dehydrogenase. A l l of you have been instrumental i n making t h i s thesis both enjoyable and possible. x INTRODUCTION 1.1 Model Membrane Systems A l l b i o l o g i c a l membranes contain a v a r i e t y of phospholipids, varying amounts of s t e r o l s and a wide array of proteins. The a b i l i t y of l i p i d s to assume a b i l a y e r structure i s dictated by the amphipathic character of membrane l i p i d s . These l i p i d s contain a polar or hydrophilic headgroup region and a nonpolar or hydrophobic region. Much of the research i n b i o l o g i c a l membranes involves the use of model membrane systems which contain naturally occurring or synthetic l i p i d species of i n t e r e s t . Generation of model membrane systems requires three steps. F i r s t there i s the i s o l a t i o n or chemical synthesis of a given l i p i d . Second, an appropriate model system must be constructed containing that l i p i d and l a s t l y incorporation of a non - l i p i d component to determine i t s influence on the l i p i d or v i c e versa. Hydration of a dry phospholipid f i l m r e s u l t s i n the formation of structures c a l l e d liposomes (Bangham et a l , 1965). The study of liposomes began with Bangham i n the mid-1960's. Bangham's pioneering work has resulted i n an extensive use of liposomes as models of b i o l o g i c a l membranes. Liposomes can carry water soluble agents i n t h e i r aqueous compartments and l i p i d soluble agents i n t h e i r b ilayers and can be prepared from a v a r i e t y of phospholipids thus producing v e s i c l e s of d i f f e r i n g properties. Major components which are generally used include 1 egg phosphatidylcholine, cholesterol and charged amphiphiles such as phosphatidic acid (negative charge) or stearylamine (positive charge) to provide p a r t i c u l a r properties to the whole v e s i c l e as a r e s u l t of the net charge. 1.2 Preparation and Properties of Liposomes Liposomes are spontaneously formed as multilamellar v e s i c l e s (MLVs) upon hydration of a dried phospholipid f i l m . Multilamellar v e s i c l e s ( f i g . 1) are composed of a series of concentric b i l a y e r s interrupted by narrow aqueous spaces. MLVs are quite heterogeneous ranging i n size from about 80nm to lOu (Kaye, 1981) and being e a s i l y prepared are frequently u t i l i z e d for defining the properties of l i p i d s . Use of MLVs i s mostly r e s t r i c t e d to physical studies on b i l a y e r organization and the motional properties of individual l i p i d within a membrane structure ( C u l l i s et a l , 1985). Small unilamellar v e s i c l e s (SUVs) ( f i g . 2) can be produced by sonicating MLVs with either a probe or bath-type sonicator. These SUVs are characterized by diameters less than 50nm and a very high degree of membrane curvature (ie outer to inner monolayer phospholipid r a t i o i s generally greater than 1) re s u l t i n g i n packing r e s t r a i n t s . Some sonicated v e s i c l e systems are also metastable and tend to fuse into larger v e s i c l e s (Parente and Lentz, 1984). This can r e s t r i c t the use of SUVs for physical studies on various properties of membrane l i p i d s . 2 Large unilamellar v e s i c l e s (LUVs) of diameters greater than lOOnm can be generated by a variety of techniques including reverse phase and solvent evaporation (Szoka et a l , 1980). The reverse phase and solvent evaporation techniques, however, are both tedious and time-consuming as organic solvents must be c a r e f u l l y removed. In a recent work (Hope et a l , 1985), a procedure was developed for the rapid generation of LUVs ( f i g . 3) v i a extrusion of preformed MLVs through polycarbonate f i l t e r s under a moderate pressure. Advantages of the extrusion procedure includes i t s rapid preparation time and i t s use in generation of large unilamellar v e s i c l e s from a wide variety of l i p i d species and mixtures. These LUVETs (large unilamellar v e s i c l e s by extrusion technique) can be generated at high l i p i d concentrations and exhibit r e l a t i v e l y high trapping e f f i c i e n c i e s . 1 . 3 Rationale for Drug Encapsulation i n Liposomes I n i t i a l l y , the goal of liposome encapsulation involved enzymes for replacement therapy i n genetic deficiency diseases. Since then a wide range of compounds have been successfully entrapped i n liposomes with the aim of modifying t h e i r behavior i n the body. These compounds include a n t i b i o t i c s ( p e n i c i l l i n ) (Gregoriadis, 1973), antimonials used i n leishmaniasis (New et a l , 1978; Alving, 1982; Alving and Schwartz, 1985) and hormones such as i n s u l i n (Patel et a l , 1976). These aims were broadened to include a considerable emphasis on anticancer drugs 3 F i g . l Freeze-fracture electron micrographs of egg PC at 50 mg/ml concentration. (A) MLVs prepared i n 150mM NaCI, 20mM Hepes at pH 7.4. (B) MLVs i n the same buffer containing 25% (v/v) gl y c e r o l . MLVs were subjected to 5 freeze-thaw cycles. (C) FATMLVs were prepared by subjecting MLVs to 5 freeze-thaw cycles in the absence of g l y c e r o l . Bar = 200nm. The micrographs were reproduced with permission from M.J. Hope. 4 Fig.2 Freeze-fracture electron micrographs of egg PC SUVs at lOmg/ml concentration. SUVs were prepared in 150mM NaCl, 20mM Hepes at pH 7.4 by sonication of the MLV preparation. Bar = 2 00nm. The micrographs were reproduced with permission from M.J. Hope. 5 Fig. 3 Freeze-fracture electron micrographs of FATMLVs passed 2 0 times through f i l t e r s of various pore s i z e s . V e s i c l e s were prepared from egg PC at lOOmg/ml. The pore sizes of the f i l t e r s employed were A) 400nm, B) 200nm, C) lOOnm, D) 50nm, and E) 30nm. The bar represents 150nm and a l l panels e x h i b i t the same magnification. Electron micrographs were reproduced with permission from M.J.Hope. 6 (Weinstein and Leserman, 1984; Weinstein, 1984; Poznansky and Juliano, 1984). The d i r e c t administration of these drugs can be hampered by immunological reactions, development of drug resistance and uptake of drug by non-diseased tissues often leading to serious side e f f e c t s . As liposomes are amphipathic by nature i t was believed that both polar, hydrophilic drugs as well as nonpolar, hydrophobic drugs could be entrapped within the aqueous compartment or within the b i l a y e r . For polar drugs the entrapment depends on the s o l u b i l i t y of the drug i n water and on the volume of water encapsulated per mass of l i p i d . For l i p o p h i l i c drugs, maximal incorporation into liposomes simply depends on the amount of l i p i d and the s o l u b i l i t y of the drug i n the l i p i d . There are many possible reasons for the use of liposomes as drug c a r r i e r s i n vivo. F i r s t l y , they are non-toxic, non-immunogenic and biodegradable. In p a r t i c u l a r , liposomes often protect the host against liposome contents. This i s b e n e f i c i a l i f the drug, i n the free form, has toxic side e f f e c t s on non-diseased tissues. A liposome can also be used to concentrate the drug as i t can carry hundreds to thousands of drug molecules compared to only a few molecules of drug which can be d i r e c t l y coupled to small molecular c a r r i e r s such as antibodies or hormones. Liposomes tend to c i r c u l a t e in the bloodstream much longer than most free drugs. This i s es p e c i a l l y true of v e s i c l e s which are negatively charged or less than lOOnm i n diameter (Juliano and Layton, 1980). 7 There are numerous problems and l i m i t a t i o n s of liposomes as drug c a r r i e r s i n vivo. A considerable percentage of injected liposomes are removed from the c i r c u l a t i o n by r e t i c u l o e n d o t h e l i a l c e l l s i n the l i v e r , spleen and elsewhere. Thus a f a i r proportion of liposomally encapsulated drug never reaches i t s intended s i t e . Another problem involves the i n a b i l i t y of liposomes to escape the c i r c u l a t o r y system because of b a r r i e r s such as the endothelial c e l l layer and basement membrane i n the c a p i l l a r i e s (Weinstein,1984). Thus liposomes are u n l i k e l y to reach c e l l s of s o l i d tumours. Also a variety of other factors such as uniformity of liposome siz e , s t e r i l i t y and s t a b i l i t y must also be taken into account i n any liposome preparation which i s intended for pharmaceutical application. 