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Transmembrane pH gradients in liposomes: drug-vesicle interactions and proton flux Harrigan, Paul Richard 1992

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TRANSMEMBRANE pH GRADIENTS IN LIPOSOMES:DRUG-VESICLE INTERACTIONS AND PROTON FLUXbyP. RICHARD HARRIGANB.Sc.(Hons) University of British Columbia, 1985M.Sc. University of British Columbia, 1987A THESIS SUBMITI’ED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDepartment of BiochemistryWe accept this thesis as conformingto the required standardTHE OF BRITISH COLUMBIAApril 1992© P.R. Harrigan, 1992Signature(s) removed to protect privacyIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of (51oc/’eM( 5’74(SignatureThe University of British ColumbiaVancouver, CanadaDate AM1--( /s /9L\DE-6 (2/88)Signature(s) removed to protect privacyABSTRACTThis thesis examines the properties of large unilamellar lipid vesicles (LUVs)having transmembrane pH gradients (ApH). These pH gradients Induce protonmovement across the membrane and can result in accumulation of amines intothe vesicles. A major focus of the thesis is to develop a quantitative approach todescribe the response of drugs which are lipophilic amines, as well as protons, tothese pH gradients.Large, stable transmembrane pH gradients (ApH) of up to 3.5 units areshown to be detectable in LUVs by examining the transmembrane distribution of[14]C-methylamine. This approach is subject to artefacts in situations where theinterior buffering capacity is low, where the interior vesicle volume changes due toosmotic effects, or where probe redistribution is too slow to be practical. Theseproblems are generally easily overcome in liposomal systems.It is demonstrated that transmembrane pH gradients provide a practicalmethod of entrapping drugs in lipid vesicles. It is shown that the anticancer drugdoxorubicin accumulates into LUVs with an acidic interior via permeation of theneutral form of the drug. The critical dependence of translocation rates on pH,temperature and lipid composition allow manipulation of drug loading and releaseto achieve desired characteristics. A model incorporating vesicle volume, bufferingcapacity, drug partitioning and other factors is shown to describe theaccumulation of doxorubicin in response to a zpH. This results in the conclusionthat more than 95% of the encapsulated doxorubicin is partitioned into the innermonolayer for a 100 nm vesicle. Representative basic drugs from different drugclasses also accumulate into vesicles in response to an acidic interior, althoughthe extent of uptake varies considerably.Finally, the unique nature of the transbilayer movement of protons (orequivalents) was examined in well buffered lipid systems with large (3 unit) pHgradients. Development of a transmembrane electrical potential had a half-time ofabout 12 mm in EPC LUVs at 25°C, with an activation energy near 11 kcal/mol,11near the activation energy of water transport. Further, model membrane systemswere developed which exhibited stable membrane potentials without induced pHgradients. or stable pH gradients without induced membrane potentials.inTABLE OF CONTENTSABSTRACT IiTABLE OF CONTENTS ivLIST OFTABLES ixLIST OF FIGURESABBREVIATIONS USED .xiiACKNOWLEDGEMENTS xivCHAPTER 1 INTRODUCTION1.1 BACKGROUND 11.2 CHEMICAL AND MATERIALS PROPERTIES OF LIPIDPREPARATIONS 31.2.1 STRUCTURE OF PHOSPHOLIPIDS AND CHOLESTEROL. . . . 31.2.2 MATERIALS PROPERTIES OF LIPOSOMES 51.2.3 LIPID PHASES 51.2.4 EFFECTS OF CHOLESTEROL 61.3 PRODUCTION AND USE OF LIPOSOMES 61.3.1 MULTILAMELLARVESICLES (MLVs) 71.3.2 SMALL UNILAMELLAR VESICLES (SUVs) 91.3.3 LARGE UNILAMELLAR VESICLES (LLJVs) 101.3.4 OSMOTIC PROPERTIES OF EXTRUDED LIPOSOMES 121.3.5 TRAPPED SOLUTES AND TRAPPEDVOLUME DETERMINATIONS 121.3.6 SOME RELEVANT PARAMETERSCONCERNING LIPOSOMES 131.4 SOLUTE PARTITIONING AND PERMEABILITY 141.4.1 PARTITION COEFFICIENTS 14iv1.4.2 PERMEABILITY OF NON-ELECTROLYTES 161.4.3 PERMEABILITY OF WATER 191.4.4 PERMEABILITY OF IONS 201.4.5 PERMEABILITY OF PROTONS 221.5 TRANSMEMBRANE ION GRADIENTS 251.5.1 PANDzpH 251.5.2 MEASUREMENT OF ApH AND t’I’ IN LIPID VESICLES 261.5.3 SIGNIFICANCE OF ACID-BASE CHARACTERISTICS OFCOMPOUNDS 281.6 DRUG TRAPPING IN LIPOSOMES 301.6.1 PASSIVE ENTRAPMENT TECHNIQUES 301.6.2 ACTIVE TRAPPING TECHNIQUES 311.7 THESIS OUTLINE 32CHAPTER 2. MEASUREMENTS OF TRANSMEMBRANE pH GRADIENTS INLUVS2.1 INTRODUCTION 332.2 MATERIALS AND METHODS 342.2.1 MATERIALS 342.2.2 LIPID VESICLE PREPARATION 352.2.3 PARTICLE SIZE DETERMINATIONS 352.2.4 DETERMINATION OF ENTRAPPED SOLUTE 352.2.5 EFFECT OF INITIAL SOLUTEDISTRIBUTION ON ENTRAPPED SOLUTE 362.2.6 VESICLE VOLUME DETERMINATIONS 362.2.7 DETERMINATION OF LIPID CONCENTRATIONS 372.2.8 GENERATION AND MEASUREMENT OFTRANSMEMBRANE ION GRADIENTS 382.3 THEORETICAL CONSIDERATIONS 392.4 RESULTS 41V2.4.1 VESICLE CHARACTERISTICS.412.4.2 MEASUREMENTS OF EpH 452.5 DISCUSSION 60CHAPTER 3. DRUG UPTAKE INTO LIPOSOMES IN RESPONSE TO pHGRADIENTS3.1 INTRODUCTION 643.2 MATERIALS AND METHODS 663.2.1 MATERIALS 663.2.2 LIPID VESICLE PREPARATION 663.2.3 DETERMINATION OF DOXORUBICIN UPTAKE LEVELS.. . .663.2.4 DOXORUBICIN FLUORESCENCE STUDIES 673.2.5[13C]-NMR STUDIES 673.2.6 CRYO-ELECTRON MICROSCOPY 673.2.7 DRUG UPTAKE “SURVEY” EXPERIMENTS 683.2.8 OTHER ANALYTICAL PROCEDURES 683.2.9 KINETIC ANALYSIS 693.2.10 EQUILIBRIUM ANALYSIS 723.3 RESULTS 743.3.1 KINETICS OF DOXORUBICIN UPTAKE DETERMINED BYFLUOROMETRIC TECHNIQUES 743.3.2 pH DEPENDENCE AND ACTIVATION ENERGY OFDOXORUBICIN ACCUMULATION INTO LUVs 763.3.3 PARTITION COEFFICIENTS AND COUPLINGCHARACTERISTICS ASSOCIATEDWITH DOXORUBICIN UPTAKE 763.3.4 [13C1 NMR STUDIES ON DOXORUBICIN UPTAKE 783.3.5 MORPHOLOGICAL FEATURES OF LUVs FOLLOWINGDOXORUBICIN ACCUMULATION 813.3.6 DRUG UPTAKE “SURVEY” 833.3.7 CLASS 1. DRUGS WHICH EXHIBIT PARTIAL BUT STABLEviUPTAKE .833.3.8 CLASS 2. DRUGS WHICH EXHIBIT PARTIAL UPTAKE ANDSUBSEQUENT RELEASE 873.3.9 CLASS 3. DRUGS WHICH EXHIBIT NO APPARENTRESPONSE 883.3.10 CLASS 4. DRUGS WHICH ARE TOTALLY ACCUMULATED. .883.3.11 PARTITION COEFFICIENTS AND COUPLING OF OTHERAMINES 883.4 DISCUSSION 913.4.1 DOXORUBICIN ENTRAPMENT IN RESPONSE TO A zpH... .913.4.2 ApH ACCUMULATION OF OTHER COMPOUNDS 94CHAPTER 4. RATES AND ACTIVATION ENERGIES OF PROTON FLUXACROSS LIPID BILAYERS4.1 INTRODUCTION 964.2 MATERIALS AND METHODS 984.2.1 PREPARATION OF LIPID SAMPLES 984.2.2 GENERATION OF pH GRADIENTS 984.2.3 GENERATION OF MEMBRANE POTENTIALS 994.2.4 DETERMINATION OF PROBE UPTAKE INTO LUVs 994.2.5 CALCULATION OF MEMBRANE PQTENTIALS AND pHGRADIENTS 994.3 THEORETICAL CONSIDERATIONS 1004.4 RESULTS 1014.4.1 DETECTION OF PROTON FLUX 1014.4.2 INFLUENCE OF LIPID COMPOSITIONON PROTON FLUX 1034.4.3 ACTIVATION ENERGIES ASSOCIATEDWITH PROTON FLUX 1034.4.4 DETECTION OF PROTON FLUX IN RESPONSETO MEMBRANE POTENTIALS 110vii4.4.5 LUVs EXHIBITING A 1xP OR pH,BUT NOT BOTH 1104.5 DISCUSSION 111CHAPTER 5. SUMMARIZING DISCUSSION 114REFERENCES 117viiiLIST OF TABLES1-I NAMES AND STRUCTURES OF SOME COMMON FATTY ACIDS 41-11 RELEVANT PARAMETERS CONCERNING UNILAMELLAR LIPOSOMES. .142-I.[14C1-GLUCOSE AND[14C1-CITRATE VOLUMES OF 100 NM EXTRUDEDVESICLES 433-I. EXTENT AND STABILITY OF ACCUMULATION OF VARIOUS DRUGS BYVESICLES EXHIBITING A pH GRADIENT (ACIDIC INTERIOR). .. .843-IT PARTITION COEFFICIENTS OF DRUGS EXAMINED 904-I. PROTON FLUX RATES AND ACTIVATION ENERGIES 106ixLIST OF FIGURES1-1. ELECTRON MICROGRAPHS OF MLV AND FATMLV VESICLEPREPARATIONS 81-2. ELECTRON MICROGRAPHS OF EXTRUDED VESICLES OF DIFFERENTSIZE 111-3. SOLUBILITY-DIFFUSION MODEL OF MEMBRANE PERMEABILITY.... 181-4 REPRESENTATION OF PROTONS CROSSING LIPID BILAYERS BY ACARRIER MECHANISM 241-5. EFFECT OF TRANSMEMBRANE pH GRADIENTS ON THE EQUILIBRIUMDISTRIBUTION OF WEAK BASES 292-1 ENTRAPMENT OF 300 mM CITRATE BUFFER IN FATMLVs AS AFUNCTION OF FREEZING TIMES 422-2. DEPENDENCE OF SOLUTE TRAPPING ON INITIAL SOLUTEDISTRIBUTION 442-3 EFFECT OF LIPID COMPOSITION ON METHYLAMINE DISTRIBUTIONS. .472-4. METHYLAMINE RESPONSE IN VESICLES WITH DIFFERENTBUFFERING CAPACITIES AND OSMOTIC STRENGTH 492-5. EFFECT OF EXTERNAL METHYLAMINE CONCENTRATION ONMETHYLAMINE UPTAKE 522-6. DETERMINATION OF pH GRADIENTS OVER A RANGE OF pH 532-7. MEASUREMENT OF LARGE pH GRADIENTS 552-8. TRANSMEMBRANE DISTRIBUTIONS OF RADIOLABELLED PROBES INEPC LUVs FOR VESICLES WITH A BASIC INTERIOR 562-9. DETERMINATION OF pH GRADIENTS (INTERIOR BASIC) EMPLOYING[14C]-ACETATE 582-10. THE RELATIONSHIP BETWEEN TRANSMEMBRANE TPP GRADIENTSAND MeNH3GRADIENTS 593-1. STRUCTURE OF DOXORUBICIN 653-2 MODEL OF INTERACTIONS OF DOXORUBICIN WITH LUVs IN THExPRESENCE OFAApH. 713-3 DOXORUBICIN ACCUMULATION INTO LUVs IN RESPONSE TOTRANSMEMBRANE pH GRADIENTS 753-4. EFFECTS OF EXTERNAL pH AND TEMPERATURE ON KINETICS OFDOXORUBICIN ACCUMULATION 773-5. RELATIONSHIP BETWEEN RESIDUAL pH GRADIENT ANDDOXORUBICIN ACCUMULATION 793-6. EFFECT OF TRANSMEMBRANE pH GRADIENTS ON THE[13C]-NMRSPECTRA OF VESICLES INCUBATED WITH DOXORUBICIN 803-7. CRYOELECTRON MICROSCOPY OF DOXORUBICIN LOADED ANDDOXORUBICIN FREE VESICLES 823-8. UPTAKE OF TIMOLOL BY EPC VESICLES 853-9. UPTAKE OF QUINIDINE BY EPC AND EPCCHOLESTEROL VESICLES 863-10. RELATIONSHIP BETWEEN RESIDUAL pH GRADIENT AND DRUGACCUMULATION IN VESICLES WITH A TRANSMEMBRANE pHGRADIENT 894-1. GENERATION OF MEMBRANE POTENTIALS IN RESPONSE TO 3 UNITACIDIC OR BASIC PH GRADIENTS 1024-2. COMPARISON OF THE EFFECTS OF CHOLESTEROL ON PROTON ANDWATER FLUX 1044-3. COMPARISON OF THE EFFECTS OF TEMPERATURE ON PROTON ANDWATER FLUX 1054-4. GENERATION AND STABILITY OF pH GRADIENTS IN RESPONSE TOTRANSMEMBRANE K GRADIENTS 1084-5. VESICLES WITH STABLE MEMBRANE POTENTIALS BUT NO pHGRADIENTS AND VICE-VERSA 109XlABBREVIATIONS USEDApH Transmembrane pH gradientTransmembrane electrical potentialchol CholesterolCHES 2-( N-cyclohexylamino )ethane-sulfonic acidCCCP Carbonyl cyanide m-chlorophenylhydrazoneDAPC 1 ,2-diarichinoyl-sn-glycero-3-phosphorylcholineDMPC 1 ,2-dimyristoyl-sri-glycero-3-phosphorylcholineDPPC 1 ,2-dipalmitoyl-sn-glycero-3-phosphorylcholineDSPC 1 ,2-distearoyl-sri-glycero.-3-phosphoryicholineEa Activation energyEPC egg PC (from hen egg yolk)EPPS N-(2- Hydroxyethyl)piperazme- N’-3-propanesulfonic acidESR Electron spin resonanceFATMLVs Frozen and thawed MLVsHOC1O3 Perchloric acidHEPES I 4-( 2-Hydoxyethyl)]-piperazlneethanesulfonic acidLUVs Large unilamellar vesiclesMeNH3 MethylamineMES 2-(N-Morpholmo)ethanesulfonic acidMLVs Multilamellar vesiclesNaHSO3 Sodium bisuffiteNa2SO4 Sodium sulfateNM R Nuclear magnetic resonancePC PhosphatidyicholinePL PhospholipidPLM Planar lipid membraneppm parts per millionxiipsi Pounds per square inchQELS Quasi-elastic light scatteringSA StearylamineSCN Thiocyanatesn stereospecific nomenclatureSTEM Scanning tunneling electron microscopeSUVs Small unilamellar vesiclesTNBS Trinitrobenzenesulfonic acidTMS TetramethylsilanieTPP TetraphenylphosphoniumTPB TetraphenylboronTris 2-Amino-2(hydroxymethyl)propane- 1,3 - diolGel to liquid-crystalline transition temperaturet1/2 Half-time for transportUV Ultravioletxli’ACKNOWLEDGEMENTSA large thank you must go to the many members of the Cullis lab (youknow who you are), and to Pieter both for keeping me in food and clothing and fora display of super-human patience in the production of this thesis.Also, I would like to thank God that I’m finally finished.xivCHAPTER 1 INTRODUCTION1.1 BACKGROUNDOne of the earliest insights into the biochemical nature of membranes was theobservation of Overton (1899) that the permeability of biological membranes to avariety of molecules correlated with the solubiity of the molecule in olive oil. Thisimplied that the cellular permeability barrier was lipidic. After it was establishedthat red blood cells contained enough lipid to cover the surface area of these cellstwice (Gorter and Grendel, 1925). Danielli and Davson made the suggestion thatthe lipid could be organized as bimolecular leaflets, with the protein portion ofbiological membranes spread in monolayers on each side of the bilayer (Danielliand Davson, 1935; Robertson, 1957). Subsequent demonstrations of the ability oflipids and proteins to move within the bilayer and the existence of many proteinswith transmembrane a-helices led to the ‘fluid mosaic” model of biologicalmembranes (Singer and Nicholson, 1972). In this model, peripheral and integralproteins are supported in the fluid matrix of a lipid bilayer and proteins and lipidsare able to diffuse laterally around the cell.The common picture of an inert lipid bilayer containing lumps of protein is, ofcourse, oversimplified (see Bloom et aL. 1991). For example, the two faces of thebilayer are not equal. Lipids and proteins are generally asymmetrically distributedor oriented arid chemical modifications such as glycosylation are usually confinedto one side of the membrane (Storch and Kleinfeld, 1985; Kleinfeld, 1987). Ioniccomposition, pH and electrical potential can also differ between the two sides(Kleinfeld, 1987).Other complexities of biological membranes not addressed in this modelinclude the wide differences in the lipid composition between organisms, tissuesand cells and organelles (for example, Op den Kamp, 1979; Schroeder et at 1991),and the astonishing variety of lipids and proteins found within even a singlemembrane (see Storch and Kleinfeld, 1985, van Deenen et al., 1974).1The inherent complexity of biological membranes has led many investigatorsto examine simpler model systems consisting of only a small number of definedchemical species. As mentioned above, the first model of the lipid portion of themembrane was olive oil. Later developments included planar lipid membranesconsisting of a lipid bilayer in an organic solvent which separates two accessiblecompartments (see Muellar, 1962). Another model membrane system wasestablished by Bangham (1965), based on the observation that when manyphospholipids are hydrated they spontaneously form sealed systems (“liposomes”),which consist of lipid bilayers separated by water layers in an ‘onion-skin”arrangement. Simpler unilamellar systems can also be prepared. These systemsare widely used to model the lipid portion of biological membranes.These lipid vesicles have also turned out to be useful as drug deliveryvehicles. One goal in the design of liposomal delivery systems is to deliver drugspecifically to diseased tissues, using targeted lipid-based systems. While suchsystems are not yet achievable, the therapeutic properties of many drugs can beimproved by administration in an encapsulated form (for example, see Gregoriadis,1976). Liposomes have several desirable properties, including a great flexibilitywith respect to size and biodistribution properties, biodegradability and a relativelack of toxicity, (Weinstein, 1984). The therapeutic benefit likely results from thealtered pharmacokinetics and biodistribution of the entrapped drug (Ostro andCullis, 1989; Mayer et al., 1990; Rahman et al., 1980) due to the removal ofliposomes (containing the drug) from the circulation by the organs of thereticuloendothelial system. In particular, the liver and spleen accumulate much ofa typical intravenously injected liposome preparation, resulting in a passivetargeting to these organs (Gregoriadis. 1988). There also appears to be preferentialaccumulation of “untargeted liposomes” in tumours and at sites of inflammation(Ogihara et al., 1986; Morgan et al., 1985). There are several other applications ofliposomal encapsulation (Weinstein, 1984), including extending the duration ofdrug action, for example, by reducing diffusion of local anaesthetics from the site of2interest (Gregoriadis, 1988).A partial list of liposomal drugs presently undergoing human clinical trialsincludes liposomal anti-fungal agents (Lopez-Berestein, 1988), liposomalanticancer agents (Rahman et al., 1986; Creaven et al., 1990) and liposomalimmunomodulators (Sone et al., 1980). In the case of amphotericin B (an antifungal agent) and doxorubicin (an anti-cancer agent), drug toxicity is reduced whileefficacy is maintained or increased (Ostro and Cullis, 1989).A major limitation to the practical application of liposomes for drug delivery isthe difficulty in entrapping sufficient material, due to the submicron diameters ofthese liposomes. One approach which can increase the efficiency of drugentrapment is to employ transmembrane pH gradients (ApH) across the bilayer.For example, weakly basic drugs can accumulate in vesicles with an acidic interior.The generation, measurement and stability of the ipH in liposomes, as well as theaccumulation of some common pharmaceuticals into vesicles in response to a ApHare examined in this thesis. In addition, the transport of proton equivalents inresponse to these pH gradients is investigated.1.2 CHEMICAL AND MATERIALS PROPERTIES OF LIPID PREPARATIONSThe characteristics of lipid systems can be conveniently divided into theirchemical properties, which describe the characteristics of individual molecules,and their materials properties, which depend on the cooperative interactions oflarge numbers of molecules (Gruner, 1987). The properties under investigation inthis thesis are generally the materials properties of mixtures of phospholipids,sterols (where present) and water rather than their respective chemical properties,though some of the chemical properties of these compounds should be considered,as indicated below.1.2.1 STRUCTURE OF PHOSPHOLIPIDS AND CHOLESTEROLThis section will deal only with the lipids used in this thesis, which are mainlyphosphatidyicholine (PC) and cholesterol. The chemical properties of other lipids3have been extensively reviewed elsewhere (Small. 1986). Phosphatidyicholine is themost common phospholipid in eukaryotic plasma membranes, a zwitterioncomposed of a glycerol-phosphate ester with a choline headgroup and two acylchains esterified to the sn-i and sn-2 positions (Small, 1986). It is generally drawnas a circle (representing the headgroup) with two wavy lines (representing the acylchains). Table i-I indicates the more common fatty acids found in eukaryotic PCs.In general, the sn-i position tends to be a saturated fatty acid, while the sn-2 tendsto be unsaturated (Small. 1986). For example, the fatty acid side chains of eggderived PC (EPC) are 16:0(32% mol %), 18:0(15%); 18:1 (3 1%) and 18:2 (16%)(Blok at aL, 1974a), where the number preceding the colon refers to the number ofcarbons of the fatty acid and the number following it refers to the number of doublebonds. Other fatty acids are present in EPC in trace amounts (Blok at aL, 1974a).it is striking that there is an extremely large number of structural possibilities evenfor a single phospholipid such as PC.Table 1-I Names and structures of some commonfatty acids.No of CarbonAtoms Structural Formula NameSaturated fatty acids12 CH3(C2)10H lauric acid14 ( 2 myristic acid16 CH(C140 palmitic acid18 3(2)6H stearic acid20 CH(C180 arachidic acidUnsaturated fatty acids16 CH3(CH2)5CH=CH(CH)7C02H palmitoleic acid18 (7CH=CH(CHO oleic acid18 ()4CH=CHCH6 linoleic acid18 (CH=CHCH0 linolenic acid20 CH3(CH2)4( CH=CHCH2)4(CH20H arachidonic acid4Cholesterol is the major neutral lipid component of eukaryotic plasmamembranes. Part of the molecule has a rigid steroid structure, with amphipathiccharacteristics due to the 3-f3-hydroxyl group on one end of the molecule.1.2.2 MATERIALS PROPERTIES OF LIPOSOMESPerhaps the most important materials property of phospholipid-watermixtures is the tendency of the lipid to form bilayers. This property is a result ofthe “hydrophobic effect” (Tanford, 1980), wherein the ordering of water moleculeswhich would have to occur if the lipid acyl chains were exposed to the waterprevents this exposure, driving the formation of micelles, bilayers, or otherstructures. An important point to note is that materials properties can bedetermined by the methods used to produce the final structures, so it is importantto define the conditions used, and to emphasize the distinctions betweenapparently similar systems. For example, different preparations of the same lipidmolecules often have radically different drug trapping properties, whilepreparations with different chemical compositions can behave similarly.1.2.3 LIPID PHASESThe organization of lipid molecules is described in terms of the lipid phase.The gel-liquid crystalline phase transition in the presence of excess water is one ofthe best characterized properties of phospholipids (Silvius, 1982; Marsh, 1991).Upon heating, aqueous dispersions of a saturated phospholipid such as DPPC canundergo transitions from a highly ordered Lf3 phase (gel-like), through a “ripple”phase, to a La liquid-crystalline phase, characterized by considerably less order inthe hydrocarbon region (Chapman, 1975; Silvius, 1982). The gel to liquidcrystalline transition is characterized by the transition temperature generallymeasured by differential scanning calorimetry (DSC) (Chapman, 1967; Chapman,1975). T is influenced by the degree of hydration, lipid head group and acyl chaincomposition, the presence of cholesterol and a variety of other factors. As anexample. the T of DPPC is 4 10C (Silvius, 1982). Increased acyl chain length5increases T. DSPC, which has two more carbons per acyl chain than DPPC, has aphase transition temperature of 58° (Small, 1986). Unsaturation in the acyl chainsreduces the transition temperature, to below 0°C for EPC, with an average of aboutone unsaturation per acyl chain (Chapman, 1975). Since living organisms aregenerally at a constant temperature, pressure and hydration state, it is unlikelythat these phase changes have direct physiological relevance (Bloom et al., 1991).Other lipid phases which are perhaps less well known are the non-bilayerphases. For example, an unsaturated phosphatidic acid will adopt a non-bilayerhexagonal H11 phase when exposed to divalent cations (Cullis, et al., 1983; Tate etat, 1991). These non-bilayer phases have been postulated to have biologicalsignificance, especially with regard to membrane fusion (Cullis and de Kruijff,1979). Most biological membranes contain a significant proportion of non-bilayerforming lipids (Cullis and de Kruijff, 1979).1.2.4 EFFECTS OF CHOLESTEROLCholesterol has varied and subtle effects on membrane properties. Additionof cholesterol to saturated PC progressively decreases the enthalpy of the gel-liquidcrystalline phase transition until, at about 30 mol % cholesterol or higher. thetransition can no longer be detected by DSC (Chapman, 1975). Cholesterolincreases the order of the acyl chains of PCs which are above their phase transitiontemperature and decreases order for PCs which would otherwise be in the gel state(Gennis, 1989). The permeability of bilayers containing cholesterol generallydecreases as acyl chain order increases (Jam, 1980). Cholesterol can also affectthe bilayer to non-bilayer phase preferences of phospholipids (Bally et at., 1983).Finally, cholesterol can have a “condensing” effect on PC, such that the volume of amixture of PC and cholesterol is less than the volume of the two componentsseparately (Hyslop et at., 1990; Demel et at. 1968).1.3 PRODUCTION AND USE OF LIPOSOMESLipid bilayers which have formed seated structures able to encapsulate water6soluble material are known as liposomes. There are basically three liposome types:multilamellar vesicles (MLVs); small unilamellar vesicles (SUVs); and largeunilamellar vesicles (LUVs ) (see Hope et al.. 1986; Szoka and Papahadjopoulos,1980). Both the vesicle type and the method of preparation influence theproperties of the resulting systems.1.3.1 MULTILAMELLAR VESICLES (MLVs)MLVs spontaneously form when phospholipids are added to water (Banghamet al., 1965; Bangham, 1983). MLVs are very heterogeneous, with a widedistribution in both size and number of bilayers. They consist of concentric lipidbilayers and water layers in an onion-skin configuration and are typically greaterthan 1000 nm in diameter. MLVs have internal aqueous volumes of 2-3 L/molephospholipid, but, because they have an unequal distribution of solute across theirlamellae (Gruner et al., 1985; Perkins et al., 1988) the amount of solute entrappedis much lower than expected, corresponding to apparent internal volumes near 1 Lper mol phospholipid. MLVs which exhibit higher solute entrapment may beprepared by ether evaporation from an ether-buffer-lipid mixture (Gruner et al..1985), or by repeated freeze-thawing of MLVs in liquid nitrogen (termed Frozenand Thawed MLVs, or F’ATMLVs) (Mayer et al., 1985a; Westman et al., 1982).FATMLVs are also heterogeneous in size and contain unique intravesicularstructures such as vesicles within vesicles and vesicles between lamellae. Closelypacked lamellae are rare in FATMLVs in comparison to “normal” MLVs (Hope et al.,1986).Freezing MLVs to form FATMLVs may involve more than a simpleequilibration of solutes. Two reports have suggested that the internal soluteconcentrations achieved inside these FATMLVs can actually exceed the externalconcentrations in which the vesicles are prepared (Chapman, et al., 1990;Chapman et al, 1991) due to the freezing leading to locally high soluteconcentrations near the bilayer. Conversely, one must be aware that7Figure 1-1. Electron micrographs of MLV and FATMLVvesicle preparations.EPC MLV (a) and FATMLV (b) lipid vesicles were prepared as inSection 2.1 and examined by freeze-fracture electronmicroscopy. The bar represents 100 nm and the arrowhead thedirection of shadowing. Photographs courtesy of M.J. Hope8many agents act as cryoprotectants (Crowe and Crowe, 1988), preventing MLVdisruption during freeze-thaw and hence reducing solute entrapment in FATMLVs.The size, heterogeneity and presence of multiple bilayers of MLV preparationscomplicate the interpretation of experiments designed to examine transbilayertransport or fusion (Hope et at, 1986). Their relatively large size also results inrapid clearance of these vesicles from the circulation upon intravenous injection(Gregoriadis, 1988), reducing the chemotherapeutic potential of these preparations.There have been a variety of procedures developed to make unilamellar lipidvesicles to overcome these disadvantages of MLVs.1.3.2 SMALL UNILAMELLAR VESICLES (SUVs)Small unilamellar vesicles (SUVs) can be produced by sonication of MLVs(Huang. 