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Loading of doxorubicin into liposomes by forming Mn2+ drug complexes Cheung, Chung-Ling 1998

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LOADING OF DOXORUBICIN INTO LIPOSOMES BY FORMING Mn 2 +-DRUG COMPLEXES by CHUNG-LING CHEUNG B.Sc. (Honours), The University of Calgary, 1996 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Biochemistry and Molecular biology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1998 © Chung-Ling Cheung, 1998 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. The University of British Columbia Vancouver, Canada Department DE-6 (2/88) Abstract Doxorubicin has been encapsulated into liposomes with a transmembrane pH gradient. In this thesis, a new procedure for loading doxorubicin into large unilamellar vesicles (LUVs) is characterized and compared to loading using the ionophore A23187 with MnSCvcontaining liposomes. It is shown that doxorubicin can be loaded into LUVs composed of sphingomyelin/cholesterol (55/45 mole/mole) in response to a transmembrane MnSC^ gradient in the absence of a transmembrane pH gradient. Complex formation between doxorubicin and Mn 2 + is found to be a driving force for doxorubicin uptake. Uptake levels approaching 100 % can be achieved up to a drug-to-lipid molar ratio of 0.5 utilizing an encapsulated MnSC^ concentration of 0.30 M. In vitro leakage assays show excellent retention properties over a 24 hour period. The possible advantages of a liposomal formulation of doxorubicin loaded in response to entrapped MnS04 are discussed. ii Table of Contents Abstract ii Table of Contents iii List of Tables v List of Figures vi Abbreviations viii Acknowledgements ix Dedication x Chapter 1 Lipids and Doxorubicin 1.1 Introduction 1.1.1 Background information on phospholipids 1 1.1.2 Lipid polymorphism 5 1.1.3 Transition temperature of lipids 8 1.1.4 Effects of cholesterol on lipid bilayer 10 1.2 Liposomes 1.2.1 Lamellarity of liposomes 12 1.2.2 Liposomes as drug carriers 15 1.2.3 Passive and active targeting of liposomes 16 1.3 Doxorubicin 1.3.1 Characteristics of doxorubicin 17 1.3.2 Encapsulation of doxorubicin 19 i i i 1.4 Use of 100 nm-Egg Sphingomyelin/Cholesterol (55/45 mol%) liposomes for drug encapsulation 25 1.5 Thesis Objective 26 Chapter 2 Encapsulation of doxorubicin by forming drug-metal complexes 2.1 Introduction 27 2.2 Materials and Methods 28 2.3 Results 2.3.1 Remote loading of doxorubicin into MnS 04-containing liposomes using the ionophore A23187 33 2.3.2 Encapsulation by forming drug-metal complexes 36 2_|_ 2.3.3 Complex formation between doxorubicin and Mn ions 38 2.3.4 State of doxorubicin inside Mn2+-containing liposomes 41 2.3.5 Effects of external pH and entrapped Mn 2 + levels on complex dependent doxorubicin accumulation 42 2.3.6 The pH of the entrapped MnS0 4 solution has minimal effect on doxorubicin uptake 46 2.3.7 Doxorubicin is stably entrapped after accumulation in response to a Mn 2 + gradient 48 Chapter 3 Discussion 50 Bibliography 56 List of Tables Table 1: Structures of common saturated and unsaturated fatty acids List of Figures Figure 1: Schematic representation of a phospholipid molecule 1 Figure 2: Structures of common phospholipid headgroups 3 Figure 3: Molecular geometry and morphological structures of lipids 7 Figure 4: Schematic representation of lipid bilayers during phase transition 9 Figure 5: Structure of cholesterol 10 Figure 6: Schematic representation and freeze-fracture micrographs of MLV, LUV and SUV 14 Figure 7: Structure of doxorubicin 18 Figure 8: Loading of doxorubicin into liposomes with a transmembrane pH gradient 21 Figure 9: Loading of doxorubicin into liposomes with a transmembrane ammonium sulfate gradient 22 Figure 10: Structure of ionophore A23187 23 Figure 11: Loading of doxorubicin using the A23187 ionophore method 24 Figure 12: (A) Time course of doxorubicin uptake using A23187 into MnS04-containing LUVs 34 (B) Final pH gradient established after doxorubicin loading in the presence of A23187 34 (C) Time course of doxorubicin uptake in the absence of A23187 into MnS04-containing LUVs 34 Figure 13: Equilibrium levels of doxorubicin uptake into LUVs containing MnS0 4 in the presence and absence of A23187 37 Figure 14: (A) Effect of Mn 2 + on the absorption spectrum of doxorubicin 39 (B) Effect of pH on doxorubicin-Mn complexes 39 (C) Effect of Mn 2 + on the pKa of the sugar amine of doxorubicin ..39 (D) Absorption spectra of unencapsulated doxorubicin and encapsulated doxorubicin loaded into MnS04-containing liposomes in the presence or absence of A23187 39 Figure 15: Effect of extraliposomal pH on doxorubicin uptake in the absence of A23187 43 Figure 16: Effects of the intraliposomal MnS04 concentration on doxorubicin uptake in the presence and absence of A23187 45 Figure 17: Effect of pH of the encapsulated MnSCu on doxorubicin uptake in the absence of A23187 47 Figure 18: In vitro leakage assay of liposomal doxorubicin 49 2+ Figure 19: Chemical structure of doxorubicin-Mn complex 51 Figure 20: Schematic representation of reactions involved in the loading of doxorubicin in response to a transmembrane Mn 2 + gradient 53 Abbreviations Ac: cross sectional area of fatty acyl chains A H : cross sectional area of lipid headgroup H H : hexagonal phase 3H-CHE: 3H-Cholesteryl hexadecyl ether i.v.: intravenous LUVs: large unilamellar vesicles MLVs: multilamellar vesicles PA: phosphatidic acid PC: phosphatidylcholine PE: phosphatidylethanolamine PG: phosphatidylglycerol ApH: pH gradient pH;: intraliposomal pH pH0: extraliposomal pH PI: phosphatidylinositol pKa: -log acid ionization constant pKo: -log dissociation constant PS: phosphatidylserine SUVs: small unilamellar vesicles T m : mid-point phase transition temperature v/v: volume-to-volume ratio Acknowledgements Acknowledgement.... or express appreciation of.... I find this section in every book I have read and all seminars I have attended. Every author or presenter gives a long list of names, an incredibly long list, to readers or audiences who enjoy sharing the appreciation for the contributions they have made. But among them, there are a few who could not care less about any of them. During my two-year stay at the University of British Columbia, I have met a lot of incredibly nice people who provided me with tremendous help. So I want to take this opportunity to acknowledge their contributions and hope those readers (if any!) would take a minute to share my appreciation toward them. First of all, I would like to sincerely thank Dr. Pieter R. Cullis, my supervisor, for giving me the opportunity to work, learn, and enjoy life in his laboratory. I am also grateful for Dr. M. Bally and Dr. R. Molday's participation in my graduate committee and their invaluable advice. In addition, I am thankful for everyone whom I have worked with in the laboratory (in alphabetical order): Allison, Angel, Conrad, Dave, Dora, Elisabeth, Ismail, Ken, Kim, Lenore, Lome, Norbert, and especially Hanneke Leenhouts and Tyler Sun for their guidance and inputs in my project. I also want to thank NSERC for providing me a graduate studentship, MRC of Canada for funding this project, Dr. Trip for giving me the opportunity to work as a T.A., Sharon Krowchuk for her assistance in the department, Gary, Emily, and Thuan for their company in the everlasting graduate laboratory course, and last but not least, Dr. R. Barton for teaching me three very interesting courses in the graduate program. ix Dedication This thesis is dedicated to King, my father May, my mother and my sister Rita