Arcadio Chonn, Sean C. Semple and Pieter R. Cullis. (Received 5 June 1991) Key words: Liposome; Chromatography; Complement; Blood protein

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1 Bichhnica et Biphysica Acre, 1071) (! 991 ) ~i) 1991 Elsevier Science Publishers B.V. All rights reserved /91/$ BBAMEM Separatin f large unilamellar lipsmes frm bld cmpnents by a spin clumn prcedure" twards identifying plasma prteins which mediate lipsme clearance in viv Arcadi Chnn, Sean C. Semple and Pieter R. Cullis Department f Bichemistt3,, The Unicersity f British Clumbia, Va,wut'er (Canada) (Received 5 June 1991) Key wrds: Lipsme; Chrmatgraphy; Cmplement; Bld prtein In rder t facilitate the islatin f iipsmes frm bld cmpnents, we have develped a simple and rapid prcedure cmbining chrmatgraphic and centrifugal methds. This 'spin clumn' prcedure was used t islate lipsmes frm incubatin mixtures with human serum r frm the bld f CDI mice after intravenus administratin f lipsmes. An advantage f this prcedure is that prcessing times are fast (typically minutes) such that the islatin prcedure can be dne in the absence f chelatrs r ther cagulatin inhibitrs which may affect prtein/lipsme interactins. Furthermre, several samples can be analyzed tgether and small sample vlumes can be prcessed. In additin, we shw that this spin clumn prcedure can be emplyed t islate large unilamellar vesicles averaging 100 nm in diameter frm lipprteins and plasma prteins. The applicability f this spin clumn prcedure in studying prtein/lipsme interactins is demnstrated by quantitating the amunt f human cmplement cmpnent C3 hund per iipsme using a C3 cmpetitive ELISA assay after incubatin with human serum. The prteins assciated with the recvered lipsmes were further analyzed by cnventinal SDS-plyacrylamide gel electrphresis. We shw that egg phsphatidyichline/chlesterl (55:45, tl/tl) r egg phsphatidylchline / chlesterl / dileylphsphatidylserine (35: 45: 20, tl / tl) lipsmes islated frm the circulatin f CDI mice within minutes f administratin have distinct, cmplex prfiles f assciated prteins. By islating circulating large unilamellar lipsmes using the spin clumn methd and characterizing the prteins assciated with their membranes, this prtein fingerprinting apprach will expedite identifying prtein interactins which affect lipsme stability and clearance in viv. Intrductin Large unilamellar vesicles (LUVs) are widely used fr lipsme-based carrier systems [1,2]. As with all iipsme systems, hwever, LUVs are rapidly cleared frm the circulatin, having half-lives ranging frm Abbreviatins: C3, cmplement cmpnent C3; ELISA, enzyme-linked immunsrbent assay; CH, chlesterl; CL, bvine heart cardilipin; DOPA, dileylphsphatidic acid; DOPS, dileylphsphatidyiserine; EPC, egg phsphatidylchline; EPG, egg phsphatidylglycerl; PI, plant phsphatidylinsitl; LDL, lw density lipprteins; LUV, large unilamellar vesicle; SDS-PAGE, sdium ddccyl sulphate-plyacrylamide gel electrphresis; VLDL, vet3, lw density lipprteins. Crrespndence: A. Chnn, The University f British Clumbia, Department f Bichemistry, Faculty f Medicine, 2146 Health Sciences Mall, Vancuver, B.C., Canada V6T IZ3. minutes t hurs. The interactins lipsmes encunter in the circulatin are believed t significantly influence their clearance and inherent stability in bld via at least tw mechanisms. First, the interactins with lipprteins, cmplement and serum albumin have been shwn t result in lipsme membrane destabilizatin leading t leakage f cntents (fr reviews, see Refs. 3-6). This has been attributed t a net transfer f lipid t lipprteins r t the frmatin f pres in the membrane in the case f cmplement prteins. Secnd, lipsme interactins with cmplement, fibrnectin and immunglbulins have been suggested t mediate lipsme clearance by the fixed and free macrphages f the reticulendthelial system, as lipsmes cated with these prteins exhibit enhanced uptake by cultured macrphages [7-9]. The factrs which mediate lipsme psnizatin in viv are prly understd, particularly fr large uni-