1969). The size of SUVs produced by sonication depends upon lipidcomposition, varying from 20-30 nm for EPC vesicles to 50 nm for cholesterol-containing systems (Johnson, 1973). Vesicles in this size range may also beprepared using a “French press”, essentially a high pressure chamber with anarrow orifice (Szoka and Papahadjopoulos, 1980). SIJVs have such a high degreeof membrane curvature that there is as much as 3-fold more surface area for theouter monolayer of the vesicle than for the inner monolayer (see Table 1-TI). Thishigh curvature may contribute to the different properties of SUVs compared toLUVs. For example, SUVs prepared from lipid mixtures can have inherentasymmetric lipid distributions while larger vesicles do not (Lentz, et al., 1980).SUVs can also spontaneously fuse to form larger systems (Parente and Lentz,1984). Differences in binding properties between the inner and outer faces of thesevesicles may also occur (CafIso, 1989). Finally, permeability coefficients for iontransport across SUVs may be 1-2 orders of magnitude smaller than forcorresponding larger systems (Deamer and Bramhall, 1986; Perkins and Cafiso,1986).The mmiscule interior volumes (-0.2-0.5 tL/tmo1 lipid) and relative instability9of SUVs make them poor model membranes and also inappropriate for drugdelivery. In order to overcome these inherent disadvantages of SUVs, severalprocedures have been developed to make unilamellar vesicles of a larger size.1.3.3 LARGE UNILAMELLAR VESICLES (LUVs)Large unilamellar vesicles (LUVs) can be made by ethanol injection (Kremer et al.,1977), ether infusion (Deamer and Bangham, 1976), reverse phase evaporation(Szoka and Papahadjopoulos, 1978), or detergent dialysis (for example, Mimms etal., 1981; Madden, 1986). In these methods, lipid is solubilized in organic solventor detergent, followed by injection of the mixture into buffer. The solvent ordetergent is removed by evaporation at the time of hydration (ether method), byreduced pressure (reverse phase method), by dilution (ethanol injection method) orby dialysis (detergent dialysis). LUVs prepared by these techniques have averagediameters of 50 to 200 nm and trapped volumes of between 1-3 L per mol lipid(Szoka and Papahadjopoulos. 1980). These preparation techniques have thedisadvantages that they are time-consuming. tend to result in heterogeneousvesicle populations, may not be applicable to all lipids and can contain residualdetergent or organic solvents (Parente and Lentz, 1984).A more recent method of preparing LUVs without these disadvantagesinvolves direct extrusion of multilamellar vesicles (Hope et al., 1985) through filterswith a small pore size. Olson et al., (1979) demonstrated that vesicles produced byreversed-phase procedures were more homogeneous in size after low pressureextrusion through polycarbonate filters. Cullis and co-workers (Hope et al., 1985;Mayer et al., 1986) demonstrated that direct extrusion of MLVs or FATMLVsthrough polycarbonate filters (pore size 100 nm) results in vesicles with averagediameters of 90-110 nm as judged by freeze fracture electron microscopy andquasi-elastic light scattering techniques. The vesicles are unilarnellar as judged byfreeze fracture and NMR techniques (Hope et al., 1985). This method for producingvesicles has been discussed in detail elsewhere (Hope et al., 1986). It should be10Figure 1-2. Electron micrographs of extruded vesiclesof different size.Vesicles were prepared by extrusion of FATMLVs through(A) 400 nm (B) 200 nm (C) 100 nm, (D) 50 nm (E) 30 nmpore size polycarbonate filters and examined by freezefracture electron microscopy. The bar represents 150 nmand the magnification is the same in all photos.Photographs courtesy of L.D. Mayer.11noted that vesicles extruded through filter pore sizes 200 nm or larger haveincreasing amounts of contaminating multilamellar systems (Mayer et al, 1986c).1.3.4 OSMOTIC PROPERTIES OF EXTRUDED LIPOSOMESLUVs are not necessarily spherical. Two lines of evidence suggest vesiclesproduced by extrusion under iso-osmotic conditions are nearly, but not quite,spherical. Cryo-electron microscopy (B. Mui, unpublished data) shows thatextruded vesicles are slightly oblate. and the internal aqueous volumes of thesevesicles increase slightly when the vesicles are incubated in a hypo-osmoticexterior (Chapter 2).This non-sphericity may be a result of vesicle distortion during the extrusionprocess. From simple geometrical constraints, an initially non-spherical vesiclecannot become spherical without increasing its internal volume. The “roundingup” would dilute the contents of the vesicle and generate an osmotic gradientwhich would oppose the volume increase. Transmembrane osmotic gradients(outside hypo-osmotic) below —800 mOsm, result in vesicle swelling to a maximumsize, at which point the vesicles are spherical. At higher osmotic gradients ahydrostatic pressure gradient develops equal to the osmotic pressure difference(see Kedem and Katchaisky, 1958). Greater than -800 mOsm hypo-osmotic(outside) gradients results in vesicle rupture and resealing in 100 nm EPC vesicles(B. Mui, Personal Communication). Hyper-osinotic (outside) conditions would beexpected to result in vesicle shrinkage in proportion to the osmotic gradient as aresult of the permeability of the bilayer to water (Section 1.4.3).1.3.5 TRAPPED SOLUTES AND TRAPPED VOLUME DETERMINATIONSThe trapped” or internal aqueous volume is one of the most importantparameters defining a lipid vesicle preparation. It is generally determined bypreparing the liposomes in trace amounts of an impermeant water solublecompound such asI22NaJ or [‘4C} inulin and determining the amount of radiolabelentrapped. The trapped volume can be calculated assuming that the proportion of12entrapped solute reflects the volume inside the vesicles. As noted earlier, this isnot always the case, since MLVs prepared by hydratmg phospholipids in anaqueous medium exclude solute from their interior (Gruner, et al., 1985). Thissolute exclusion is a property of the process of liposome formation and does notdepend on the specific solute used (if it is impermeant). Subsequent osmoticgradients across the membrane can result in vesicle volume changes, which canalso introduce errors into vesicle volume determinations.An alternative method of determining vesicle volumes is to examine thevolumes outside the vesicles and calculate the internal volumes from a knowledgeof the lipid concentration. This approach to determining vesicle volume givesaccurate measurements of the aqueous volume of liposomes (Perkins et al., 1988),but gives no indication of the amount of solute captured. Solute capture can be amore important parameter if it is the trapped molecule which is of interest, as fordrug delivery, for example.1.3.6 SOME RELEVANT PARAMETERS CONCERNING LIPOSOMESTable 1-Il gives an indication of some of the parameters to consider whenusing unilamellar liposomes. These are based on calculations which assume thevesicles are idealized spheres with a bilayer thickness of 5 nm and a lipid surfacearea of 0.6 nm2/molecule (Deamer and Bramhall, 1986). For example, a 100 nmdiameter EPC unilarnellar vesicle (typically used in these studies) would beexpected to contain on the order of 90,000 phospholipid molecules per vesicle, witha slightly higher number on the outer monolayer than the inner. It should beappreciated just how small “large” unilarnellar vesicles actually are. Each 100 nmvesicle has an interior volume of only about 10-19 L.Perhaps the most important considerations from Table 1-Il are the largenumber of vesicles produced by small amounts of phospholipids and the highsurface area:volume ratio of liposomes. A milligram of PC has a surface area ofapproximately 2000 cm2 and results in about 1013 100 nrn diameter vesicles with13an Internal volume of only about 2 giL. This high surface to volume ratio allows thedetection of the flux of compounds with low permeability coefficients (Deamer andBramhall, 1986).Table 1-Il Relevant parameters concerning unilamellar liposomesVesicle Outer inner Outer: Total # of Aqueous % Volume Surface:Diameter monolayer Monolayer Inner Lipids per Vesicles per Volume which Is Volume(nm) (lipids/yes) (lipids/yes) Ratio Veslcle 1mole PL (1/mol) lipid (m2/tLl20 1790 450 4.0 2240 2.68x1014 0.14 87.5 3.0040 7180 4040 1.7 11220 5.37x10’ 0.76 57.8 0.5660 16160 11220 1.4 27380 2.20x1013 1.44 42.1 0.2980 28720 21990 1.3 50710 1.19x10’3 2.13 33.0 0.20100 44880 36350 1.2 81230 7.41x10’2 2.83 27.1 0.15200 179520 162020 1.1 341540 1.76x10’2 6.33 14.3 0.07These parameters were calculated assuming that allvesicles are perfect spheres of the stated diameter with 5urn bilayer thickness and a surface area of 0.60nm2/ phospholipid molecule.It can also be seen that the lipid bilayer itself can be a significant proportionof the total volume of the liposome. Therefore, solute interactions with the lipidbilayer must be considered, as discussed in the next section.1.4 SOLUTE PARTITIONING AND PERMEABILITY1.4.1 PARTITION COEFFICIENTSPartitioning and binding of ligands to membranes can be analysed in severalways. In the simplest case, the membrane can be treated as a bulk “oil” phase,with a partition coefficient K* defined as14K*Cm/Cw (1-1)where Cm and C, are the concentrations of a given compound in the membraneand in the water phases respectively. More realistically, the bilayer cannot alwaysbe expected to behave as a thin isotropic oil, but can be treated as a two-dimensional solvent. Hence, partitioning can also be expressed as a surfacephenomenon13=Nm/Cw (1-2)where the partition coefficient is 13 (in cm) and Nm is the surface concentration ofbound ligand. If Nm is divided by d, the bilayer thickness, one obtains the value ofthe partition coefficient K* above. Alternatively, another partition coefficient,usually also called K (in mol/cm3), can be defined, wherein Nm is divided by o, thedepth of the membrane into which drug is bound. All three partition coefficientsare commonly used (Gennis, 1989).A key parameter determining partition coefficients (as well as transport ratesand activation energies) appears to be the number of intermolecular hydrogenbonds which a diffusing molecule can form in water, since these bonds cannot bere-formed in the bilayer (Cohen, 1975a,b). This relation breaks down aboveapproximately 10 hydrogen bonds (Bangham and Hill, 1986). Each potentialhydrogen bond changes the activation energy of transport by about 4 kcal (Cohen,1975a), approximately the energy of formation of a hydrogen bond. Other“incremental changes in free energy” (which predict the effects of varioussubstituents on partitioning) have been estimated for partitioning into bilayers(Diamond and Katz, 1975).There has been considerable effort put into finding the best oil model to mimica membrane (Walter arid Gutknecht, 1984; Diamond and Katz, 1975), butessentially any low dielectric medium provides a reasonable membrane model to afirst approximation. The standard oil for which partition coefficients are15determined is octanol. Octanol:water partition coefficients are widely available andappear to be surprisingly useful predictors of membrane:water partition coefficientsfor non-electrolytes (for example, see Walter and Gutknecht, 1984). Unfortunately,partition coefficients of acids and bases are generally not determined separately forthe charged and uncharged species of these compounds. Most commonly,partition coefficients for acids and bases are reported under unbuffered orunspecified conditions, rendering it impossible to separate the relativecontributions of two molecular species of the compounds (Strichartz, et al., 1990).1.4.2 PERMEABILITY OF NON-ELECTROLYTESNet solute flux (J, in units of moles 1) can be described by a permeabilitycoefficient (P. which generally has units of cms1), whereJ=PA(AC) (1-3)where A is the surface area and AC is the concentration gradient which drivessolute flux. Typical values of P range from 1O-1O cm s for compounds whichrapidly cross bilayers (e.g. water or urea), to 10-10 cm s’ for moderately permeablemolecules (e.g. glucose) to 10-13 cm s’ for “impermeable’ ions such as sodium andpotassium (Walter and Gutknecht, 1984: Deamer and Bramhall, 1986).Assuming a first order process for net diffusion of a compound out of aunilamellar liposome of constant interior volume it can be shown that to a firstapproximationln {C(t)/C(0)} = -kt (1-4)where C(t) is the internal solute concentration at time t. C(0) the initialconcentration, t is time and k the first order rate constant.Considering the efflux of solute from an LUV of radius r, it follows thatP=kr/3 (1-5)16The half-time of transport (t1 /2 can be obtained easily from the rate constant:t112=.693/k (1-6)Half-times for efflux out of 100 nm diameter vesicles employing thepermeability coefficients above are in the millisecond range for water, a few hoursfor glucose and several days for potassium.Permeability properties can be described by of a “solubility-diffusion” model.In this model the rate limiting step is diffusion across the bilayer and interfacialresistance is negligible (Gennis, 1989). HenceP=KD/d (1-7)where D is the diffusion constant within the membrane and d is the width of thebilayer. Both K (the partition coefficient) and D (the diffusion coefficient) areaverage values, which are not necessarily constant across the bilayer(Diamond and Katz, 1975). There is a good correlation between oil:water partitioncoefficients and lipid bilayer permeability over at least 7 orders of magnitude. ageneralisation known as “Overtons rule’ (Walter and Gutknecht, 1984). Thus, if oneknows the permeability of one or two small solutes to a given membrane, thepermeability of other small solutes can be reasonably approximated (see Cohen,1975; Walter and Gutknecht, 1984).It has been suggested that lipid solubility alone does not determine thepermeability of all compounds, since some very small molecules may permeatefaster than predicted by their partition and diffusion coefficients (Lieb and Stein,1986). This behavior has been suggested to be due an ability of the smallmolecules to “fit” into small transient defects in the lipid acyl chain packing (Lieband Stein, 1986).More glaring exceptions to Overton’s rule occur with larger molecules. Ifphospholipid membranes do deviate from the behavior expected of thin oil layers,17SSS S4SSSSSIIIIIIIIIIIISS*SSFigure 1-3. Solubility-diffusion model ofpermeability.If the [nterfacial energy barrier is small, the interfacialconcentration of a given compound can be assumed to be inequilibrium (as described by the partition coefficient. K) with theaqueous concentration. The rate limiting step of permeation isdiffusion across the lipid acyl chain region of the bilayer. Thedepth of binding ô is not to scale.K[mJSIII[I]K[o]< >Outside*S44-S L’m,S44Vm0’Insidemembrane18one might expect the deviation to be more pronounced when the size of thediffusing molecule is significant compared to the dimensions of the bilayer.Phospholipids such as PC, for example, are relatively large molecules which haveextremely high partition coefficients, but their rates of diffusion across the bilayer(“flip-flop”) are very slow (Hauser and Barratt, 1973).1.4.3 PERMEABILITY OF WATERPerhaps the most important solute to consider is water. Determinations ofwater permeability monitor the rate of exchange of water under isotonic conditionsor in response to an osmotic gradient (F) (Finkeistein, 1987; Ye andVerkman, 1989; Benga et al., 1990). These values do not have to be the same.Different values of D arid F imply that water transport is occurring throughaqueous channels (Fettiplace and Haydon, 1980; Dearner and Bramhall, 1986).After appropriate corrections for unstirred layer effects (wherein flux through“unstirred” water at the membrane:water interface can be slower than flux throughthe membrane itself - see Finkeistein, 1987 for discussion and appropriatecorrection procedures) D and F in lipid bilayers are on the order of iO-3 cm s*with activation energies generally between 8 and 13 kcal/mol (Fettiplace andHaydon, 1980). These values are much higher than the activation energy of 4.6kcal/mole expected for bulk diffusion of water through aqueous pores (Fettiplaceand Haydon, 1980, Finkeistein, 1987). The same is not necessarily true forbiological membranes. Red blood cell membranes, for example, appear to haveprotein dependent pores which can be poisoned by mercury containing compounds(Macey and Farmer, 1970). Water transport through red cells decreases about 5-fold upon treatment with mercurials and the activation energy of water movementrises from 3-5 kcal/mol to 11.5 kcal/mole (Benga et al., 1990).There are two main models for water transport across protein-free modelmembranes, one based upon the solubility-diffusion model described above and theother based on water entering transient defects which spontaneously form in the19bilayer (Deamer and Bramhall, 1986). For example, water permeability increasesdramatically in vesicles composed of lipids which are at the phase transitiontemperature, where these defects are expected to be most common (Deamer andBramhall, 1986). These models are not mutually exclusive.1.4.4 PERMEABILITY OF IONSThe permeability of bilayers to small ions such as or Na+ is very low in theabsence of lonophores, though proton/hydroxide flux appears to be an exception(see below). Permeability coefficients for these ions are on the order of 10-12 to10-14 cm s (Bangham et al., 1965: Hauser et a!. 1973). These slow rates aregenerally attributed to the large energy barrier (‘Born’ energy) encountered uponentering the low dielectric constant medium of the bilayer, where the Born energy,w, of transferring an ion from water to a hydrocarbon can be approximated (in cgsunits)w = e2 / 2hCr (1-8)where e is the ionic charge, r the ionic radius and hc the dielectric constant of thehydrocarbon (Parsegian, 1969). For example, a 0.2 nm radius monovalent ionpassing into a 7 nm thick slab of hydrocarbon with a dielectric constant of 2 mustpass an energy barrier of about 40 kcal/mol (Parsegian, 1969). One of the fewtests of this hypothesis (Dilger and McLaughlin, 1979) showed that the presence ofchiorodecane (expected to raise the membrane dielectric constant) increased thethiocyanate permeability of planar lipid membranes several thousand fold,consistent with Born energy considerations.While Born energy is a reasonable framework in which to view the lowpermeabifity of membranes to ions, the critical values of the membrane dielectricconstant and the ionic radius used to calculate the energy barrier are not knownwith precision (D. Deamer, personal communication). For example, if the dielectricof a membrane and the ionic radius were both half of the values used in the20calculations used above, the calculated Born energy barrier would rise to above160 kcal/mol and the predicted ion flux rate would be about 10100 slower. Thus,the observation that sodium has a permeability coefficient about three orders ofmagnitude greater than that predicted on the basis of Born energy considerations(Hauser at al., 1973) may not necessitate alternative mechanisms to describe iontransport across bilayers, due to the sensitivity of these calculations to theseparameters.One observation which is not consistent with simple Born energyconsiderations is that membranes are generally much more permeable to anionsthan to cations. For example, tetraphenylboron anion (TPB-) is several orders ofmagnitude more permeable than the corresponding tetraphenyiphosphoniumcation (TPP), even though these molecules have similar hydrophobicity andhydrogen bonding abilities (Flewellmg and Hubbell, 1 986a). An “internal dipolepotential of about 250 mV, which favours anions, has been suggested to be thebasis of the higher permeability of anions than cations (Fleweuing and Hubbell,1 986b). A portion of the internal dipole is believed to be due to the alignment ofthe ester linkages