A novel method for the efficient entrapment of calcium in large unilamellar phospholipid vesicles

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1 Bichimica et Biphysica Acta, 1025 (1990) Elsevier BBAMEM A nvel methd fr the efficient entrapment f calcium in large unilamellar phsphlipid vesicles Jeffrey A. Veir 1 and Pieter R. Cullis 1,2 1 Department f Bichemistry, University f British Clumbia, Vancuver and 2 TheCanadian Lipsme Cmpany Ltd., Nrth Vancuver (Canada) (Received 20 Nvember 1989) (Revised manuscript received 1 March 1990) Key wrds: Inphre A23187; Transmembrane ph gradient; ph gradient; Phsphatidylchline vesicle; Large unilamellar vesicle; Catin uptake A technique fr the efficient entrapment f high cncentratins f Ca z+ in large unilamellar phsphlipid vesicles (LUVs), using the carbxylic acid antibitic inphre A23187 (calcimycin) is demnstrated. It is shwn that rapid A23187-mediated entrapment f Ca 2+, crrespnding t essentially 100% sequestratin f the extravesicular catin may be achieved fr egg ylk phusphntidylchline LUVs (100 nm) in the presence f a transmembrane prtn gradient (acidic interir). Interir-exterir cncentratin catin gradients f ver 400-fld may be readily achieved, with interir Ca 2+ cncentratins in excess f 250 ram. It is shwn that the extent and efficiency f the A23187-mediated uptake prcess is affected by the intravesicular buffering capacity and the extravesicular Ca 2 + cncentratin in a manner that is cnsistent with a Ca2+-H + exchange prcess. In the absence f a ph gradient, r the presence f a reversed gradient (basic interir), nly backgrund levels f catin uptake are detected. The driving frce fr A23187-mediated uptake f Ca 2+ is shwn t depend n the intravesicular prtn pl rather than n a chelatin prcess. This prtcl prvides a nvel methd fr the efficient entrapment f high cncentratins f Ca 2+ and ther catins in phsphlipid vesicles. Intrductin We have previusly demnstrated that transmembrane electrchemical gradients (A~k and ApH) can result in the rapid and efficient accumulatin f a variety f lipphilic catins int the intravesicular space f large unilamellar phsphlipid vesicles (LUVs) [1-3]. This can result in extremely high interir cncentratins f certain lipphilic catinic drugs (fr example, dxrubicin), in cmbinatin with trapping efficiencies appraching 100%. In additin it has been shwn that asymmetric transbilayer distributins f simple acids that exhibit weak acid r base characteristics, as well as certain acidic phsphlipids, can be induced in LUVs experiencing a transmembrane ph gradient [4,5]. In this regard the lipphilic inphre antibitic A23187 (calcimycin) is capable f divalent catin transprt acrss bilgical and mdel membranes, with an verall electrneutral transprt arising frm a M 2+ fr 2H exchange [6-9]. In view f the previus findings, it was cnsidered that Ca 2+ transprt mediated by the carbxylic inphre shuld respnd t a transmembrahe ph gradient. In particular fr LUVs with an acidic interir, the presence f A23187 shuld allw the exchange f interir H ins fr exterir Ca 2+ ins, resulting in net Ca 2+ accumulatin. In this reprt we shw that A23187-mediated Ca 2+ accumulatin int LUVs can be driven by transmembrane ph gradients, resulting in trapping efficiencies f up t 100% and interir Ca 2 + cncentratins which are ver tw rders f magnitude greater than initial extravesicular Ca 2 cncentratins. The results are cnsistent with a Ca 2-2H exchange prcess. Abbreviatins: LUV, large unilamellar vesicle; EPC, egg phsphatidylchline; Hepes, 4-(2-hydrxyethyl)-l-piperazineethanesulphnic acid; EDTA, ethylenediaminetetraacetic acid. Crrespndence: P.R. Cullis, Department f Bichemistry, University f British Clumbia, 2146 Health Sciences Mall, Vancuver, B.C., V6T 1W5, Canada. Materials and Methds Egg ylk phsphatidylchline (EPC) was purchased frm Avanti Plar Lipids (Pelham, AL), and used withut further purificatin after verificatin f purity by thin-layer chrmatgraphy. The inphre A /90/$ Elsevier Science Publishers B.V. (Bimedical Divisin)