1.4 Factors A f f e c t i n g Encapsulation of Drugs i n Liposomes Previous studies have shown that highly polar hydrophilic drugs such as cytosine arabinoside or p e n i c i l l i n are encapsulated at lower e f f i c i e n c y than less polar hydrophobic drugs such as actinomycin D or doxorubicin (Fendler, 1980). This i s most l i k e l y due to the fact that polar drugs are contained only i n the internal aqueous compartment while nonpolar drugs may be d i s t r i b u t e d between the aqueous compartment and l i p i d membrane compartment of the liposome. This behaviour can be demonstrated by b r i e f l y sonicating liposomes containing entrapped polar or l i p o p h i l i c drugs. The polar molecules are quickly and completely released as the 8 liposome membrane was disrupted while the l i p o p h i l i c drug remains associated with the liposome membrane (Stamp et a l , 1979) . Since a large v a r i e t y of techniques are available for the preparation of liposomes (Hope et a l , 1986) the s e l e c t i o n of the encapsulation protocol i s based on the p a r t i c u l a r demands a r i s i n g from the type of drug to be entrapped and the b i o l o g i c a l e f f e c t s desired. One of the most popular techniques for drug encapsulation involves passive entrapment which r e l i e s on the a b i l i t y of liposomes to capture a cer t a i n aqueous trap volume during v e s i c l e formation. Passive drug entrapment can be applied to the 3 types of liposomes previously described. Sonicated v e s i c l e s may be e a s i l y prepared and v i r t u a l l y any l i p i d composition can be employed. However, drawbacks to using t h i s system for drug encapsulation include the low trapping e f f i c i e n c i e s from 1-5% (Szoka and Papahadjopoulos, 1978; Mayhew et a l , 1984) and that sonication may degrade entrapped molecules such as proteins (Lelkes, 1984). Large unilamellar v e s i c l e s have been used because of t h e i r larger aqueous trap volume enabling superior drug to l i p i d r a t i o s and trapping e f f i c i e n c i e s to be obtained compared to most SUVs and MLVs. Methods for producing LUVs which have gained widespread popularity for entrapping drugs i n LUVs include solvent evaporization/vaporization techniques (Szoka et a l , 1978) and the extrusion technique (Hope et a l , 1985 ; Mayer et a l , 1986). F i n a l l y multilamellar v e s i c l e s 9 have also been employed as drug c a r r i e r s because maximal drug retention can be obtained due to the increased number of lamellae the drug must cross to reach the exterior. However, the major problem t y p i c a l l y experienced with MLVs i s the low aqueous trapped volumes and trapping e f f i c i e n c i e s obtained for t r a d i t i o n a l MLV dispersions. Techniques including solvent evaporation (Gruner et a l , 1985) or freeze-thawing MLVs (Mayer s et a l , 1985) have resulted i n an increase i n the aqueous trap volume of multilamellar systems. Based on trapping e f f i c i e n c y i t i s possible that passive entrapment may not provide an e f f i c i e n t method for entrapment. Amphiphilic compounds are d i f f i c u l t to encapsulate and r e t a i n i n liposomes since they can r e a d i l y permeate through l i p i d b i l a y e r s . Alterations i n l i p i d compositions of liposomes have been used to enhance encapsulation e f f i c i e n c y and decrease release rates by enhancing i o n i c interactions between drug and charged l i p i d components. The use of ion gradients offers a more general means for encapsulation of amphiphilic drugs such as doxorubicin. This process i s analagous to the a b i l i t y of various probes of membrane potentials and transmembrane proton gradients to d i s t r i b u t e across l i p i d b i l a y e r s i n response to t^l or ApH. Studies have shown that l i p o p h i l i c cations such as the drug safranine can be accumulated by LUVs exhibiting a K potential (Bally et a l , 1985). In these systems the membrane potentials were established by forming LUVETs i n potassium glutamate and subsequently exchanging the untrapped potassium glutamate for 10 NaCl. The ionophore valinomycin, which i s s p e c i f i c for + potassium, was used to move K out and generate a membrane pot e n t i a l ( i n t e r i o r negative). Bulk accumulation of antineoplastic agents (Mayer et al,1985,1986; Bally et a l , 1985) and biogenic amines (Nichols and Deamer, 1976; Bally et a l , 1988) inside liposomes can be obtained by incubating the drug i n the presence of v e s i c l e s + + ex h i b i t i n g K or H gradients. Although both types of gradients induce e f f i c i e n t entrapment of l i p o p h i l i c cations, drug uptake i n response to ApH most l i k e l y w i l l be of more p r a c t i c a l use. This i s due to the fact that -dependent encapsulation requires the use of exogenous ionophores, which are usually t o x i c , whereas ApH-dependent uptake does not. The use of ion gradients i s an extremely useful method for entrapment as the transmembrane ion gradients not only accomplishes e f f i c i e n t drug encapsulation but also decrease the rate of drug e f f l u x from the v e s i c l e s . As previously mentioned, doxorubicin i s c l a s s i f i e d as an amphipathic cation. I t i s among the most widely used antineoplastic agents which i s e f f e c t i v e against a broad spectrum of tumors. However, cardiotoxic side e f f e c t s l i m i t the dose which can be administered. Thus i t i s thought that liposome encapsulation of doxorubicin may provide s i g n i f i c a n t therapeutic benefit while decreasing the dose-limiting t o x i c side e f f e c t s by a l t e r i n g the pharmacokinetics of the drug. 11 1.5 Doxorubicin 1.5.1 Doxorubicin-Structure D o x o r u b i c i n , i d e n t i f i e d by Arcamone e t a l i n 1969, i s one o f t h e most i m p o r t a n t a n t i t u m o u r agents i n c l i n i c a l use t o d a y . I t d i s p l a y s a c t i v i t y a g a i n s t a wide range o f human m a l i g n a n c i e s , i n c l u d i n g a v a r i e t y o f s o l i d tumours. D o x o r u b i c i n ( f i g . 4) c o n s i s t s o f a t e t r a c y c l i n e r i n g s t r u c t u r e a t t a c h e d t o an u n u s u a l s u g a r , daunosamine, by a g l y c o s i d i c l i n k a g e . The s u g a r has a p r i m a r y amine w h i c h has a pKa—9 and i s t h e r e f o r e p r e d o m i n a n t l y p r o t o n a t e d a t p h y s i o l o g i c a l pH's. C y t o t o x i c a g e n t s o f t h e a n t h r a c y c l i c a n t i b i o t i c c l a s s a l l have quinone and h y d r o q u i n o n e m o i e t i e s on a d j a c e n t r i n g s t h a t p e r m i t them t o f u n c t i o n as e l e c t r o n - a c c e p t i n g and - d o n a t i n g agents 1.5.2 Doxorubicin-Mechanism of Action S t u d i e s s u g g e s t t h a t d o x o r u b i c i n b i n d s t o n u c l e a r DNA and i t s c onsequent i n h i b i t i o n o f DNA r e p l i c a t i o n and RNA t r a n s c r i p t i o n i s c o n s i d e r e d t o be one o f t h e major mechanisms o f c y t o t o x i c i t y ( P o t m e s i l e t a l , 1984). However, t h e e x a c t n a t u r e o f t h i s p r o c e s s remains u n c l e a r and s e v e r a l e x p l a n a t i o n s o f t h e sequence o f e v e n t s l e a d i n g t o DNA damage have been s u g g e s t e d . The damage caused by d o x o r u b i c i n may be r e l a t e d t o t h e f o r m a t i o n o f f r e e r a d i c a l s . E l e c t r o n s from NADPH a r e t r a n s f e r r e d t o t h e quinone m o i e t y o f d o x o r u b i c i n , most l i k e l y due t o t h e i n t e r a c t i o n s between one o f t h e a n t h r a c y c l i n e r i n g s o f 12 Fig.4 Structure of Doxorubicin 13 doxorubicin and the f l a v i n component of NADPH:cytochrome P-450 reductase (Bachur et a l , 1982). Another explanation suggests that the d i s t o r t i o n of the DNA h e l i x caused by drug i n t e r c a l a t i o n may activate endonucleases which r e s u l t s i n DNA strand s c i s s i o n (Ross et a l , 1979, 1981). Recent studies (Potmesil et a l , 1984) have provided evidence that both mechanisms may occur but that t h e i r detection depends on the concentration of doxorubicin. At high drug concentrations, greater than the peak plasma l e v e l achievable a f t e r i . v . bolus, i t has been shown that DOX-mediated free r a d i c a l s cause d i s c e r n i b l e DNA damage. At lower doxorubicin l e v e l s , more relevant to c l i n i c a l use, another type of i n t e r a c t i o n between drug and DNA seems to occur which i s independent of the f r e e - r a d i c a l mechanism. 1.5.3 Therapeutic Uses and C l i n i c a l T o x i c i t y of Doxorubicin Doxorubicin has been found e f f e c t i v e against acute leukemias and malignant lymphomas. When i t i s used concurrently with other anticancer drugs ( c i s p l a t i n , cyclophosphamide), i t has been found successful i n tr e a t i n g carcinoma of the ovary and non-Hodgkin's lymphoma. I t i s also one of the most active single agents for treatment of metastatic adenocarcinoma of the breast, carcinoma of the bladder and neuroblastoma (DiMarco, 1975; Calabresi et a l , 1985) However, serious toxic manifestations l i m i t the t o t a l dose of doxorubicin which can be administered. Acute t o x i c i t y 14 involving myelosuppression i s a major dose-limiting complication with leukopenia reaching a maximum during the second week of therapy and recovering by the fourth week. Stomatitis, g a s t r o i n t e s t i n a l disturbances and alopecia are also common acute side e f f e c t s but are reversible. Tissue necrosis may develop i f the intravenous i n j e c t i o n misses the vein. Cardiomyopathy i s a unique c h a r a c t e r i s t i c of anthracycline a n t i b i o t i c s . Two forms of cardiomyopathies may occur, the f i r s t type i s an acute form which i s characterized by abnormal changes i n the ECG. This form i s b r i e f and rarely presents a serious problem. However, the second type i s a chronic, cumulative dose-related t o x i c i t y which often r e s u l t s i n congestive heart f a i l u r e that i s 2 unresponsive to d i g i t a l i s . A maximum t o t a l dose of 550 mg/m should not be exceeded as the mortality rate above t h i s dose i s in excess of 50% (Minow et a l , 1977; Bristow et a l , 1978; Myers, 1982; Wiemann and Calabresi, 1985). 1 .5.4 D i s t r i b u t i o n and Metabolism of Doxorubicin Once injected intravenously, doxorubicin i s cleared rapidly from the plasma with a t r i p h a s i c disappearance curve. There i s a rapid uptake of doxorubicin by the heart, kidneys, lungs, l i v e r , spleen but the drug does not appear to cross the blood-brain b a r r i e r . Doxorubicin i s mainly metabolized i n the l i v e r and excreted i n the b i l e . A s i g n i f i c a n t portion of doxorubicin i s excreted unchanged and the rest appears to be converted into multiple metabolites including adriamycinol (Myers, 1982; Myers et a l , 1984; Wiemann and Calabresi, 1985). 