Chapter 1 Lipids and Doxorubicin 1.1 Introduction 1.1.1 Background information on phospholipids Biological membranes play an important role in all living cells. Its semi-fluidity and selective permeability are unique properties that allow membranes to constrain cellular constituents. These qualities originate from the amphipathic nature of phospholipid molecules. As illustrated in Figure 1, the polar region of all phospholipids is comprised of the headgroups, and the nonpolar characteristics originate from the fatty acyl chains. Polar headgroup Non-polar fatty acyl chain Figure 1. Schematic representation of a phospholipid molecule 1 Phospholipids can be composed of a huge variety of headgroups and fatty acyl chains. Figure 2 summarizes the common phospholipid headgroups found in cell membranes: phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), cardiolipin, and sphingomyelin. These headgroups can be attached to various fatty acyl chains, which vary in length and degree of unsaturation, to give membranes a wide range of properties. Some common examples of acyl chains are illustrated in Table 1. Both the headgroups and the acyl chains play a critical role in determining the morphology, fluidity, transition temperature, and function of membranes. 2 Structure Name F i g u r e 2. Structures of common phospholipid headgroups Table 1. Structures of common saturated and unsaturated fatty acids Saturated fatty acids Laurie acid Myristic acid Palmitic acid Stearic acid Arachidic Behenic CH3-(CH2)io-CH3-(CH2)l2-CH3-(CH2)l4-CH3-(CH2)i6-CH3-(CH2)i8-CH3-(CH2)20-COOH COOH COOH COOH COOH COOH Unsaturated fatty acids Monoimsaturated (all cis-double bonds): Palmitoleic acid Oleic acid Vaccenic acid CH3-(CH2)5CH=CH(CH2)7-COOH CH3-(CH2)7CH=CH(CH2)7-COOH CH3-(CH2)5CH=CH(CH2)9-COOH Polyunsaturated (all cis-double bonds): Linoleic acid Linolenic acid Arachidonic acid CH3-(CH2)4CH=CHCH2CH=CH(CH2)7-COOH CH3-CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7-COOH CH3-(CH2)4(CH=CHCH2)3CH=CH(CH2)3-COOH 4 1.1.2 Lipid polymorphism In aqueous environment, lipid aggregates can adopt various morphological structures. Depending on the molecular geometry, lipids can form micelles, bilayers or the hexagonal (HH) structures (Cullis et al., 1983). The ratio between the cross sectional area of the headgroup (AH) and the fatty acyl chains (Ac) is a very important parameter in determining the geometry of lipid molecules. (i) Micelle-forming lipids: Lipid molecules with a large ratio of headgroup-to-acyl chain cross sectional area (>1) exhibit an inverted conical geometry (Figure 3A). Lysolipid (lipid with only one hydrocarbon acyl chain) and detergent molecules have shapes resembling an inverted cone, and they adopt the micelle morphology in aqueous solutions. (ii) Bilayer-forming lipids: Lipids with a uniform headgroup-to-acyl chain cross sectional area ratio (from 0.5 to 1) are cylindrical in shape (Figure 3B). Their aggregates spontaneously adopt the bilayer morphology in aqueous environment. PA, PC, PG, PI, PS, cardiolipin, sphingomyelin, and digalactosyldiglyceride are examples of bilayer-forming lipids. 5 (iii) Hii-forming lipids: Lipids which possess a small headgroup-to-acyl chain cross sectional area ratio (<0.5) exhibit a conical geometry. These lipid aggregates adopt the Hn morphology upon hydration (Figure 3C). Unsaturated PE, cardiolipin in the presence of Ca , PA with Ca at pH < 6, PA without Ca 2 + at pH < 3, monogalactosyldiglyceride, and PS below pH 4 are lipids that belong to this category. Lipid polymorphism is highly dependent on all types of lipid molecules that are present. Due to differences in the molecular geometry, different lipids can complement each other, thus altering the overall morphology. Biological membranes are composed of a wide range of lipids, therefore lipid polymorphism is an important factor in determining the properties of different membranes. 6 < > Ac F i g u r e 3. Molecular geometry and morphological structures of lipids 7 1.1.3 Transition temperature of lipids Extensive hydrophobic interactions between fatty acyl chains minimize their contact with the aqueous environment by orienting the hydrophilic lipid headgroups toward the aqueous media. Most interactions between adjacent lipid molecules occur at the hydrophobic acyl chain region, and temperature is an important determinant in the degree of interaction between the acyl chains. At low temperature, the molecular movement of acyl chains is impeded. This causes lipid molecules to stay closer together, resulting in an increase in the van der Waals interactions between fatty acyl chains. The increased interaction leads to a rigid bilayer structure known as the gel state (Figure 4A). In the gel state, the tightly packed lipid molecules hinder the passage of molecules through the bilayer. However as temperature rises, the increase in the molecular motion between acyl chains causes lipid molecules to remain further apart. This reduces the strength of van der Waals interactions, thus resulting in an increase in the membrane fluidity. When temperature is high enough and fluidity is sufficient to allow lipid molecules freely to translocate longitudinally, membrane-permeable solutes can pass through lipid bilayers readily. This is referred to as the liquid-crystalline state (Figure 4B). During the transition between the gel and liquid-crystalline state, the temperature at which 50 % of the transition has taken place is known as the T m , or the mid-point transition temperature. 8 Figure 4. Schematic representation of lipid bilayers during phase transition The Tm of various lipid bilayers is determined by a number of factors. The headgroup, fatty acyl chain, pressure, temperature, ionic strength, and pH all play a role in determining the T m (Gennis, 1989). Headgroups, which interact extensively with one another, increase the energy required to distant the lipid molecules in the lipid crystalline state, thus raising the Tm. An increase in pressure elevates the T m by enhancing the stability of the gel state. The pH and the presence of certain divalent cations could also affect the interaction between neighbouring headgroups. With Ca ions at appropriate pH, the Tm of anionic bilayer-lipids increases due to a decrease in repulsion between headgroups. In addition, the length and the degree of unsaturation of the fatty acyl chains also have a huge impact on the T m . In general, for saturated fatty acyl chains, long acyl 9 chains increase the T m by increasing van der Waals interaction between lipid molecules. In the case of unsaturated fatty acyl chains, cis-double bonds introduce kinks which disrupt the packing of the lipid bilayer. This disruptive effect reduces the interaction between acyl chains and consequently lowers the T m . 