of the phospholipids along the plane of the bilayer, while the restmay be a result of oriented water molecules (Flewelling and Hubbell, 1986a,1986b). Adding the compound phloretm to the bilayers increases the permeabilityof cations and decreases the permeability of anions, apparently altering themembrane dipole potential (Perkins and Cafiso, 1987a). A “total potential” model ofmembranes, which includes Born energy, dipole potential, “image energy”(additional energy resulting from the finite thickness of the bilayer), and neutralenergy terms (encompassing all other factors) has been proposed to explainobserved ionic permeabilities (Flewelling and Hubbell, 1 986a,b).In vivo, ion permeation is controlled by proteins. In model membranes, ionicselectivity is generally provided by ionophores, such as valinomycin. Valinomycindramatically increases the permeability of unsaturated PC bilayers to K ions (Bloket a!., 1974b; Bangham, 1983). If there is an initial potassium ion gradient across21the bilayer, the result is a net movement of K+ ions across the bilayer. Thisionophore is therefore “electrogenic”, since it promotes the net movement of chargeacross the bilayer. Other ionophores which are specific for different ions can also beused (see Section 1.4.5). An alternative way in which some ions may crossmembranes is through the formation of neutral “ion-pairs”. In a low dielectricmedium, some ions can complex to form stable neutral species. For example,thiocyanate and a nitroxide analog of TPP+ have been shown to be much morepermeable in the presence of agents which can form a neutral, hydrophobic ion-pair(Gutknecht and Walter, 1982).1.4.5 PERMEABILITY OF PROTONSBecause of the importance of the proton electrochemical gradient in biologicaltransport and energy transduction, proton transport across bilayers deservesspecial consideration. According to the “chemiosmotic hypothesis” (Mitchell, 1961)biological ATP-synthesis is coupled to the proton electrochemical potential, orprotonmotive force (p.m.f.). This led to interest in determining the permeability ofmembranes to protons and to a considerable degree of controversy.It should first be noted that the term “protons” as used here does not refer toH+ or hydronium (H30+) ions. Like other ions, protons are expected to be solvatedin aqueous solution, although the exact number of solvating molecules is notknown. There may be as many as 20 or 21 water molecules of hydration around afree proton and the protonic charge can likely delocalise around this water cluster(Yang et al., 1991). For most purposes it is convenient to define Hiaq) as“protons”. Consideration of proton flux across membranes forces a furtherbroadening of the effective definition of “proton”. This is because it is difficult todistinguish between the movement of protons in one direction and the movement ofhydroxyls in the opposite direction (Nichols and Deamer, 1980). Therefore the term1net was developed to describe the undefined combination of these two events(Nichols and Deamer, 1980) for small gradients around pH 7.It is also important to distinguish between the electrogenic movement (which22results in the generation of a transmembrane electrical potential) and nonelectrogenic movement of protons (which is electrically neutral) (Cafiso andHubbell, 1983). If one has a probe which measures the change in the internal pHof vesicles, it is difficult to tell whether the change in internal pH arises from“proton” movement, or from the net redistribution of other compounds in responseto a ApH (see Section 1.5.3). The system can be best described only if bothelectrogenic and non-electrogenic proton flows can be defined.Nichols and Deamer first noted that proton transport in bilayers wasanomalous (Nichols and Dearner, 1978; Nichols and Deamer, 1980; Nichols et al.,1980; Deamer and Nichols, 1983). The key features of this anomaly are thatproton permeability is greater than other small ions by orders of magnitude (forsmall gradients near pH 7), and that the permeability of protons appears todecrease as the size of the proton gradient increases. A wide range of protonpermeabilities have been reported since, ranging from 1O to i0 cm s(Gutknecht, 1984; Gutknecht, 1987a; Elamrani and Blume, 1983; Cafiso andHubbell, 1983, Perkins and Cafiso, 1986; Deamer, 1987, Gutknecht, 1987b, Nagle,1987, Perkins and Cafiso, 1987b, and references therein). This suggests a millionfold disagreement! However, much of this disagreement disappears when it istaken into account that calculated values of net cannot be compared unlessexperimental conditions are similar. This surprising conclusion is a result of thefact that initial proton flux across bilayers is relatively independent of the size ofthe proton gradient and is generally found to be about 10-13 to 10-15 mol cm’ s(for reviews, see Deamer, 1987, Gutknecht, 1987b, Nagle, 1987, Perkins andCafiso, 1 987b). The available data are therefore not consistent with the simplediffusion mechanism of a charged species over an energy barrier (Perkins andCafiso, 1987b; Deainer, 1987). The fact that proton conductance is relativelyconstant over a wide range of pH (i.e. of proton gradients) clearly implicates somesort of carrier mechanism as the rate determining step in proton flow (Deamer,1987).23Figure 1-4 Representation of protons crossing lipid bilayersby a carrier mechanism.Protons (or equivalents) cross bilayers at a rate which isindependant of the proton concentration gradient. Themechanism by which protons cross the bilayer (represented bythe funnel) is unknown.H H+HHH H24Several possible mechanisms have been put forward to explain the two keyfeatures of proton flux, which are that it is relatively independent of the size of theproton gradient and it is faster than other small cations. Many of these modelsdepend on aggregates of intra-membrane water as key transporting agents, byanalogy with the high proton conductance of ice (Nagle and Morowitz, 1978;Nichols and Deamer, 1980; Deamer, 1987; Miller, 1987; Nagle, 1987 (threemodels!); Deamer and Nichols, 1989). These models share the common factor thatprotons can effectively “hop” down a chain of water molecules via a rearrangementof the water molecules, resulting in a higher mobility of protons than other ions.Others suggest that weak acid protonophores present in low levels in lipidpreparations are responsible for the majority of proton flux across membranes(Gutknecht, 1984; Gutknecht, 1987b).1.5 TRANSMEMBRANE ION GRADIENTS1.5.1 AW AND zpHA transmembrane electrical potential (z\’P) is established across a membraneby the uncompensated transmembrane movement of a charged species. If only onepermeant monovalent ion is present, the equilibrium A’I can be estimated from theNernst equation:=- RT ln(C/C0) (1-9)Fwhere R is the gas constant, T is the temperature, F is the Faraday constant and Cand C0 are the internal and external concentrations of the permeant species,respectively. This equation makes the approximations that pressure, activitycoefficient, and other differences between the internal and external compartmentsare small (Nobel, 1991).For the special case of protons, the protonmotive force (p.m.f.) is defined as25zero when, at equilibrium, at 25°C,bP=--60ApH (1-10)where iW is in mV.It should be emphasized that while the p.m.f. can decay to zero as protonsleak out of vesicles to achieve their electrochemical equilibrium, a sizeable, stablei\pH and an opposing 1M1’ can remain. Note also that membrane potentials andtransmembrane pH gradients occur widely biologically, for instance in nerve cells,mitochondria, and chioroplasts.1.5.2 MEASUREMENT OF ApH AND &P IN LIPID VESICLESThe accurate measurement of transmembrane potentials and pH gradients iscentral to membrane bioenergetics (Rottenberg, 1979; Nicholls and Rial, 1989).From the viewpoint of this thesis, these techniques are important both in“accounting” for protons (Chapter 2 and 3) and in determining rates of protonmovement (Chapter 4). Therefore, the conditions required for these determinationsin lipid vesicles are discussed below.Many techniques to measure AW and ApH have been developed, includingtechniques which monitor the distribution of radiotracers (Rottenberg, 1979, 1984),ESR of spin-labelled probes (Cafiso and Hubbell, 1978a, 1978b, 1982),[31P]-NMR(Redelmeier et aL, 1989, Moon and Richards, 1973), fluorescence of pH sensitivecompounds (Nichols and Deamer, 1980, Bramhall, 1985), and other methods(Rottenberg, 1989) of determining the fate of the appropriate probes. Not all ofthese techniques have a strong theoretical basis (Rottenberg, 1989). In addition,these procedures generally require specialized equipment and are usually restrictedto ApH values of 2 units or less.The equilibrium formalism shown below is pertinent to any probe whichredistributes in response to membrane potentials and is based on a modeldeveloped by Cafiso and Hubbell (1978a), and Rottenberg (1984). A similar26formalism can be adapted for ipH probes (see Chapter 3). Consider the behavior ofa probe of &P in response to a membrane potential as indicated in Fig. 1-3. Atequilibrium, the electrochemical potential of the probe must be the same in allcompartments. Thus, for a symmetric membrane where the probe partitions intonarrow interfaces at the interior and exterior surfaces of a vesicle and where thereare negligible surface potentials, the distribution of probe molecules in the fourregions denoted in Figure 1-3 can be calculated as follows. If the number ofunbound, unencapsulated molecules, N0, is taken to be 1, then from the definitionof the partition coefficient, the relative number in the outer membrane region (Nm0)isNm0 mo/V0 (1-11)Since the equilibrium transmembrane concentrations of the ionic probe aredetermined by the presence of a AW (see Eq. 1-6), the relative number inside thevesicles, N, is:N = (V1/0)eW1lT (1-12)and the number at the inner interface (Nm1), is therefore:Nm1= KVmi N1 = KV (V1/0)e’’/RT (1-13)vi v1The most important feature evident from these equations is that thedistribution of molecules in all compartments will vary with the membranepotential. Thus, if the technique used to measure the equilibrium distributions ofprobe monitors the aqueous population (for example, if the potential probe isK+, which has a very small partition coefficient) or monitors the membranebound population (as in ESR studies of lipophilic probes), the determination of zq’27is relatively simple. However, among the most commonly used AW (and z\pH) probesare hydrophobic fluorescent compounds which undergo changes in fluorescence inresponse to AM’ (or ApH) (Rottenberg, 1989). Only total fluorescence changes can bemeasured and these changes can arise from any or all compartments. It is oftendifficult to predict what fluorescence behavior to expect of these probes as afunction of AM’. Furthermore, AM’ probes must not be directly affected by thepresence of a pH (and vice-versa), vesicle volume changes, temperature and otherfactors unless appropriate corrections can be made.These considerations mean that caution must be employed when interpretingmeasures of AM’, tpH, or proton permeability. The continued successful use ofchanges in the fluorescent properties of these membrane potential probes isperhaps partly due to several self-cancelling errors (Rottenberg, 1989).1.5.3 SIGNIFICANCE OF ACID-BASE CHARACTERISTICS OF COMPOUNDSA great many biologically interesting compounds are weak acids or bases.The equilibrium constant (Ka) of a weak base is:KaHB/BH (1-14)where H is the proton activity, B is the activity of the unionized base and BH isthe activity of protonated base and Ka is the dissociation constant. To a firstapproximation activity coefficients are not required and activities can be replacedby concentrations. The relative concentrations of the neutral and protonated weakbase at a given pH are therefore described by the Henderson-Hasselbach equation:pH=pK + log {[B]/1BHi} (1-15)If the dissociation constants (and activity coefficients) for a weak base are thesame on both sides of a vesicle membraneKa = [H] LB]1/[BHi = [Hi0[B]0/[ H1 (146)28[D[rI=1O // [Dl-r]=10000H÷”118 ( ( PK=81[D]=1 [D]=1pH? pH4Outside InsideI L\PH IFigure 1-5 Effect of transmembrane pH gradients on theequilibrium transbilayer distribution of weak basesThe neutral form of the weak base equilibrates across thebilayer As a result of reprotonation in the low pH environmentof the vesicle interior a transmembrane gradient of the weakbase develops At equilibrium the concentration gradient of thebase equals the transmembrane proton gradient if pK>pH. Thearrows reflect net movements under the conditions described inChapters 2 and 3 the numbers indicate the relativeconcentrations of the species29where the subscripts and refer to inside and outside the vesicle, respectively. Theuncharged species of a given compound tends to be much more membranepermeable than the corresponding charged species (Crofts, 1967; Rottenberg, 1979),so if the compound resides in the aqueous space, the equilibrium concentration ofthe neutral species will be the same on both sides of the membrane (Figure 1-5). IfpK >> pH0 >> pHi, the weak base distribution will therefore reflect the pH gradient:[BH1/[BH]0 = (1-17)The above analysis is included to emphasize that the equilibriumtransmembrane distributions of all acids and bases which are not zwitterions andwhich are membrane permeable in the neutral form are expected to be influencedby the presence of a transmembrane pH gradient. This fact can be exploited formeasuring pH gradients (Chapter 2) and for loading drugs into vesicles (Chapter 3).Since membrane potentials can induce transmembrane pH gradients and vice-versa (Bramhafl, 1985; Redelmeier, 1989), the distribution of other ions can affectthe distribution of a wide range of compounds either directly or by inducing a ApH.Finally, some weak acids are significantly permeable in both their chargedand uncharged forms (McLaughlin and Dilger, 1980). These compounds (forexample, CCCP) are known as protonophores, since they can shuttle protonsacross the membrane (Kasianowicz et al., 1984).1.6 DRUG TRAPPING IN LIPOSOMES1.6.1 PASSIVE ENTRAPMENT TECHNIQUESAs mentioned earlier, liposomes have potential as drug delivery vehicles (forreview, see Ostro and Cullis, 1989). Drugs can be “passively” encapsulated intoliposomes by simply preparing vesicles in a solution containing the desiredcompound (Taylor et al., 1990). However, the resulting low efficiency of drugentrapment and low drug:lipid ratios could require unacceptably high levels of lipid30to be injected into patients (and could also result in waste of expensive drugs). Forexample, a preparation of 10 mg/mL EPC 100 nm diameter vesicles canencapsulate only about 1% of a given hydrophiic drug by passive entrapment. A“dehydration-rehydration” protocol can slightly improve the efficiency of drugentrapment (see Kirby and Gregoriadis, 1984a; 1984b). Leakage of encapsulateddrugs during storage could also limit the usefulness of such passively loadedpreparations. Not surprisingly, hydrophobic compounds show greater associationwith lipid vesicles than hydrophilic ones (Stamp and Juliano, 1979).A variety of parameters, such as the ratio of encapsulated drug to the lipiddose can have profound effects on liposomal drug disposition in vivo (Mayer, et al.,1990a). Passive drug loading protocols, however, do not permit simple,independent variation of these parameters. These problems can be overcome with“active” loading techniques as indicated in the next section.1.6.2 ACTIVE TRAPPING TECHNIQUESTo overcome the problems of inefficient drug entrapment, low drug:lipid ratiosand poor drug retention, other techniques have been developed for liposomal drugformulations. For example, the particularly high affinity of the drug doxorubicinfor cardiolipin has been exploited to produce liposomal doxorubicin preparationswith high drug trapping efficiency (Rahman et al., 1980). Alternatively, anotherliposomal doxorubicin preparation (detailed in Chapter 3) is based upon aprocedure whereby the drug is encapsulated in liposomes in response totransmembrane pH gradients. This occurs essentially as outlined for weak basesin Section 1.5.3 (Mayer et al., 1990a; Mayer et a!., 1986a; Mayer et al., 1986b). Asimilar protocol was earlier shown to entrap catecholamines in liposomes (Nicholsand Deamer, 1976).This zpH loading technique has a key advantage in that it is potentiallyapplicable to loading a wide range of compounds which are lipophilic weak acids orbases, including a large proportion of commonly used pharmaceuticals. zpH31loading allows independent variation of many liposomal parameters and can resultIn much higher drug:lipid ratios than can be achieved by passive drug entrapment(Mayer, 1986b). Drug leakage from the vesicles is also much slower (Mayer,1 986b). Further, as the transmembrane distribution of the drug is determined bythe proton gradient, it is possible to control the rate of drug release by altering thepH gradient.1.7 THESIS OVERVIEWChapter 2 presents an investigation of the accuracy of the techniquesemployed to measure zpH and z’I’ for LUVs with a variety of lipid compositions. Itis shown that transhilayer distributions of the base methylamine accuratelyindicate ApH under a variety of conditions. In turn, if the vesicle volume,concentration and interior buffering capacity are known, these can be used topredict the level of accumulation of methylamine.The interactions of a variety of drugs with LUVs having transmembrane pHgradients are examined in Chapter 3 and the results compared with thosepredicted on the basis of the analysis of Chapter 2. The technique of ApH loadingis shown to be applicable to a wide range of drugs, although drugs areaccumulated to much different extents. An extension of the model derived inSection 2.1, incorporating the ability of drug to partition into the membrane can beused to explain the different levels of drug accumulation.Finally, in Chapter 4, proton flux and associated activation energy inresponse to a 3 unit transmembrane pH gradients are examined under variousconditions. The activation energy of proton flux is found to be about 11 kcal/mol.Under appropriate conditions, proton flux is not exceedingly rapid and pHgradients do not decay quickly, allowing the generation of systems with a stableApH and no AW, and vice-versa.32CHAPTER 2. MEASUREMENTS OF TRANSMEMBRANE pH GRADIENTS IN LUVs2.1 INTRODUCTIONIt is well known that weak acids and bases can undergo a net redistributionacross bilayers in response to transmembrane pH gradients (Crofts 1966; Crofts,1967; Rottenberg, 1979). Recent work has characterized the influence oftransbilayer pH gradients in large unilamellar vesicle (LUV) systems on thetransbilayer distributions of many of these compounds, including a variety of drugs(Nichols and Deamer, 1976; Bally et al., 1985; Bally et al., 1988; Madden et al.,1990), ions such as calcium (Viero and Cullis, 1990), modified peptides(Chakrabarti et al., 1992), fatty acids (Hope et al., 1987), and phospholipids(Redelmeier et a!., 1990, Eastman et al., 1991). An ability to measure the pHgradients (ApH) present across LUV membranes is clearly central to these andother investigations. A number of methods of measuring tpH in LUVs have beendeveloped, based on early work demonstrating that the transmembrane pHgradients in organelles such as chloroplasts and mitochondria could be measuredby determining the transmembrane distribution of weak bases such as ammonia(Deamer et a!., 1972; Rottenberg, 1979; Deamer, 1982). Similar procedures usinglipophiic ions can be used to indicate iW (Rottenberg, 1989). As emphasized byRottenberg, z\pH values determined by probes which can partition into themembrane can be misleading (Rottenberg, 1989; Section 1.5.2).The transmembrane distribution of trace amounts ofL14C1-methylamine canbe used to determine pH in LUVs (see Section 1.5.3). Due to the high permeabilityof the uncharged form of methylamine, rapid equilibration across the membraneoccurs (Rottenberg, 1989). Protonation of the neutral form in the low pHenvironment of the vesicle interior results in a net accumulation of probe to achievean equilibrium where the probe concentration gradient reflects the pH gradient.Separation of trapped probe from untrapped probe and the subsequentdetermination of entrapment allows the proton gradient to be measured. For33liposomes with acidic interiors, it is shown that protocols employing radiolabelledmethylarnine in conjunction with gel filtration procedures to remove untrappedmethylarnine provide accurate measures of ApH in most situations. However, thereare situations in which the accuracy of this technique of ApH measurement can becompromised.First, as indicated above, to achieve equilibrium, the neutral form of the probemust readily permeate the vesicle bilayer, leading to possible errors for relativelyimpermeable membranes. Second, the ratios of trapped to free[14C]-MeNH3inliposomes are usually determined by removing exterior (untrapped) probeemploying centrifuged gel filtration mini-columns (Redelmeier et al., 1989).However, once the vesicles enter the gel matrix the system is not at equffibrium,resulting in possible efflux of the probe from the vesicles while the vesicles are onthe column. Finally, the protonation of I‘4C]-MeNH2on arrival in the vesicleinterior consumes a proton. Thus, the ApH is affected by the probe itself. Theextent to which these and other factors can compromise the accuracy of ApHmeasurements are examined in this Chapter, procedures which avoid thesedifficulties are described. Further, techniques to determine the membranepotentials (zW’) induced in response to these pH gradients are also evaluated.Finally, it is of interest to determine the maximum transmembrane pH gradientwhich these vesicles can withstand.2.2 MATERIALS AND METHODS2.2.1 MATERIALSBuffers and other chemicals used were purchased from Sigma Chemical Co.