2 110 was btained frm Calbichem (Calgary) and [14C] methylamine and 45CAC12 were purchased frm New England Nuclear. All ther reagents were analytical grade r equivalent. Large unilamellar vesicles were prduced by extrusin [10] f frzen and thawed lipid dispersins thrugh 0.1 #m plycarbnate filters (Nuclepre), emplying an extrusin device (Lipex Bimembranes, Vancuver). Vesicles prepared in this manner had trapped vlumes f 1.5 #l/#ml phsphlipid, emplying [14C]inulin as an aqueus marker, and an average diameter f 90 nm as measured by quasi elastic light scattering and freezefracture electrn micrscpy [10]. Phsphlipid cncentratins were determined by analysis f lipid phsphrus as previusly described [11]. Vesicle size was rutinely determined by quasi elastic light scattering using a Nicmp Mdel 200 Laser Particle Sizer [12]. Transmembrane ph gradients were generated by initially preparing EPC LUVs in the presence f a lw ph buffer (300 mm citrate, ph 4.0, unless indicated therwise). Untrapped buffer was then remved by passing the LUVs thrugh a Sephadex G-50 clumn pre-equilibrated with the external neutral ph buffer (300 mm sucrse, 20 mm Hepes (ph 7.4) r 150 mm NaC1, 20 mm Hepes (ph 7.4) unless indicated therwise). The experimental prtcl invlved adding a defined amunt f A23187 (in a chlrfrm stck slutin) t a test tube. The slvent was remved under a stream f nitrgen fllwed by incubatin at lw pressure. The apprpriate extravesicular buffer system cntaining CaC12 (0.5 mm unless indicated therwise) and a trace amunt f 45Ca (1.0 #Ci/ml) was added t the test tube, and the dispersin was mixed thrughly. T this was added the vesicle suspensin (2.0 mm phsphlipid) at a time which marked the start f the experimental time curse. All experiments were cnducted at 25 C unless indicated therwise. Vesicle entrapped Ca 2+ was separated frm extravesicular Ca 2+ by gel filtratin. Aliquts (100 #1) were withdrawn at apprpriate times and extravesicular Ca 2+ remved by passage ver 1 ml Sephadex G-50 mini spin clumns, pre-equilibrated and washed in the apprpriate buffer as previusly described [10]. Within 20 s, the spin clumns were eluted by centrifugatin at 2500 g fr three minutes. The LUVs and assciated Ca 2 were subsequently analyzed fr 45Ca by liquid scintillatin cunting emplying a Packard 2000 CA liquid scintillatin cunter, and fr lipid phsphrus [11]. All data shwn are representative f multiple experiments (n > 3). All experiments were perfrmed abve the gel t liquid crystalline transitin temperature f the lipid. The magnitude f the transmembrane ph gradient was determined by measurement f the equilibrium transmembrane distributin f the weak base [14C]- methylamine. Methylamine was added t the LUVs (typically 2.0 mm phsphlipid) t achieve a cncentratin f 1/~M cntaining 1 #Ci/ml f [14C]methylamine. At apprpriate time intervals aliquts (100 #1) were withdrawn, and untrapped prbe remved emplying 1 ml Sephadex G-50 mini spin clumns as previusly described [10]. The trapped prbe was determined by liquid scintillatin cunting, and