15 1.6 Rationale for Doxorubicin Encapsulation i n Liposomes The ultimate goal of a liposomal-encapsulation of doxorubicin i s to maintain or improve the antitumour properties of the drug while s i g n i f i c a n t l y decreasing the t o x i c side e f f e c t s . The predominant toxic e f f e c t s of concern are the myelosuppression and the chronic c a r d i o t o x i c i t y . As liposomes are taken up mainly by re t i c u l o e n d o t h e l i a l c e l l s of the l i v e r and spleen the entrapment of doxorubicin i n liposomes may reduce the amount which i s taken up by the heart and decrease the c a r d i o t o x i c i t y . In t h i s thesis, a novel "active" trapping procedure for doxorubicin was developed using liposomes with a pre-established transmembrane pH gradient. This system has been characterized by examining the variables which may influence uptake and release of doxorubicin including cholesterol content, inte r n a l buffering capacity, temperature and v e s i c l e s i z e . In addition, a technique for rapid assaying of the amount of entrapped doxorubicin w i l l be discussed as i t has both research and broader c l i n i c a l applications. I t i s also hoped that t h i s active trapping procedure w i l l apply to the entrapment of other l i p o p h i l i c c a t i o n i c drugs such as daunomycin and v i n c r i s t i n e . 16 2 . Uptake of Doxorubicin into Large Unilamellar Vesicles i n Response to a pH Gradient 2 . l Introduction Previous studies have shown that doxorubicin can be accumulated into large unilamellar v e s i c l e s made up of egg PC i n response to a K generated membrane potential created by the ionophore valinomycin (Mayer et a l . , 1986). In t h i s study, i t has been demonstrated that doxorubicin can be accumulated i n LUVs which exhibit a pH gradient i n the absence of ionophores. The pH gradient i s established by hydrating the v e s i c l e s i n pH 4.0 buffer and exchanging for an outside buffer of pH 7.5. This encapsulation procedure which r e l i e s on the a b i l i t y of doxorubicin to accumulate into liposomal systems i n response to a transmembrane pH gradient (inside acidic) allows doxorubicin to be loaded into preformed liposomes immediately p r i o r to use, thus eliminating s t a b i l i t y problems. Trapping e f f i c i e n c i e s approaching 100% are rea d i l y achieved and drug to l i p i d r a t i o s 3- to 10-fold higher than obtained for previous formulations are straightforward. Various parameters such as l i p i d composition, temperature, i n t e r i o r buffering capacity and v e s i c l e s i z e were examined i n order to elucidate the mechanism of doxorubicin uptake into v e s i c l e s . 17 2 . 2 Materials and Methods 2.2.1 Materials Egg phosphatidylcholine was obtained from Avanti Polar Lipids and was greater than 99% pure. Cholesterol, c i t r i c acid, lyso phosphatidylcholine, NaCl, Hepes and TX-100 from Sigma Chemical Company. Doxorubicin was obtained from the Cancer 3 14 Control Agency (Vancouver, B.C.). H-DPPC, C-citrate and 14 C-methylamine were purchased from New England Nuclear. 2.2.2 Preparation of large unilamellar v e s i c l e s Vesicles of various sizes were prepared by the extrusion technique according to the protocol of Hope et a l (1985). A dry l i p i d f i l m of egg PC or egg PC:cholesterol was hydrated i n c i t r a t e buffer and vortexed to produce multilamellar v e s i c l e s between 25 and 2 00 umole phospholipid/ml. The r e s u l t i n g dispersion was then frozen i n l i q u i d nitrogen and thawed i n a o 40 C water bath. A t o t a l of f i v e freeze-thaw cycles was usually employed. These frozen and thawed MLVs or FATMLVs were then transferred into a device (produced by Sciema Technical Services and sold through Lipex Biomembranes,Inc.) which allowed the extrusion of the FATMLVs through standard 25mm polycarbonate with pore sizes ranging from 0.2 to 0.015u (Nucleopore Corp., Pleasanton, CA). Unless otherwise stated, two stacked f i l t e r s were employed and extrusion was repeated 10 times. 18 The si z e d i s t r i b u t i o n s of the extruded liposomal systems were determined by qua s i - e l a s t i c l i g h t (QEL) scattering u t i l i s i n g a Nicomp Model 200 Laser P a r t i c l e Sizer with a 5mW Helium-Neon Laser at an exc i t i n g wavelength of 632.8 nm. QEL scattering employs d i g i t a l autocorrelation to analyze the fluctuations i n scattered l i g h t i n t e n s i t y generated by the d i f f u s i o n of v e s i c l e s i n solution. The measured d i f f u s i o n c o e f f i c i e n t i s used to obtain the average hydrodynamic radius and hence the mean diameter of the v e s i c l e s . 2.2.3 Generation of the pH gradient The pH gradient (inside acidic) was generated by forming the LUVs i n c i t r a t e buffers pH 4.0 ranging i n concentration from lOmM to 1M and subsequently exchanging the untrapped buffer for 150mM NaCI, 20mM Hepes pH 7.5 buffer (Hepes buffered saline, HBS) employing Sephadex G-50 5ml columns equilibrated i n HBS. A faster alternate method which involved d i l u t i n g the pH 4.0 v e s i c l e s i n sa l i n e (2X) and t i t r a t i n g the exterior pH to 7.5 using NaOH was also employed . The pH gradient of these systems 14 was determined u t i l i s i n g C-methylamine as the pH probe. One 14 uCi/ml of C-methylamine was added to v e s i c l e s exhibiting pH gradients and allowed to equ i l i b r a t e for 15 minutes. Aliquots were passed down Sephadex G-50 columns to remove untrapped 14 14 C-methylamine. L i p i d phosphorous and C-methylamine content of liposomes were assayed before and af t e r gel f i l t r a t i o n . 19 L i p i d phosphorous was quantitated using the method prescribed by 14 Fiske and Subbarow (1975). C-methylamine was quantitated using a United Technologies Packard Tri-Carb 2 000 Series Liquid S c i n t i l l a t i o n Analyzer. The pH gradient was determined by c a l c u l a t i o n of i n t e r i o r and exterior proton concentration according to the following equation: pH gradient = l o g 1 Q [ H + ] ^ + [H ] L J o 2.2.4 Doxorubicin uptake i n response to pH gradients Doxorubicin i n either solution or powder form was added to LUV dispersions of defined concentrations i n the presence or o absence of a pH gradient. The mixture was then heated at 60 C for 10 minutes with intermittent vortexing to ensure a l l powdered doxorubicin was i n solution. After 10 minutes, the non-sequestered drug was removed by loading aliquots of the solution onto a Sephadex G-50 column packed i n 1ml disposable syringes (Pick, 1981). L i p i d and drug were then assayed. L i p i d concentrations were determined by l i q u i d s c i n t i l l a t i o n counting 3 to quantitate H-DPPC (0.05uCi/ umole l i p i d ) or by phosphorous assays (Fiske and Subbarow, 1925). Doxorubicin was quantitated following the mixing of an aliquot of the column eluant with 1% t r i t o n X-100 to disrupt the v e s i c l e s and release the trapped drug. The absorbance of t h i s solution i s monitored employing a Shimadzu UV-160 spectrophotometer and doxorubicin uptake i s expressed i n nmoles doxorubicin/umole phospholipid or t o t a l l i p i d . 20 2.2.5 Passive entrapment of doxorubicin Doxorubicin was trapped passively by preparing v e s i c l e s i n 150mM NaCI, 20mM Hepes buffer containing the drug. The non-sequestered drug was removed by passing aliquots of the solution over 1ml Sephadex G-50 columns. L i p i d and drug concentrations were then assayed according to the protocol i n section 2.2.4. 2.2.6 Trap volume determinations Trap volumes were determined by preparing multilamellar 14 v e s i c l e s i n the presence of 1 uCi/ml of C-citrate i n the c i t r a t e buffer pH 4.0 and the LUVETS were made according to the procedure i n section 2.2.2. Aliquots were then loaded onto a Sephadex G-50 1ml spin column and v e s i c l e s eluted by centrifugation of t h i s column at 500xg for 3 minutes. Samples obtained before and af t e r the G-50 column were assayed for l i p i d 14 phosphorous and C-citrate was determined using a l i q u i d s c i n t i l l a t i o n counter. Trapped volumes calculated are expressed as u l of trapped volume per umole of phospholipid or t o t a l l i p i d . 21 2.3 R e s u l t s 2.3.1 Temperature dependence of doxorubicin uptake As described i n the protocol of doxorubicin uptake i n o section 2.2.4 the sample was heated to 60 C for 10 minutes. This temperature was arrived at a f t e r experiments involving uptake at various temperatures showed uptake to be temperature dependent for v e s i c l e s composed of egg PC:cholesterol i n a 55:45 mole percent r a t i o . Comparative uptake studies were performed at o 21, 37 and 60 C over a time course of 120 minutes to determine the k i n e t i c s of doxorubicin uptake. F i g . 5 demonstrates that the rate and e f f i c i e n c y of drug encapsulation i s extremely temperature dependent for liposomal systems containing cholesterol. Incubation of doxorubicin i n the presence of liposomes exhibiting a transmembrane pH gradient of 3.5 units o (inside buffer of 300mM c i t r a t e at pH 4.0) at 21 C r e s u l t s in only 50% trapping e f f i c i e n c y . Increasing the temperature of o incubation above 37 C y i e l d s doxorubicin trapping e f f i c i e n c i e s of approximately 100%. This value i s achieved within 90 minutes o o at 37 C and 5 minutes at 60 C. 22 Fig. 5 Temperature dependence of doxorubicin uptake i n Egg PC:cholesterol LUVETs prepared as indicated i n section 2.2.2 with 300mM c i t r a t e pH 4.0 inside and 150mM NaCI, 20mM Hgpes pH 7.5 outside. The temperatures of incubation were (•) 21 C, (%) 37 C and ( A ) 60°C. 30 60 90 120 Time (min) 23 2.3.2. E f f e c t of cholesterol on doxorubicin uptake The i n c l u s i o n of cholesterol ( f i g . 