(St. Louis, Missouri) unless otherwise stated. Radiolabels were supplied by NewEngland Nuclear (Mississauga, Ontario). Benzene, methanol and other solventswere purchased from BDH (Vancouver, B.C.). All phospholipids were obtainedfrom Avanti Polar Lipids, (Birmingham, Alabama).342.2.2 LIPID VESICLE PREPARATIONCholesterol was incorporated (where noted) in lipid samples by colyophilization from benzene:methanol (70:30, v/v). Multilamellar vesicles (MLVs) ofthe stated lipid composition were prepared by adding the indicated buffer (generally300 mM citrate buffer, pH 4.0 or 300 mM CHES, pH 9.0) to the dry lipid powder attemperatures above the lipid gel-liquid crystalline phase transition temperature.Samples were vortexed for 5 mm, then subjected to 5 cycles of freezing (> 3 mm inliquid nitrogen) and thawing in order to produce “FATMLVs” (Mayer et al., 1985a),unless otherwise indicated.Large unilamellar vesicles were prepared by extruding these MLVs orFATMLVs 10 times through two stacked 100 nm filters as previously described(Hope et al., 1985). For saturated lipids, vesicle extrusion was performed above thegel-liquid crystalline phase transition temperature employing a thermally jacketedextrusion apparatus (Lipex Biomembranes, Vancouver, Canada). Vesicles preparedby this method are unilamellar, with diameters of approximately 100 nm (Hope etal., 1985; Nayar et al., 1989).2.2.3 PARTICLE SIZE DETERMINATIONSVesicle diameters of some samples were examined by freeze-fracture (Hope etal., 1985), or cryo-electron microscopy. Vesicle size was routinely determined byquasi-elastic light scattering (QELS) at about 0.1 mM lipid (Mayer et al., 1986a)with a Nicomp Model 200 Laser Submicron Particle Sizer (Nicomp Instruments,Goleta, CA) using a 5 nW Helium-Neon Laser at a wavelength of 632.8 nm. QELSanalyses fluctuations in scattered light intensity generated by the diffusion ofparticles in solution. The measured diffusion coefficient is used to obtain theaverage hydrodynarnic radius and hence the mean diameter of the particles.2.2.4 DETERMINATION OF ENTRAPPED SOLUTEIn general, solute entrapment was determined by preparing vesicles in thepresence of 2 iCi/mL of the indicated impermeable trap marker. Unencapsulated35marker was removed by passing 250 1L of the sample through a 5 mL SephadexG-50 gel filtration column([14C1-citrate or[14]C-Histidine marker), or SepharoseCL-4B([3H]-inulin). In the case of MLV or FATMLV preparations, unencapsulatedprobe was removed by 5 cycles of washing and centrifugation in a Fischermicrofuge at 9000 rpm for 3 mm. Trapped marker was quantified by liquidscintillation counting and phospholipid by phosphate assay (see below).The amount of trapped citrate buffer was established by adding 10 Ci/mL[14C]-citrate to the 300 mM citrate buffer used for hydration and determiningcitrate entrapment by gel filtration after freeze-thaw and extrusion through 100 nmpore size filters.2.2.5 EFFECT OF INITIAL SOLUTE DISTRIBUTION ON ENTRAPPED SOLUTEEPC vesicles (extruded through the indicated filter pore size) derived fromMLVs as well as from FATMLVs were prepared as above in 150 mM NaCl 20 mMHEPES, pH 7.5, containing 5 iCi/mL[3H]-inulin. Vesicles were washed andcentrifuged in a Fischer microfuge at 9000 rpm for 3 mm, the buffer removed andthis cycle repeated 3 times to produces MLVs or FATMLVs with the solute markerpresent only inside the vesicles. This preparation was subsequently extruded andthe levels of solute remaining inside the vesicles determined as above. Similarresults were obtained using[‘4C]-Histidine as the trap marker.Alternatively, the marker was added after the preparation of multilamellarvesicles (MLV or FATMLV) but before the extrusion procedure, resulting in systemswhere the marker is initially present only outside the vesicles.2.2.6 VESICLE VOLUME DETERMINATIONSThe interior volume of extruded vesicles (in a hypo-osmotic medium) wasdetermined by preparing vesicles in 300 mM citrate, pH 4.0, exchanging theexternal buffer for 150 mM NaCl, 20 mM HEPES, 0.2% azide containing 10 i.tCi/mL[14C]-glucose and incubating the preparations for 24 h at 45°C. The half-time forglucose permeation in 100 nm EPC:cholesterol (55:45; mol:mol) LIJVs is — 1 h at3645°C (B. Mui, unpublished data). Entrapped [14C1-glucose was determinedemploying Sephadex G-50 gel filtration to separate free from entrapped material asabove. The amount of entrapped glucose is expressed as an equivalent volume,assuming that the probe distribution reflects the volume distribution.In general, the experiments presented in this thesis are done underconditions where the interior of the vesicles are hyperosmotic with respect to theexternal medium (generally 600 mOsm inside, 300 mOsm outside), such thatvesicles are at their maximum size. It should be noted that drug loading inresponse to pH gradients (see Chapter 3) results in an increase in the internalosmolarity of the vesicles. Under the conditions used, the osmotic gradientremains below 800 mOsm.2.2.7 DETERMINATION OF LIPID CONCENTRATIONSLipid concentrations were generally determined by analysis of lipidphosphorus as described previously (Fiske and Subbarow, 1925; Bottcher et al.,1961). Aliquots containing between 0.02 and 0.2 tmole phospholipid were digestedin 0.6 mL of 70% HC1O3 for at least 1 hr. After cooling, 7.0 mL of ammoniummolybdate reagent (0.22%, w/v, ammonium molybdate in 2% H2S04,w/v) and 0.6mL of Fiske-Subbarrow reagent (30 g NaHSO3, 1 g Na2SO3and 0.5 g bis 1-amino-2-napthol-4-sulphonic acid in 200 mL water were added. Subsequently, thesamples were heated for 20 mm at 100°C and the absorbance at 815 nm wasdetermined after cooling. The amount of phospholipid was calculated by comparingthe absorbance to a standard curve.On some samples, cholesterol assays (Rudel and Morris, 1973) were alsoperformed. 1.0 mL of glacial acetic acid containing o-pthalaldehyde were added tolipid samples. After 10 min, 2.0 mL concentrated H2S04was added and theamount of cholesterol determined optically by reference to a standard curve.Cholesterol determined in the final vesicle preparations corresponded to theoriginal proportions added, indicating that the neither component is preferentially37lost during vesicle preparation. This assay was also used when samples containedsignificant amounts of non-lipid phosphorous.2.2.8 GENERATION AND MEASUREMENT OF TRANSMEMBRANE IONGRADIENTSThe pH gradient was imposed by passing 300 iL of the vesicles down a 10 mLSephadex G-50 column equilibrated with the appropriate external buffer, usually150 mM NaC1, 20 mM HEPES, pH 7.0. These vesicles were quickly diluted into thesame buffer containing 0.5 tCi[14C]-MeNH3/mL(or other radiolabel) to a finallipid concentration of 1-3 mM. The samples were incubated at the indicatedtemperatures and at appropriate times 100 tL aliquots were removed and passeddown 1 mL Sephadex G-50 mini-columns which were centrifuged for 3 mm at 2000x g (Penefsky, 1977). Entrapped probe was determined using liquid scintillationcounting and phospholipid concentrations determined using a modified phosphateassay described above. The ratios of entrapped to free concentrations of probe weredetermined employing the measured internal aqueous volumes indicated in Table2-I for 100 nm vesicles.For some samples, transmembrane probe distributions were determinedusing a “centrifree” equilibrium binding analysis apparatus (Amicon, Danvers, MA,USA). After a 30 mm incubation in the presence of the radiolabel under theconditions indicated above, 1 mL samples were placed in the upper filter chamberand the apparatus was centrifuged at 1500 x g for 5 mm. Probe distributions werethen determined as indicated above.Measures of membrane potential using[3H1-TPP or[14C]-Iabelled SCN wereobtained in a similar manner as with[‘4C]-MeNH3.For comparative purposes,electrical potentials are often expressed here as Iog(Iprobe1/[probe]0).Nocorrections for probe binding were made to these values, since these were small(see Results).382.3 THEORETICAL CONSIDERATIONSThe pH of the LUV interior can be influenced by the influx of probe (whichconsumes a proton) and by proton efflux to set up a AW in electrochemicalequffibrium with the ApH. First consider the influence of amine accumulation onthe interior proton concentration. For a buffer such as citrate with three acidicgroups, it is straightforward to show that the concentration of the buffer in theneutral, fully protonated, form ([B]), in the singly deprotonated form ([WI) and so onare related to the total buffer concentration [B]tot via the relations[B] = [BtOt] /fiHj (2-1)[B-] = K1/[H] x [Btot] /fiH) (2-2)[B2] =K12/([H]) x IBt0t] /fiH) (2-3)[W3] =K123/([Hi) x [Btot] /fiH) (2-4)wherefiH) = (1 + K1/[Hj +K12/[H] +K123/[H+I) (2-5)and K1, K2 and K3 are the dissociation constants of the titratable groups. Theproton concentration is indicated by [Hf], and activity coefficients are omitted.Each (neutral) methylamine that moves across the vesicle membraneconsumes a proton as it is reprotonated in the vesicle interior. By charge balance,the final concentration of charged amine in the vesicle interior ([AH]1)can thus beexpressed as[AH]1 = (MW]) +2(A[B]) +3(MW]) (2-6)39where ALB1 represents the change in the internal concentration of the singlycharged buffer (i.e. {LBjfmal - [Bjinitiai}) as a result of drug accumulation, and soon.At equilibrium, assuming that the charged form of methylamine does notpartition appreciably into the vesicle bilayer, the final inside:outside methylamineconcentration gradient must obey the relation[AlI+eq =_____(2-7)[AH+]eq0where the subscripts and refer to the inner and outer environments,respectively. Further, if [H]0 and [H]>> Ka, where Ka is the dissociationconstant of methylamine (pK 10.6), then[AH]0 = LA]tot - (V1/0)[AWl1 (2-8)where [A]tot is the total (initial) concentration of methylamine, V1 is the interior(trapped) volume of the LUVs and V0 is the external volume. Thus, combiningequations 2-7 and 2-8, we obtain[H+10 [Al-l]q (2-9)([A]tot- (V1/0)[AH+]eq1)By substituting Equations 2-1 to 2-5 in Equation 2-6 the value of [H+]e canbe calculated by an iterative process.A second factor which can reduce the internal proton concentration resultsfrom the initial efflux of protons from the acidic interior to the exterior environmentto set up an opposing electrical potential (A’I’). This equilibrium is described by theNernst relationA’P = -RT/F(ApH) (2-10)40where R is the gas constant, T the temperature and F is the Faraday constant. Thenumber of proton equivalents required to set up this equilibrium can be estimatedfrom the membrane capacitanceQ=CAmAP (2-11)where Q is the charge in coulombs, Am is the area of the membrane and C is themembrane capacitance. The number of protons released is given by N(Hi = Q/ewhere e is the elementary unit of charge (1.6 x 10-19 coulomb).2.4 RESULTS2.4.1 VESICLE CHARACTERISTICSThe vesicle systems used most commonly in this thesis are 100 nm diametervesicles prepared by hydrating powdered lipid in 300 mM citrate, pH 4.0, (to makeMLVs) followed by 5 freeze-thaw cycles in liquid nitrogen (to make FATMLVs) andextrusion through 100 nm pore size filters (to make LUVs). The amount ofentrapped buffer is a critical parameter in these investigations, so the distributionof the citrate buffer was examined at each step of the vesicle preparation. Repeatedcycles of freezing and thawing in liquid nitrogen increases the level ofencapsulation of 300 mM citrate buffer in multilamellar vesicles (Fig 2-1) in a time-dependent fashion. This increase in encapsulation is consistent with theobservations of Gruner (1985) and Perkins et al. (1988) that MLVs do not haveequilibrium solute distributions and with those of Mayer et al. (1985a) that freezethawing can increase encapsulation efficiency. It can be seen that the vesiclesmust be maintained in liquid nitrogen for a surprisingly long time (>3 mm) in orderfor the citrate to becomes maximally entrapped in FATMLVs. This likely reflectsthe ability of these high concentrations of citrate buffer to act as a cryoprotectant(data not shown).Because of the higher solute entrapment of FATMLV-derived extruded vesicles,410.600.50 _L0.40a.0.30I- /0.20 1 -I,Co0.10C)o.oo I1 2 3 4 5Freezing Time (mm)Figure 2-1 Entrapment of 300 mM Citrate Buffer inFATMLVs as a function of freezing timesEPC MLVs were prepared m 300 mM citric acid pH 4 0containing 10 tCi/mL[-14]C-citrate as a solute marker. Vesiclesamples (in plastic “cryovials’) were plunged into liquid nitrogenand thawed in a 40° water bath for five cycles of the indicatedfreezing times. Unencapsulated buffer was removed bycentrifugation and the amount of solute entrapped determined(see Methods)42Table 2-I.[4C1-g1ucose and[14C1-citrate volumes of 100 nmdiameter extruded vesicles.Lipid Composition [14 1-Glucose [14C]-CitrateSpace (jiL/mol) Space (iL/,mol)Egg PC 1.78 ± 0.4 (n=8) 1.50 ± 0.1 (3)EPC:Cholesterol 0.98 ± .28 (9) 0.841 ± 0.09 (3)(55:45 mol%)The amount of entrapped [‘4C1-glucose was determined afterincubating vesicles at 45° with 10 Ci/mL[‘4Ci-glucose. Citratebuffer space was determined by preparing vesicles in 300 mMcitrate, pH 4.0, freezing in liquid nitrogen for 5 five mm cyclesand extruding through 100 nm filters. The number ofexperiments is given by n.vesicles were always subjected to freeze-thawing cycles (>3 mm freezing time)before extrusion. As seen in Table 2-I, the resulting 100 nm diameter vesicles havemaximal aqueous trapped volumes (as reflected by[‘4C1-glucose encapsulation) of1.8 L/mol lipid for EPC vesicles and 1.0 L/mol for EPC/cholesterol (55:45,mol:mol). The amounts of citrate buffer entrapped in these vesicles corresponds to1.5 and 0.84 L/mol lipid respectively. These values for aqueous volumes andbuffer entrapment are used in the simulations presented in Chapter 2 and 3.It should be noted that the citrate buffer leaks only very slowly from thesevesicles, even in the presence of the imposed pH gradient (greater than 80 %retention after 2 h at 60° for EPC vesicles incubated with a transmembrane pHgradient, results not shown). Other characteristics of these extruded vesicles areindicated in Hope et al., (1985).As indicated earlier, extruded lipid vesicles prepared from MLVs show greatlyreduced solute entrapment compared to those prepared from FATMLVs. It ispossible that these different degrees of solute entrapment result from thedifferences in the initial solute distribution of MLVs and FATMLVs, or from430E00I0aI002I00 200 400 800- 800Filter Pore Size (nm)Figure 2-2. Dependence of Solute TrappingSolute Distribution.on InitialSolute entrapped per !mol lipid after extrusion, where 13H]-mulin was added to preformed FATMLVs (•) or MLVs (A).Alternatively, the marker was present only inside the FATMLVs(0) or MLVs (t.) before extrusion. Also indicated is the amount ofsolute entrapped when vesicles have an equal initial solutedistribution (0).44physical differences in the resulting extruded vesicles themselves.. For example,MLV-derived extruded vesicles could contain internal lamellae which are notpresent in FATMLV-derived vesicles.To distinguish between these possibilities, the effects of initial solutedistribution on the amount of encapsulated solutes were examined as a function ofthe filter pore size for both MLV-derived and FATMLV-derived extruded vesicles(Fig. 2-2). Vesicles prepared from MLVs and from FATMLVs behave similarlyduring the extrusion procedure. In both cases, solute equilibration between theintravesicular and external media does not occur as a result of the extrusionprocess (Fig. 2-2), despite a more than 10-fold reduction in vesicle size uponextrusion.These observations strongly suggest that the extremely low encapsulationefficiency of MLV-derived LUVs (Mayer et al., 1986a) results mainly from the initialsolute exclusion of MLVs and that extruded vesicles prepared from MLVs orFATMLVs are otherwise similar. The majority of the solute lost during extrusionoccurs on the first pass through the filters (results not shown). Once these smallervesicles are formed, they appear to be able to pass through the polycarbonatefilters (with a pore size equal to their diameter) without suffering further loss ofcontents.2.4.2 MEASUREMENTS OF tpHMeasurements of transmembrane pH gradients (zpH) are central to thisthesis. Therefore, a first series of experiments were directed towards examining theinfluence of lipid composition and temperature on the zpH indicated by thetransmembrane distribution of[14C-methylamine. Vesicles composed of EPC,DPPC (16:0/16:0 PC), DSPC (18:0/18:0 PC) and DAPC (20:0/20:0 PC) alone and incombination with cholesterol (PC:cholesterol, 55:45 mol:mol) were thereforeprepared by extrusion. A tpH of 3 units (inside acidic; pH1 = 4.0, pH0 = 7.0) wasimposed, as indicated in Methods. Note that the filter pore size used in the45experiments of Fig 2-3 was 200 nm, due to difficulty extruding the dispersions ofthe highly saturated lipids through filters with smaller pore size.Lipid composition can have a profound effect on the zpH reported.Specifically, whereas the zpH reported for the EPC vesicles accurately reflects theapplied ApH, little or no apparent pH gradient is detected in the vesicles composedof the saturated lipids DPPC, DSPC or DAPC (Fig. 2-3). In the case of DAPC, thehalf-time for methylamine accumulation is > 24 h at 20° C (results not shown). Theapparent lack of the methylamine response to the imposed zpH may initially beattributed to the impermeable, gel state nature of those vesicles at the incubationtemperature (21°C), which would be expected to reduce the permeability of theneutral form of methylamine. This interpretation is supported by the resultspresented in Figs 2-3(b) and 2-3(c). The presence of 45 mol % cholesterol, whicheliminates the gel-to-liquid crystalline transition and increases the motion availableto previously gel state phospholipids, results in equilibration of methylamine whichis nearly complete by 30 mm for the DPPC, DSPC and DAPC systems (Fig. 2-3(b)).Alternatively, heating the vesicles to temperatures above their gel-to-liquidcrystalline transition temperature (‘Ps) should also result in rapid equilibration. Asshown in Fig. 2-3(c), this is the case for the DPPC (Tc 41°C) and DSPC (Tc 58°C)vesicles when incubated at 60°C, where rapid transbilayer equilibration ofmethylamine is observed within 5 mm. It is interesting to note that the presence ofgel state lipid per se does not prevent methylamine equilibration, as methylamineaccurately reports the imposed ApH in the DAPC vesicles at 60°C, some 15°C belowthe T of this phospholipid (Nayar et al., 1989). Further, the[14C1-methylaminedistributions report a 3 unit pH gradient for all vesicle types incubated at 60° Cand indicate that this ApH is stable for at least 1 h at this temperature in all lipidcompositions tested.It is straightforward to show that the percentage of the probe that isaccumulated for a given tpH obeys the relation:464.’0I-IC’,IzC.,2:zFigure 2-3 Effect of lipid composition on methylaminedistributions.(A) Methylamine distributions were determined as described InMethods for vesicles containing 300 mM citrate (pH 4.0)extruded through 200 nm ifiters and subsequently incubated in150 mM NaC1, 20 mM HEPES, pH 7.0 at 21°C. Vesicles werecomposed of: EPC (.); DPPC (ê; DSPC (A); and DAPC (‘). (B)Methylamine response determined as In (A). but vesiclescontained 45 mol % cholesterol. (C) Methylamine response forvesicles (in the absence of cholesterol) incubated at 370 (opensymbols) or 60° (filled symbols). The symbols are the same as Inpart (A).3210a— •—--0 10 20 303200 10 20 30C3210300 10 20Time (mm)471 O1PH[PL]V.01 xlOO%/o entrapped (2-12)1 + 1OAPH[PL]Viwhere [PLI is the lipid concentration and V1 is aqueous volume per mol of lipid.Thus, under the experimental conditions employed here (2 mM lipid, 11Ci/mL methylamine) detection of a 3 unit pH gradient involves the accumulationof 67% of the probe, for a trap volume of 1 L/mol lipid. Given the initial externalconcentration of the radiolabelled MeNH3 as 1 Ci/mL, or 21 tM (specific activity= 48 mCi/mmol) this indicates a final interior probe concentration of 7 mM. TNBSassay of the radiolabel confirmed that the probe was of the indicated specificactivity (results not shown). As each methylamine accumulated consumes aproton on arrival in the vesicle interior, it is clear that the vesicle interior must bereasonably well-buffered in order that radiolabelled methylamine provides anaccurate measure of the initial ApH. This effect is illustrated in Fig. 2-4a, where itis found that at interior citrate concentrations below 50 mM (under iso-osmoticconditions) the accuracy of the zpH reported by methylamine is increasinglycompromised for a pH gradient which was initially 3 units.As indicated under Methods, the presence of a pH gradient (inside acidic)across the vesicle bilayer will also induce a membrane potential (z\W; insidenegative) due to the efflux of H+ ions. The effective interior concentration ofprotons [N(H+Hefc lost to establish electrochemical equilibrium can be written as[N(W1-)]eff = 3.7 x 10 ApHid, for a membrane capacitance C = 1 pF/cm2,where dis the vesicle diameter in cm and ApH is the equilibrium pH gradient. For a 100nm diameter LUV with a ApH 3.0, this corresponds to a loss of 1.1 mM protonequivalents of buffering capacity. Thus, the loss of buffering capacity to set up A’I’would not be expected to compromise the measurement of ApH to the extentcaused by the accumulation of the radiolabelled methylamine. This conclusion can480________a).0010.ta).000.I-’0)00I—’a).000.Ca).000.0Citrate Concentration (mM)Figure 2-4. Methylamine response in vesicles withdifferent buffering capacities and osmotic strength.The effects of internal buffering capacity (A) and osmoticgradients (B) on the apparent transmembrane pH gradients andmembrane potentials. EPC:Chol (55:45: mol:mol) vesicles (2mM, 100 nm diameter) were prepared at the indicatedconcentration of citrate, pH 4.0. These vesicles were incubatedfor 30 mm with (A) an iso-osmotic NaCI-HBS buffer (pH 7.0) or(B) 150 mM NaC1, 20 mM HEPES, pH 7.0. Buffers contained 1.01tCi/mL[14C]-methylamine (21 jtM MeNH3) to determine ApH(•) or 1.0 iCi/mL [‘H1-TPP (26 nM TPP) to determine ‘P (j.The solutions also contained 5.0 1M CCCP to speed developmentof tW. The dotted lines represent theoretical curves derived fromthe model described in Methods.I’A_4- ---.----------AII /I,0 10 20 30 40 50 300Citrate Concentration (mM)32103210A A-/4’_: —B0 10 20 30 40 50 30049be tested by measuring the induced z’I’ employing labelledtetraphenylphosphonium((3H1-TPP) as a probe of zS’I’. In this regard, as each TPPmoves into an LUV in response to i\M’, an H+ ion is released to re-establish AW, sothere is a 1:1 stochiometry between TPP+ accumulation and internal protons“consumed’, as for MeNH3+. However,[3H]TPP+ is available at specific activities(39 Ci/mmol) which are nearly 1000-fold higher than those of[‘4C]-MeNH3.As aresult, under the standard initial conditions of 1 tCi/mL TPP to measure AW, thefinal interior TPP+ concentration (and thus the concentration of proton equivalentsconsumed) is only 8.5 tM. Thus, as shown in Fig. 2-4a, measurement of the z\pHinduced M’ using[3H]-TPP can be a more accurate method of measuring zpH thanthe methylamine procedure for low (< 50 mM citrate) internal buffering capacities.It should be noted that errors in determining the amount of entrappedMeNH3+or in determining the aqueous trapped volume can result in large errors inthe estimates of ApH if zpH is small. An example of this is given by the ApH data ofFig 2-4(b), where the exterior aqueous buffer is maintained as 150 mM NaCl, 20mM HEPES, while the interior citrate concentration is varied. At lower interiorcitrate concentrations the LUVs will shrink due to the osmotic imbalance to achievean equilibrium volumeveq= v (H1/fl0) (2-13)where V and fl indicate the initial interior volume and osmolarity,respectively and 110 indicates the osmolarity of the exterior medium. As a result,less MeNH3will be accumulated to satis1,’ the relationship [MeNH3]/[MeN+]0= [H+]1/[W10. If no correction is made for the change in volume, an apparent ApH(ApHaPP) will be measured which is less than the actual zpH according to therelationApFIapp zpH - log(1T/1) (2-14)50This volume correction would apply only to hyper-osmotic (outside) gradientssince these vesicles do not swell beyond the maximal values seen in Table 2-I. It isinteresting to note that the measured TPP distribution is less affected by vesicleshrinkage. This is likely related to the ability of this probe to partition in thevesicle membrane (see below).The influence of the internal citrate concentration and external (initial)MeNH3 concentration on the levels of internalized MeNH3 in 100 nm diameterEPC:cholesterol LUVs at equilibrium and the derived ApH values are illustrated inFig. 