1.1.4 Effects of cholesterol on lipid bilayer Cholesterol, the most common sterol found in membranes, is a compact, rigid hydrophobic entity with a polar hydroxyl group (Figure 5). When incorporated into lipid bilayer, this molecule integrates in between adjacent phospholipid molecules. The hydroxyl group of cholesterol is positioned next to the carbonyl ester of lipid headgroups, and the rigid hydrophobic rings are located in the vicinity of the acyl chains. The hydrophobic portion of cholesterol interacts extensively with the hydrocarbon chains, while the polar hydroxyl group does not interact with the lipid headgroups (Gennis, 1989). HO Figure 5. Structure of cholesterol 10 A unique characteristic of cholesterol is its ability to alter the fluidity of membranes. Cholesterol increases the membrane fluidity at temperatures below T m , while it decreases the fluidity at temperatures above T m . The presence of cholesterol in lipid bilayers at 30 mol% or higher can decrease the enthalpy of the phase transition to 0 kcal/mol, hence the sharp phase transition between the gel and liquid crystalline states at T m is eliminated (Estep et al., 1979). The rationale behind this unique property lies in the rigidity of the cholesterol molecule. In the gel state, the rigid structure of cholesterol disrupts the interaction between acyl chains, thus increasing the fluidity. However, in the liquid-crystalline state, rigid cholesterol molecules constrain the molecular movement of acyl chains. This increases the level of interaction between acyl chains, thus decreasing the fluidity. Therefore at 30 mol% or higher, cholesterol can eliminate phase transition. Through eliminating phase transition, lipid bilayers remain intact over a wide range of temperature. This enhanced integrity allows effective retention of entrapped materials within liposomes. 11 1.2 Liposomes 1.2.1 Lamellarity of liposomes Liposomes are lipid vesicles consisting of an internal compartment. Hydration of bilayer-forming lipids leads to the spontaneous formation of multilamellar vesicles (MLVs) (Figure 6A). M L V s consist of multiple lipid vesicles, resembling "vesicles within vesicles" or "concentric onion rings". With a lipid-filled interior, M L V s possess a low entrapped volume (-0.5 uL/umol lipid) despite their huge external diameter (up to 10 jim). To increase the entrapped volume, various techniques have been used to make unilamellar vesicles. Techniques such as sonication, injection of ethanolic solution of lipids into aqueous phase, and French Press shearing can create small unilamellar vesicles (SUVs; Figure 6C) with a diameter of less than 50 nm (Huang, 1969; Barenholz et al., 1979; Gennis 1989). Although these techniques produce unilamellar lipid vesicles with uniform and controllable sizes, SUVs possess a very small entrapped volume (0.5 to 1.0 uL/umol lipid) because of their small diameter (Stamp and Juliano, 1979; Szoka and Papahadjopoulos, 1980). In addition, the small diameter also leads to a high degree of surface curvature which makes SUVs prone to fusion. This intrinsic instability has led to the use of unilamellar vesicles with a greater diameter. Several methods have been used to make large unilamellar vesicles (LUVs) with diameters ranging from 50 to 500 nm (Figure 6B), including detergent dialysis, infusion and reverse phase evaporation, and fusion methods (Gennis, 1989). In 1985, Hope et al. have devised high pressure extrusion techniques to make LUVs. By extruding through polycarbonate filters with known pore 12 sizes, LUVs with defined diameter can be produced. This method is very convenient and gives more reproducible liposome size than other techniques. The advantages of LUVs are their resistance to fusion and large entrapped volumes, with 1.5 uX/umol lipid for 100 nm vesicles. Therefore, LUVs are unilamellar membrane models optimal for encapsulating a wide range of chemicals. 13 F i g u r e 6. Schematic representation and freeze-fracture rfiicrographs of MLV, LUV and SUV. The bar (black) in micrograph A represents 200 nm in length. 14 1.2.2 Liposomes as drug carriers The selective permeability of lipid bilayers gives liposomes the ability to retain charged and polar entities within their internal aqueous compartment. Being enclosed by lipids, entrapped chemicals are isolated from the external environment, thus minimizing any undesirable interactions with extraliposomal materials. This ability to isolate encapsulated chemicals, together with the biological compatibility of natural lipids, make liposomes excellent carriers for intravenous administration of many chemical reagents. Due to the toxic side effects of many antitumour reagents, intravenous administration of these drugs in their free forms was performed at low levels. But after encapsulation methodologies were available, higher doses can be administered because of the reduced toxic side effects achieved through encapsulation within LUVs . Encapsulated doxorubicin inside LUVs has shown superior therapeutic activity against tumours over their free forms (Mayer et al., 1990; Masood et al., 1993). Hence, LUVs can be employed to deliver high levels of encapsulated drugs while minimizing undesirable side effects. 15 1.2.3 Passive and active targeting of liposomes Targeting is a characteristic which delivers materials to specific tissues. Liposomes have been shown to be able to preferentially accumulate at tumour tissues through passive and active targeting. (i) Passive targeting: 100 nm LUVs are relatively large entities in serum. In circulation, these LUVs are too large to penetrate into healthy tissues. However, the vasculature at tumour tissues is not perfectly formed, thus 100 nm LUVs can penetrate through blood vessels and accumulate in tumours (Bally et al., 1990). This characteristic of 100 nm liposomes is known as passive targeting. Therefore, passive targeting increases the anti-tumour efficacy of encapsulated drugs in comparison to their free form. (ii) Active targeting: Advancement in coupling techniques makes the coupling of various ligands onto liposomes possible. With the appropriate ligands on their surface, liposomes can be actively targeted to specific tissues expressing certain receptors. As a result, ligand-conjugated lipid vesicles would preferentially accumulate at tissues expressing the corresponding ligand receptors. Lee and Low (1995) have demonstrated the accumulation of folate-conjugated liposomes in cells overexpressing folate receptors. 16 With the ability to actively and passively target to specific tissues, the anti-tumour therapeutic activity of encapsulated drugs increases. Therefore, the use of liposomes as a drug carrier is very beneficial for intravenous applications. 1.3 Doxorubicin 1.3.1 Characteristics of doxorubicin Doxorubicin is a potent antineoplastic agent isolated from Streptomyces peuceticus. As shown in Figure 7, doxorubicin is an amphipathic molecule with a hydrophilic amino sugar moiety and a hydrophobic anthraquinone portion. Because of the various functional groups on this antibiotic, doxorubicin can be charged or neutral depending on the pH of the environment. The p K a of the sugar amine is 8.15, while the p K a of the phenolic hydroxyl group is 10.1 (Bouma et al., 1986). Hence at pH between 8.15 and 10.1, doxorubicin is neutral. The neutral form of doxorubicin can complex with various metal ions. As reported by Bouma et al. (1986), neutral doxorubicin forms complexes with Fe 3 + (18.0), C u 2 + (12.7), T h 4 + (10.3), Tb 3 + (7.2), Pb 2 + (7.0), M n 2 + (7.0), Fe 2 + (6.5), Z n 2 + (4.5), M g 2 + (3.7) and C a 2 + (3.3) with relatively high affinity (the p K D of these doxorubicin-metal complexes are shown in parentheses). 17 Figure 7. Structure of doxorubicin Doxorubicin can bind to DNA, lipid, and proteins (de Wolf et al., 1993). The ability of doxorubicin to interact strongly with these biological molecules can inhibit DNA replication, RNA transcription, signal transduction, and protein activity, thus making doxorubicin an effective agent against a wide variety of human tumours. However, intravenous administration of doxorubicin is associated with severe toxic side effects such as cumulative dose-limiting cardiotoxicity and myelosuppression (Blum and Carter, 1974; Benjamin, 1975; Herman et al., 1983; Rahman et al., 1986; Bally et al, 1990). Therefore, the encapsulated form of doxorubicin has been employed for intravenous administrations in order to reduce the undesirable side effects. 