6) i n the b i l a y e r has a very important modulatory e f f e c t on the phase t r a n s i t i o n behaviour of b i l a y e r s composed of homogeneous phospholipids (Poznansky and Juliano, 1984). Cholesterol i s often used to increase the r i g i d i t y of the b i l a y e r and to reduce leakage of entrapped molecules from phosphatidycholine-containing v e s i c l e s . Cholesterol has been included i n these liposomal systems to improve t h e i r a p p l i c a b i l i t y as drug delivery vehicles. I t has been shown that an incorporation of cholesterol of greater than 30 mole percent into the liposomal b i l a y e r can s i g n i f i c a n t l y reduce the tendency of serum components such as lipoproteins to disrupt liposomes (Weinstein, 1984; F i n k e l s t e i n and Weissmann, 1979; Kirby et a l , 1980). Studies i n which cholesterol composition i n v e s i c l e s ranged from 0 to a maximum of 45 mole percent showed that cholesterol did not greatly influence the o ApH-dependent uptake of doxorubicin at 60 C. The v e s i c l e s were composed of egg PC:cholesterol (100:0 mol%), egg PC:cholesterol (85:15), egg PC:cholesterol (67:33) and egg PC:cholesterol (55:45). At a drug to l i p i d r a t i o of 0.3:1 the trapping e f f i c i e n c i e s obtained for the d i f f e r e n t systems were a l l greater than 90% (Table 1). 24 F i g . 6 S t r u c t u r e of c h o l e s t e r o l 25 Table 1. Uptake of doxorubicin at various cholesterol concentrations. Vesicles were prepared according to the protocol i n section 2.2.2. Separation of free from liposomally encapsulated doxorubicin was accomplished using G-50 columns. % Cholesterol Before Sep'n* After Sep'n* %Trapping e f f . 0 336 301 90 15 320 291 91 33 298 272 91 45 289 287 99 * Before and a f t e r separation values are expressed as nmole DOX/umole l i p i d Table 2. E f f e c t of lyso PC on uptake of doxorubicin. Vesicles were prepared according to the protocol i n section 2.2.2 except that the pH gradient was established by t i t r a t i n g the exterior pH to 7.5 with IM NaOH and the i n t e r i o r buffer was 150mM c i t r a t e at pH 4.0. % lyso PC Before Sep'n* After Sep'n* %Trapping e f f . 0 289 287 99 1 288 281 98 5 222 221 100 * Before and af t e r separation values are expressed as nmole DOX/umole l i p i d . 26 2.3-3 E f f e c t of lyso PC on doxorubicin uptake The e f f e c t of addition of lyso PC into egg PC:cholesterol v e s i c l e s was examined to determine i f uptake was affected by the presence of small quantities of lyso PC. The structure of lyso PC i s i d e n t i c a l to that of egg PC except that only one f a t t y acyl chain i s ester-linked to the g l y c e r o l backbone instead of two chains for egg PC. I t i s known that the ester linkage between the f a t t y acid chains and the g l y c e r o l backbone of the phosphatidylcholine i s susceptible to acid hydrolysis. Since the i n t e r i o r buffer i s c i t r a t e at pH 4.0, i t was postulated that lyso PC may be produced. This increase i n lyso PC may r e s u l t i n an increase i n v e s i c l e permeability due to the micellar and s o l u b i l i z i n g character of lyso phospholipids ( Weiner, 1987). It has been found that the presence of lyso PC at a c i d i c pH markedly increased the permeability of mucosal c e l l s i n the d i s t a l ileum ( Bolin et aJL, 1981) . Thus experiments were done to determine i f lyso PC increased v e s i c l e leakiness such that uptake was decreased due to rapid e f f l u x of doxorubicin a f t e r uptake. The systems that were examined included lyso PC:EPC:cholesterol i n the following mole percent r a t i o s a) 1:51:48, b) 5:47:48 . I t was found that the l i p i d composition did not influence the ApH-dependent uptake of doxorubicin. Both systems achieved a trapping e f f i c i e n c y of approximately 100% as shown i n Table 2. 27 2.3.4 E f f e c t of doxorubicin concentration on v e s i c l e permeability and s t a b i l i t y The actual e f f e c t of doxorubicin on liposome permeability i s not well characterized. Determination of v e s i c l e leakiness 14 and s t a b i l i t y was accomplished using C - i n u l i n as a trap volume and permeability probe. Inulin i s a neutral molecule (molecular weight of approx. 5000) which i s not normally permeable to membranes. In t h i s experiment the egg PC:cholesterol was hydrated i n Hepes buffered saline pH 7.5 containing luC i 14 C-inulin/ml of buffer. The v e s i c l e preparation was sized through 0.2um f i l t e r s and then passed down a 5ml LKB Ultrogel column and trap volume was determined. Liposomes that were passed down the u l t r o g e l columns were then incubated with various concentrations of doxorubicin from 0.1 to 40mM and a saline control at room temperature over a time course of two 14 hours. Table 3 shows that leakage of C-inu l i n from the ves i c l e s i s n e g l i g i b l e for samples incubated with up to 40mM doxorubicin. These re s u l t s suggest that the presence of doxorubicin does not s i g n i f i c a n t l y a l t e r the membrane permeability. The e f f e c t of passively entrapped doxorubicin on the membrane permeability and trapped volume of FATMLVs was also studied. The l i p i d f i l m was hydrated i n Hepes buffered saline 14 containing l u C i C-inulin/ml of buffer and 34mM doxorubicin. Trap volumes were determined by taking aliquots of the solution 28 Table 3. E f f e c t of external doxorubicin concentration on v e s i c l e leakiness. Vesicles were prepared according to the protocol i n ^section 2.2 and 2.3.4. Leakage of probe i s expressed as DPM C-inulin/umole PC. Time (min) External 15 45 60 120 [DOX](mM) 0 5630 5920 6244 6217 0.1 5810 6195 6287 5677 1.0 5473 5436 5872 5470 5.0 5803 5682 6074 5870 10.0 5654 6138 6157 5474 20.0 5955 5774 5755 5739 40. 0 6461 5731 6172 6187 Table 4. Trap volumes of v e s i c l e s with i n t e r n a l l y and externally incubated doxorubicin. The aqueous trap volume of the v e s i c l e s were determined according to the protocol i n section 2.2.6. Trap volume* Before After Externally added DOX 2.33 2.30 Passively entrapped DOX 4.84 4.72 (FATMLVs)+ * Trap volume values are expressed as u l buffer/umole l i p i d + For the passively entrapped drug FATMLVs were used instead of v e s i c l e s extruded through 0.2um f i l t e r s . 29 a f t e r passive entrapment and af t e r d i a l y s i s of the v e s i c l e s over 14 24 hours to remove untrapped C-inulin. Quantitation of the i n u l i n and phosphorous and trap volume determinations were completed as described i n section 2.2.6. Table 4 shows the trap volumes obtained for the passively entrapped doxorubicin and externally added doxorubicin. The passively entrapped doxorubicin does not appear to a f f e c t v e s i c l e s i z e or i n t e g r i t y as the trap volumes did not change a f t e r d i a l y s i s . 30 2.3.5 E f f e c t o f b u f f e r i n g c a p a c i t y on d o x o r u b i c i n uptake I n t h e c h a r a c t e r i z a t i o n o f ApH-dependent d o x o r u b i c i n uptake i t was h y p o t h e s i z e d t h a t t h e b u f f e r i n g c a p a c i t y o f t h e i n t e r i o r b u f f e r p l a y s a l a r g e r o l e i n upta k e . T h i s h y p o t h e s i s was f i r s t t e s t e d by d e t e r m i n i n g t h e b u f f e r i n g c a p a c i t y o f t h e v a r i o u s c o n c e n t r a t i o n s o f c i t r a t e b u f f e r s t h a t were t o be used. As shown by f i g . 7 t h e r e i s a l i n e a r r e l a t i o n s h i p between i n c r e a s i n g c i t r a t e c o n c e n t r a t i o n and e n t r a p p e d p r o t o n p o o l . The e n t r a p p e d p r o t o n r e s e r v e s f o r 10,50,100,300 and 500mM c i t r a t e systems were c a l c u l a t e d by t i t r a t i n g each o f t h e s e b u f f e r s w i t h NaOH t o pH 7.5. D o x o r u b i c i n t r a p p i n g e f f i c i e n c i e s i n v e s i c l e s c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f c i t r a t e b u f f e r (10-500mM) were examined t o a s s e s s t h e r o l e o f t h e i n t e r i o r p r o t o n p o o l on dr u g u p t a k e . Q u a n t i t a t i o n o f d o x o r u b i c i n u p t a k e was a c c o m p l i s h e d u s i n g two v e s i c l e systems. One o f whi c h was LUVs d e r i v e d from MLVs i n t h e p r e s e n c e o f i n c r e a s i n g c o n c e n t r a t i o n s o f c i t r i c a c i d as w e l l as from f r o z e n and thawed MLVs (FATMLVs). The FATMLVs have l a r g e r aqueous t r a p volumes (Mayer e t aJL.,1985) and t h e r e f o r e i n c r e a s e d amounts o f e n t r a p p e d c i t r i c a c i d . F o r an i n i t i a l d r u g t o l i p i d (wt:wt) o f 0.3:1, i n c r e a s i n g t h e c i t r a t e c o n c e n t r a t i o n from 10 t o lOOmM produces an i n c r e a s e i n t h e d o x o r u b i c i n t r a p p i n g e f f i c i e n c i e s f o r b o t h MLVs and FATMLVs ( f i g . 8 ). I t was a l s o n o t ed t h a t t h e t r a p p i n g e f f i c i e n c y f o r v e s i c l e s produced from MLVs a r e l o w e r t h a n t h a t o b s e r v e d f o r l i p o s o m e systems produced from FATMLVs u s i n g c i t r a t e 31 Fig. 7 The rel a t i o n s h i p between entrapped proton concentration and c i t r a t e concentration. T i t r a t i o n of various concentrations of c i t r a t e with NaOH were conducted to pH 7.5. 2000 L a 4-4-0 100 200 300 400 500 [ C i t r a t e ] (mM) 3 2 F i g . 8 The influence of trap volume and buffering capacity on doxorubicin uptake. Vesicles were prepared according to section 2 . 2 . 2 with the indicated concentrations of c i t r a t e buffer. LUVETs of 0 . 2 u m diameter were prepared from ( A ) FATMLVs and (•) MLVs. [ C i t r a t e ] (mM) 3 3 concentrations between 10 and 3 00mM. These r e s u l t s suggest that doxorubicin accumulation i n response to a transmembrane pH + gradient causes a decrease of the inter n a l H pool and t h i s reduction may r e s u l t i n decreased drug trapping e f f i c i e n c i e s i f entrapped buffering capacities are not adequate. 