2-5(a) and 2-5(b) respectively. At high initial concentrations of externalMeNH3 (10 mM), extremely high levels of internalized MeNH3 can be achieved(300 nmol/i.tmol lipid) using internal citrate concentrations of 300 mM. The solidlines indicate the theoretical behavior expected on the basis of the analysispresented in Methods, employing the measured trapped buffer of 0.84 L/mol (Table2-I) with no adjustable parameters. An important ramification of the observedagreement with theory is that the uptake of any simple weak base can be predictedon the basis of the buffering capacity, or alternatively, that the buffering capacitycan be measured by determining methylamine distributions at higher methylamineconcentrations.A final situation which would be expected to compromise the accuracy of the[14C]-MeNH3technique for measuring zpH concerns the influence of higherinternal and external pH values. The equilibrium relation [H]1/[ i0=[MeNH3ii/IMeN10holds only for LHi [H]0 << K. where Ka is thedissociation constant of the weak base. As the exterior and interior pH approachKa, the apparent t\pH (zpHaPP) is related to the actual z\pH (pHal) by the relationzpHaPP = pHrea1- log (1 + Ka/LH]o) (2-15).This would lead to a decrease in the measured ApH as [Hi0 approaches Ka.More importantly, as the interior pH is raised, the proportion of internalized amine51300.00L.0.C0.00C.0)00C’00EE0.0.0CE0AA‘%. ô.. —.2..__‘S—-_‘.-::--—_......... A A•s_. .—_._ . :——.‘-..- .•__‘---.__ .U.1•-I • I •00. 2 4 6 8 10__External Probe Concentration (mM)AB A300.-.— —— — — —— — — — —— _.200..———‘—————— .____—__.- - - - - - - - - - - -U100 • ‘-——ii Uf4—.--- - - - -0 . I . I0 2 4 6 8 10External Probe Concentration (mM)• Figure 2-5. Effect of external methylamine concentrationon methylamine uptake.EPC:cholesterol (55:45; mol:mol) LUVs (100 nm diameter, 4 nmlipid) were prepared in 50 mM, 100 mM (U). 200 mM (.) or 300mM citrate (A), pH 4.0 (see Methods), and the vesicles incubatedwith the indicated concentration of methylamine, containing 0.5Ci/mL14CJ-.methylamine. Part (A) shows the effect of probeaccumulation on the apparent pH: part (B) indicates theamount of accumulated MeNH3. The dotted lines represent thetheoretical behavior predicted by the model described inMethods.52-___—A—__A__-—-—A-_-A3 Aa)0L.a..External pHFigure 2-6 Determination of pH gradients over a range ofpHEPC LUVs (100 nm) were prepared in 200 mM citrate, 200 mMMES or 200 mM HEPES. at pH 3.0. 4.0, 5.0, 6.0. or 7.0 (closedsymbols). This buffer was exchanged for an external buffer of150 mM NaC1. 20 mM MES. 20 mM HEPES and 20 mM CHESwith the appropriate pH required to maintain a 3 pH unitdifference between the internal and external buffers.Transmembrane distributions of I14C1-MeNH3were determinedafter 30 mm Incubations at room temperature (.); transbilayerdistributions of[3HJ-TPP (A) were determined after 30 mm inthe presence of 10 tM CCCP.53which is in the neutral, membrane permeable, form will be increased, leading tothe probability of increased probe leakage during the spin column separation. Thiswifi also lead to lower measures of ApH. As shown in Fig. 2-6, the influence ofthese effects becomes noticeable at exterior pH values of 9.0 or higher for 100 nmEPC LUVs exhibiting a ApH of 3 units. As may be expected, detection of theinduced AW employing[3H]-TPP is not subject to such limitations and provides anaccurate measure of eq (and thus, the equilibrium zpH) at exterior pH values upto 9.5.The results to this point indicate that theL14C1-MeNH3probe in combinationwith the spin column procedure provides a convenient and accurate measure ofApH (up to 3 units) assuming that conditions allowing the equilibrium transbilayerequilibration of the neutral form are observed and that the interior environment issufficiently well-buffered. A further point of interest concerns the magnitude of theApH which can be generated and measured. In this regard, it is experimentallyconvenient to work in a region where the maximum probe entrapment, at themaximum ApH, corresponds to 75% or less than the total amount of probeoriginally present in solution. For larger pH gradients this, in turn, limits theamount of phospholipid that can be employed. To detect a pH gradient of 5 unitswhile only accumulating 75% of the probe would require using only 0.03 mMphospholipid, which could result in lipid phosphorus assay errors after the spincolumn. An alternative technique, which becomes progressively more accurate athigh ApH values, involves equilibrium filtration to separate vesicles from theexternal buffer (see Methods). As shown in Fig. 2-7, both the equilibrium filtrationand the spin column procedure are in good agreement for imposed ApH gradientsfrom 1-5 units. The maximum pH gradient which 100 nm diameterEPC:cholesterol (55:45 mol:mol) LUVs can maintain is approximately 3.7 units.This is also indicated by the transmembrane distributions of[3H1-TPP (Fig. 2-7).The techniques discussed above relate to measurements of pH gradients invesicles with an acidic interior. It is of interest to determine whether similar spin54570I—’- 7o 4 7 07o‘ICi 3.0 2o 7IC.-. 0i_____________________________Applied pH GradientFigure 2-7. Measurement of large pH gradients.EPC:chol vesicles prepared in 300 mM citrate, pH 4.0 wereincubated in 150 mM NaC1 and 20 mM each of citrate, MES,HEPES, EPPS and CHES buffers with pH values in the range 4.5to 9.0. The ipH was determined by the transmembranedistribution of I-4Cj-methy1amine using spun mini-columns (.1or “centrifree” filters (0) after a 30 minute incubation at roomtemperature. E3H1-TPP distributions () werç determined in thepresence of 5 M CCCP. as indicated in Methods. The dottedline represents the size of the Imposed gradient.55-0-o1 PI--I —21I—1/çwC) I_•_-.‘ ... ._4o I.-0 -10 1 2 3 4Time (hrs)Figure 2-8. Transmembrane distributions of radiolabelledprobes in EPC LUVs for vesicles with a basic interior.Apparent transmembrane distributions of radiolabelled benzoicacid (A), acetylsalicylic acid (s), acetic acid (•). and mevalonicacid () were determined employing gel filtration in vesiclescontaining 300 mM CHES. (pH 9.0) incubated in 150 mM NaCl,20 mM MES (pH 6.0) with 0.5 iCi/mL of the indicated probe.Positive (interior) membrane potentials induced in response tothese pH gradients were determined by the redistribution ofI’4C]-thiocyanate in EPC vesicles using the gel filtrationseparation procedure (v).56column procedures can be applied to determine zpH in vesicles with a basicinterior employing radiolabelled weak acids as the ipH probes. Such studies werepursued for 100 nm EPC vesicles experiencing a 3 unit pH gradient (pHi = 9.0; p1-I0= 6.0) utilizing as probes[-4C]-labelled benzoic, acetylsalicylic, acetic andmevalonic acid. As shown in Fig. 2-8, the measured transmembrane distributionsof benzoic and acetylsalicylic acid do not reflect the imposed ApH, whereas thetransbilayer distribution of acetic acid significantly underestimates the pHgradient. Mevalonic acid appears a useful indicator of ApH for vesicles with basicinteriors, however, a long (2 hr) incubation time to achieve equilibrium is requiredat 20°C. The most rapid and accurate indication of ApH is given by the membranepotential indicator 114C]-thiocyanate in the presence of CCCP, which gives atransmembrane distribution commensurate with the induced W (inside positive)expected for a 3 unit pH gradient.The response of[1-4C]-acetate was further examined in order to understandthe basis of the behavior exhibited in Fig. 2-8. As for MeNH3,a logical possibilityis that entrapped acetate is released during the spin column procedure. Theinclusion of cholesterol or the substitution of DSPC for EPC would be expected toreduce such leakage. As shown in Fig. 2-9(a),[14C]-acetate provided a muchimproved measure of ApH for vesicles with these lipid compositions. Alternatively,as shown in Fig. 2-9(b), the equilibrium filtration procedure can be usefully appliedto achieve accurate measures of ApH even for the EPC system.A final point of investigation concerned the relation between ipH as measuredby probes such as methylamine and the induced AW measured by probes such asTPP+. Clearly, in the absence of other factors, the transbilayer concentrationgradients detected by MeNH3 and TPP resulting from a given ApH and induced iW’,respectively, should be the same at equilibrium. However, other workers havereported that TPP exhibits a significant membrane-water partition coefficient(Flewelling and Hubbell, 1986a,b). This would be expected to increase the insideoutside concentration gradient for TPP+, for a given A’I, due to the small aqueous57Applied pH GradientFigure 2-9. Determinationemploying [14C]-acetate.of pH gradients (interior basic)(a) Transmembrane distributions of labelled acetate weredetermined as in Fig. 2-8 for 100 nm diameter LUVs composedof EPC rn); EPC/cholesterol (55:45; mol:mol) (a), orDSPC/cholesterol (55:45; mol %) (A). (b) The transmembranedistribution of [‘4C]-acetate as determined by equilibriumcentrifugation in EPC (100 nm) LUVs. These vesicles wereincubated in 150 mM NaCI, 20 mM CHES, 20 mM HEPES and20 mM MES, with the pH adjusted to values between 9.0 and6.0 and the transrnernbrane distribution of 0.5 tCi/mL 1acetate measured by “centrifree” filters as indicated in Methods.The dotted line is a imear regression, with a slope of 0.94.0Cu-D0I0.Cu.000.C)0CuCu4-.a,C.,CuCuCUCuUCu0)032103211 2 3Applied pH Gradient4‘-B ——--—--N0 1 2 3 45815001200Aoa 900— .sst ::External TPP Concentration (mM)Figure 2-10. The relationship between transmembraneTPP gradients and MeNH3gradients.A 3.0 unit pH gradient (interior acidic) was created inEPC/cholesterol (55:45; mol:mol) LUVs (100 nm diameter)containing 300 mM citrate (pH 4.0) in the presence of 5 tMCCCP. Transmembrane TPP (s) and MeNH3 (1) gradients weredetermined as a function of the external TPP concentration aftera 30 mm incubation at 25°C as indicated in Methods.59volume to membrane volume ratio in the vesicle interior (see Section 1.5.2 ofIntroduction). As shown in Fig. 2-10, behavior corresponding to such effects canbe observed in 100 nm EPC LUVs exhibiting a I\pH of 3 units (inside acidic). Themeasured ratios [TPPi/[TPP] are consistently larger than the[MeNH3i/[MeN0ratios over a wide range of external TPP+ concentrations.However, the increased inside-outside ratios lead to a relatively small overestimateof AW (— 7 mV), indicating that the effects of TPP partition can be neglected underthe conditions observed in this work.2.5 DISCUSSIONThe results presented here give insight into factors influencing theencapsulation of compounds by lipid vesicles and into the validity of measurementsof transmembrane pH gradients and membrane potentials in liposomal systems.The major factors considered in the measurement of ion gradients were lipidcomposition, interior buffering capacity, osmotic gradients, the absolute magnitudeof the ApH, the measurement of tpH in vesicles with a basic interior and, withrespect to measuring the induced membrane potential, the influence of probepartitioning into the lipid bilayer. These aspects are discussed in turn.The lipid composition can strongly influence the measured ApH determined byweak bases such as MeNH3+. As illustrated here, liposomes composed ofsaturated, gel state, lipids can exhibit apparent i\pH values which are substantiallyless than the actual gradient. Clearly, some minimum level of permeability of theneutral form of the probe through the membrane is required to allow equilibrium tobe achieved within a reasonable time frame. As indicated earlier (see Introduction),amine uptake in response to zpH can be treated as a simple first order process,described by the relation IAH(t)11 = EAH(eq)](1et) where (AH(t)]1 is the interiorconcentration of the amine at time t and [AH(eq)] is the equilibrium interior amineconcentration at long incubation times. Uptake data of the type presented in Fig.2-3 can be utilized to obtain the rate constant (k) associated with the uptake60process. Thus, an approximate measure of the mimmum permeability coefficientfor MeNH3+required can be determined from the rate constant determined fromthe data of Fig. 2-3(b) for DPPC:cholesterol (55:45) at 20°C. This yields a value k =2 x i0 sec’. It is straightforward to show that for unilamellar vesicles, this rateconstant is related to the permeability coefficient P of the neutral form ofmethylamine via the relation P= kVoLHio/(AmKa) where V0 is the total externalaqueous volume, Am is the membrane area and Ka is the dissociation constant ofMeNH3(pK=lO.6). Assuming an area per phospholipid of 0.60 nm2, thisindicates a permeability coefficient for the neutral form of 7 x i0 cm/sec or largeris required. As indicated in Results, a brief incubation at an elevated temperature(e.g. 60°C) increases P for all the systems studied to the extent that equilibrium isachieved within 5 mm, without compromising the ipH.The accuracy of the zpH detected across LUV membranes by radiolabelledprobes such as MeNH3 is a sensitive function of both the interior bufferingcapacity of the vesicles and measures of the interior trapped volume. As detailedhere, interior citrate concentrations of 20 mM or higher are necessary to accuratelydetect pH gradients of 2 units or higher for[14C]-labelled MeNH3 (specific activity48 mCi/rn mol). The need for such high interior buffering capacities can bereduced by using probes with higher specific activity. In this regard, it is oftenconvenient to employ probes of t\1P with higher specific activity (such as TPP), todetect the iI induced in response to zpH as a more accurate measure of ipH. Asindicated here, the ability of TPP to partition into the lipid bilayer does introduce aslight overestimate of zW and therefore tpH. This is relatively minor under theconditions employed here, approximately 0.1 pH units. An important general pointis that for pH gradients of 3 units or more in 100 nm vesicle systems interiorcitrate buffering concentrations in excess — 20 mM are required in order that thepH gradient is not significantly dissipated by proton efflux required to form zW.Further, the presence of osmotic gradients which lead to vesicle shrinkage cancause significant underestimates of the tpH present.61The fourth point of discussion concerns the maximum pH gradients that canbe achieved. A major thrust of this work has concerned the accurate measurementof relatively large pH gradients of 3 units or more. The results presented here forEPC/cholesterol (55:45) indicate a maximum ApH of - 3.7 units, corresponding to aAW of 220 mV. An inability to generate larger pH gradients and induced membranepotentials is likely due to electrical breakdown of the bilayer (El-Mashak andTsong, 1985). The filtration centrifugation procedure is a sensitive technique formeasuring very large tpH values and results give zpH values close to those usingthe spin column approach.With regard to the measurement of tpH in vesicles with a basic interior, asuitable probe for use with the gel filtration procedure is not readily identified.Most of the probes investigated (acetic, benzoic and acetylsalicylic acids) are poorindicators of the ApH in such situations, apparently because they leak from thevesicles during the separation procedure. Alternatively, while mevalonic acid doesnot leak from the vesicles during separation, long equilibration times areinconvenient. More accurate procedures are provided by the filtrationcentrifugation method or by measuring the A’P induced in response to ApH.The final point of discussion concerns the measurement of the AW induced inresponse to ApH employing cationic probes such as TPP+, and the influence ofprobe partitioning into the membrane. As indicated above, this partitioning doesnot introduce large errors (approximately 0.11 pH units, or 7 mV). However it is ofinterest to compare the value of the partition coefficient which may be calculatedfrom this data with previous reports. Specifically, employing the formalismdeveloped by Cafiso (Cafiso and Hubbell, 1978a) and Rottenberg (Rottenberg,1984), a partition coefficient (= 1 x 10-6 cm can be calculated for TPP in thisEPC: cholesterol LUV system. This is in reasonable agreement with previous valuesof 6x107 cm in POPC MLVs (Altenbach and Seelig, 1985) and 4x106 cm forsonicated EPC vesicles (Flewelling and Hubbell, 1986a,b) using standardtechniques. In passing, the underestimate of AW reported by Nakazato al. (1988)62employing TPP in cholesterol-containing systems is likely due to a kinetic effect.The small amounts of the protonophore CCCP present in the experiment shown inFig. 2-4 ensure rapid development of the induced A’P.63CHAPTER 3. DRUG UPTAKE INTO LIPOSOMES IN RESPONSE TO pHGRADIENTS3.1 INTRODUCTIONThe technique of loading drugs into liposomes in response to a zpH (insideacidic) provides a dramatically improved method of drug entrapment compared tothe conventional procedure of preparing the liposomes in a solution of drug. Drugtrapping efficiencies approaching 100% can be readily achieved (Mayer et al.,1986b; Mayer et al., 1990b). Resulting drug:lipid ratios can be an order ofmagnitude higher than can be achieved using conventional procedures. The ipHloading procedure also markedly enhances drug retention properties (Mayer et al,1990a). These considerations are of considerable importance in liposomal drugdesign (Mayer, 1986b). A ApH-loaded liposomal formulation of the anti-cancerdrug doxorubicin (Fig. 3.1) is currently in advanced clinical trials (Creaven et al.,1990). The liposomally entrapped form of this drug shows reduced cardiac toxicitycompared to free drug, while drug efficacy is maintained or enhanced (Mayer et al.,1989; Mayer, 1990a; Balazsovits et al., 1990).Certain aspects of zpH-dependent drug loading into vesicles remain unclear.For example, several catecholamines, antineoplastic agents and other drugs havebeen shown to accumulate inside lipid vesicles in response to pH gradients acrossthe bilayer (Nichols and Deamer, 1980, Bally et al., 1988; Mayer et al., 1986b) andalso in response to transmembrane potassium gradients (Bally et al, 1988; Mayeret al., 1985c). Since transmembrane potentials generate a ApH and vice-versa, it isnot always clear which driving force is responsible for the uptake of a particularcompound (see de Kroon et al., 1989; Chakrabarti et aL, 1992; Mayer et al., 1985c;Mayer et al., 1986b; Bally et al., 1988). Further, the relationships between theamount of drugs accumulated into LUVs in response to a ApH, the internalbuffering capacity and the residual pH gradient have not been adequatelyexamined. For example, it has been noted that the transbilayer concentrationgradients of doxorubicin achieved considerably exceed the residual transbilayer64Figure 3-1. Structure of doxorubicin. The PKa àf theprimary amine is 8.6 at 37°C. -CH3 0NH2-65proton gradient. (Mayer et al., 1990b). It is of interest to develop a morequantitative understanding of these and other factors which determine the extentof drug encapsulation in liposomes in response to ipH, which is developed in thisChapter for doxorubicin.In addition, it is of interest to establish the generality of this “active” zpHdependent method of loading drugs into lipid vesicles employing a variety of ammocontaining drugs. This was performed for representative examples from severaldifferent drug classes including antineoplastics, local anaesthetics andantihistamines.3.2 MATERIALS AND METHODS3.2.1 MATERIALSDoxorubicin and epirubicin were obtained from Adria Laboratories of Canada,Mississauga, Ont. while mitoxantrone was purchased from Cyanamid Canada Inc.,Montreal, Que. Codeine and pethidine were supplied by Abbott Laboratories Ltd,Downsview, Ont. The radiolabels 17-14C]dopamine 17,814C ]- imipramine wereobtained from Amersham., while Ibenzene ring-3H]-chlorpromazine (23 Ci/mmol),[3H]-piocarpine, [4-3H1-propranolol (19 Ci / mmol), Icarboxyl-’4C]-lidocaine(48 mCi/mmol),14C1-methylamine (46 mCi/mmol) and [14C]-ethanolamine camefrom NEN. The Liposome Company, Inc. N.J. provided14C]-timolol.3.2.2 LIPID VESICLE PREPARATIONUnless otherwise stated all experiments were performed using eggphosphatidyicholine (EPC) or EPC:cholesterol (55:45 mol%) vesicles prepared in300 mM citrate pH 4.0 as described in Section 2.1, including five freeze-thaw cyclesin liquid nitrogen.3.2.3 DETERMINATION OF DOXORUBICIN UPTAKE LEVELSVesicle associated doxorubicin was determined (after separation of66unentrapped drug) by measuring the absorbance at 480 nm in a 1% Triton X-100solution, which resulted in vesicle disruption and drug release. At extremely lowdrug concentrations, internal doxorubicin was measured using[14C]-doxorubicin(0.25 mCi/mL) as a radiolabelled marker.3.2.4 DOXORUBICIN FLUORESCENCE STUDIESDoxorubicin fluorimetry was performed at 480 nm (emission 590 nm)employing a Perkin-Ehner LS-50 fluorimeter. When doxorubicin is incubated withlipid vesicles with an acidic interior, the fluorescence intensity decreases in a timedependent manner which correlates with drug uptake (see Results). These effectslikely reflect the accumulation of drug into the interior monolayer of the LUVs (seeDiscussion). LUVs which did not exhibit a transmembrane pH gradient had littleeffect on the fluorescence intensity of the doxorubicin, either at pH 4.0 or at pH 7.0(data not shown).3.2.5 [‘3C1-NMR STUDIESDoxorubicin was made up in solutions of 300 mM citrate, pH 4.0, or in 150mM NaC1, 20 mM HEPES, pH 7.0. EPC LUVs either with or without a pH gradientwere added to these solutions to achieve a final concentration of 12 mM lipid andthe final volume adjusted to 4.0 mLwith D20. The proton decoupled[‘3C]-NMRspectra were obtained by employing a Bruker MSL 200 spectrometer operating at50.3 MHz. Free induction decays corresponding to 62,000 transients was obtainedby using a 10 s 62° pulse, a 1 s interpulse delay and a 220 ppm sweep width. Anexponential multiplication corresponding to 5 Hz was applied to the free inductiondecay prior to Fourier transformation. The chemical shift is referenced to externalTMS.3.2.6 CRYO-ELECTRON MICROSCOPYCryo-electron microscopy was performed as previously described (Frederik etal., 1991). Samples containing the LUVs were placed on a 700 mesh gold EM grid67and the excess blotted off with filter paper. The grid was plunged into liquid ethanecooled to -190 °C and transferred to a Gatan 126 cold stage at liquid