18 1.3.2 Encapsulation of doxorubicin The amphipathic nature of doxorubicin makes it an excellent candidate to be encapsulated. Various encapsulation methods rely on the sugar amine of doxorubicin; because when protonated, its positive charge enables doxorubicin to be retained in the internal aqueous compartment of liposomes. Once encapsulated inside liposomes composed of strong bilayer forming lipids, doxorubicin is also retained very efficiently inside liposomes (Lasic et al., 1995). Therefore, doxorubicin is a suitable agent to be encapsulated. Both passive and active encapsulation methods have been devised for doxorubicin. Passive encapsulation methods, such as the hydration of dried lipid film with doxorubicin solution (Rahman et al., 1986; Amselem et al., 1990) and the incorporation of negatively charged lipids into the liposomal composition (Crommelin and von Bloois, 1983) have been used initially. However, these passive entrapment methods can only achieve low levels of drug encapsulation. Improvement in the encapsulation efficiency can be achieved using active encapsulation methods. Since only the neutral form of doxorubicin can pass readily through the lipid bilayer, doxorubicin has been loaded with a transmembrane proton gradient (Mayer et al., 1986, 1993; Haran et al., 1993; Harrigan et al., 1993). Upon encountering the intraliposomal pool of protons, neutral doxorubicin becomes protonated. The protonated form of doxorubicin is positively charged and is impermeable through the lipid bilayer, thus it remains inside the liposomes. In addition, doxorubicin has been reported to form a gel with SO42" or 19 precipitate inside liposomes, hence this characteristic further enhances the retention property of doxorubicin (Haran et al., 1993; Lasic et al., 1995). Two of the methods to actively load doxorubicin through a transmembrane proton gradient include the low pH citrate and the ammonium sulfate methods. The mechanisms behind these methods are shown in Figure 8 and 9. In Mayer's low pH citrate method (Figure 8), unprotonated neutral doxorubicin in the pH 7.4 extraliposomal media can pass through the lipid bilayer and penetrate into the liposomal interior. Once inside the liposomes, doxorubicin becomes protonated in the low pH environment and thus is retained inside the liposomes. A citrate buffer at pH 4.0 is used as the source of protons because of its strong buffering capacity. At this pH, the three carboxylic acid groups of citrate, with pK a values of 3.06, 4.74 and 5.40, enable one citrate molecule to protonate up to two doxorubicin molecules inside the liposomes. In Haran's ammonium sulfate method (Figure 9), neutral ammonia can go to the outside by dissociating a proton from the entrapped positively charged ammonium ion, thus building up a transmembrane proton gradient. Neutral doxorubicin from the extraliposomal media can be protonated in the intraliposomal space upon encountering the pool of protons. One molecule of ammonium sulfate (i.e. two molecules of NH^) can lead to the protonation of two doxorubicin molecules within liposomes. Both the low pH citrate and the ammonium sulfate methods could encapsulate high level of doxorubicin close to 100 % efficiency. 20 Extraliposomal space Intraliposomal space PH 7.4 pH 4.0 F i g u r e 8. Loading of doxorubicin into liposomes with a transmembrane pH gradient 21 Extraliposomal space pH 7.4 Intraliposomal space pH 7.4 F i g u r e 9. Loading of doxorubicin into liposomes with a transmembrane ammonium sulfate gradient 22 Recently, weakly basic drugs such as vincristine and ciprofloxacin have been shown to be actively encapsulated into MnSCvcontaining liposomes in the presence of the ionophore A23187 (Fenske et al., 1998). The structure of A23187 is shown in Figure 10. This calcium ionophore in its dimeric form is capable of translocating divalent cations such as Ni 2 + , Co 2 +, Zn 2 + , Mn 2 + , Mg 2 + , Ca 2 + , Sr2+, and Ba 2 + across the lipid bilayer in exchange for two protons (Chapman et al., 1987). Me Figure 10. Structure of ionophore A23187 The role of A23187 in this method is to establish an acidic liposomal interior by exchanging one Mn 2 + ion for the inward movement of two protons. Therefore, each entrapped Mn ion results in the exchange of two protons to the internal compartment, which consequently leads to the encapsulation of two drug molecules. This method has been studied in this thesis and found to also load doxorubicin efficiently. The mechanism of the A23187 method in loading doxorubicin is illustrated in Figure 11. 23 Extraliposomal space pH 7.4 EDTA-Mrr 2+ EDTA A23187 Intraliposomal space pH ~ 4 s o 4 2 -M n 2 + H + S 0 4 2-2-s o . H + H + S0 4 2 ' Mn 2+ Dox-NH, EDTA EDTA-Mn: Dox -N r i + Dox-NH2 < • Dox-NH 3+ F i g u r e 1 1 . Loading of doxorubicin using the A23187 ionophore method 24 1.4 Use of 100 nm-Egg Sphingomyelin/Cholesterol (55/45 mol%) liposomes for drug encapsulation Egg Sphingomyelin: Sphingomyelin is a natural phospholipid present in many animal cell and bacterial membranes (Gennis, 1989). In combination with cholesterol, sphingomyelin can form rigid bilayer structure due to strong van der Waals interactions between the lipid acyl chains and cholesterol (Mcintosh et al., 1992). The tight interaction between sphingomyelin and cholesterol results in superior retention properties of egg sphingomyelin/cholesterol (55/45 mol%) liposomes over DSPC/cholesterol (55/45 mol%) lipid vesicles for the antitumour drug vincristine (Webb, 1995). Cholesterol: Besides eliminating the phase transition at 30 mol% or higher (see 1.1.4), the incorporation of cholesterol into liposomes also increases the circulation lifetime of lipid vesicles (Patel et al., 1983; Semple et al, 1996). Cholesterol reduces the disruptive effects of plasma proteins on the lipid bilayer, thus enhancing the integrity of liposomes. Cholesterol has also been reported to impart bilayer rigidity (Kulkarni et al., 1995). The enhanced rigidity and stability induced by the presence of cholesterol are additional reasons for the inclusion of cholesterol in liposomes used for drug encapsulation. 25 100 nm in diameter: 100 nm-liposomes have been shown to exhibit extended circulation half-life and they are very effective against tumours (Mayer et al., 1989). Therefore, for optimal drug retention, 100 nm-liposomes composed of egg sphingomyelin/cholesterol at 55/45 mol ratio are the choice for encapsulating doxorubicin. 1.5 Thesis objective The enhanced drug cytotoxicity reported for various doxorubicin-metal complexes (Gutteridge, 1984; Muindi et al, 1984; Hasinoff, 1989; Greenaway and Dabrowiak, 1982; Hasinoff et al., 1989) has led to the development of a liposomal doxorubicin loading technique that relies on the formation of drug-metal ions complexes. The high affinity between doxorubicin and various metal ions provides the driving force necessary for the active drug loading process. Among all these metal ions, Mn 2 + is chosen in this study because of two reasons: Mn 2 + can form complex with doxorubicin and can also be used to load doxorubicin in the presence of the ionophore A23187. Therefore MnSCu-containing liposomes are used to load doxorubicin in order to compare the drug loading effectiveness of the complex formation method with the pH gradient method using the A23187 ionophore. 