2 216 lamellar lipsmal systems. This is because f the difficulty in islating LUVs frm bld cmpnents such as lipprteins and recvering sufficient amunts t analyze by cnventinal SDS-plyacrylamide gel electrphresis. Much early wrk invlved multiiamellar ~csicles r snicated vesicles which were recvered frm plasma mixtures by ultracentrifugatin fllwed by multiple washes r by gel filtratin chrmatgraphy, respectively [3,4,10,11]. N cnvenient prcedure fr islating LUVs frm plasma cmpnents has been available. Such a technique is imprtant t study the prtein interactins LUVs experience in the circulatin. These interactins are likely t differ frm thse bserved fr multilamellar and snicated vesicles as vesicle size affects lipsme stability [12,13]. In this paper we describe a methd fr the rapid islatin f LUVs frm plasma cmpnents. We have applied this methd t islate lipsmes frm incubatin mixtures with human serum and mre significantly, frm the bld f mice injected intravenusly with lipsmes, in the latter case the lipsmes are expsed t the entire bilgical milieu, i.e. they are in cntact with the immune system, bld cells, cagulatin prteins, endthelial cells and physilgical in (ntably Ca -'+ and Mg 2+) cncentratins. The applicability f this islatin prcedure fr studying prtein/ lipsme interactins is demnstrated by quantitating the amunt f C3 bund t varius aninic r neutral LUVs after incubatin with human serum. Further, as lipsmes bearing a net surface charge are rapidly cleared frm the circulatin [12] and are ptent activatrs f the cmplement system [14], we had previusly prpsed that lipsme,.assciated C3b may play a significant rle in receptr-mediated lipsmal uptake. We have therefre determined here whether lipsme cmpsitins which exhibited rapid clearance kinetics bund greater amunts f C3. In additin, the prteins assciated with the recvered lipsmes were analyzed by cnventinal SDS-plyacrylamide gel electrphresis and visualized by a sensitive silver stain prtcl. Our findings indicate that iipsmes islated frm the circulatin f CDI mice within minutes f intravenus administratin have a cmplex prfile f prteins assciated with their membranes which is dependent n the lipid cmpsitin, and that iipsmes which are cleared mre rapidly bind significantly mre C3b. Methds and Materials Preparatin f lipsmes. Large uniiamellay vesicles (LUVs) were prepared by extrusin f freeze-thawed muitilamellar vesicles thrugh tw stacked 100 nm plycarbnate filters (Nuclepre, Pleasantn, CA) using an extrusin device (Lipex Bimembranes, Vancuver, Canada) as described in detail elsewhere [15]. Lipsme suspensins were 20 mm ttal lipid in is- tnic Hepes-buffered saline (HBS; 20 mm Hepes (ph 7.4), 145 mm NaCl) sterilized using Syrfil 0.22 /zm filters (Nuclepre). The size f the extruded vesicles was determined by quasi-elastic liqht scattering analysis n a NICOMP Mdel 270 Submicrn Particle Sizer (NICOMP Instruments, Santa Barbara, CA) t be 100 :1:30 nm. The lipsmes were radilabelled by incrprating the nn-exchangeable, nn-metablizeable marker [3H]chlesterylhexadecyl ether (10 ~Ci/30 /xml lipid) t fllw the bidistributin f the lipsmes in mice and t quantitate the cncentratin f the recvered lipsme suspensins [16]. The specific activity f the lipsmes was determined by measuring the radiactivity cntent using standard liquid scintillatin cunting methds n a Hewlett Packard Tri-C~,'b 2000CA liquid scintillatin analyzer and the phsphlipid cntent