the phsphlipid cncentratin by phsphate assay [11]. Transmembrane ph gradients were calculated accrding t the relatinship ApH = lg[meam]i/meam] [13]. Results Initial transprt experiments were designed with the aim f establishing whether a transmembrane ph gradient culd drive A23187-mediated uptake f Ca 2+. As shwn in Fig. 1, the LUVs experiencing a transmembrahe ph gradient (ph 7.4 utside, ph 4.0 inside), exhibit a remarkable ability t accumulate Ca 2+ in the presence f A23187 (50 #g/ml). Within tw minutes Ca 2 is laded t interir levels f 220 nml Ca2+/#ml phsphlipid, r 148 mm (given a trapped vlume f 1.5 #l/#ml phsphlipid [10]), cmpared t an initial extravesicular Ca 2+ cncentratin f 0.5 mm. This crrespnds t ver 9996 entrapment f extravesicular Ca 2+. Crrespnding experiments cnducted n vesicle systems experiencing n ph gradients, ph 4.0 inside and utside (see Fig. 1), r ph 7.4 inside and utside (see belw), shwed nly backgrund levels f Ca 2 uptake, typically less than 5 nml Ca2+/#ml phsphlipid. Experiments cnducted n vesicular systems experiencing a reversed ph gradient (basic interir) again resulted in nly backgrund Ca 2 uptake (<2 nml Ca2+//~ml phsphlipid, results nt shwn). The data f Fig. 1 als shws that after the initial rapid accumulatin f Ca 2+, a time-dependent leakage f the entrapped catin ccurred. As much as 60% f the laded Ca 2 + was released frm the LUVs within tw hurs. In this regard, the specificity f A23187 fr Na + is much less than that f Ca 2 [14]. Hwever, the relatively high extravesicular Na + cncentratin (150 mm) with respect t Ca 2+ (0.5 mm) initially present in the extravesicular space culd cnceivably result in significant A23187-mediated Na + uptake. This wuld serve t cntribute t dissipatin f the internal prtn pl via Na+-H + exchange. Tw ptential methds f reducing this dissipatin invlve decreasing the cncentratin f the A23187, r remving the extravesicular Na +. Fig. 1 shws the effect f decreasing the inphre cncentratin t 0.1 #g/ml n the rate f Ca 2 lading int the LUVs. The results indicate that the rate f uptake was substantially slwer at lwer inphre cncentratins. Hwever, greater than 99% entrapment was still achievable, but ver a lnger perid f time. N leakage at the lwer inphre cncentratin was

3 111 ~, 250 J 200 E 150 & N ',-, 2.5. m t-. m-----m m <I ~ A,-F---A, -& 20.,: 6 8,6 12 Time (minutee) Fig. 1. Time-curse f the accumulatin f Ca 2+ int EPC LUVs (100 urn) experiencing a transmembrane ph gradient and 0.5 mm external Ca 2+ in the presence f 50 #g/ml (e) and 0.1 #g/ml () A EPC LUVs (2.0 mm phsphlipid) were prepared in 300 mm citrate buffer (ph 4.0) and the untrapped (exterir) buffer exchanged fr 150 mm Na2SO 4 and 20 mm Hepes (ph 7.4). Ca 2+ uptake was quantitated as described in Materials and Methds. The slid triangle indicates inphre mediated catin uptake in the absence f a transmembrane ph gradient (ph 4.0 in and ut), as described in Materials and Methds. bserved ver a perid f several hurs, in cntrast t the situatin at high A23187 levels. Data supprting a direct rle f extravesicular Na + in dissipatin f the transmembrane ph gradient is shwn in Fig. 2. A Na+-free vesicle system was used, in which the external Na2SO 4 was replaced with sucrse (300 mm) and the internal and external ph was adjusted with arginine (free base). As shwn in Fig. 2 the Na+-free LUVs exhibited n significant Ca 2+ leakage after the initial uptake even at high A23187 levels. These results are cnsistent with an ability f the inphre t transprt Na + as well as Ca 2+, which