nitrogentemperatures using a Reichart Jung Universal Cryo-Fixation system. The samplewas visualised using a Zeiss EM1OC STEM.3.2.7 DRUG UPTAKE ‘SURVEY” EXPERIMENTSLarge unilamellar vesicles (1 mM lipid) were incubated with the drug (0.2 mM)in 300 mM NaCl, 20 mM HEPES pH 7.5 at 25°C unless otherwise stated. Theseratios were selected such that a redistribution similar to that seen withmethylamine would result in approximately 50% of the drug being accumulatedinside the vesicles. At various times up to 2 hours, aliquots (100 tL) of the mixturewere taken and vesicles separated from unentrapped drug by centrifugationthrough a 1 mL “minicolumn” of Sephadex G-50. Lipid and drug were quantifiedas described below.Using radiolabelled methylamine as an indicator of bpH, at least a 3.0 pH unitgradient across the vesicle membrane was measured in the absence of drug for allpreparations.3.2.8 OTHER ANALYTICAL PROCEDURESPilocarpine, chlorpromazine, timolol, proprariolol, imipramine, lidocaine,ethanolamine and dopamine were quantified using tracer quantities of the(3H]- or[14C]-radiolabel.Physostigmine was assayed by fluorescence spectroscopy employing an SLMAminco SPF 500C spectrofluorometer following solubilisation of the vesicles in 60%ethanol (v/v). The excitation and emission wavelengths used were 305 and 350 nmrespectively. Quinacrine, chloroquine, quinine and quinidine were also quantifiedfrom their fluorescence using excitation and emission wavelengths of 420 nm and505 nm; 335 nm and 375 nm; 335 nm and 365 nm; and 350 nm and 390 nmrespectively.Vinblastine and vincristine were assayed using UV spectroscopy from their68absorbances at 262 nm and 297 nm, respectively, following solubilisation of thevesicles in 80% ethanol. Codeine was also measured by UV spectroscopy at220 nm in this case after solubflisation in 40 mM octyl-13-D-glucopyranoside.Mitoxantrone was quantified from its absorbance at 670 nm following solubilisationof the vesicles in 2% Triton X-100.Diphenhydramine was assayed by gas-liquid chromatography using aHP 9850 gas chromatograph fitted with a Chromatographic Specialties DB-225(25% cyanopropylphenyl) capillary column. The helium carrier flow rate was1 mL min’ and detection was by flame ionization. An internal standard,methylpentadecanoate, was used to quantify diphenhydramine following itsextraction from the aqueous sample in diethyl ether and its separation from eggphosphatidylcholine by thin layer chromatography.Trarisbilayer pH gradients were quantified employing the weak basemethylamine(14C-labelled) as described previously (see Chapter 2).3.2.9 KINETIC ANALYSISDrug distributions in the presence of a transmembrane pH gradient aredescribed using a four compartment model as indicated in Fig. 3-2, based upon amodel developed by CafIso and Hubbell (1978a, 1978b) to describe the behavior (atlow concentrations) of several spin-labelled ESR probes of t\pH and iW’. Thecompartments will be denoted by the subscripts o (regions outside the vesicles), I(the vesicle interior), m0 (in the outer monolayer into which drug partitions), andm (the inner monolayer into which drug partitions). Under the assumption thatonly the neutral form of the drug traverses the bilayer, it follows thatd[D]tot PA0 m ([D]0 - [D11) (3-1)dt V0where [D10t0t is the total exterior concentration of drug (including charged,uncharged, free and membrane bound species), P is the permeability coefficient of69the neutral form, Am is the area of the membrane, V0 is the external aqueousvolume and [D10 and ID]1 are the concentrations of the neutral form of the drug.Assuming that the drug dissociation constant Ka is similar for the free andmembrane associated drug, and the membrane:water interface is at equilibrium,[Dim0 can be expressed as:[D]tot[Dim0 0 (3-2)1/K + [H]0 + (Vm0/)+ [H]0 (Vm0/)Kawhere K is the membrane:water partition coefficient for the charged form of thedrug, [H+10 is the exterior proton concentration and Vm is the volume of themembrane. Using a solubiity-diffusion model of permeation (Section 1.4.2), underthe assumption that [H]0>> Ka (since the external pH is 7 and the pKa ofdoxorubicin is 8.6), and that V0 >> Vm, it follows from Equation 3-2 thatd([D] tot) KKDAm a [D]0t0tdt dmVowhere D is the diffusion coefficient and dm is the width of the bilayer. This resultsin the relation[D(t)]otot = [D(eq)]0t0t ekt (34)where k is the rate constant associated with the process and k = KD/d AmKa/(Vo)[H+]o).As the interior drug concentration must obey the relation [D]0t0t ([Dl0tot -[D]1t0t)Vo/V,we obtain[D(t)]t0t = [D(eq)11t0t (1et) (3..5)70DH+ ‘SISSLIFigure 3-2 Model of the interaction ofphospholipid LUVs in the presence of a zpH.doxorubicin withOnly the uncharged form of the drug is able to translocate thebilayer. The volumes Vm1 and Vm0 into which the charged drugpartitions are not drawn to scale. For clarity, arrows indicatingequilibria are omitted.DH OWFrBufferVm,sb\OutsideI fpHInsIde -71where [D(eq)1 is the equilibrium interior drug concentration. Thus, according to thisanalysis, the rate constant of drug accumulation would be expected to be inverselydependent on the external hydrogen-ion concentration.3.2.10 EQUILIBRIUM ANALYSISA model of equilibrium transmembrane drug distributions in the presence ofa ApH requires calculation of the number of drug molecules in the outer and inneraqueous compartments (V0 and V, respectively), and the number of drugmolecules in the outer and inner monolayers of the membrane (Vmo and Vmj).Assuming that the membrane-water partition coefficient (K) of the charged form of—-the drug (dcnoted by the superscript ) is the same on the inside and outside of thevesicleN V N.V.K = mo 0 = mi 1 (3-6)V N V Nmo o mi iSimilarly, assuming that the drug dissociation constant (Ka) is the same inboth the aqueous and membrane phase:_________= (N0/V)[H1= (Nmi/Vmi)[Hji (Nmo/Vmo)[HjoKa— (37)N’V N ‘V N ‘V N +/\7i ‘ 1 0 ‘ 0 mi ‘ mi mo moBy definition,Nt0t= N1 + Nmi + N +Nmi (3-8)Ntot N++N ++N +N0 0 mo 0 mowhere N0t and N0t0t are the total number of internal and external drug moleculesrespectively.From relations 3-6 to 3-8 and making the well justified assumption thatsurface potentials due to the charged drug partitioning into the membrane aresmall (due to the high ionic strength of the interior buffer)72Ky KVI Jj+ )1 [Hj1 + + miNt0t Ka KaVi V1V0 [H10 + 7mo [Hi0 + 1 + ‘mO)N0t0t \T Ka Kao V0Where [Hi1>> [H10>> Ka (the conditions usually employed are pH = 4, pH= 7, pK — 8.6 for doxorubicin), and at low lipid concentrations, where Vm <<V0 SOthat the partition coefficient of the drug obeys the relation K << Vo/Vmo, it can bereadily shown that the ratio of vesicle internalized drug to external drug isapproximated by:N1t0t= (1 + KV/V1) IHi1— * (39)N0t0t (VO)( 1 + ‘mo ) IFI10V0We define K* as the apparent bulk partition coefficient of the drug into themembrane whereK*= KVmo/Vm (3-10)Eq. 3-9 simplifies torni.tot IH+1.I K*V(1 + m * (3-11)tot V. FH+1Jo 1 1 Jowhere [D]0t0t is the total free concentration of drug and [D]1t0t the effective vesicleassociated “concentration” of the drug (that is, the total amount of drug associatedwith the LUVs divided by the LUV volume).The proton concentration gradient [Hi1/[ 10can be readily determined bymeasuring the transmembrane distribution of radiolabelled methylamine (seeChapter 2), and the amount of accumulated drug ([D1t0t) can be determined in a73similar manner, allowing the ratio [D]1t0t/[D]ot to be calculated. In turn, thisallows the partition coefficient K* to be calculated using Eq. 3-11.At higher external drug or probe concentrations, the analysis by this model iscomplicated by the effects of drug accumulation on the pH gradient which causesthe accumulation. Obviously, if sufficient amounts of an acid or base redistribute,the pH on at least one side of the membrane (and therefore the pH gradient) will bealtered. These effects are discussed in more detail in Chapter 2 for methylamine.If drug accumulation is well “coupled” to the pH gradient (in the sense that nonspecific proton loss is minimal), both [DH]1 and [H]1 can be calculated for a givenset of conditions with a knowledge of drug partition coefficient. In particular, asdetailed in Chapter 2, the variation of internal pH expected for an internal buffer of300 mM citrate as a function of methylamine uptake can be reasonably modelledusing the equations of Section 2.1.3.3 RESULTS3.3.1 KINETICS OF DOXORUBICIN UPTAKE DETERMINED BY FLUOROMETRICTECHNIQUES.In previous studies of drug accumulation into LUVs with acidic interiors,assays of entrapment have involved the separation of entrapped drug andsubsequent assays of entrapped material (Mayer et aL, 1990b). For kinetic studies,particularly on systems exhibiting rapid uptake, a more convenient method ofmonitoring doxorubicin accumulation was to monitor doxorubicin fluorescence.This is illustrated in Fig. 3-3 for uptake into EPC:cholesterol (55:45) LUVs (100 nmdiameter) in response to a transmembrane pH gradient (pH 4.0, pH0t7.0). Asmall increase in fluorescence is observed on addition of LUVs to the doxorubicinsolution (200 M doxorubicin) which is followed by an exponential time-dependentdecrease in fluorescence to an equilibrium value. (The small initial fluorescenceincrease is not detectable in cases where the drug is accumulated more rapidly).The kinetics of fluorescence decrease correlate well with doxorubicin accumulation74o.E •_ 130_‘_,-.--D.-o./ 105150. /.100•.•::><.Ol “ .-30 0o 0 10 20 30 ii..Time (mm)Figure 3-3 Doxorubicin accumulation into LtJVs in responseto transmembrane pH gradientsEPC:cholesterol vesicles (100 nm diameter) with an interior 300mM citrate buffer, pH 4.0, were added tà a doxorubicin.containing solution (200 [IM final drug concentration) at pH 7.0and the amount of . vesicle assàciated doxorubicm wasdetermined as in Methods (•). Doxorubicin association withvesicles without a transmembrane pH gradient is also shown atpH 4 (D) and at pH 7 (tJ. Also shown is the fluorescence decreaseof doxorubicin incubated with vesicles in the absence of atransmembrane pH gradient (line). The temperature in thecuvette was 33.4°C, the lipid concentration was 1.5 mM andexternal pH was 7.0.75.assayed by the chromatographic separation procedure. Both data sets can befitted using the first-order kinetic analysis indicated in Methods, with a rateconstant (k) of 3x103.3.2 pH DEPENDENCE AND ACTIVATION ENERGY OF DOXORUBICINACCUMULATION INTO LUVsThe kinetic model developed in Methods predicts that the rate constant ofdoxorubicin uptake should be proportional to the external proton concentration forthe transbilayer movement of the neutral form. Thus a plot of log k vs the externalpH should be a straight line with a slope of unity. The rates of doxorubicin uptakeinto EPC and EPC:cholesterol LUVs were determined over the pH range pH0 = 5.7to pH0 = 8. As shown in Fig. 3-4(a), plots of the log of the rate constant derivedfrom this data show the expected linear dependence on pH, with slopes of 1.08 and0.96 for EPC and EPC:cholesterol LUVs respectively. This provides strong evidencefor the transbilayer movement of the neutral form of the drug.Rates of doxorubicin accumulation appear to be markedly temperaturedependent, given the drastically enhanced rate of uptake at 60° compared to 20°(Mayer et al., 1990b). Rate constants derived from uptake data over thetemperature range 5°C to 55°C demonstrate high activation energies as shown inthe Arrhenius plots of Fig. 3-4(b). It is interesting to note that different activationenergies are observed for different lipid systems, where Ea = 38 kcal/mol for theEPC:cholesterol (55:45) LUV system, whereas Ea = 28 kcal/mol for the EPCsystems.3.3.3 PARTITION COEFFICIENTS AND COUPLING CHARACTERISTICSASSOCIATED WITH DOXORUBICIN UPTAKEAs indicated in Methods, an ability of doxorubicin to partition into the lipidbilayer can result in inside/outside drug concentration ratios which significantlyexceed the inside/outside drug concentration ratios. In particular, a plot of[drug]/[drug]0vs [H]th/[H]OUt should reveal a straight line with a slope of76CFigure 3-4. Effects of external pH and temperature on thekinetics of doxorubicin accumulation.(A) EPC (.1 or EPC:cholesterol (.) vesicles prepared in 300 mMcitrate pH 4.0 were incubated with 200 i.tM doxorubicin at pH7.0 and the effects of pH on the rate constant of accumulationdetermined by monitoring the rate of fluorescence change as inFig. 3-3. The temperature was 21°C with EPC vesicles, or 53°Cwith EPC:cholesterol vesicles. (B) The effect of temperature onthe rate constant of doxorubicin accumulation were monitoredby fluorescence changes (closed symbols) or spin columns (opensymbols) for EPC (•) or EPC:cholesterol LUVs (•) as indicated inMethods.-- 770706.00 6.50 7.00 7.50 8.00External pH0—1-2-3-4—1-3-5-7-93.00 3.15 3.30 3.45l/T (IC1) (xl000)3.60K*Vm/Vi. Drug uptake into 100 urn EPC:cholesterol LUVs (2 mM lipid) exhibiting a3 unit pH gradient (pH 4 in, pH 7 out) containing 300 mM citrate was thereforeexamined over initial exterior doxorubicin concentrations up to 14 mM.Corresponding interior proton gradients were determined usingL14C-rnethylamine.A plot of the interior/exterior drug concentration ratios vs the residual protongradient is shown in Fig. 3-5(a), revealing a linear dependence with a slope of 24. A100 nm diameter LUV is expected to have Vm/Vj = 0.37, assuming a bilayerthickness of 5 nm (see Section 1.3.6), leading to an estimated doxorubicin partitioncoefficient K* of 65.It is of interest to compare this value of K* with that determined by moreclassical procedures. This requires an estimate of the amount of drug binding tothe vesicles with no applied ApH. A filter centrifugation technique was used toestimate the partition coefficient of doxorubicin for EPC:cholesterol LUVs at pH 4.0for a range of lipid concentrations and yielded an estimated K* of 74 forEPC:cholesterol vesicles (results not shown).The data of Fig. 3-5 can also be employed to determine how well doxorubicinuptake is coupled to the interior buffering capacity, which provides a measure ofthe non-specific leakage which may be induced by drug accumulation. Asindicated under Methods, for a well “coupled’ system, the values [H+]1 and[drug1 after drug uptake can be calculated from a knowledge of the partitioncoefficient K*, the initial drug concentration arid the other vesicle parameters asdescribed in Chapter 1. A plot of the theoretical doxorubicin and pH gradients vsthe observed values is shown in Fig. 3-5(b). It may be noted that the theoreticaland observed values agree reasonably well, suggesting that little non-specificleakage has occurred from these vesicles.3.3.4 [‘3C] NMR STUDIES ON DOXORUBICIN UPTAKEIt is straightforward to calculate that for a K* value of 64, over 95% of thedoxorubicin accumulated into 100 nm LUVs will be partitioned into the inner7800I00C><0C,0Figure 3-5. RelatIonship between the residual pHgradients and doxorubicin accumulationEquilibrium doxorubicin and methylamine concentrationgradients were determined by incubating EPC:cholesterolvesicles containing 300 mM citrate, pH 4.0 at 600 for 20 mmwith the indicated concentration of doxorubicin. (A) Therelationship between the equilibrium concentration gradient ofdoxorubicin and the residual pH gradient, as determined by theconcentration. gradient of methylamine. The slope of the linearregression (solid line) is 24. (B) The dotted lines were generatedusing the model presented in Methods, using the parametersdetermined in Chapter 1 and the apparent partition coefficientdetermined in (A) above. Experimental results presented areequilibrium doxorubicin (.) or methylamine (.) concentrationgradients.16001200BOO40000 20 40 60[MeNHJJ[MeNHJt4433.. ——221i..100-10 1500.Drug. Concentration79aL-b -— -.-—-c. LJJLdeILkL200 - i80 10 i40 020 000 8C 60 4C 20ppFigure 3-6. Effect of transmembrane pH gradients on the[1”C]-NMR spectra of vesicles incubated with doxorubicin.Natural abundance[13C1 NMR spectra of: a) free doxorubicin (4mM) at pH 7.0: b) EPC vesicles; c) doxorubicin encapsulated inEPC vesicles in response to a transmembrane pH gradient: d)doxorubicin incubated with EPC vesicles without a pH gradient(pH 4 inside and outside the vesicles): and e) as in (d). but at pH7 inside and out). Data collected with the assistance of K.F.Worig.80monolayer of the LUV bilayer. As a result, it would be expected that the motionalproperties of the accumulated drug would be restricted in comparison with theexternal free drug. This was tested by monitoring the [‘3C1 NMR behavior ofdoxorubicin before and after uptake. In order to detect the natural abundance[-3C] NMR spectrum arising from doxorubicin within a reasonable time frame,relatively high drug concentrations (4 mM) must be employed. In turn, thisrequires higher LUV concentrations (12 mM) in order that 90% or more of theavailable drug is accumulated in response to the EpH. Therefore, in these samplesthere Is a much higher level of background binding (in the absence of a zpH) thanin the previous results presented. Again, essentially all of the drug present isencapsulated in those vesicles with a transmembrane pH gradient.The I 13C] NMR spectra arising from free doxorubicin and EPC alone areshown in Figs 3-6a and 3-6b respectively. Additional resonances arise from theHEPES and citrate buffers. Interestingly, essentially no difference can be detectedbetween the[13C]-NMR spectra of EPC vesicles alone (3-6b) arid EPC vesicles whichhave accumulated doxorubicin in response to a bpH (3-6c). This is perhaps mostclear for those resonances between 100 and 200 ppm, and may indicate that thedoxorubicin is immobilized in the bilayer. Incubation of doxorubicin in thepresence of LUVs without a pH gradient (Fig 3-6d and e) reveals a broadening ofthe doxorubicin resonances, particularly at pH 7but this broadening does notresult in complete signal disappearance. It should be noted that the doxorubicinsample incubated at pH 7 (inside and out) was clumped and aggregated after 24hours of signal accumulation, which may account for some of the signal reductionof this sample.3.3.5 MORPHOLOGICAL FEATURES OF LUVs FOLLOWING DOXORUBICINACCUMULATIONThe experiments to this point show that doxorubicin can be accumulated intoLUVs to high levels in response to a ApH and that this behavior can be understood81Figure 3-7. Cryoelectron microscopy of doxorubicin-free anddoxorubicin loaded vesicles.100 nm diameter EPC:cholesterol vesicles with atransmembrane pH gradient were incubated at 600 for 15 mm inthe (a) absence or (b) presence of 200 iM doxorubicin as detailedin Methods. The bar represents 150 nm. Photographs takenwith the assistance of J.J. Wheeler.82on the basis of a model whereby the large majority of the encapsulated drug isassociated with the inner monolayer of the vesicles. This raises questionsconcerning the inner monolayer. For example, assuming a doxorubicin cross-sectional area of only 0.3 nm2 (half the area of PC), this would still correspond toan increase in surface area of 30%. The LUV morphology following doxorubicinuptake was investigated employing cryo-electron microscopy. An internal structurebisecting the length of the vesicles is commonly observed in vesicles which haveaccumulated doxorubicin, resulting in a “coffee-bean” appearance (Fig. 3-7(b)).This feature is absent in control vesicles which are not loaded with drug (Fig. 3-7(a)).3.3.6 DRUG UPTAKE “SURVEY”It is of interest to compare the tpH-dependent behavior of doxorubicin withother drugs. The ApH response of a variety of drugs examined under “standard”conditions (1 mM 100 nm diameter EPC vesicles, internal buffer 300 mM citrate,pH 4.0, 200 .tM drug, 20°) is summarized in Table 3-I. Four drug categories can bedefined on the basis of their uptake characteristics: first, drugs which show partialbut stable uptake; second, drugs which show partial uptake and then release;third, compounds which do not redistribute in response to a proton gradient; andfinally, compounds which show essentially complete accumulation. While thesecategories can be expected to encompass a continuous spectrum of uptakebehavior, in the majority of cases the assignment of a drug to a particular categorywas clear. Representative examples from these four classes of uptake will bediscussed in turn. In the absence of pH gradients (pH 4 inside and outside thevesicles, or pH 7 inside and outside the vesicles), only low background levels ofdrugs were associated with the lipid for all compounds tested (results not shown).3.3.7 CLASS 1. DRUGS WHICH EXHIBIT PARTIAL BUT STABLE UPTAKE.The uptake of timolol, an example of this class of drugs, is shown in Fig. 3-8.After 30 mm, timolol is taken up to about 100 nmoles/imo1e lipid (about 50% of83Table 3-I. Extent and stability of accumulation of various drugsby vesicles exhibiting a pH gradient (acidic interior)Drug Class Uptake at Uptake at15 mm (nmol 2 hr (nmol/!Amol lipid) /imol lipid)Mitoxantrone4 Antineoplastic 200 198Epirubicin 201 200Daunorubicin “ 200 204Doxorubicin “ 202 203Vincristine “ 178 130Vinblastine ‘I 175 1 127Lidocalne Local anaesthetics 87 87Chlorpromazine “ 98 96Dibucaine 194 176Propranolol Adrenergic antagonists 198 187Timolol It 95 97Quinidirie5 Antiarrythmic agents 203 74Pilocarpine Cholinergic agents <1 <1Physostigmine “ <2 <1Dopamine Biogenic amines 1802 177Serotonin “ 80 78Imipramine Antidepressant 182 188Diphenhydrainine6Antihistamine 1761 87Quinine Antimalarial 1481 81Chloroquine5 1041 88Quinacrine5 Antiprotozoan 2 71Codeine Analgesic <1 <1Footnotes: 1, maximum uptake taken at 5 mm; 2, maximum uptake takenat 30 mm; 3, maximum uptake taken at 90 mins; 4, data collected by L.C.L. Tal, 5,data collected by M.B. Bally, 6, data collected by C.P.S. Tilcock.84- 200C.o 150E— 100 I- .oE•Q 500 50 100Time (mm)Figure 3-8. Uptake of timolol by EPC vesicles.Timolol (200 M) was incubated with vesicles exhibiting a protongradient (pH 4.0 in/pH 7.5 out) (•): or with control vesicles withno pE-I (pH 4.0 in/pH 4.0 out)(’. or (pH 7.5 in/pH 7.5 out)(D).-- 850.0E—.4)CC0•0,C)0ECTime (mm)0.0,C)Figure 3-9.vesicles.Uptake of qulnldlne by EPC and EPC:cholesterolQuinidine (200 tM) was incubated with 1 mM EPC (a) orEPC:cholesterol (b) (55:45 mol:mol) 100 nm diameter vesicles.Drug uptake into the vesicles (0) and residual zpH () weredetermined as detailed in Methods..