26 Chapter 2 Encapsulation of doxorubicin by forming drug-metal complexes 2.1 Introduction Doxorubicin is a potent antineoplastic agent active against a wide range of human cancers. However, treatment with doxorubicin is associated with severe toxic side effects which include cumulative dose-limiting cardiotoxicity and myelosuppression (Blum and Carter, 1974; Benjamin, 1975; Herman et al., 1983; Rahman et al., 1986; Bally et al., 1990). When administered by intravenous (i.v.) injection, the use of liposomes to encapsulate doxorubicin can reduce toxic side effects without decreasing drug potency (Rahman et al., 1980, 1986; Herman et al., 1983; Gabizon et al., 1985; Mayer et al., 1989; Szoka, 1991; Vaage et al, 1997; Daemen et al., 1997). Doxorubicin is an amphipathic weak base consisting of an anthraquinone moiety (the aglycone part) and an amino sugar. Previous work has shown that doxorubicin and other weak bases can be accumulated into vesicles with an acidic interior as a consequence of the transmembrane pH gradient (Mayer et al., 1986, 1993; Harrigan et al., 1993). Recently, a new method for drug loading has been developed using LUVs containing Mn 2 + in the presence of the ionophore A23187 (Fenske et al., 1998). The ionophore A23187 translocates one Mn 2 + ion to the outside of the LUV in exchange for the inward movement of two protons, thereby generating a pH gradient (interior acidic) across the bilayer which consequently drives drug uptake. Efficient loading of 27 vincristine and ciprofloxacin has been demonstrated using this method (Fenske et al., 1998). In this thesis, Fenske's ionophore method was used to also load doxorubicin. However, it was found that doxorubicin can be efficiently loaded into liposomes with a transmembrane Mn 2 + gradient in the absence of ionophore via the formation of intravesicular doxorubicin-Mn complexes. It is shown that complex formation between doxorubicin and Mn 2 + provides a strong driving force for doxorubicin accumulation inside liposomes. 2.2 Materials and Methods Materials: Egg sphingomyelin was purchased from Northern Lipids Inc. (Vancouver, BC). Cholesterol and the calcium ionophore A23187 were obtained from Sigma Chemical Co. (Mississauga, ON). Doxorubicin hydrochloride (Adriamycin) was manufactured by Pharmacia & Upjohn (Don Mills, ON). 14C-Mevalonic acid (RS-[2-14C]Mevalonic acid, DBED salt) was purchased from Amersham Canada Limited (Oakville, ON). H-Cholesteryl hexadecyl ether (3H-CHE) was produced by NEN Life Science Products (Boston, MA). 14C-Methylamine (14C-methylamine hydrochloride) was purchased from DuPont (Boston, MA). Sterile mouse serum was obtained from Cedarlane Laboratories Limited (Hornby, ON). Nuclepore polycarbonate filters (25 mm 0.1 urn pore size) were obtained from Costar Scientific Corporation (Toronto, ON). All chemicals were reagent grade. 28 Methods: Preparation of 100 nm large unilamellar vesicles ( L U V s ) : Liposomes were prepared by the freeze-thaw extrusion method as described previously by Hope et al. (1985). Throughout this study, 100 nm diameter large unilamellar vesicles consisting of sphingomyelin/cholesterol (55/45 molar ratio) were used, because they have been shown to exhibit excellent retention properties (Webb et al., 1995). Briefly, mixtures of sphingomyelin and cholesterol (55/45 mole ratio) were dissolved in 2-methylpropan-2-ol at 60 °C. A trace amount of 3H-CHE was added to achieve a final activity of 0.05 u.Ci/umol lipid, and the solution was then frozen in liquid nitrogen. This frozen material was lyophilized for at least 4 h under high vacuum to remove the organic solvent. Unless specified otherwise, the lyophilized lipid mixture was then hydrated at 50 mM with a buffer containing 0.30 M MnSC>4 and 30 mM Hepes (pH 7.4) in a 60 °C water bath. This suspension was subjected to five freeze-thaw cycles by alternating between liquid nitrogen and a 60 °C water bath with vigorous vortexing between cycles. Subsequently, the hydrated lipid mixture was extruded 10 times through two layers of polycarbonate filters with pore size of 0.1 urn at 60 °C using a water-jacketed extruder (Lipex Biomembranes Inc., Vancouver). Phospholipid content of the LUVs was measured employing Fiske-Subbarow phosphate assay (Fiske and Subbarow, 1925). The phospholipid content, together with the H dpm from H-CHE lipid marker, was used to determine the specific activity of the liposomes (expressed as dpm/nmol total lipid including cholesterol). The specific activity of liposomes was used for quantification of the lipid concentration. 29 Doxorubicin uptake experiments: A transmembrane Mn gradient was generated across the LUVs by exchanging the extraliposomal buffer using Sephadex G-50 spin columns (Harrigan et al., 1993). Subsequently, the lipid concentration was determined by liquid scintillation counting. Unless specified otherwise, all experiments involving the ionophore A23187 used buffer containing 0.30 M sucrose, 20 mM Hepes and 15 mM EDTA at pH 7.4; experiments performed in the absence of the ionophore employed buffer containing 0.30 M sucrose and 20 mM Hepes at pH 7.4. The transition temperatures of sphingomyelins range from 40.5 to 57 °C (Marsh, 1990); as a consequence, all uptake experiments were performed at 60 °C in order to permit efficient partitioning of the drug into and across the lipid bilayer. When present, A23187 was used at a concentration of 0.1 |_ig ionophore/umol lipid and LUVs were incubated with the ionophore at 60 °C for 5 min prior to the addition of doxorubicin. The amount of doxorubicin used in each experiment varied depending on the desired drug-to-lipid molar ratio. After the addition of doxorubicin, the lipid concentration was 5 mM. Samples of this mixture were taken at specific time points, applied onto spin columns and centrifuged at 760 x g for 2 min. This step removes the unencapsulated doxorubicin, leaving liposomes with encapsulated doxorubicin in the eluant. In order to determine the drug-to-lipid molar ratio, part of the eluant was subjected to liquid scintillation counting to assay for the amount of lipid, while another part was added to a solution of 1.0 % Triton X-100, 2.0 mM EDTA and 20 mM Hepes (pH 7.5) for the quantification of doxorubicin by absorption spectroscopy at 480 nm (e = 1.06 x 104 M"1 cm"1 (De Wolf et al., 1993)). The total drug-to-lipid molar ratio (100 % value) in the uptake mix was 30 determined at specific time points by the same procedure but omitting the spin column centrifugation step. Determination of p H gradients: To determine the pH gradient (inside acidic) present across the lipid bilayer, a trace amount of 14C-methylamine (Rottenberg, 1979) was added to the uptake cocktail (final activity = 0.2 u.Ci/mL). The doxorubicin uptake experiments were performed as described above, except dual 3 H - and 14C-radioisotope liquid scintillation counting was used. Assuming a trapped volume of 1.5 uL per umol lipid for 100 nm diameter LUVs (Hope et al., 1985; Veiro and Cullis, 1990), intraliposomal and extraliposomal methylamine concentrations could be determined. The pH gradient (ApH) can then be calculated by (Rottenberg, 1979; Harrigan et a l , 1992): ApH = log {[H+]inSide/[H+]outside}= log {[methylamine]inside/[methylamine]outside} For the determination of ApH in LUVs with a basic interior, 14C-mevalonic acid was used instead of 14C-methylamine (Harrigan et al., 1992). Absorption spectra: The effect of Mn on the doxorubicin absorption spectrum was determined by mixing various concentrations of MnS04 with doxorubicin (54 u M final concentration) in 100 m M Hepes (pH 7.4). Absorption spectra from 350 to 700 nm were taken using a Shimadzu UV160U Spectrophotometer. Then, at saturating concentration of Mn2 +, the pH dependence of the doxorubicin-Mn2+ complex was also 31 studied. 