cntributes t the dissipatin f the transmembrane ph gradi- ~" E 200- :L E 150- i O4 m~m m--- m/m e ~ O ~ 0,A 20 4' 6 8' 16,20 Time (minutes) Fig. 2. Time-dependent A23187-mediated uptake f Ca 2+ int EPC LUVs (100 nm) experiencing a transmembrane ph gradient (exterir ph 7.4, interir ph 4.0), in the presence f 50 #g/ml A23187 and 0.5 mm external Ca 2+, fr LUVs (2.0 mm pbsphlipid) cntaining Na + in the external medium (150 mm Na2S4, 20 mm Hepes) (e) and LUVs that cntain n Na + in the external medium (300 mm sucrse, 20 mm Hepes) (m) (see Materials and Methds fr details). The slid triangle indicates Ca 2+ uptake in the absence f a transmembrane ph gradient (ph 4.0 in and ut) fr LUVs in the absence f Na ' 4: d 8'0 loo 1~ Time (minutes) Fig. 3. Magnitude f the transmembrane ph gradient as measured by [14C]methylamine (see Materials and Methds), fr EPC LUVs (100 nm) at a final phsphlipid cncentratin f 2.0 mm, in the absence f inphre (O); EPC LUVs in the presence f A23187 (1.0/~g/ml) and 0.5 mm external Ca 2+ fr LUVs cntaining Na in the external medium (150 mm Na2SO4, 20 mm Hepes) (A), and LUVs cntaining n Na + in the external medium (300 mm sucrse, 20 mm Hepes) (m). ent. The Na+-free LUVs in the absence f a transmembrane ph gradient, r in the presence f a reverse ph gradient (basic interir) indicated n significant Ca 2 uptake (see Fig. 2). In rder t cnfirm that exterir Na + resulted in the dissipatin f the transmembrane ph gradient, the ph prbe [14C]methylamine was used t measure the residual ph gradient acrss the Na+-free and Na+-cntain - ing LUVs after Ca 2 uptake (see Materials and Methds). At high inphre cncentratins (50 #g/ml) after tw hurs n residual transmembrane ph gradient was detectable acrss the LUVs with exterir Na, whereas a ph gradient f apprximately 2.0 units was measured acrss the Na free vesicle system. The relatinship between the magnitude f the residual transmembrane ph gradient, and A23187-mediated Ca 2 uptake was als investigated at lw inphre cncentratins (1.0 #g/ml). As indicated in Fig. 3 fr EPC LVUs in the absence f inphre (Na+-cntaining LUVs), a stable transmembrane ph gradient f 2.8 units was bserved. In the absence f inphre similar values were als btained using the Na+-free LUVs (results nt shwn). In the presence f inphre (1.0 /~g/ml) the magnitude f the ph gradient fr the Na free LUVs was 2.2 units, and remained effectively cnstant ver tw hurs. The initialy decrease in ph is due t the rapid inphre mediated Ca2+-H+ exchange acrss the bilayer, where 100% entrapment ccurs within tw minutes. In the case f the Na+-rich LUVs, hwever, the magnitude f the ph gradient was determined t be 1.9 after tw minutes, and further decreased as a functin f time. The latter bservatin is likely due t Na+-H + inphre mediated exchange. The effects f the initial extravesicular Ca 2 cncentratin n A23187-mediated catin uptake and the crrespnding trapping efficiencies are depicted in Fig.

4 ~ E 250- E 200-.~ 15. ~ ~--0- j e 501 N O l '0,, /~ f2 f6 2 External C 2+ cncentratin (ram) Fig. 4. The effect f initial extravesicular Ca 2+ cncentratin n A23187 (10 #g/ml) mediated Ca 2+ uptake (e) and percentage trapping efficiency (), int EPC LUVs (100 nm) experiencing a transmembrane ph gradient (300 mm sucrse, 20 mm Hepes (ph 7.4) in the external medium and 300 mm citrate (ph 4.0) in the internal medium). Measurements were made at tw hurs, and the phsphlipid cncentratin was 2.0 ram. I 6 ~ "u -60 ~ -2 "~? 