--=0.0,4,50 100Time (mm)•00.0EC)C•0C0•0,4)0ECEPC:CHOL200 o o150100500—032‘1050 10086available drug. This represents a 650 fold concentration gradient(interior/exterior), in reasonable agreement with the measured residual protongradient (2.7 pH units). Other drugs which exhibit partial but stable uptake arequinacrine, chiorpromazine, lidocaine, ethanolamine, serotonin and chioroquine.3.3.8 CLASS 2. DRUGS WHICH EXHIBIT PARTIAL UPTAKE AND SUBSEQUENTRELEASE.Qulnidme uptake into liposomes having a ApH is shown in Fig. 3-9. Initially,EPC vesicles rapidly accumulate virtually all available quinidine under thestandard conditions employed. This uptake is unstable and within 30 mm about50% of the drug has been released from the vesicles (Fig. 3-9(a)). This release isnot associated with any apparent structural change in the vesicles, such as fusionor aggregation, although the drug appears to cause a decay of the pH gradient.Prior to the addition of quinidine, 14C]-MeNH3distributions indicate a stablepH gradient of greater than 3 units, which rapidly dissipates upon addition of thedrug. It appears that the high internal quinidine concentrations generatedincrease the permeability of the membrane, leading to dissipation of the pHgradient. To test this hypothesis quinidine uptake was examined in vesiclescomposed of EPC:cholesterol. Cholesterol reduces the destabilizing effect ofquinidine on the bilayer as shown in Fig. 3-9(b). Following quinidine uptake thereis an initial decrease in the pH gradient (due to net proton binding by drugaccumulated within the vesicles) but the level of drug accumulation and theresidual pH gradient are then stable over the two hour incubation period. Otherdrugs which are released from egg phosphatidyicholine vesicles following uptakeare quinine, diphenhydramine, vinblastme and vincristine. The leakage rates varyconsiderably. Vesicles loaded with vincristine and vinbiastine lose about 27% ofinitially sequestered drug over two hours. As would be expected, this loss isassociated with a corresponding reduction in residual ipH as determined usingmethylamine. A similar decrease in the proton gradient is observed as quinine anddiphenhydramine are released from egg PC vesicles.873.3.9 CLASS 3. DRUGS WHICH EXHIBIT NO APPARENT RESPONSE.Drugs in this class include codeine, pilocarpine and physostigmine. The lackof uptake of these compounds may be explained if the compounds cause a majorIncrease in membrane permeability resulting in complete dissipation of the pHgradient. This is not the case, however, at least for physostigmine and codeine(results not shown). Under the conditions used to assess drug uptake (200 tMdrug) only a small decrease in measured tpH was observed. At least in the case ofphysostigmine, the neutral form of the drug may simply not be sufficientlypermeable that zpH accumulation can be detected.3.3.10 CLASS 4. DRUGS WHICH ARE TOTALLY ACCUMULATED.A final set of drugs showed complete accumulation in response to theimposed ApH, including doxorubicin, dau norubicin, epirubicin, propranolol,dopamine, dibucaine and imipramine. Accumulation of these drugs in EPCvesicles was generally rapid and complete, with little or no release observed overtwo hours. In the absence of a pH gradient (vesicle interior and exterior bothpH 4.0 or pH 7.5), only low levels of background binding are observed, representingless than 5% of the drug present (results not shown).3.3.11 PARTITION COEFFICIENTS AND COUPLING OF OTHER AMINESIt was of interest to ascertain whether the behavior of the different drugs seenin Table 3-I reflects different values of the partition coefficient K*. The equilibriumlevels of drug uptake and the residual pH gradients (indicated by methylaminedistributions) were therefore examined and the partition coefficients (K*) calculatedemploying the same procedures as used for doxorubicin. The partition coefficientsobtained range from 0 to> 200 and are summarized in Table 3-Il, along with theiroctanol:water partition coefficients. For compounds which showed sufficiently slowaccumulation, rates and activation energies of transport were also determined.Plots of the residual drug and pH gradients, as well as the theoretical behaviorexpected for a “well-coupled” system using these estimated K* values are shown in88Iaa1043 ‘o‘2 ‘a.’a’— I.0Ethariolamine1.a£2z4-ai0DopamineDibucaineEPC:cholesterol (55:45 mol%) vesicles prepared in 300 mMcitrate, pH 4.0 were incubated with the indicated drugs at, pH7.0 and equffibrium pH gradients (.) and drug gradients J) weredetermined as described -in Methods. The dotted lines aresimulations based on the models presented in Sections 2.1 and3.1, using the partition coefficients shown in Table 3-Il,assuming no non-specific proton leakage.Tim ol 0)C4-a0Lidocainea”• a”2 •a— —• a4 3: I0 2 4 ( I ISDrug Concentration20 2 4 4 I ICa0.a0a0a0.—2_2010Drug Concentrationr.——‘b’•’•__.-_____._-t\“1• “‘.-‘--.-.-----“-““-.___...,..•10.aCax0.aCaza.aCa0 00 2 4- 4 I ICDrug ConcentrationI mip ramin e0 2 4 I I 10Drug Concentration.-aa.aCa0 2 4 I 8 10-Drug Concentration•— — — —— 2‘••a0 Z 4- 1 I IC- Drug.ConcentratlonFIgure 3-10. Relationship between residual pH gradIent and- drug accumulation in vesicles with a transmembrane pHgradient. ---- 89Table 3-11 Partition Coefficients of Drugs ExaminedDrug Drug K* Octanol:WaterClass PartitionCoefficientLidocalne Local 0 0AnaestheticEthanolamine- 5 0Timolol 13-blocker 4 0Dopamine Biogenic 50 800AmineDoxorubicin Anti-cancer 64 13Imipramine Antidepressant 30 39,000Dibucaine Local 170 25,000Anaesthetic90Fig. 3-10. In general, drug uptake and residual pH appear to be reasonably wellpredicted by theory, indicating that the internal pH is coupled to drug uptake.Imipramine and dibucairie are exceptions, in that the uptake of these compoundsare poorly modelled by these calculations.3.4 DISCUSSION3.4.1 DOXORUBICIN ENTRAPMENT IN RESPONSE TO A zpHThe results presented for doxorubicin clarilr the mechanism of accumulationof the drug into LUVs with an acidic interior, demonstrate the high level of drugpartitioning into the inner monolayer in the loaded systems and explore theconsequences of high levels of drug accumulation into the membrane both inregard to permeability and morphological consequences.With regard to the mechanism of uptake, the kinetic studies demonstrating alinear dependence of the rate constant of accumulation on the external protonconcentration unambiguously establish that the neutral form of doxorubicin is thetransported species. This conclusion is in accord with Frezard and GamierSuillerot (1991) who examined doxorubicin movement in the absence oftransmembrane pH gradients. While consistent with the large body of literatureshowing that the neutral forms of weak acids and bases are generally themembrane permeable species (Rottenberg, 1979; Rottenberg, 1989), theseconclusions are important for two reasons. First, results from at least two groupshave been previously interpreted to implicate transbilayer diffusion of the charged(protonated) species of several weak bases (Bally et aL, 1988; Bally et a!., 1985; deKroon et al., 1989), in part because the ApH apparently could not account for theamount of internalized drugs observed. (Mayer et al., 1986b; Mayer et al., 1985c).Secondly, in combination with the observed activation energies, the kinetics of drugaccumulation and release properties can now be quantitatively described. Amongother applications, this has utility in the design of liposomal drug delivery systems,particularly with regard to loading and regulated release properties. For example,91under the loading conditions employed to obtain the data of Fig. 3-3, the half-timeof uptake is 3 mm; one can then obtain a generalized rate constant for doxorubicmnuptake ask (T, pH0, [PL]) = 2 x 10(pH7) [PL]exp 62.3 (1-307/T) 51 (3-12)where T is in °K and [PLI is the lipid concentration in mM. From this relation itcan be calculated that at 60°C, pHo = 7, uptake is extremely rapid, as k = 2x104(t112 4 sec), while at 20°C uptake is much slower, k = 1x104(t112 = 110 mm).It is interesting to note that after rapid entrapment at an elevatedtemperature, subsequent release at room temperature will be considerably slower,even though the effective lipid concentration for liposomally entrapped drug isconsiderably higher (—300 mM for a 100 nm LUV). Thus, a maximal rate constantof k = 3-8x105(t112 18 hr) would be expected for doxorubicin efflux fromEPC:cholesterol LUVs at 20°C, pH1 4.0 assuming that the pH gradient is dissipatedand that all drug which leaks out is immediately removed from the vicinity of theLUVs.The high activation energies associated with the transbilayer permeation ofthe neutral form of doxorubicin are also of interest. Briefly, previous studies on theApH dependent transport of phospholipids which are weak acids (Redelmeier, et al,1990; Eastman et aL, 1991) and (derivatized) peptides which are weak bases(Chakrabarti et aL, 1992) have demonstrated high activation energies, in the rangeof 30 kcal/mol. Similar results are shown here for doxorubicin. Activationenergies can be rationalised on the basis of the number of hydrogen bonds whichmust be broken and not reformed upon passing into the hydrocarbon (Lieb andStein, 1971), multiplied by the energy associated with hydrogen bond breaking (—3-5 kcal/mol), or on the basis of molecular size. It is straightforward to see howactivation energies in the range of 30 kcal/mol could be achieved for doxorubicingiven its large size and its high potential for hydrogen bond forming. It should be92noted, however, that the observed activation energies are likely overestimates. Thisis because the PKa of doxorubicin Is strongly temperature dependent (Frezard andGarnier-Suilerot, 1991). If the pK of doxorubicin decreases by 0.5 pH units overthe range 5° to 37° (Frezard and Garnier-Suilerot, 1991), the Ea determined wouldoverestimate the actual activation energy by approximately 7 kcal. The increase inEa observed in EPC:cholesterol vesicles implies that the presence of cholesterol inthe bilayer adds a considerable barrier to doxorubicin movement.The results of the equilibrium uptake studies show that the extremely highlevels of internalized doxorubicin achieved can be accounted for by the ability ofdoxorubicin to partition into the LUV membrane in a ApH-dependeril manner.Given the high ratio of membrane to aqueous volume of 100 nm vesicles (see Table1-TI of the Introduction) any significant membrane partition would result in druguptake levels considerably higher than those predicted if partitioning effects.It is somewhat surprising that the lipid bilayer can maintain a permeabilitybarrier when one monolayer is exposed to effective drug concentrations in therange of 300 mM. Such concentrations could lead to detergent effects, or totransbilayer packing differentials, either of which could be expected to causemembrane disruption. As indicated in Methods, these levels of drug associationwould be expected to lead to at least a 30% expansion of the inner monolayer,which is difficult to reconcile with normal LUV bilayer structure.Doxorubicin encapsulated in LUVs in response to pH gradients results in achange in vesicle morphology, as determined by cryo-electron microscopy. It is notcertain what the ‘linet’ running through the centre of vesicles which haveaccumulated doxorubicin (see Fig. 3-7) represents. The fact that this morphologyis not observed for LUVs experiencing a pH gradient in the absence of drugssuggests that it arises from accumulation of doxorubicin and its partition into theinner monolayer. As noted earlier, the inner monolayer is expected to be forced toundergo a significant increase in surface area, which may not be compatible withnormal LUV structure. The observed morphology may therefore result from excess93lipid “blebbed” from the inner monolayer to reduce the surface area of the innermonolayer. Alternatively, since some of these compounds may be above theirsolubifity in the internal media (Madden et al., 1990), drug precipitation may occurin addition to partitioning. These possibilities are presently being examined.It is interesting to note that the value of K* obtained for doxorubicin here ismuch larger than would be expected on the basis of its octanol/water partitioncoefficient (—13), but it is similar to the value determined for DMPC bilayers (—40)(see Burke and Tritton, 1985).The model described in Methods can also be used to predict drugdistributions for smaller or larger vesicles in a similar way. For spherical vesiclesat low lipid concentrations (where K*Vm/Vo <<1), Eq. 3-11 can be written as[DH]1= 1 + K* (r3 - (r-d)3)_____(3-13)[DH0] (r-d)3 [Hjowhere d is the bilayer thickness and r is the vesicle radius. Thus the drugconcentration gradient will exceed the pH gradient by a factor related to thegeometry of the vesicles for the range of LUV diameters commonly used (30-200nm). In addition, reduced vesicle radius also reduces the internal bufferingcapacity. Thus, the observations of Mayer et al., (1990b) that there was apparentlyno relationship between the residual pH gradient and the amount of doxorubicinaccumulation can be understood on the basis of equation 3-10 and the fact thatthe amount of internal buffer varies with vesicle size.3.4.2 zpH ACCUMULATION OF OTHER COMPOUNDSIt is clear from Table 3-Il that the ability of pharmaceutical agents toaccumulate within lipid vesicles exhibiting a proton gradient is a fairly generalphenomenon (Table 3-I). While the majority of drugs examined redistribute inresponse to a ApH it is interesting to note that the extent and stability of uptakevaries considerably. Timolol, lidocaine, chlorpromazine, serotonin, chloroquine94and quinacrine redistribute in rough agreement with the pH gradient, whiledoxorubicin, dopamine. dibucaine and imipramine, among others, attain gradientsconsiderably in excess of the proton gradient.The behavior of other drugs can also be rationalised on the basis of thepartitioning model employed. Drugs such as lidocaine or timolol have smallpartition coefficients and dissipate the pH gradient at approximately the sameexternal concentration as a comparable amount of the probe methylamine (seeChapter 2). The transmembrane concentration gradients therefore mirror theproton gradients. In contrast, drugs such as dopamine, imipramine or dibucainewhich have larger partition coefficients are accumulated to a much greater extent(at the same initial external concentration). Thus their apparent transbilayerconcentration gradients considerably exceed the residual proton gradient and theresidual pH gradient of the vesicles is dissipated at a lower external concentrationof drug than if the drug has a low partition coefficient.Finally, it is instructive to compare the behavior of the compounds studiedhere with that of compounds with extremely large partition coefficients, such ascertain phospholipids and fatty acids. It has been previously shown that thesemolecules redistribute in accordance with the proton gradient (Redelmeier et al.,1989). This is consistent with Eq. 3-9, where the observable parameters are thenumber of molecules on each side of the bilayer, that is:Nm = Vm1 [H] (3-14)Nm0If [H01 and IH1]>>Ka for these molecules, their partition coefficients aresufficiently high that the compounds reside entirely in the membrane and theirtransbilayer distributions again reflect ApH.95CHAPTER 4. RATES AND ACTIVATION ENERGIES OF PROTON FLUX ACROSSLIPID BILAYERS4.1 INTRODUCTIONAs mentioned in Chapter 1, reported values of the proton-hydroxidepermeability1net of unmodified lipid bilayers extend over a very wide range. Thisis mainly because the initial rate of proton flux is largely independent of thegradient which drives the flux (see Figure 4-1 and also Nichols and Deamer, 1989;Gutknecht, 1984; Perkins and Cafiso, 1986; Gutknecht, 1987b; Deamer, 1987;Perkins and Cafiso, 1 987b). Hence, there is an strong dependence of calculatednet values on the size of the proton gradient imposed. Much of the reporteddiscrepancies in the values reported for net can be accounted for by differences inthe pH, and the pH gradient, as well as the lipid composition employed in thevarious studies (Perkins and Cafiso, 1986). Proton flux also appears to be sensitiveto low levels of chloroform (Perkins and Cafiso, 1986), lipid oxidation (Perkins andCafiso, 1986) anaesthetics (Barchfeld and Deamer, 1988; Rarnes and Cafiso, 1990),and alcohols (Barchfeld and Dearner, 1985). Furthermore, there are reports thatthe direction of proton flux affects proton permeation (Norris and Powell, 1990),and that the buffering capacity of the lipids must be included in any analysis ofproton permeation which relies on the measurement of ApH (Grzesiek and Dencher,1986).The independence of initial flux with respect to the size of the imposed protongradient implicates some form of carrier mechanism in proton movement acrosslipid bilayers (Deamer, 1987). The physical nature of this carrier is unclear,though it appears to be uniquely available to protons (Deamer, 1987). In planarlipid bilayers, Gutknecht has produced considerable evidence that low levels offatty acid contaminants are responsible for the majority of proton flux (Gutknecht,1987b). In planar bilayers, the sensitivity of proton flux to albumin (which bindsfatty acids), to phioretin (which is believed to alter the membrane dipole potential)and to exogenous fatty acids suggests that most of the proton conductance is due96to low levels of fatty acids acting as protonophores (Gutknecht, 1 987b).In lipid vesicles, while electrogenic proton movement remains relativelyindependent of the size of the proton gradient, it is not altered by the addition ofphioretin or long chain fatty acids (Perkins and Cafiso, 1 987a). Apparently twoseparate phenomena are being examined in the two systems (Perkins and Cafiso,1987b). These arid other results seen in liposomal systems are not consistent withthe rate limiting step of proton flux being the simple diffusion of a charged species(Perkins and Cafiso, 1987b; Deamer, 1987). One probable mechanism of protonflux across vesicle bilayers involves coupling to water movement (see Nagle, 1987;Nichols arid Deamer, 1989 for reviews). However, there is presently no definitiveevidence to support a water-based mechanism. Structured water has not beenfound in membranes (Conrad and Strauss, 1985). Indeed, such evidence would bedifficult to find, given that the structured water chains or “wires” would be expectedto exist only transiently (Nagle, 1987). In addition, replacing H20 with D20 doesnot appear to affect proton flux across the bilayer (Perkins and Cafiso, 1 987b).Theoretically, current-voltage relationships could shed light on the mechanism ofproton permeation, (Nagle, 1987), but both linear and superlinear current-voltagerelations have been described for liposomal systems (Perkins and Cafiso, 1986;O’Shea et al., 1984; Deamer, 1987; Krishnamoorthy and Hinkle, 1984).Activation energies often help to elucidate underlying mechanisms. Relativelyfew studies have examined the effects of temperature on rates of proton movementexcept at the lipid phase transition temperature, where there is a discontinuity inArrhenius behavior (Bramhall, 1985; Elamrani and Blume, 1983). Kineticmeasurements of proton movement in lipid vesicles are most often made bymonitoring pH sensitive probes located in a poorly buffered vesicle interior. Thishas the practical disadvantages that the interpretation of results is complicated bythe titration of internal buffers and that non-electrogenic movement of protonscannot be distinguished from electrogenic flux (Cafiso and Hubbell, 1983).Additionally, these experiments may be poorly suited to situations in which the97temperature is varied, due to the extreme sensitivity of these experiments totemperature dependent changes in the pK of the reporter molecule. Since the pKaof many compounds decreases with increasing temperature, it is possible that thereported values of the activation energy of proton flux are overestimates.An alternative approach is to monitor the development of a membranepotential across vesicles in response to a ApH where protons are the only relativelypermeable ion (Cafiso and Hubbell, 1983). In this case, only electrogenic protonmovement is detected. Additionally, one can monitor the development of a pHgradient in response to a ‘I’, as utilised here.4.2 MATERIALS AND METHODS4.2.1 PREPARATION OF LIPID SAMPLESCholesterol, cholesterol sulfate and phloretin were incorporated (where used)in lipid samples by co-lyophilization from benzene:methanol (70:30 v/v). Palmiticacid and stearylamine were added to the liposome mixture from a concentratedstock solution in ethanol. Vesicle preparation was otherwise conducted asindicated in Chapter 2.1.4.2.2 GENERATION OF pH GRADIENTSUnencapsulated buffer was removed employing Sephadex 0-50 gel filtrationcolumns equilibrated with 3 mM of the internal buffer at the initial pH.Transmembrane pH gradients were imposed by diluting the vesicles 8-fold (to afinal lipid concentration of approximately 2 mM) in buffer solutions containing theappropriate ApH or zP probes and ionophores. The external buffer pH was notsignificantly altered upon the addition of the vesicles. Where employed, thepotassium ionophore valinomycin was used at a concentration of 0.5 pg per mollipid and the protonophore CCCP was used at a concentration of 10 tM. Theexternal buffer contained 0.5 Ci/mL of the appropriate membrane potential probe(TPP for negative interior potentials; SCN for positive interior potentials). Unless98otherwise stated, the external buffer was 100 mM K2S04,20 mM HEPES (pH 7.0)or 100 mM K2S04,20 mM MES (pH 6.0).4.2.3 GENERATION OF MEMBRANE POTENTIALSK diffusion potentials were created by entrapping 100 mM K2S04, 1 mMHEPES, pH 7.0 in vesicles as indicated above. The external buffer was thenexchanged for 100 mM Na2SO4,20 mM HEPES, pH 7.0 containing 0.1 mM K2S04using Sephadex 0-50 columns. The membrane potential was generated by the 10-fold dilution of the vesicles into an identical external medium which also contained0.5 ig valinomycin per jtmol lipid.4.2.4 DETERMINATION OF PROBE UPTAKE INTO LUVsVesicle associated probe was separated from free probe by gel filtrationchromatography employing 1 mL syringes which had been previously filled withSephadex 0-50 (50-150) equilibrated in the appropriate external buffer as indicatedin Section 2.1. Aliquots of vesicles (0.1 mL) were loaded and immediately eluted bycentrifugation at 500g for at least 3 mm. The associated timing errors areestimated to be as high as 30 sec.4.2.5 CALCULATION OF MEMBRANE POTENTIALS AND pH GRADIENTSQuantitation of vesicle associated probe was performed by liquid scintillationcounting and phospholipid analysis as previously described (Section 2.1;Redelmeier et al., 1989). Briefly, membrane potentials can be calculated bymembrane potential probe distributions according to:RTz’IJ(mV) = - log(C/C0) (4-1)Fwhere C1 and C0 represents interior and exterior TPP+ or SCN- concentrationsand R, T and F have the definitions used in Section 1.5. The pH gradients wereestimated in an analogous manner employing methylamine (see Chapter 1).994.3 THEORETICAL CONSIDERATIONSIf the membrane is described as a simple capacitor of capacitance C, andthere is an approximately linear current-voltage relationship, as determined byPerkins and Cafiso (1986), the kinetics of development of a membrane potential ofvoltage V in response to an applied ApH obey the relation Q = CV, where Q is thecharge on the capacitor (corresponding to the number of protons which havecrossed the membrane). From the relation I = dQ/dt = V/R, it follows thatdV = V (4-2)dt RCwhere R is the specific membrane resistance and C the specific membranecapacitance (approximately 1 iF/cm2).This has the solutionV= Veq (1- ekt (43)where k = 1 /RC, where k is the rate constant associated with the buildup ofthe voltage. The product of k, the first order rate constant and the membranecapacitance, yields the conductance. Thus, measures of EM’ following imposition ofa ipH were fitted to the equationA’P(t)1 = A’P(eq)i (1 - et) (44)where zW(t) is the accumulated probe at time t and A’IJ(eq) is the equilibriumamount of accumulated probe (determined by adding 10 tM CCCP to allowequilibration of protons). A 7 mV correction factor was included to account for thesmall overestimate of z’I’ due to the partition coefficient of TPP (see Figure 2-10,Section 2.5).The generation of a z\pH in response to an imposed AP is slowed by thepresence of internal buffers. A set of equations has been derived to describe the100effect of proton flux on the interior pH of vesicles containing buffers in the absenceof membrane potentials (Whitmarsh, 1987). Such equations could potentially beused to model the systems in Figure 4-5b, but the solution is difficult for a triproticbuffer such as citrate.4.4 RESULTS4.4.1 DETECTION OF PROTON FLUXThe movement of uncompensated charge across a lipid bilayer generates atransmembrane electrical potential. In Fig. 4-1, transmembrane pH gradients werecreated across the vesicles, and the subsequent development of a membranepotential can be measured by examining the transmembrane distributions of thelipophiic cations TPP (internal negative potential) or SCN- (internal positive) asindicated in Figure 1. In each case, a potential near 180 mV develops with a halftime of 12 mm in EPC vesicles at 25°C. The interior of these LUVs are sufficientlywell buffered that their internal pH does not change significantly due to protoninflux or efflux to establish the membrane potential (see Chapter 2).There are several lines of evidence that the rate of development of themeasured membrane potential in these systems is limited by proton movementrather than rates of equilibration of the TPP and SCN- probes. First, low (10 tM)concentrations of the proton ionophore CCCP dramatically increase the rate ofdevelopment of zSI’ and the addition of 100 mM ammonium acetate (which depletesthe transmembrane pH gradient) causes the rapid dissipation of the membranepotential (Figure 1). Second, TPP has a permeability coefficient of 10 to 10-8cms’ and an activation energy of about 20 kcal/mol in EPC vesicles (Flewellingand Hubbell, 1986a). These rates are 2-3 orders of magnitude faster than the ratesseen here, while the activation energy is about 8 kcal/mol higher than observedhere (see below). Third, if valinomycin is used to induce a membrane potential inresponse to a transmembrane pH gradient, TPP+ distributions reflect a 180 mV101>E>2180120600Figure 4-1. Generation of membrane potentials in responseto 3 unit acidic or basic pH gradients.100 nm EPC vesicles containing 300 mM citrate, pH 4.0 (panela) or 300 mM CHES pH 9.0 (panel b) were prepared as describedin Methods. A 3 unit zpH was imposed across the vesicles byincubation in 100 mM K2S04, 20 mM HEPES, pH 7.0containing 0.5 i.tCi/mL[3H]-TPP (panel a); or 100 mM K2S04,20 mM MES, pH 6.0 containing 0.5 iCi/mL[14C]-SCN (panelb). 