400 mM MnS04, 24 uM doxorubicin and 100 mM of various buffers were used: MES as the buffer for pH 5.2 and 5.6, Pipes buffer for pH 6.0, 6.4 and 6.8 and Hepes was used at pH 7.2, 7.5, 7.8 and 8.0. Spectra of doxorubicin loaded in the presence or absence of A23187 were compared with the spectrum of unencapsulated doxorubicin in the presence of empty liposomes. In vitro leakage assay: Doxorubicin was loaded at a drug-to-lipid ratio molar of 0.5 (mol/mol) in the presence or absence of A23187. Each uptake mixture was incubated for 70 min at 60 °C after which it was applied onto spin columns to remove unencapsulated doxorubicin. After centrifugation, an equal volume of sterile mouse serum was added followed by incubation at 37 °C for up to 24 h. The final concentration of liposomes was 1 mM. Aliquots were taken out at specified time points for the determination of doxorubicin retention. 32 2.3 Results 2.3.1 Remote loading of doxorubicin into MnSCVcontaining liposomes using the ionophore A23187 Previous studies (Fenske et al., 1998) have shown that amino-containing drugs 2+ such as vincristine and ciprofloxacin can be loaded into liposomes exhibiting a Mn gradient in the presence of ionophore A23187 and external EDTA. In this thesis, this method has been characterized and found to load doxorubicin efficiently. The kinetics of doxorubicin loading into 100 nm diameter LUVs at 60 °C is shown in Figure 12A. The transbilayer pH gradient was also measured, demonstrating an initial pH gradient of 3.4 units (inside acidic). Upon drug addition, this gradient was quenched within 5 min. However, the ApH was later re-established, rising to 2.4 units at t = 90 min. The magnitude of the re-established ApH varied depending on the drug-to-lipid molar ratio used in the experiment (Figure 12B). The disappearance and subsequent recovery of the acidic gradient was accompanied by marked colour changes: the solution was initially red in colour (t = 0) and then turned purple within 5 min. After 45 min, the colour changed back to red. The transient purple colour could not be due to the change of the internal pH, since doxorubicin turns purple only at pH values higher than 8 (results not shown). The 1 4 C -mevalonic acid data show that no basic pH gradient was generated throughout the entire uptake process (Figure 12A). This means that the highest intraliposomal pH at any point during drug uptake was equivalent to the extraliposomal pH (i.e. pH 7.4). 33 Figure 12 (A): Time course of doxorubicin uptake using A23187 ( • ) into LUVs containing MnS04 (0.30 M). The acidic ( O ) or basic ( • ) nature of the LUV interior was determined during the uptake process using 14C-methylamine and 14C-mevalonic acid as ApH probes, respectively. The colour of the uptake mixture was indicated on top of the diagram. Uptake was performed at a drug-to-lipid molar ratio of 0.53. (B): Final pH gradient (interior acidic) re-established after loading at various drug-to-lipid molar ratios in the presence of A23187. Final pH gradients were determined using 1 4 C -methylamine after an equilibrium interior pH had been established. (C): Time course of doxorubicin uptake in the absence of A23187 ( • ). The pH of the LUV interior was monitored using 14C-methylamine ( O ) and 14C-mevalonic acid ( • ) respectively. The colour of the uptake mixture was indicated on top of the diagram. Uptake was performed at a drug-to-lipid molar ratio of 0.45 with an A23187 concentration of 0.1 p.g/umol lipid. 34 0 15 30 45 60 75 90 105120 Time (min) 3.5 — i 0.00 0.25 0.50 0.75 1.00 1.25 1.50 Drug-to-lipid ratio 0 15 30 45 60 75 90 Time (min) Figure 12 2.3.2 Encapsulation by forming drug-metal complexes Interestingly, doxorubicin uptake as shown in Figure 12A continued between 5 to 30 min despite the absence of any acidic gradient. This suggested that the pH gradient was not required for doxorubicin uptake. To test this hypothesis, uptake of doxorubicin was examined in the absence of the ionophore A23187. The kinetics of doxorubicin uptake in the absence of A23187 (Figure 12C) are very similar to those in the presence of A23187 (Figure 12A). Only background levels of methylamine or mevalonic acid were found entrapped inside liposomes during uptake in the absence of A23187, indicating that no pH gradient was generated during the uptake process. The intraliposomal and the extraliposomal pH values were stable at pH = 7.4 throughout the uptake process. The solution turned from red to purple within 5 min after uptake had begun, and it stayed purple thereafter. Doxorubicin was loaded at various drug-to-lipid molar ratios in the presence or absence of A23187, and the loading efficiencies are shown in Figure 13. Both systems demonstrated similar doxorubicin loading efficiencies. Nearly 100 % drug uptake could be achieved up to a drug-to-lipid molar ratio of approximately 0.5. At higher drug-to-lipid molar ratios, the efficiency of drug uptake decreased. Therefore, in order to detect possible differences in uptake levels between different systems, a drug-to-lipid molar ratio of approximately one was used in subsequent experiments which require distinctions between loading efficiency. 36 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 Drug-to-lipid ratio Figure 13. Equilibrium levels of doxorubicin uptake into LUVs containing MnS0 4 in the presence ( • ) or absence ( • ) of A23187 at various drug-to-lipid molar ratios. All uptake experiments were performed at 60 °C and were continued until the uptake levels became constant (60 to 120 min). LUVs used for both systems were composed of Egg Sphingomyelin/Cholesterol (55:45) and contained 0.30 M MnS0 4 titrated to pH 7.4 by 30 mM Hepes. 37 2.3.3 Complex formation between doxorubicin and Mn ions In order to explore the mechanism of doxorubicin uptake into liposomes exhibiting a transmembrane M n 2 + gradient but no pH gradient, the possible formation of doxorubicin-Mn2+ complexes was investigated using absorption spectroscopy. Absorption spectra of doxorubicin at pH 7.4 in the presence of various concentrations of MnSC>4 are shown in Figure 14A. A gradual red-shift was observed in the spectra upon the addition of M n 2 + , and the colour of the solution turned from red to purple at higher M n 2 + concentrations. Doxorubicin (54 uM) was found to be saturated with M n 2 + at approximately 400 mM MnS04. Figure 14B shows the change in absorption spectra of the doxorubicin-Mn2+ complex (at the saturating concentration of Mn 2 + ) when titrated from pH 5.2 to 8.0. At pH 5.2, most of the complex is dissociated, resulting in an absorption spectrum resembling the characteristic absorption peak for free doxorubicin which has a peak at approximately 480 nm. Between pH 6.0 and 7.8 the spectrum was red-shifted, resulting in a spectrum characteristic of the doxorubicin-Mn complex. A further increase in pH beyond 8.0 caused precipitation of M n 2 + . The absorbance of the doxorubicin-Mn complexes varies significantly over a wide range of wavelengths between pH 5.2 to 8.0, with the most drastic changes at 580 nm. Since complex formation only occurs when the sugar amine is neutral, so the colour change is indicative of the degree of protonation of the sugar amine. When plotting the change in A580 versus the pH, the apparent pKa of the doxorubicin sugar amine at saturating M n 2 + concentration was determined to be approximately 6.6 (Figure 14C). Figure 14. (A): Absorption spectra of doxorubicin in the presence of various concentrations of MnSCu. Doxorubicin (54 (Jvl) was mixed with 0, 2, 10, 50, 100, 200, 400 and 500 m M M n S 0 4 . A l l solutions were buffered at pH 7.4 by 100 m M Hepes. The colour of the uptake mixture was indicated in parentheses. (B): Absorption spectra of 2+ doxorubicin-Mn complexes at various pH values. MnSC>4 (400 mM) and doxorubicin (24 uM) were buffered using 100 m M concentrations of the following buffers: MES pH 5.2-5.6; Pipes pH 6.0-6.8 and Hepes pH 7.2-8.0. The colour of the uptake mixture was indicated in parentheses. (C): Plot of absorbance (Asso) vs. pH. The p K a of the 2+ doxorubicin sugar amine at saturating concentration of Mn was determined from the inflection point of the curve (pK a = 6.6). (D): Absorption spectra of unencapsulated doxorubicin in the presence of empty liposomes ( ), and of encapsulated doxorubicin, loaded either in the presence of A23187 ( ) or the absence of A23187 ( ). A l l spectra were obtained at a drug-to-lipid molar ratio of 0.5. 