250 / O0 "6 / 200 // " " ' ~ " 80 *=,50.60 c 8 / 5,,~ -20 ~ O, I 0 ~ , Internal citrate cncentratin (ram) Fig. 5. The effect f internal citrate cncentratin n Ca 2+ uptake ( ); and percentage trapping efficiency () int EPC LUVs (100 nm) experiencing a transmembrane ph gradient (300 mm sucrse, 20 mm Hepes (ph 7.4) in the external medium and 300 mm citrate (ph 4.0) in the internal medium), in the presence f A23187 (10 #g/ml) and 0.5 mm external Ca 2+. Measurements were made at tw hurs, and the phsphlipid cncentratin was 2.0 ram. 4. All data reprted were cllected after tw hurs. At lw initial extravesicular Ca 2+ cncentratins ( mm), greater than 99% trapping efficiency was bserved at the phsphlipid cncentratin used (2.0 mm). This indicated essentially a cmplete sequestratin f the external Ca 2+ pl. This crrespnded t entrapped Ca 2 levels f between 2.3 and 200 nml per #ml phsphlipid. Under these experimental cnditins a residual transmembrane ph gradient was detected at the cmpletin f the time curse (tw hurs) (results nt shwn). At higher extravesicular Ca 2 cncentratins ( mm), hwever, the trapping efficiency decreased significantly, and catin uptake levels plateau at abut 350 nml Ca2+/gml phsphlipid. N residual transmembrane ph gradient was measured under these experimental cnditins, which limits further catin uptake. Deleers and Malaisse bserved similar enhancement in 4SCa exchange diffusin acrss multilamellar lipsmal membranes experiencing n ph gradient at increasing cncentratins f external calcium [151. The ph-dependent uptake f Ca 2+ mediated by A23187 was further studied t determine the influence f internal buffering capacity. The effects f changing the intravesicular buffering capacity frm 10 mm t 500 mm citrate at a cnstant extravesicular Ca 2+ cncentratin (0.5 mm), n Ca 2+ uptake and the trapping efficiency are shwn in Fig. 5. All data reprted were cllected after tw hurs. The sucrse cncentratin in the extravesicular buffer was als adjusted t be issmtic with respect t the vesicle interir. The data f Fig. 5 indicated that a lw citrate cncentratins ( < 300 mm), the buffering capacity limited A23187-mediated catin uptake, and trapping efficiencies were well belw 100%. Furthermre, n residual transmembrane ph gradient was measured at these lw citrate cncentratins at the cnclusin f the experimental time curse. At higher internal citrate cncentratins, hwever, the available extravesicular Ca 2 limited uptake, and greater than 99% trapping efficiencies were achieved. A residual transmembrane ph gradient f apprximately tw units was still present at the higher citrate cncentratins (> 300 mm), after maximal uptake levels had been attained (results nt shwn). Furthermre, under cnditins where external calcium was in excess (> 2.0 mm), maximum A23187-mediated Ca 2+ uptake was prprtinal t the intravesicular buffering capacity (results nt shwn). The effect f temperature n A23187-mediated Ca 2 uptake in the presence f a transmembrane ph gradient is shwn in Fig. 6. The inphre cncentratin was decreased t 0.05 gg/ml, in rder t fllw the kinetics. The results shw a strng temperature dependence. Despite the relatively lw inphre cncentratin used (0.05 #g/ml) maximal catin entrapment was achieved 200t! ~ 1 < ~ Q -, -,-, ( Time (minutes) Fig. 6. The effect f temperature n Ca 2+ accumulatin int EPC LUVs (100 nm) experiencing a transmembrane ph gradient (300 mm sucrse (ph 7.4) in the external medium and 300 mm citrate (ph 4.0) in the internal medium), and in the presence f A23187 (0.05 #g/ml) and Ca 2+ (0.5 mm), at 15 C (); 26 C (A); 36 C (r~), 45C (); and 60 C (A).