10 1M CCCP was present in samples indicated by (s).Ammonium acetate (100 mM final concentration) at the externalpH was added at the time indicated by the arrows. Alsoindicated is the development of membrane potential inEPC:cholesterol systems (•). Solid lines represent fits to the datausing Equation 4-3.-1020 30 60- 90Time (mm)1200 30 60 90 120Time (mm)potential within 2 mm or less (results not shown). Finally, micromolarconcentrations of the ion-pairing agent TPB- do not increase the apparent rate ofpotential development monitored by TPP (results not shown).4.4.2 INFLUENCE OF LIPID COMPOSITION ON PROTON FLUXProton flux rates across the membranes of EPC LUVs at 25°C can be fitted asa first order processes with rate constants of 9x104s1, (half-time approximately12 mm) (Fig 4-la and 4-ib). This corresponds to an initial proton current of 100picoampere/cm,similar to that obtained by Perkins and Cafiso (1986) for largeunilamellar vesicles. The rate of development of the A’1’ is much slower inEPC:cholesterol (55:45 mol%) LUVs (t112 of 70 mm) (Figure 1).The effects of cholesterol content on proton flux in extruded POPC:cholesterolLUVs are indicated in Fig. 4-2. These results are compared with data determinedby Koenig et al (1991) for water movement through similar vesicles. This providesa direct comparison of proton versus water flux for the same lipid preparations.Increasing membrane cholesterol content from 0 to 40 mol % decreases water andproton permeation rates approximately 8-fold (Fig. 4-2).The effects of lipid composition and exogenously added compounds such asfatty acids, stearylamine and phioretin on the rates and activation energies ofproton flux are also indicated in Table 4-I. Briefly, the addition of fatty acid,stearylamine, or phloretin do not appear to influence the rate of electrogenic protonmovement greatly. Higher concentrations of amines and fatty acids diminish thetransmembrane ApH, as determined by the distribution of [14C1-methylamine(results not shown). Inclusion of cholesterol and cholesterol sulfate in the bilayerdecrease the rate of proton movement (Table 4-I).4.4.3 ACTIVATION ENERGIES ASSOCIATED WITH PROTON FLUXA central objective of this investigation was to examine the effects of lipidcomposition and other factors on the activation energy (Ea) of proton flux (Figure 4-3; Table 4-I). Activation energies of 11±2 kcal/mol (40-50 kJ/mol) can be1034.’le-03 5e+038e-04 4e+03.2 o6e-04 -. 3e+03E ‘c E1 5e-04 “S 2e÷034) A.. ‘. 4.’:2 20Cholesterol (mol%)Figure 4-2. ComparIson of the effects of cholesterol onproton and water flux.100 nm vesicles containing the indicated amounts of cholesterolwere prepared as in Figure 4-1(a). The rate constants of protonflux (•) are indicated as a function of cholesterol content at 25°C. Also indicated are comparable data for water flux reportedfor these vesicles by Koenig et al. (1990) (A)104-7 8U) A 7-8 1o a4-’o-9- 0..\S 53.00 3.25 3.50 3.75 4.00l/T x 1000 (K1)Figure 4-3 Comparison of the effects of temperature onproton and water flux100 nm POPC:chol (55:45 molô/o) extruded vesicles with a 3 unitipH were prepared as in Methods and the rate of development ofa membrane potential was determined as in Fig. 4-1, as afunction of temperature (•). Also indIcated are comparable datafor water flux reported for these vesicles by Koenig et al. (1990)105Table 4-I. Proton Flux Rates and Activation Energies.Lipid System zpH k Ea(in:out) (25°) (kcal/mol)EPC 4/7 9.4x10 12.8EPC 9/6 8.8x104 12.1EPC:chol(55:45) 4/7 1.3x104 11.3EPC:chol-Sulfate (55:45) 4/7 1.4x10 11.3EPC:chol (55:45) 6/9 2.5x104 12.4EPC:chol (55:45) 9/6 1.5x104 11.4EPC:chol:Phloretin(50:45:5) 4/7 2.1x104 12.6EPC:chol:(55:45) 6/9 2.6x104 11.+ 2 iM Palmitic AcidEPC:chol:(55:45) 9/6 2.6x104 11.8+ 2 1M StearlyamineDMPC:Chol (55:45) 4/7 1.1x1O 9.9DPPC:Chol (55:45) 4/7 1.Ox1O 10.1Transmembrane pH gradients (3 units) were imposedacross well buffered vesicles of the indicated compositionas indicated in Methods and the time course ofdevelopment of a AP was examined using [3H1-TPP(inside acidic) or[14C]-SCN (inside basic) as a function oftemperature.106calculated from Arrhenius plots of the rate constants associated with proton flux(Figure 4-3). The Ea of proton flux determined for POPC vesicles containingdifferent amounts of cholesterol (up to 40 mol%) ranged from 12 to 14 kcal/mol(data not shown). The Ea determined for water flux in these vesicles by Koenig et al(1991) was in the range of 11 to 16 kcal/mol.The activation energies determined were independent of the direction of theimposed pH gradient and the range of pH examined within the limits of accuracy ofthe technique (Table 4-I). While both cholesterol and cholesterol sulfate slow therate of proton flux, they do not markedly affect the activation energy.4.4.4 DETECTION OF PROTON FLUX IN RESPONSE TO MEMBRANEPOTENTIALSProton movements can also be detected by creating an initial electricalpotential by the addition of valinomycin to vesicles with a transmembrane Kgradient and measuring the subsequent development of a transmembrane pHgradient (Redelmeier et at, 1989). The time course of the development of the pHgradient is complicated by the effects of internal buffers, which slow thisequilibration. Early reports (Hope et al., 1985; Redelmeier, 1989) indicated thatthe resulting pH gradient does not appear to come to electrochemical equilibriumwithin the 4 hour time course of the experiments using EPC vesicles. In addition,the combination of valinomycin and CCCP resulted in the rapid (<5 mm) decay ofboth the tpH and zW due to an increase in the Na ion permeability of thesemembranes (Redelmeier et al., 1989). In contrast, the pH gradient and membranepotential are stable over several hours in the presence of both of these ionophores ifthe membrane is composed of EPC:cholesterol, rather than EPC alone (Fig.4-2).The Ea of proton movement measured by examining the initial rate ofaccumulation of j14C1-methylamine in response to an electrical potential is 11 kcal,approximately the same as that determined for the development an electricalpotential in response to a pH gradient (results not shown).107E0a0a.CI-.0ESA 1000-fold K gradient was imposed across 100 nmEPC:cholesterol (55:45 mol%) vesicles which contained 1 mMHEPES buffer pH 7.0. The AW was monitored using[3H1-TPP4, while the development of the ApH was measured using thedistribution of [14C1-methylamine (•). The open symbols refer tosamples which contain 10 iM (final concentration) CCCP.31801206000.00 0.50 1.00 1.50 2.000Time (hours)Generation and stability of pH gradients intransmembrane K+ gradients.Figure 4-4.response to108Figure 4-5 Vesicles with stable membrane potentials but nopH gradients and vice-versaVesicles were prepared in 100 mM K2S04 1 mM HEPES pH 7 0and incubated in 300 mM sucrose, 10 mM Tris, 10 mM HEPES,pH 7 0 contatnmg 1 iig valinomycm to mduce a membranepotential (panels (a) and (c)) or prepared in 300 mM citrate 10mM K2S04 pH 4 0 and incubated in 100 mM K2S04 20 mMHEPES pH 7 0 containing 1 .ig vahnomycm (panels (b) and (d)Membrane potential (.) and pH gradients I) were determined bythe distribution of TPP and methylamine, respectively. Panels(c) and (d) also contained 10 uM (final concentration) CCCP.Data for (a) and (c) used with permission of T E Redelmeier1093 18012060-- A0 -_0.00 0.50 1.00 1.50 2.00Time (hours)C— ——I I I-.2 ApH A’fr(units) (my)032 ApH(units) (my)32 ApH(units)032 ApH(units)180A4 120(my)60180120(mV)60U0 1 2 3Time (hours)180 D120.60-.------n - I I —000 0.50 1.00 1.50 2.0000Time (hours) Time (hours)1 2U34.4.5 LUVs EXHIBITING A AW OR pH, BUT NOT BOTHA final area of investigation is the development of model membrane systemswhich have a stable membrane potential, yet exhibit no detectable transmembranepH gradient and vice-versa. LUVs with these (non-equilibrium) properties can beused to examine the relative contributions of iI1 and ApH to a variety of transportprocesses, such as those described in Chapter 3.Tris, a weak base, dissipates induced transmembrane pH gradients whenpresent at 10 mM in the external buffer with LUVs exhibiting a K diffusionpotential (Figure 4-5a). The nearly 170 mV (inside negative) AW which isestablished in the presence of valinomycin is stable (Figure 4-Sa) and close to thatobserved in Fig 4-3. However, no AW-induced zpH can be detected by MeNH3distributions. This observation has been previously documented by Sone et al.,(1980), who also proposed that Tris is transported in the neutral form anddissipates the pH gradient upon reprotonation in the vesicle interior.Similarly, the presence of valinomycin/K+ for vesicles having an imposed pHgradient prevents the generation of a transmembrane electrical potential. If thesevesicles are sufficiently well buffered, the transmembrane pH gradient can bemaintained for several hours (Figure 4-5b). Even after 6 h, a gradient of more than2 pH units can be detected, with no detectable z’I’ (results not shown). In bothcases, the stability of the imposed gradients is expected to depend upon the netflux of proton equivalents. For example, about 225 nmol/iimol lipid of protonequivalents must move into the vesicles to negate the applied K gradient in Fig 4-5(a), and 900 nmol/jtmol lipid of proton equivalents are required to titrate theinterior buffer in Fig 4-5(b) from pH 4.0 to 7.0. Since the initial proton flux isabout 2x10’ mol/sec per imol lipid, (corresponding to a current 100 pA/cm2,assuming a surface area of 2000 cm2/ tmol lipid), the dissipation of these gradientscan be expected to take as long as 100 h.Low levels of the proton ionophore CCCP cause the time dependentdissipation of the potential and the pH gradient (Figure 4-Sc and 4-5d). CCCP also110leads to electrochemical equilibrium for all time points.4.5 DISCUSSIONThe uncompensated movement of protons across liposomal membranesgenerates a transmembrane potential, at a rate which depends upon the flux ofprotons or proton equivalents. As shown in Table 4-I, proton conductance asdetermined by lipophilic ion distributions is independent of pH (i.e. the size of theproton gradient), consistent with the observations of many previous workers thatthe value of the apparent proton permeability coefficient varies with theexperimental conditions. Furthermore, in contrast to the conclusions of Norris andPowell, (1990) (who examined the effect of proton movement on the internal pH oflipid vesicles), electrogenic flux of protons does not depend upon the direction ofthe pH gradient.The results presented here employ LUV systems, whereas electrogenic protonflux has been well characterized by CafIso and coworkers for smaller (1-2 unit) pHgradients around pH 7 in sonicated vesicles (SUVs) of about 25 nm diameter. Theyhave shown a linear current-voltage relationship (at least for 2 unittransmembrane pH gradients) and that there is little effect of phloretin and fattyacids on electrogenic proton flux in SUVs. It is possible, however, that proton fluxin SUVs is anomalous due to their extremely small size and highly curved nature.For example, proton movement in SUVs differs from that seen in planar lipidbilayers (Perkins and Cafiso, 1987b), and is about an order of magnitude slower insonicated vesicles than LUVs of identical composition (Perkins and Cafiso, 1986).However, the experiments shown here indicate that SUVs and LUVs exhibitqualitatively similar characteristics with respect to proton movement, as indicatedby the lack of effects of phioretin and fatty acids seen in Table 4-I, though theinitial proton current is larger in the LIJVs.With regard to the activation energies associated with proton flux, there havebeen three previous reports measuring the Ea of proton movement through111unmodified lipid bilayers which are not at the phase transition temperature. Onereport determined an Ea of proton flux of 9.6 kcal/mol (Grzesiek and Dencher,1986). In two other studies, the high activation energies reported (17-20 kcal) wereinterpreted as supporting the hypothesis that permeation of protons proceeds viahydrogen-bonded water molecules. However, reported activation energies of waterflux through bilayers are generally somewhat lower (8-15 kcal/mol) (Finkelstein,1987).The similarity of the activation energies of water and proton movement acrossbilayers suggests that the processes may be related. Indeed, the effects ofcholesterol content on proton and water flux also appear to correlate well (Fig 4-3b). This provides supportive, but not conclusive, evidence of a relationshipbetween water and proton flow.The lack of equilibration between zpH and z\’P previously reported (Redelmeieret at., 1989) for vesicles having transmembrane potassium gradients (in thepresence of valinomycin) was puzzling in light of the high permeability coefficient ofprotons, generally given in the range of i03 to i0 cm/s. Even in the presence ofinternal buffers (pK between 4 and 7) at up to 100 mM, an induced ApH inequilibrium with the imposed &-P would be expected within seconds using thesepermeability coefficients. In fact, an internal buffer as poor as 1 mM HEPES issufficient to retard the development of t\pH (Fig 4-3). These results emphasize theimportance of the experimental conditions employed to monitor the apparentpermeability of protons.Additionally, it should be noted that the shape of the ApH curve generated inresponse to A’I’ cannot be adequately modelled using an adaptation of theequations described in Whitmarsh (1987), describing the effect of internal bufferson the rate of change of pH on the internal pH of vesicles, regardless of the value ofk (the rate constant) chosen. This is possibly due to the non-specific movement ofother species (ions or buffers) during the long time course of these experiments,likely augmented by the presence of valinomycin.112The markedly different behavior of EPC and EPC:cholesterol vesicles in thepresence of the combination of valinomycin and CCCP is also surprising (Fig.4-4).The combination of valinomycin and CCCP cause the rapid dissipation of AM’ andApH in EPC vesicles by increasing membrane permeability to Na ions, but thepresence of cholesterol prevents this decay. It can be calculated that the increasein Na permeability required to destabilise the AM’ can be quite small: from the dataof Redelmeier et al. (1989), the Na permeability coefficient in the presence ofvalinomycm and CCCP is on the order of 10-12 cm/s in EPC LUVs.One of the ramifications of a significant proton flux is that vesiclepreparations with a AM’ will generally develop a ApH and vice-versa, as protonsreach their equilibrium. The results illustrated in Figure 4-5 demonstrate modelmembrane systems which exhibit a stable pH gradients and no observedmembrane potentials and vice-versa. External concentrations of Tris rapidlyredistribute across the bilayer in response to ApH in a similar manner as manyother weak bases. This prevents the formation of a large pH gradient. In ananalogous manner, the valinomycin/K+ combination prevents the development ofan induced AM’ in vesicles with an imposed ApH, while the high buffering capacityof the vesicle interior allows the pH gradient to be relatively stable. The systemsdemonstrated in Fig.4-5a and b are also useful in distinguishing AMJ-dependentfrom ApH-dependent transport processes and are also potentially useful tools fordetermining whether agents can act as protonophores, since an increase in netproton flux leads to dissipation of the electrical potential and pH gradient. As anexample, incubation of weakly basic drugs with a 180 mV A’P, but no detectableApH (the vesicle systems shown in Fig. 4-5a) results in much lower levels of drugaccumulation than seen in response to 3 unit ApH, consistent with drugaccumulation being in response to ApH, as opposed to AM’ (T. Redelmeier, 1989b).113CHAPTER 5. SUMMARIZING DISCUSSIONThis thesis has been focussed on the measurement of ApH and iM’ in LUVsystems, the development of realistic models to describe the uptake of these probesand lipophiic amines into LUVs exhibiting a zpH, and finally on the flux of protonsthrough bilayers.With regard to the first area, for LUVs with an acidic interior, determination ofthe equilibrium transbilayer distributions of radiolabelled methylamine employinga gel filtration procedure provides a reliable procedure to measure ApH providedthat transbilayer equilibration rates are sufficiently rapid, and that interiorbuffering capacities are sufficiently high. In situations where this accuracy iscompromised, equilibrium centrifugation techniques or techniques to measuremembrane potentials induced by the pH gradient provide straightforwardalternatives. The expected intra-vesicular pH can be reasonably predicted afterprobes are accumulated utilizing several parameters: vesicle diameter and volume;vesicle and probe concentrations; amount of encapsulated buffer and buffer pKa(s);and the initial internal and external pH.In the second set of studies, the uptake of the anti-cancer drug doxorubicininto large unilamellar vesicles (LUVs) exhibiting a transmembrane pH gradient(inside acidic) was investigated in considerable detail, using both kinetic andequilibrium approaches. It was shown that doxorubicin accumulation into thevesicles proceeds via permeation of the neutral form of the drug. The criticaldependence of translocation rates on pH and lipid composition suggest ways tomanipulate drug loading and release in a predictive manner. The extent of drugaccumulation at equilibrium was analysed in terms of a model which incorporatesdrug partitioning into the interior monolayer of the vesicles and also takes intoaccount the influence of internalized drug on the interior buffer. The extent ofaccumulation can be rationalised on the basis of the partition of the drug in thevesicle interior, with a partition coefficient of 64 estimated for EPC:cholesterolbilayers. For a 100 nm vesicle, this indicates that more than 95% of the114encapsulated drug is partitioned into the inner monolayer. The accumulation ofdoxorubicin appears to be well coupled to the pH gradient. The behavior of anumber of other compounds was examined in terms of the above model. Finally,the drug trapping efficiency and high drug:lipid ratios achieved using ApH liposomeloading are of considerable practical value.The conductance and activation energy of the transbilayer movement ofproton equivalents were examined for a variety of lipid systems with large (3 unit)pH gradients. Lipid vesicles (100 nm diameter) with well-buffered interiorsgenerated transmembrane potentials which could be monitored using the lipophilicmembrane potential probes TPP or SCN-. Development of the potential had ahalf-time of about 12 mm in EPC LUVs at 25°C, with an activation energy near 11kcal/mol. The rate and activation energy of proton movement were not affected bythe addition of phloretin, fatty acids, or stearylamine, or by direction of theimposed pH gradient. The incorporation of cholesterol and cholesterol sulfate intothe LUV bilayer decreased the rate of proton flux. Proton movements were alsoexamined in vesicles with transmembrane potassium diffusion potentials, bymonitoring the development of a ApH using methylamine. Finally, modelmembrane systems were developed which exhibited stable membrane potentialswithout induced pH gradients, or stable pH gradients without induced membranepotentials.There are several obvious directions for continued work in this area. Thetherapeutic benefits of liposomal encapsulation of drugs in response to a ApH, suchas those listed in Table 3-Il, provides a rich research area. Since it appears thatApH-loaded loaded liposomal doxorubicin has clinical advantages of reducedtoxicity over the free drug, similar benefits could be expected for doxorubicinanalogs such as epirubicin and daunorubicin.The design of novel ApH-loaded drugs is also simplified by the considerationsof Chapter 3. 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