2.3.6 E f f e c t of varying i n i t i a l drug to l i p i d r a t i o s At high i n i t i a l drug to l i p i d r a t i o s (greater than 0.3:1) doxorubicin trapping e f f i c i e n c i e s are observed to decrease. For these high drug to l i p i d r a t i o s the transmembrane pH gradient may be depleted thus l i m i t i n g uptake. Studies were undertaken to optimize trapping e f f i c i e n c i e s and d r u g : l i p i d r a t i o s for EPC:cholesterol (55:45) containing 300mM c i t r a t e pH 4.0 as well as to i d e n t i f y conditions which maintain the transmembrane pH gradient subsequent to doxorubicin entrapment. Drug to l i p i d r a t i o s (wt:wt) were varied from as high as 2:1 down to 1:10 (where i t was known that trapping e f f i c i e n c i e s of 100% could be rea d i l y achieved). I t was found that between drug to l i p i d r a t i o s of 1:10 and 1:3 there was no e f f e c t on doxorubicin trapping e f f i c i e n c i e s and values of 100% were achieved i n t h i s range ( f i g . 9)• Increasing the i n i t i a l drug to l i p i d r a t i o above 1:3 resulted i n entrapped doxorubicin:lipid r a t i o s as high as 1:1. However, trapping e f f i c i e n c i e s decrease s i g n i f i c a n t l y as the i n i t i a l d r u g : l i p i d i s increased above 1:2. Measurements 14 of pH gradients using C-methylamine as the probe (protocol 34 section 2.2.3) were done to determine the e f f e c t of high drug to l i p i d r a t i o s on the trapping e f f i c i e n c i e s . The decreased trapping e f f i c i e n c i e s observed for the high drug to l i p i d r a t i o s can be r a t i o n a l i z e d i n fig.10 where the transmembrane pH gradient a f t e r encapsulation i s monitored as a function of i n i t i a l drug to l i p i d r a t i o . Controls involving v e s i c l e s with pH gradients of 3.5 units and no gradients were performed to determine the v a l i d i t y of the method and determine the background binding of the methylamine. I t was found that systems exhibiting drug to l i p i d r a t i o s below 1:3 maintain transmembrane pH gradients i n excess of 2.0 units. Such pH gradients would predict [dox]^ /[dox] t r a t i o s greater than 100 and thus trapping e f f i c i e n c i e s greater than 99% as i s observed experimentally. However, doxorubicin entrapment does deplete + some of the int e r n a l H pool i n a l l systems as the ApH values calculated were always lower than the i n i t i a l l y imposed ApH of 3.5. The e f f e c t becomes most pronounced for i n i t i a l drug to l i p i d r a t i o s greater than 1:2 where the residual pH gradient f a l l s below 1.5 and corresponding trapping e f f i c i e n c i e s also decrease accordingly ( f i g . 9,10) In a comparative study the same drug to l i p i d r a t i o s were examined but instead of 3 00mM c i t r a t e as the internal buffer 1M c i t r a t e ( pH 4.0) was employed to increase the buffering capacity and perhaps maintain the pH gradient of the higher i n i t i a l drug to l i p i d r a t i o s . The increased buffering capacity did have a s i g n i f i c a n t e f f e c t on increasing trapping e f f i c i e n c i e s as 100% 35 F i g . 9 The e f f e c t of varying i n i t i a l l i p i d t d r u g r a t i o s on trapping e f f i c i e n c y of doxorubicin i n v e s i c l e s e x h i b i t i n g a transmembrane pH gradient. V e s i c l e s were prepared according to secti o n 2.2 and incubated with various amounts of doxorubicin. A comparison of trapping e f f i c i e n c i e s between v e s i c l e s containing 300mM c i t r a t e pH 4.0 (•) and IM c i t r a t e p H 4.0 (A) at the d i f f e r e n t d r u g : l i p i d r a t i o s was examined. l i p i d : d rug (mg l i p i d i l m g DOX) Fig. 10 Comparison between residual transmembrane pH gradients of v e s i c l e s with int e r n a l buffers of 300mM c i t r a t e pH 4.0 (•) and 1M c i t r a t e pH 4.0 (A) at d i f f e r e n t i n i t i a l d r u g : l i p i d j a t i o s . Determination of pH gradients involved the use of C-methylamine according to the protocol i n section 2.2.3. l i p i d : drug (mg l i p i d : lmg DOX) 37 e f f i c i e n c i e s were possible for drug to l i p i d r a t i o s as high as 1:1 and maintenance of pH gradients also improved for these r a t i o s ( f i g . 9). In p a r t i c u l a r , v e s i c l e s containing 300mM c i t r a t e at d r u g : l i p i d 1:1 the trapping e f f i c i e n c y achieved was 86% with no pH gradient remaining while at IM c i t r a t e the trapping e f f i c i e n c y was increased to 97% with a ApH of l . l l remaining a f t e r entrapment (fig.10). 2.3.7 E f f e c t of v e s i c l e size and drug to l i p i d r a t i o s on uptake Previous studies have shown that decreasing v e s i c l e size w i l l increase the longevity of liposomes i n c i r c u l a t i o n (Abra and Hunt, 1981; Senior and Gregoriadis,1982). In the case of liposomal doxorubicin smaller liposomes may increase the e f f i c a c y of the drug compared to free drug which i s cleared very rapid l y from the plasma. However, smaller liposomes have reduced trap volumes which lowers the internal buffering capacity perhaps l i m i t i n g drug uptake. The e f f e c t of v e s i c l e size and drug to l i p i d r a t i o on doxorubicin uptake and maintenance of the pH gradient a f t e r uptake were examined. The v e s i c l e s were sized according to the protocol i n section 2.2.2 and extruded 10 times through a double stack of either 100, 50, or 30 nm f i l t e r s . The sizes and trap volumes (Mayer et al,1985) of v e s i c l e s generated through these f i l t e r s are given i n Table 5 . An i n i t i a l drug to l i p i d r a t i o of 1:10 for a l l 3 systems gave 100% trapping e f f i c i e n c i e s while maintaining a pH gradient greater than 2.0 38 units. However as the drug to l i p i d r a t i o was increased to 1:5 the systems s t i l l achieved high trapping e f f i c i e n c i e s but the residual pH gradient dropped dramatically i n the VET 5 Q and V E T ^ Q systems. At a r a t i o of 1:3 the trapping e f f i c i e n c i e s started to decrease as v e s i c l e size decreased and there was no remaining pH gradient i n each system (fig.11,12). These r e s u l t s suggest that the reduced trap volume decreases the int e r n a l pool of protons thereby l i m i t i n g the a b i l i t y of these systems to maintain the pH gradient a f t e r doxorubicin encapsulation. 39 Table 5. Size d i s t r i b u t i o n of v e s i c l e s extruded through the indicated f i l t e r s izes. Vesicles were prepared according to the procedure outlined i n section 2.2. F i l t e r s i z e (nm) QEL size (Gaussian analysis) (nm) 100 93.8-25.3 50 64.7±14.4 30 55.2±14.2 40 F i g . 11 E f f e c t of l i p i d : d r u g r a t i o on the e f f i c i e n c y of trapping of doxorubicin i n various sized v e s i c l e s . Vesicles were prepared according to the protocol described i n section 2.2 and were extruded through (•) lOOnm f i l t e r s , (•) 50nm f i l t e r s or (A) 3 0nm f i l t e r s . 100 -o c 01 4-4-UJ m c Q. Q. O L 2 3 4 5 6 7 8 9 l i p i d j d r u g (mg l i p i d : lmg DOX) 41 Fig. 12 The influence of l i p i d to drug r a t i o on the maintenance of the pH gradient i n smaller v e s i c l e systems. Determination of the remaining pH gradient a f t e r doxorubicin uptake was calculated according to protocol i n section 2.2.3. Vesi c l e s were extruded through (•) lOOnm, (•) 50nm or (•) 30nm f i l t e r s . 3 l i p i d : drug (mg l i p i d : lmg OQX) 42 2 . 4 Discussion The r e s u l t s presented here provide new information on the mechanism of doxorubicin uptake i n liposomes exhibiting a transmembrane pH gradient (inside a c i d i c ) . The proposed mechanism whereby doxorubicin i s accumulated into LUV systems (Fig. 13) involves the penetration of doxorubicin i n the neutral form. The unprotonated form of the drug would be expected to be in equilibrium with the protonated form and accumulation of doxorubicin should therefore follow the Henderson-Hasselbach type of transmembrane d i s t r i b u t i o n . Upon reaching the inner aqueous compartment the drug i s subsequently reprotonated. With t h i s model of uptake, one would expect uptake to be largely dependent upon buffering capacity of the i n t e r i o r buffer and the trap volume of the v e s i c l e s . As reprotonation of the amine f u n c t i o n a l i t y would deplete the inner aqueous medium of protons, an i n t e r i o r buffer with a larger buffering capacity and/or a v e s i c l e system with a larger trap volume i s expected to accumulate a greater amount of doxorubicin. This model i s supported by the r e s u l t s obtained when varying concentrations of c i t r a t e buffer were employed. Increasing the c i t r a t e concentration from 10 to lOOmM produced increases i n doxorubicin trapping e f f i c i e n c i e s for v e s i c l e s derived from MLVs and FATMLVs. As predicted, the trapping e f f i c i e n c i e s for v e s i c l e s derived from MLVs were lower than that observed for liposomes produced from FATMLVs at the equivalent 43 Fig-13 Proposed mechanism of doxorubicin uptake i n LUVETs e x h i b i t i n g a transmembrane pH gradient. P H 7.5 c i t r a t e concentrations between 10 to 300mM. The increased aqueous trapped volumes of the FATMLV-derived v e s i c l e s leads to increased amounts of internal c i t r i c acid thereby increasing the i n t e r i o r buffering capacity. The inc l u s i o n of cholesterol and lyso PC did not appear to a l t e r the ApH-dependent uptake of doxorubicin. However, the addition of cholesterol does influence the temperature dependence of uptake. This influence correlates well with the suggested role of cholesterol i n c o n t r o l l i n g membrane f l u i d i t y . Studies involving various i n i t i a l d r u g : l i p i d r a t i o s supports the proposed mechanism of uptake. As the i n i t i a l concentration of doxorubicin was increased, the pH gradient remaining a f t e r drug uptake decreased i n a proportional manner. This suggests that increasing the i n i t i a l drug concentration depletes the i n t e r i o r buffering capacity thus increasing the inte r n a l pH and eliminating or decreasing the pH gradient. From these studies the maximum drug to l i p i d r a t i o which exhibits properties appropriate for therapeutic use ( >98% trapping e f f i c i e n c y ) appears to be approximately 0.3:1. This value i s 4.3 to 75 f o l d higher than drug to l i p i d r a t i o s obtained for previous liposomal doxorubicin preparations (Table 6). This drug to l i p i d r a t i o i s obtained i n conjunction with trapping e f f i c i e n c y approaching 100% whereas ex i s t i n g entrapment techniques t y p i c a l l y y i e l d trapping e f f i c i e n c i e s of 50% or less (Table 6). The use of higher drug to l i p i d r a t i o s i s po t e n t i a l l y important i n two aspects of liposomal drug therapy. 