5 113 within 90 min at 60 C. The temperature dependence f the initial rate fr transprt reflects an Arrhenius activatin energy f 12.8 kcal/ml. Thus, even with a significant reductin in the amunt f inphre, which culd be f imprtance in in viv applicatins, high catin uptake levels may still be rapidly achieved at elevated temperatures. At the lwer temperatures used (< 36 C) 100% entrapment was nt achieved within the tw hur time curse. In this regard, it shuld be nted that under these experimental cnditins there are nly fur A23187 mlecules per vesicle. In rder t attain high internal Ca 2+ levels (apprx. 150 ram) bserved in the abve experiments at 45 and 60 C, each inphre must translcate a large number f Ca 2+ ins. Fr example, at an inphre cncentratin f 0.05 #g/ml, an initial Ca 2+ cncentratin f 0.5 mm and 2.0 mm LUV phsphlipid, it may be readily calculated that each inphre mlecule has t underg apprximately translcating events in rder t achieve 100% entrapment. Mauk and Gamble (1979) [16] have previusly demnstrated that (in the absence f a transmembrane ph gradient) in the presence f a suitable chelatr (fr example nitriltriacetic acid r ethylenediaminetetraacetic acid (EDTA)) in the intravesicular space, an increase in the A23187-mediated uptake f catins can be bserved. The chelatr prvides a strng driving frce fr mediated catin uptake. In rder t distinguish between entrapment by chelatin and catin uptake due t membrane prtn gradients detailed abve, experiments were cnducted in the absence f a ph gradient (ph 7.4 in and ut) and the presence f 300 mm citrate (ph 7.4) r 300 mm EDTA (ph 7.4) in the vesicle interir. In each case the extravesicular buffer was 300 mm sucrse, 20 mm Hepes (ph 7.4). The data f Fig. 7 illustrate that citrate, unlike EDTA, lacks the ability t act as a driving frce fr the A23187-mediated '~" E 200- I) E 150- & "~ I00- c~ ,-D~O, s 8',60 12 Tlml (minutes) Fig. 7. The effect f internal buffer n A23187 (10 pg/ml) mediated accumulatin f Ca 2+ in EPC LUVs (100 nm) in the absence f transmembrane ph gradient. 300 mm citrate (ph 7.4) (<3), and 300 mm ethylenediaminetetraacetic acid (ph 7.4) (O). In bth cases the external buffer was 300 mm sucrse, 20 ram Hepes (ph 7.4) and the phsphlipid cncentratin 2.0 ram. lading f Ca 2 + int LUVs. This suggests that citrate is nt a sufficiently strng chelating agent t drive A23187-mediated catin uptake. As a final pint it is imprtant t nte that thrughut all the abve experiments n change in light scattering, (measured at 450 nm), r increase in vesicle size (as detected by quasi elastic light scattering) was detected fr the vesicle suspensins used. This indicates that n significant vesicle-vesicle fusin r aggregatin was ccurring during the time curse f the experiments. Furthermre, n lipid degradatin was apparent as detected by thin-layer chrmatgraphy [17]. This eliminates the pssibility f fatty acid r lysphsphatidylchline break dwn prducts acting as an additinal surce f inphretic activity. Discussin The results f this investigatin clearly demnstrate that carbxylic inphretic mlecules such as A23187 tgether with LUVs experiencing a transmembrane ph gradient (acidic interir) may be used t achieve a rapid and efficient entrapment f Ca 2+. Uptake levels crrespnding t essentially cmplete sequestratin f the extravesicular catin were readily attainable. Under the cnditins emplyed here, this can result in intravesicular Ca 2 cncentratins in excess f ver 250 mm, equivalent t an inside-utside cncentratin gradient f mre than 400-fld. Furthermre, the prtcl may be easily ptimized t achieve even higher levels f catin encapsulatin. A23187 facilitates diffusin f catins acrss the lipid bilayer by a carrier-mediated mechanism. The A23187-Ca 2+ cmplex has been clsely characterized, and it is generally accepted that Ca 2 crsses the membrane cmplexed with tw mlecules f A23187 [6-9]. The carbxylic grup f the inphre participates in the electrically neutral exchange f catins fr prtns. At ph values abve the pk a f the inphre (pk a) = 6.7) [18], the prtns dissciate frm the carbxyl grup, and frmatin f the catin carbxylate is favured. At ph values belw the pka, the carbxylate will reprtnate, resulting in the release f the catin. This is supprted by the bservatins f Liu and Herman (1978) [19] wh shwed that essentially n cmplexing f Ca 2 by A23187 ccurs belw ph 4.0, whereas binding reaches a maximum at apprximately ph 7.4 [20]. The mechanism f A23187-mediated Ca 2+ accumulatin in respnse t a transmembrane ph gradient is directly related t the mde f actin f the inphre. LUVs experiencing a transmembrane ph gradient (acidic interir), with the extravesicular aqueus phase at a ph f 7.4, favurs frmatin f a 2 : 1 electrneutral cmplex f the aninic frm f the inphre with the catin. The acidic vesicle interir which pssesses a