39 i — i — i — i r 350 400 450 500 550 600 650 700 Wavelength (nm) -2-0 o c co jQ o CO < 0.55 - , 0.50 -0.45 -0.40 -0.35 -0.30 -0.25 -0.20 0.15 -0.10 0.05 -0.00 4 / li ) \ II ft V \ w \ \ \ \ \ \ \ '•• \ I I I I I 1 I 350 400 450 500 550 600 650 700 Wavelength (nm) Figure 14 40 2.3.4 State of doxorubicin inside Mn2+-containing liposomes The characterization of the liposomal doxorubicin loaded in the presence or absence of A23187 was also performed using absorption spectroscopy. Absorption spectra were taken after doxorubicin had been loaded into liposomes (Figure 14D). As a control, a spectrum of free doxorubicin in the presence of empty liposomes was taken. All spectra were obtained from doxorubicin and liposomes at a drug-to-lipid molar ratio of approximately 0.5. In the absence of A23187, the (loaded) doxorubicin spectrum shifted to higher wavelengths resembling a non-saturating doxorubicin-Mn2+ complex spectrum at pH 7.4. In the A23187-containing system, the spectrum of liposomal doxorubicin was identical to that of the unencapsulated doxorubicin, indicating that the doxorubicin was not complexed to Mn 2 + . 41 2.3.5 Effects of external pH and entrapped M n 2 + levels on complex dependent doxorubicin accumulation To investigate the influence of the external pH on doxorubicin uptake, the extraliposomal pH (pH0) in uptake experiments was varied while keeping the intraliposomal pH (pH;) constant. Figure 15 shows the effect of the pH 0 on the loading of doxorubicin into Mn2+-containing liposomes in the absence of A23187 using a drug-to-lipid molar ratio of 1.1. Doxorubicin uptake remained constant from pH 6.0 to 6.5 and gradually increased as the pH 0 rose from pH 6.5 to 8.0. By measuring the pH gradients during these uptake processes, it was found that the pHj became equal to the pH 0 as soon as drug uptake had begun (data not shown), regardless as to whether the initial pH gradient across the lipid bilayer was due to an acidic or basic interior. 42 100 6.0 6.5 7.0 7.5 8.0 extraliposomal pH Figure 15. Effect of extraliposomal pH on doxorubicin uptake in the absence of A23187. All uptake experiments employed 0.30 M MnS0 4 + 30 mM Hepes (pH 7.4) containing liposomes and doxorubicin was loaded at a drug-to-lipid molar ratio of 1.1. In the extraliposomal buffers, Hepes was used to buffer at pH 7.0, 7.4 and 8.0 while Hepes and Pipes were used to buffer at pH 6.0 and 6.5. 43 The effect of the intraliposomal Mn 2 + concentration on the level of doxorubicin uptake was investigated for both in the presence and absence of A23187. Liposomes with no Mn 2 + (employing 0.30 M Na2S04-containing liposomes), 0.15 M, 0.30 M, 0.45 M and 0.60 M MnS0 4 (buffered at pH 7.4 with 30 mM Hepes) were used in the uptake experiments. The results in Figure 16A show that 0.30 M Mn 2 + was sufficient to achieve a high level of drug uptake at a drug-to-lipid molar ratio of 0.96 in the doxorubicin-Mn complex formation system. A sharp increase in drug uptake was observed between 0 M and 0.15 M Mn 2 + , and a further but moderate increase was found between 0.15 M and 2 ~t~ 0.30 M. The level of drug uptake then leveled off at 85 % at Mn concentrations higher than 0.30 M. At a drug-to-lipid molar ratio of 1.0, doxorubicin uptake in the presence of A23187 showed very similar results (Figure 16B). 44 100 — 80 -4 CD B 60 Q_ 3 I 40 3 o X 0.00 100 80 cu t 60 3 tz O lo. - J i_ o X o TD 40 20 -4 T T 0.15 0.30 0.45 [MnS0 4] (M) T T 0.00 0.15 0.30 0.45 [MnSOJ (M) 0.60 B f / / * / / / y Or— 3 Zi 3 CD 0) O 2 % V-1 Q . CD' 3 0.60 Figure 16. Effects of the intraliposomal MnSC^ concentration on doxorubicin uptake. (A) shows the % doxorubicin uptake achieved in the absence of A23187. (B) shows the effects of intraliposomal MnS04 concentration on doxorubicin uptake ( • ) and on the final acidic gradient ( O ) in the presence of A23187. Doxorubicin uptake and the final acidic gradient were determined after their readings became constant (60 to 120 min). All concentrations of MnSCu solutions were titrated to and buffered at pH 7.4 with 30 mM Hepes. When no MnS0 4 was present (0 M), the LUVs contained 0.30 M Na 2S0 4 and 30 mM Hepes (pH 7.4). Uptake in both panels was performed at a drug-to-lipid molar ratio of 1.0 at 60 °C. 45 2.3.6 The pH of the entrapped MnS04 solution has minimal effect on doxorubicin uptake In order to simplify data interpretation, we employed entrapped pH 7.4 MnSCU solution to eliminate any contributions of an initial pH gradient to the doxorubicin uptake in the Mn2 +-containing system. To test whether liposomes with unbuffered MnS04 (pH 4.0) could be used for doxorubicin loading instead of the titrated MnS04 solution at pH 7.4, doxorubicin uptake into liposomes containing MnS04 encapsulated at pH 4.0 or at pH 7.4 were performed and the results are illustrated in Figure 17. In the absence of A23187, there is no significant difference in the drug loading effectiveness between the pH 4.0 and the pH 7.4 MnSCventrapped liposomes. 46 100 - f 80 -J ^ 60 -3 -j X Q 4 0 -20 - \ 0 _l I I I I I D/L = 0.49 D/L = 1.36 Figure 17. Effect of pH of the encapsulated MnSCn on doxorubicin uptake in the absence of A23187. Encapsulated MnSCM solutions were either untitrated (pH 4.0, E H ) or titrated with 30 mM Hepes (pH 7.4, i I). The drug-to-lipid molar ratios administered were 0.49 and 1.36. 47 2.3.7 Doxorubicin is stably entrapped after accumulation in response to a Mn gradient To assess the stability of the liposomal doxorubicin systems, in vitro leakage assays were performed employing 50 % (v/v) mouse serum. Doxorubicin was loaded in the presence or absence of A23187 at a drug-to-lipid molar ratio of 0.5. After removing unencapsulated doxorubicin, the liposomes loaded with doxorubicin were diluted with mouse serum and incubated at 37 °C for 24 h. The final lipid concentration in 50 % mouse serum was 1 mM. Figure 18 shows that virtually no drug leakage was observed over a 24 h period for Mn2+-containing LUVs loaded in the presence or absence of A23187. 