45 Table 6 Characteristics of liposome encapsulated doxorubicin preparations. Ref. Liposome Size Composition Ratio Drug:lipid %Trap Type (nm) (mol:mol) (wt:wt) e f f . 17 SUV 135*70 PS:PC:C 3:7:10 1:18.6 0.05:1 25 16 MLV N.D. PC:C 1:1 1:33 0.028:1 14 MLV N.D. PC 1:31.2 0.022:1 10 MLV N.D. CL:PC:C 1:4:5 1:21.2 0.039:1 62 MLV N.D. CL:PC 1:4 1:14.8 0.040:1 58 MLV N.D. PS:PC:C 3:7:10 1:23 0.040:1 42 SUV N.D. PC:C 1:1 1:14 0.066:1 15 SUV N.D. CL:C 5:2. 5 1:18 0.027:1 90 SUV N.D. CL:PC:C 1:4:2 1:21.2 0.033:1 47 SUV N.D. CL:PC:C 1:4:5 1:26.7 0.031:1 45 SUV N.D. PS:PC:C 3:7:10 1:44.2 0.021:1 22 53 SUV N.D. PC:C 7:2 1:130 0.006:1 6.6 SUV N.D. PC:C:DCP 7:2:1 1: 37 0.021:1 25.7 SUV N.D. PC:C:SA 7:2:1 1:225 0.004:1 4 . 0 47 SUV N.D. PC:C:PS 10:4:1 1:11.6 0.069:1 55 SUV N.D. PC:C:SA 10:4:3 1:18.4 0.049:1 35 22 SUV 90*2 0 CL:PC:C:SA 1:5:3.5 :2 1:12.4 0.068:1 55 43 LUV 150 PG:DC:C 1:4:5 1:30 0.031:1 50 46 F i r s t , large l i p i d doses ( i n excess of 5g for administration of 2 a t y p i c a l 60mg/m doxorubicin dose to a 60kg human) required for many liposomal doxorubicin preparations could be s i g n i f i c a n t l y reduced to reasonable l e v e l s (approximately 400mg for an equivalent human dose). Second, there are now indications (Mayer et a l , 1988) that increased drug to l i p i d r a t i o s may provide enhanced buffering of t o x i c i t y e f f e c t s . By decreasing v e s i c l e size i t was also shown that uptake at higher drug to l i p i d r a t i o s was limi t e d by reduced trap volumes and therefore reduced buffering capacities. Thus the maximum drug to l i p i d r a t i o appears to be approximately 0.1:1 for systems lOOnm or smaller. Although a lower d r u g : l i p i d r a t i o i s used i n smaller systems i t i s thought that they may be more ef f i c a c i o u s than the larger systems. As smaller liposomes remain i n the c i r c u l a t i o n longer the liposomally encapsulated drug ( s t i l l i n a b i o l o g i c a l l y active form) w i l l give prolonged exposure to tumour c e l l s and perhaps enhanced antitumour a c t i v i t y . 47 3. F a c t o r s I n f l u e n c i n g R e l e a s e o f D o x o r u b i c i n from Large U n i l a m e l l a r V e s i c l e s 3.1 I n t r o d u c t i o n The s t a b i l i t y of liposomally-encapsulated doxorubicin over an extended period of time i s of therapeutic importance i n drug delivery. An i n d i c a t i o n of liposome i n t e g r i t y a f t e r encapsulation i s the rate of release of the drug from liposomes. The reduced t o x i c i t y exhibited by liposomal doxorubicin (Mayer et a l , 1988) may be due to decreased t o x i c l e v e l s of the free drug. Release of doxorubicin i n the pH-driven encapsulation technique i s thought to be dependent on the s i z e of the pH gradient and the form of doxorubicin within the liposome. The retention of the pH gradient should depend on the trap volume of the v e s i c l e , the i n t e r i o r buffering capacity and the amount of drug that i s entrapped. The model of uptake i n chapter 2 proposes that the neutral form of doxorubicin permeates the liposome and i s protonated upon reaching the aqueous i n t e r i o r which has a pH around 4.0. Vesicles with large trap volumes should provide a larger buffering capacity as more c i t r a t e can be trapped. This increased buffering capacity may prove to be important i n the maintenance of the pH gradient. There i s also some uncertainty as to the form of doxorubicin that i s present within the v e s i c l e . Doxorubicin may be predominantly i n the protonated form with a small proportion 48 i n the neutral form. Doxorubicin may also be forming a complex with the c i t r a t e inside the v e s i c l e . I t has been shown that doxorubicin i n the presence of c i t r a t e at pH 7.0 w i l l form a pr e c i p i t a t e (unpublished observations). The purpose of t h i s chapter i s to examine the factors which govern doxorubicin release from liposomes prepared according to the protocol described i n chapter 2. These factors include the e f f e c t of varying l i p i d composition, internal c i t r a t e concentration and the i n i t i a l drug to l i p i d r a t i o s . In addition, a protocol of a colori m e t r i c assay for rapid determination of the proportion of free doxorubicin i n liposomal preparations w i l l be discussed. 3.2 M a t e r i a l s and Methods 3.2.1 Materials See section 2.2.1 3.2.2 Methods 3.2.2.1 Release Assay Release of doxorubicin, subsequent to the active trapping procedures described i n section 2.2.4, involved d i l u t i o n of the ve s i c l e s remaining af t e r uptake with 150mM NaCl, 20mM Hepes pH 7.5 to concentrations 2-10mM. These samples were then placed i n Spectrapor membrane tubing (MW 12000-14000) and dialyzed for 24 49 o hours against 1000 volumes of the Hepes buffered saline at 37 C on a Precision six place Mag-mix. Aliquots of 150ul were removed at the indicated times and passed down a 1ml Sepadex G-50 spin column to remove free doxorubicin and entrapped doxorubicin was determined as described i n section 2.2.4. 3.2.2.2 Colorimetric Assay Vesicles were prepared as i n section 2.2.2 i n 300mM c i t r a t e buffers of pH ranging from 4.0 to 7.2. The pH gradient for the active entrapment was achieved by r a i s i n g the exterior pH to 7.5 with IM Na 2C0 3. Doxorubicin was also passively entrapped by adding doxorubicin to the Hepes buffered saline p r i o r to the l i p i d hydration step. With the active entrapment procedure the transmembrane pH gradient drives the accumulation of the doxorubicin inside the v e s i c l e s such that Dox. /Dox . r e f l e c t in' out the [H ] i n / L " H + ] o u - t - (Mayer et a l , 1986). The spectrophotometric measurements of the free and liposome encapsulated doxorubicin were made with a Shimadzu UV-160 spectrophotometer. The uptake samples were d i l u t e d with HBS pH 7.5 to achieve doxorubicin concentrations between 0.05 to O.lOmM. For each preparation the following sequence of measurements were made. F i r s t , the absorbance at 600nm of the d i l u t e d sample was established as the "reagent blank". Second, the sample was a l k a l i n i z e d to pH 10.5 with 1.0N NaOH (0.02ml/l.0ml sample). Third, the spectrophotometer was zeroed against a 0.2% t r i t o n X-100 solution. Lastly, the absorbance at 600nm of the liposomal 50 doxorubicin sample to which 0.2% t r i t o n X-100 had been added was recorded (0.01ml 20% t r i t o n X-100/1.0ml sample). The r a t i o s of f r e e : t o t a l doxorubicin were calculated as the absorbance at 600nm upon NaOH addition divided by the absorbance a f t e r t r i t o n X-100 addition. 3 . 3 Results 3.3.1 E f f e c t of l i p i d composition on release of doxorubicin The i n v i t r o release of doxorubicin i n these systems displays a minor dependence on l i p i d composition. Increasing the cholesterol content i s known to decrease membrane permeability of certa i n entrapped drugs (Ganapathi et a l , 1984). Experiments i n which cholesterol composition ranged from 0 to 45 mole percent of the t o t a l l i p i d showed s i m i l a r release patterns ( f i g . 14). The systems with 0 and 15 mole percent cholesterol both showed a si m i l a r rate of release and a f t e r 24 hours, both retained 67% of the doxorubicin. With higher amounts of cholesterol, 33 and 45 mole percent, long-term release rates were slower and the ve s i c l e s maintained 77% of the doxorubicin a f t e r 24 hours. In each case, the release curves seem to follow a biphasic pattern with an i n i t i a l quick release i n the f i r s t few hours followed by a slower, steady state release. The i n c l u s i o n of lyso PC i n small quantities was used to determine the deleterious e f f e c t s , i f any, of the a c i d i c i n t e r i o r buffer. As lyso PC i s produced from egg PC by acid 51 Fig. 14 The influence of cholesterol concentration on doxorubicin release i n LUVETs. Vesicles were prepared according to sections 2.2 and 3.2. Vesicles were composed of EPC:chol i n the following mole percents : (•) 100:0, (•) 85:15, (•) 67:33 and (O) 55:45. 52 Fig. 15 The e f f e c t of lyso PC on the release of doxorubicin in LUVETs exhibiting a transmembrane pH gradient. Vesicles were prepared according to the protocol described i n sections 2.2 and 3.2. Lyso PC concentration was present i n the following molar percentages: (o) 0%, (A) 1% and (•) 5% lyso PC. 53 hydrolysis of a fat t y acid side chain, i t was thought that the presence of lyso PC might increase the permeability of the v e s i c l e s . However, results here suggest that the presence of lyso PC i n quantities as high as 5 mole percent induces no increase i n rates of release (fig.15). The two systems which were examined had the following l i p i d compositions of lyso PC:egg PC:cholesterol a) 1:51:48 b) 5:47:48 and c) control (egg PC:chol 52:48). 