6 114 high cncentratin f prtns, facilitates prtnatin f the inphre and catin release. Such a mechanism f actin is cnsistent with the experimental results f this study, whereby increasing the initial buffering capacity, r the magnitude f the transmembrane ph gradient, results in an increase in the level f inphre-mediated catin uptake. In each case the internal prtn pl is the imprtant variable. In additin the results clearly demnstrate that the predminant driving frce fr enhanced A23187-mediated catin entrapment is the presence f the transmembrane ph gradient. Chelatin is nt a majr factr in the abve experiments. This is because citrate is nt a sufficiently strng chelatr (see Fig. 7). It is likely, hwever, that the limited slubility f calcium citrate leads t frmatin f a precipitate in the LUV interir. It is f interest t cmpare the transmembrane ph gradient prcedure fr lading Ca 2 int LUVs with ther prcedures. First passive entrapment during lipsme frmatin results in lw trapping efficiencies and the maximum interir cncentratins is ften limited by slubility cnstraints [21]. The limitatins f this technique riginates in part frm the internal aqueus vlume f the lipsmes, which typically represents nly a small fractin f the ttal suspensin vlume. High encapsulated catin levels can be achieved by entrapping apprpriate chelating agents (fr example ethylenediaminetetraacetic acid, 8-hydrxyquinline and nitriltriacetic acid) in the intravesicular space [16,22-25]. The maximum amunt f entrapped catin, hwever, is limited by the interir cncentratin f the chelating agent that can be achieved, and als by the binding cnstant f the metal in t the chelating agent. It may be cncluded that the ph driven catin lading is a cnvenient prcedure. As A23187 is capable f transprting a wide range f catins [14,26,27], it is likely that the technique fr catin lading described here, may be applicable t any catinic species that can be cmplexed t the inphre [6,23,28,29]. These catins include the alkaline earth series, Fe 2+, La 3+ and trivalent radinucletides. The ability t lad high cncentratin f catin in the aqueus cmpartment f vesicles has many ptential applicatins. Fr example, mci amunts f radinucletides such as 153Gd3+ r 67Ga3+ may ptentially be laded int the interir f a small cncentratin f vesicles resulting in a high specific activity. LUVs cntaining Gd 3+ r very radiactive vesicles may be useful fr cntrast enhanced nuclear magnetic resnance imaging, and fr determining the bidistributin f vesicles in in viv studies [30,31]. Furthermre, the encapsulatin f a variety f catins including Fe 2 and La 3+ culd allw the preparatin f 'heavy' lipsmes, and the creatin f electrn dense LUVs. These lipsmes have ptential in cell separatin prtcls and as cell markers. These and ther applicatins are currently under active investigatin. Finally, it shuld be nted that all carbxylic catin inphres cntain a carbxylic acid functinal grup which deprtnates upn cmplexatin. Therefre, it is likely that ther carbxylic acid antibitic inphres such as inmycin and A-204, will respnd t transmembrane ph gradients in the same way as A Acknwledgements We wuld like t acknwledge Dr. T.E. Redelmeier fr many helpful discussins. This research was supprted by the Medical Research Cuncil f Canada (MRC). References 1 Bally, M.B., Hpe, M.J., Van Echteld, C.J.A. and Cullis, P.R. (1985) Bichim. Biphys. Acta 812, Mayer, L.D., Bally, M.B., Hpe, M.J. and Cullis, P.R. (1985) J. Bil. Chem. 260, Mayer, L.D., Bally, M.B. and Cullis, P.R. (1986) Bichim. Biphys. Acta 857, Hpe, M.J. and Cullis, P.R. (1987) J. Bil. Chem. 262, Hpe, M.J., Redelmeier, T.E., Wng, K.F., Rdrigueza, W. and Cullis, P.R. 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