48 0 4 8 12 16 20 24 Time (hours) Figure 18. In vitro leakage assay of liposomal doxorubicin using 50 % mouse serum. Liposomal doxorubicin was loaded at a drug-to-lipid molar ratio of 0.5 in the presence ( • ) and the absence ( • ) of A23187 by incubating doxorubicin with liposomes at 60 °C for 70 min. Subsequently, the liposomal doxorubicin was centrifuged through spin columns before incubating for 24 h with mouse serum at 37 °C. The lipid concentration in the mixture containing 50 % mouse serum during the leakage assay was 1 mM. 49 Chapter 3 Discussion The results presented here demonstrate a novel method for the loading of doxorubicin into liposomes. It is shown that doxorubicin uptake can be driven by a transmembrane Mn gradient, in the absence of any pH gradient. Loading procedures for doxorubicin described previously all rely on the presence of a transmembrane pH gradient to drive uptake (Culllis et al., 1997). Loading of doxorubicin in response to a transmembrane M n gradient relies on the formation of a membrane-impermeable complex with M n . In this section, we discuss the mechanism, properties, and potential applications of this loading procedure. The formation of doxorubicin-Mn2 + complexes with a high stability constant (pKo - 7.0) has been previously described (Bouma et al., 1986). Complex formation is accompanied by the changes in the absorption spectrum of doxorubicin upon titrating with M n (Figure 14A), which changes the colour of doxorubicin from red to purple. This colour change is also observed during doxorubicin loading into the liposomes in 2+ response to a M n gradient in both the presence and absence of A23187. In the presence of the ionophore A23187, however, the colour changes back to red after the doxorubicin is loaded, indicating dissociation of the doxorubicin-Mn complex after re-establishment of the pH gradient (inside acidic) by the ionophore. This was confirmed by the absorption spectrum of the liposomal doxorubicin that was loaded in the presence of A23187 (Figure 14D), which is characteristic of protonated doxorubicin. The absorption spectrum of liposomal doxorubicin loaded in the absence of ionophore is red-shifted, indicating the formation of complexes with M n 2 + . 50 For Cu , it has been reported that two doxorubicin molecules complex with one C u 2 + ion at pH 7.4 (Greenaway and Dabrowiak, 1982), and one proton is released from the aglycone portion of each doxorubicin molecule (Bouma et al., 1986). Complex formation only occurs when the sugar amine of doxorubicin is neutral (Lenkinski et al., 1983). Assuming that similar behaviour is observed for M n 2 + , a model for doxorubicin complexation with M n 2 + is suggested in Figure 19. NH2 OH O OH O OCH 3 O O O Mn2 + o H3CO O OH O Figure 19. Chemical structure of doxorubicin-Mn complex 51 Upon loading of doxorubicin into Mn -containing liposomes, complexation of two doxorubicin molecules with one Mn 2 + ion will release two protons, thereby acidifying the intraliposomal medium. This will result in the protonation of the next two doxorubicin molecules that enter the liposome interior. From the overall doxorubicin uptake scheme outlined in Figure 20, the addition of doxorubicin would equalize the pHj to the pH 0, as was found in experiments where the external pH was varied (data not shown). As long as pHj < pH 0, doxorubicin that enters the liposome interior will be protonated; protonation of doxorubicin depletes the intraliposomal protons and raises the pHj. This protonation process provides the driving force for doxorubicin uptake until pH; = pH 0. After that, uptake will take place by alternating between Mn 2 + complexation and protonation as given in Figure 20. In the opposite case, when pHj > pH 0 initially, doxorubicin that enters the liposome interior will complex with Mn 2 + . This will result in the release of protons from the aglycone region, which decreases the pHj until pHj = pH 0. Subsequently, drug uptake again will take place by alternating between complexation and protonation. In the experiments described in Figure 15, the pHj was rapidly equalized to the pH 0 within the first 5 min of drug loading. Only background levels of doxorubicin uptake were observed at pH 0 - 6.5 or lower, and drug uptake increases at higher pH 0 (Figure 15). This can be explained by the fact that doxorubicin-Mn2+ complexation is pH dependent (Bouma et al., 1986; Figure 3B) as the pH 0 equals to the pHj within 5 min of drug uptake. Half of the maximum level of doxorubicin uptake occurs at about pH 7.0, close to the pKa of the saturating doxorubicin-Mn complex (pKa = 6.6; Figure 14C). 52 Extraliposomal s p a c e pH 7.4 Intraliposomal s p a c e pH 7.4 Figure 20. Schematic representation of reactions involved in the loading of doxorubicin 2+ (Dox) in response to a transmembrane M n gradient. The hydroxyl ( -OH ) group attached to the "Dox" molecule is on the anthraquinone moiety of doxorubicin; the amine ( -NH2 or - N H 3 + ) group attached to the "Dox" molecule corresponds to the amine on the sugar moiety. 53 The discrepancy between the apparent pK a (6.6) and the pH for half-maximal (7.0) uptake may be due to the fact that the intraliposomal M n 2 + concentration is not sufficient to saturate the doxorubicin inside the liposomes. As demonstrated in Figure 14A, 50 m M MnSCu is required to half-saturate 54 u M doxorubicin. Assuming a trapped volume of 1.5 uL/umol lipid for 100 nm LUVs (Hope et al., 1985; Veiro and Cullis, 1990) and using a drug-to-lipid molar ratio of 1.0, the concentration of doxorubicin inside the liposomes will be approximately 0.5 M (80% drug uptake; Figure 13). It is apparent that the 0.30 M M n 2 + inside the liposomes cannot saturate the 0.5 M liposomal doxorubicin. Consequently, the pK a of the doxorubicin sugar amine will be lowered to below 8.15 due to the presence of M n 2 + , but not as low as 6.6 which is obtained only in the presence of saturating concentrations of M n 2 + . Under the appropriate experimental conditions, doxorubicin loading in response to a transmembrane M n gradient is very efficient. Uptake levels of 100% can be reached up to a drug-to-lipid molar ratio of 0.5 (Figure 13). When starting with a higher drug-to-lipid molar ratio (1.1), doxorubicin can even be loaded up to a final drug-to-lipid molar ratio of 0.8 (73% uptake). Overall, the efficiency of this loading procedure is comparable to that of the A23187 pH gradient loading method. Complexation of doxorubicin with several metal ions (such as Fe 3 + and Cu 2 + ) has been reported to facilitate the binding of the anthracycline to D N A , promote the peroxidation of lipids, and enhance the generation of reactive oxygen species (Bachur et al., 1977; Bouma et al., 1986; Gutteridge, 1984; Lown et al., 1982; Muindi et al., 1984). These actions inside cells promote cell death through damaging intracellular organelles. For doxorubicin-Fe3+ and doxorubicin-Cu2 + complexes, enhanced cytotoxicity has been 54 described (Gutteridge, 1984; Muindi et al., 1984; Hasinoff, 1989; Greenaway and Dabrowiak, 1982; Hasinoff et al., 1989). Therefore, a liposomal formulation of doxorubicin-Mn2+ complex could be therapeutically advantageous. In this regard, a concentration of MnSC^ (0.30 M) iso-osmotic to physiological saline solution was employed and was found to be sufficient to drive a high level of doxorubicin uptake at drug-to-lipid molar ratios as high as 1.0. Doxorubicin loaded by a transmembrane Mn 2 + gradient through the formation of drug-Mn2+ complexes has been shown to be active in vitro on HEK 293, COS-7 and J774 cells (data not shown). Therefore, doxorubicin is still active against cells after the formation of drug-Mn2+ complexes inside liposomes. Finally, in vitro leakage assays reveal that doxorubicin loaded in response to entrapped Mn 2 + exhibits excellent retention over a 24-hour incubation period at 37 °C. These properties suggested that liposomal doxorubicin formulations loaded in response to entrapped MnS04 may have promise for in vivo applications. Some of the results in this thesis have been presented in Leenhouts et al., 1997 55 Bibliography Amselem, S., A. Gabizon, and Y. Barenholz. 1990. 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