3.3.2 E f f e c t of buffering capacity on release As shown by the results i n the previous chapter the uptake of doxorubicin was profoundly affected by the i n t e r n a l buffering capacity of the v e s i c l e s . The comparison of v e s i c l e s derived from FATMLVs and MLVs showed that the v e s i c l e s derived from FATMLVs had larger trap volumes and thus larger trapping e f f i c i e n c i e s . Release experiments were performed on each uptake sample to determine the e f f e c t of the various amounts of entrapped c i t r a t e buffer. In v e s i c l e s which were derived from both MLVs and FATMLVs i t was found that increasing entrapped c i t r a t e concentration from 10 to 500mM provided greater retention of doxorubicin a f t e r 24 hours. The 0.2um diameter v e s i c l e s derived from MLVs showed a release rate which was related almost d i r e c t l y to the inter n a l buffering capacity (fig.16).As the c i t r a t e concentration i s increased from 10-500mM the doxorubicin retained increased from 42-97% a f t e r 24 hours. 54 F i g . 16 Release of doxorubicin from v e s i c l e s derived from MLVs. Multilamellar v e s i c l e s with various c i t r a t e buffers were extruded through 0.2um f i l t e r s . The sized v e s i c l e s were then prepared according to sections 2.2 and 3.2. The i n t e r i o r c i t r a t e concentrations included: (O) 10, ) 50, (•) 100, (O) 300, (O) 500mM c i t r a t e pH 4.0. 55 Fig. 17 Release of doxorubicin from v e s i c l e s derived from FATMLVs. Multilamellar v e s i c l e s containing various concentrations of c i t r a t e buffer were frozen and thawed p r i o r to s i z i n g of v e s i c l e s and then prepared according to sections 2.2 and 3.2. The internal c i t r a t e concentrations ranged from (o) 10, (A) 50, (•) 100, (O) 300 and (o) 500mm c i t r a t e pH 4.0. 56 The v e s i c l e s which were derived from FATMLVs showed a s i m i l a r release pattern to the ve s i c l e s derived from MLVs; however as the trap volume i s larger for the FATMLVs the retention was either higher or equal to ve s i c l e s derived from MLVs at equivalent c i t r a t e concentrations ( f i g . 17). This trend i s rea d i l y observed with the higher c i t r a t e concentration, 300 and 500mM, i n which v i r t u a l l y no doxorubicin i s released. These re s u l t s suggest that doxorubicin retention i s dependent upon the int e r n a l proton pool. In the case of the lower c i t r a t e + concentration buffers, the H pool i s depleted as doxorubicin accumulation occurs such that retention of doxorubicin i s decreased. With c i t r a t e at 300 and 500mM, the i n t e r i o r buffering i s maintained such that doxorubicin e f f l u x i s ne g l i g i b l e . 3.3.3 E f f e c t of varying d r u g : l i p i d r a t i o s on release Varying d r u g : l i p i d r a t i o s while maintaining the i n t e r i o r buffer concentration constant demonstrated that e f f i c i e n c y of doxorubicin uptake was dependent upon maintenance of the pH gradient . Release experiments were carr i e d out to determine the e f f e c t of the transmembrane pH gradient on doxorubicin retention. Using 300mM c i t r a t e (pH 4.0) as the inter n a l buffer, drug to l i p i d r a t i o s from 1:10 to 2:1 were used i n the entrapment protocol and residual pH gradients were measured using 14C-methylamine. I t was shown that as the i n i t i a l drug to l i p i d r a t i o was increased the residual transmembrane pH 57 Fig. 18 The e f f e c t of varying i n i t i a l drug to l i p i d r a t i o s on the retention of doxorubicin. Vesicles were prepared with an int e r n a l c i t r a t e concentration of 3 00mM c i t r a t e pH 4.0 according to the protocol i n sections 2.2 and 3.2. The i n i t i a l drug to l i p i d r a t i o s which were used included: (O) 1:10, (A) 1:5, (•) 1:3, (O) 1:2, (o) 1:1 and (x) 2:1. 100 58 gradients decreased. A release study was performed on each d r u g : l i p i d r a t i o and the results showed a s i m i l a r trend to that obtained i n the uptake study. In the low drug to l i p i d samples 1:10, 1:5, 1:3, 1:2 (wt:wt) where the remaining pH gradient exceeded 1.5 units the release was generally slow with less than 30% of the doxorubicin released a f t e r 24 hours i n each case (fig.18). However, as the drug to l i p i d r a t i o was increased to 1:1 and 2:1 the pH gradient was reduced to zero and doxorubicin release rates were increased s i g n i f i c a n t l y such that a f t e r 24 hours only 49% and 30% of the doxorubicin remained i n the v e s i c l e s with an i n i t i a l d r u g : l i p i d of 1:1 and 2:1, respectively. In a separate experiment i n which the i n t e r i o r buffer was increased to 1M c i t r a t e pH 4.0 the same drug to l i p i d r a t i o s were examined for t h e i r retention of doxorubicin a f t e r entrapment. As expected, the increased buffering capacity provided a greater maintenance of the pH gradient i n each of the d r u g : l i p i d r a t i o s . The release experiments conducted also indicated that the increased buffering capacity allowed greater retention of doxorubicin. I n i t i a l drug to l i p i d r a t i o s ranging from 1:10 to 1:2 showed greater than 95% retention a f t e r 24 hours (fig.19) At the high drug to l i p i d r a t i o s of 1:1 and 2:1 there i s approximately a 20% increase i n retention a f t e r 24 hours over the v e s i c l e s containing 300mM c i t r a t e (pH 4.0). These re s u l t s suggest that e f f l u x of doxorubicin a f t e r active entrapment i s dependent upon the remaining transmembrane pH gradient. 59 Fig. 19 The e f f e c t of varying i n i t i a l drug to l i p i d r a t i o s on doxorubicin retention in liposomal systems containing IM c i t r a t e . Vesicles were prepared with an in t e r n a l c i t r a t e concentration of IM c i t r a t e pH 4 . 0 according to the protocol outlined i n sections 2 . 2 and 3 . 2 . The i n i t i a l drug to l i p i d r a t i o s used were: (O) 1 : 1 0 , (A) 1 : 5 , (•) 1 : 3 , (O) 1 : 2 , (O) 1 : 1 , and (x) 2 : 1 . x 40 -o a * 30 -20 -10 -0 5 10 15 20 Time <hre) 60 3.3.4 Colorimetric assay As the ultimate goal of the liposomal doxorubicin formulation i s for c l i n i c a l use, i t i s imperative that t o x i c l e v e l s of free drug are not present outside of the liposomes. Thus a procedure (section 3.2.2) for determining free and v e s i c l e entrapped doxorubicin was developed which i s based on a pH-dependent spectral response. Figure 20 shows the absorbance spectra between 400 and 700nm for doxorubicin at pH 7.5 and 10.5. Increasing the pH from 7.5 to 10.5 causes a s h i f t i n the absorbance peak wavelength from 480nm to 550 and 592nm. I t i s important to note that the r e l a t i v e absorbance above 590nm for doxorubicin at pH 7.5 i s ne g l i g i b l e when compared to that obtained at pH 10.5. Because of t h i s , the o p t i c a l density a r i s i n g from the liposomes can be re a d i l y adjusted to baseline values. The pH-dependent spectral response provides the basis for determining the proportion of free and v e s i c l e entrapped doxorubicin i n liposomal preparations. At neutral pH no absorbance i s observed at 600nm, subsequent a l k a l i n i z a t i o n of the solution to pH 10.5 induces the spectral s h i f t of free doxorubicin and not v e s i c l e entrapped drug as the l i p i d b i l a y e r i s o l a t e s the encapsulated doxorubicin from the a l k a l i n e external media. The re s u l t i n g OD^„„ therefore r e f l e c t s the amount of 600 untrapped doxorubicin i n the liposomal preparation. The t o t a l concentration of doxorubicin can then be quantitated by monitoring the absorbance at 600nm af t e r the addition of t r i t o n 61 X-100 (compared to a t r i t o n X-100 blank solution). The detergent s o l u b i l i z e s the v e s i c l e s and thus exposes a l l the doxorubicin to the alkaline conditions. Using t h i s assay, the proportions of unencapsulated doxorubicin can be determined as the r a t i o of the absorbance obtained a f t e r a l k a l i n i z a t i o n with NaOH divided by that observed i n the presence of detergent. The spectroscopic analysis of liposomal doxorubicin preparations was compared to column chromatography methods which d i r e c t l y measure free and v e s i c l e entrapped drug to correlate absorbance r a t i o values to actual free dox/total dox r a t i o s over a wide range of trapping e f f i c i e n c i e s . Since a transmembrane pH gradient induces uptake of doxorubicin, EPC:Chol v e s i c l e s (inside acidic) with pH gradients of varying magnitude were u t i l i z e d to construct liposome systems with trapping e f f i c i e n c i e s ranging from 10 to 99%. Figure 21 demonstrates that the absorbance r a t i o at 600nm described here accurately represents the r a t i o of f r e e / t o t a l doxorubicin i n the v e s i c l e preparations over the f u l l range of trapping e f f i c i e n c i e s studied. This assay was also performed on v e s i c l e s i n which doxorubicin had been passively trapped during v e s i c l e formation to ensure that these results were not s p e c i f i c to liposomal doxorubicin obtained by active entrapment. As shown i n f i g . 21 the absorbance r a t i o at 600nm for t h i s sample correlates well with the f r e e / t o t a l doxorubicin value obtained by column chromatography. 62 The absorbance c h a r a c t e r i s t i c s of the spectral s h i f t also allows the r e l a t i v e amount of free doxorubicin i n liposome preparations to be assessed v i s u a l l y . Although such analysis i s c l e a r l y q u a l i t a t i v e , the presence of just 5% free drug can be detected and a dramatic color change i s observed for systems exhibiting greater than 15% free drug. Although trapping e f f i c i e n c i e s below 50% can be determined spectrophotometrically, l i t t l e difference i n the color change can be discerned by eye and therefore the e f f e c t i v e range for v i s u a l analysis of doxorubicin trapping e f f i c i e n c i e s i s 50-95%. 63 F i g . 20 The absorbance spectra between 400 and 700nm for doxorubicin solutions adjusted to pH 7.5 (a) and 10.5 (b). 64 Fig. 21 Comparison of fr e e / t o t a l doxorubicin r a t i o s with the absorbance r a t i o at 600nm before and a f t e r addition of t r i t o n X-100 to a l k a l i n i z e d liposomal doxorubicin. Doxorubicin was a c t i v e l y encapsulated (#) employing lOmM l i p i d (EPC:cholesterol, 55:45 mole percent) and 2mM drug while passive entrapment (o) u t i l i z e d 50mM l i p i d (EPC) and 2mM drug as described i n section 3.2.2-2. Spectroscopic analysis and quantitation of free and t o t a l doxorubicin by column chromatography was completed as described i n section 2.2. porcont frao DOX 65 3 .4 Discussion As shown in the previous chapter, doxorubicin can be a c t i v e l y trapped i n liposomes exhibiting a transmembrane pH gradient. The factors which govern uptake also play an important r o l e i n maintaining doxorubicin within the v e s i c l e s . Results have shown that l i p i d composition plays a minor r o l e i n doxorubicin retention. Increasing the cholesterol content of the v e s i c l e s slowed the rate of release. These r e s u l t s are consistent with those of Ganapathi et a l , 1984 i n which anionic liposomes with varying amounts of cholesterol were used to determine the i n v i t r o e f f l u x . The i n c l u s i o n of lyso PC i n small quantities proved to have no e f f e c t on doxorubicin release. Characterization of doxorubicin release from the liposomal-doxorubicin preparation was important for the development of an i n vivo system i n which retention of doxorubicin i s maximal. Factors which influenced e f f l u x rates included the buffering capacity of the v e s i c l e s and the i n i t i a l drug to l i p i d r a t i o s . The buffering capacity of the v e s i c l e s , which can be altered by using d i f f e r e n t concentrations of c i t r a t e or by changing the trap volume, plays a large role i n the maintenance of the pH gradient across the v e s i c l e s . The rate of doxorubicin leakage was slowed by increasing the trap volume within the v e s i c l e s . This was accomplished by freeze-thawing the v e s i c l e s p r i o r to extrusion. Increasing the c i t r a t e concentration also decreased the rate of doxorubicin e f f l u x . I t i s postulated that with low c i t r a t e concentrations such as lOmM, the accumulation depletes the i n t e r n a l pool of protons such that the equilibrium between DOX-NH3+ and DOX-NH2 would be sh i f t e d towards DOX-NH2 which i s capable of crossing the bil a y e r . Varying the i n i t i a l drug to l i p i d r a t i o s provided further evidence that depletion of the inter n a l proton pool r e s u l t s i n increased release rates. At lower drug to l i p i d r a t i o s the release i s generally slow due to the presence of a substantial residual pH gradient. The high drug to l i p i d r a t i o s showed much faster release of doxorubicin as the remaining transmembrane pH gradient was ne g l i g i b l e . Lastly, the protocol described for a colorimetric assay provides a simple and rapid method for determining the proportion of free doxorubicin i n liposome preparation. Advantages of t h i s procedure are that i t does not require the use of chromatography equipment or sophisticated assay procedures and can be completed within minutes. More importantly, as the liposomal doxorubicin can be assessed v i s u a l l y by t h i s procedure without the use of any s c i e n t i f i c equipment and samples can be checked p r i o r to i n vivo use to determine i f there are toxic l e v e l s of the free drug present. 6 7 SUMMARIZING DISCUSSION The studies presented i n t h i s thesis i l l u s t r a t e the u t i l i t y of liposomes as c a r r i e r s of toxic drugs such as the anticancer agent doxorubicin. There i s increasing evidence that liposome encapsulation may provide a s i g n i f i c a n t therapeutic benefit by decreasing dose-limiting toxic side e f f e c t s while maintaining or, i n some cases, increasing i t s e f f i c a c y (Mayer et a l , 1988, Bal l y et a l , 1988). Numerous independent studies from several laboratories have demonstrated that entrapment of doxorubicin i n liposomes reduces drug accumulation i n and subsequent damage to organs such as the heart (Rahman et a l , 1980 ; Gabizon et-a l , 1982) and kidney (Rahman et a l , 1985; van Hoesel et a l , 1984) While these studies have established the c l i n i c a l p o t e n t i a l of liposomally encapsulated doxorubicin, a c l e a r i n d i c a t i o n of the optimal liposome preparation to employ i s not so r e a d i l y available. The d i f f i c u l t i e s associated with i d e n t i f y i n g an optimal preparation are i m p l i c i t i n the wide range of v e s i c l e types, l i p i d compositions and drug to l i p i d r a t i o s previously employed (Table 6). In p a r t i c u l a r MLVs, LUVs and SUVs have been u t i l i z e d with l i p i d compositions incorporating various amounts of p o s i t i v e l y and negatively charged l i p i d s i n addition to phosphatidylcholine and cholesterol. The va r i a t i o n s i n l i p i d composition lar g e l y stem from the requirements for trapping doxorubicin, as systems containing only p o s i t i v e or neutral l i p i d s e xhibit low trapping e f f i c i e n c i e s and drug to l i p i d 68 r a t i o s . In liposomes containing negatively charged l i p i d s such as c a r d i o l i p i n (Rahman et a l , 1986) and phosphatidylglycerol, higher drug to l i p i d r a t i o s are achievable due to the association of the p o s i t i v e l y charged doxorubicin with the negatively charged l i p i d (Table 6). Other protocols, because of interdependence of l i p i d composition and doxorubicin entrapment precludes a systematic analysis of the c h a r a c t e r i s t i c s of liposomal doxorubicin which determine i n vivo drug t o x i c i t y and e f f i c a c y . By employing the ApH-dependent doxorubicin encapsulation with c i t r a t e buffer systems, parameters can be independently varied enabling the e f f e c t s of s p e c i f i c properties of liposomal doxorubicin to be investigated i n vivo. Parameters which were examined included the e f f e c t of cholesterol and lyso PC concentration on the entrapment of doxorubicin. In both cases the l i p i d composition did not e f f e c t the trapping e f f i c i e n c y of doxorubicin. The investigations of Chapter 2, which introduced a new encapsulation procedure, r e l i e s on the a b i l i t y of doxorubicin to accumulate into liposomal systems i n response to a transmembrane pH gradient (inside a c i d i c ) . In t h i s preparation, c i t r a t e at pH 4 . 0 provided the internal buffering as opposed to glutamic acid (Mayer et a l , 1986) which was used i n e a r l i e r experiments. The r e s u l t of t h i s buffer change was an increase i n doxorubicin uptake into liposomes. Based on t h i s observation, i t was proposed that the internal buffering capacity played a major ro l e i n the uptake of doxorubicin into liposomes i n response to a pH gradient. Thus the main focus of Chapter 2 was i n 69 determining the factors which influence doxorubicin uptake and the mechanism of uptake i n response to the pH gradient. The a b i l i t y to obtain high doxorubicin trapping e f f i c i e n c i e s appears to be c l o s e l y related to the capacity of the liposomes to maintain a s i g n i f i c a n t transmembrane pH gradient. I t was found that depletion of the v e s i c l e i n t e r n a l b uffering capacity caused by either reducing the amount of c i t r a t e or dramatically increasing the i n i t i a l drug to l i p i d r a t i o collapses the pH gradient and r e s u l t s i n lower trapping e f f i c i e n c i e s . As the v e s i c l e s i z e was decreased, a noticeable decrease i n trapping e f f i c i e n c i e s was found to occur. This was probably due to decreased trap volumes i n the smaller v e s i c l e systems and therefore a reduction i n the amount of c i t r a t e entrapped. The s t a b i l i t y of doxorubicin retention by active encapsulation also appears to be c l o s e l y r e l a t e d to the maintenance of the pH gradient. The investigations of Chapter 3 determined that reducing the amount of entrapped c i t r a t e or dramatically increasing the i n i t i a l drug to l i p i d r a t i o r e s u l t s i n a f a s t e r leakage of doxorubicin from liposomes. Manipulation of retention i s important in drug delivery as release of drug p r i o r to reaching the tumor s i t e would r e s u l t i n increased t o x i c i t y due to elevated levels of free drug i n the blood. Rapid release of doxorubicin from the liposomes may also lead to decreased e f f i c a c y as less drug reaches the tumor. 70 The colorimetric assay described i n Chapter 3 provides a simple method of determining the amount of unentrapped doxorubicin. I t was found that a l k a l i n i z a t i o n of a liposomal doxorubicin sample from pH 7.5 to 10.5 s h i f t e d the peak absorbance from 500 to 600nm. I t i s the absorbance at 600nm which determines the amount of free doxorubicin which i s present i n the preparation. The main advantage of t h i s procedure l i e s i n i t s q u a l i t a t i v e use as the occurrence of as l i t t l e as 5% free drug can be detected v i s u a l l y and a dramatic colour change can be seen with 15% free drug. The r e s u l t s of t h i s thesis demonstrates the importance of the v e s i c l e internal buffering capacity i n doxorubicin encapsulation and retention. Observations made i n t h i s thesis define other areas of research that warrant further investigation. F i r s t l y , the exact mechanism for pH gradient dependent doxorubicin encapsulation needs to be better understood and t h i s requires the determination of the form of doxorubicin which i s present i n the v e s i c l e . Secondly, while the i n vivo t o x i c i t y and e f f i c a c y of t h i s preparation has been studied (Mayer et al, 1988; Bally et a l , 1988) further analysis of the importance of various liposome properties needs to be characterized. 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