using a clrimetric (absrbance recrded at 815 nm) phsphrus assay [17]. Lipids were urchased frm the fllwing cmpanies: egg phsphatidylchline (EPC), egg phsphatidyiglycerl (EPG), dileylphsphatidylserine (DOPS), dileylphsphatidic acid (DOPA), plant phsphatidylinsitl (PI) and bvine heart cardilipin (CL) frm Avanti Plar Lipids, Pelham, AL; chlesterl (CH) frm Sigma; and [3H]chleste~lhexadecyl ether frm Amersham. These lipids were used withut further purificatin. Lipsme cmpsitins are expressed as mlar ratis. Serum and lipprteins. Human serum was prepared frm venus bld pled frm 20 healthy individuals (10 males, l0 females) and stred at -70 C. Purified very lw density lipprteins (VLDL) and lw density lipprteins (LDL)were prvided by Dr. H.P. Pritchard, Lipprtein Research Grup, Vancuver, Canada. Preparatin f spin clumns. The prcedure fr packing the spin clumns is as fllws. 1 ml tuberculit~ syringes plugged with glass wl were filled with Bi- Gel A-15m, mesh size, chrmatgraphic gel (Bi-Rad, Mississauga, Canada) equilibrated with istnic vernai-buffered saline (VBS, 10 mm sdium barbital (H 7.4), 145 mm NaCl) and centrifuged (Silencer H-103N Series bench tp centrifuge r Juan centrifuge G4.11; 2000 rpm, 2 min, 4 C) in mm glass cultui'e tubes. A series f fills and centrifugatins were dne until the bed vlume apprximated 1.0 ml. A final spin at 2000 rpm fr 5 min assured that the Bi-Gel A-15m gel was unifrmly packed and that excess buffer was remved. Spin clumn prfiles f lipsmes incubated with human serum. 100 /zl f the lipsme suspensin was incubated with 400 ~1 f pled nrmal human serum at 37 C fr 30 min. Aliquts f the lipsme/serum incubatin mixtures (50 /~i) were applied t spin clumns and immediately centrifuged (1000 rpm, 1 min, 4 C). Clumn fractins were cllected in glass culture tubes by applying 50 /~1 f VBS t the spin

3 217 clumns and centrifuging (1000 rpm, 1 min, 4 C). The lipsme cntent f the clumn fractins were assayed by determining the all radiactivity cntent f the fractins using standard liquid scintillatin cunting methds. Prtein cntent f the clumn fractins was determined using the BCA prtein assay (Pierce Chemical C., Rckfrd, IL). Briefly, 10,u.i f clumn fractins were incubated with 200 #! BCA prtein assay reagent in micrtiter plates. After an vernight incubatin at rm temperature, the absrbance (540 nm) f the slutins was read using an SLT-Labinstruments Austria EAR400AT micrtiter plate reader. As cntrls in all ur experiments, 50 p.i f 80% human serum withut lipsmes was chrmatgraphed using similar spin clumns under identical cnditins and the chmm fractins analyzed fr prtein cntent. Cm'entinal Bi-Gel A- 15m chrmatgraldty f lipsme /htnan serunt htctlbati/t mixtures. Tw ml f 100 mm EPC/CH/CL (35:45: 10) LUVs in VBS was incubated with 8 ml f nrmal human serum at 37 C fr 30 rain. "Ik) islate the lipst,mcs fi'm human serum cmpnents, the incubatin mixture was chrmatgraphed n a 2.5 x 90 cm Bi-Gel A-15m, mesh, clumn pr-equilibrated with VBS buffer at 4 C. Fractins (120 drps/fractin) were cllected at a flw rate f 30 ml/h. The clumn fractins were analyzed fr phsph;i~id cntent using a clrimetr phsphrus assay [17] and fr prtein cntent using the BCA prtein assay. Fractins were pled. Recrety f lipsmes frm ctrculatin f CDI mice. 200 p.! f the lipsmc suspensin was administered intravenusly via the drsal tail vein f CDl mice (4 mice/time pint). After 2-3 rain, 30 min, r l i~ pst-injectin, the mice were anesthetized with ether and bld withdrawn via cardiac puncture and ct~llected in ice cld 1.5 ml plyprpylene micr test tubes (Eppendrf). The bld was immediately cled t 0 C using an ice-water bath t prevent cagulatin and centrifuged (12000 rpm, 2 rain, 4 C) t pellet the bld cells. Aliquts f the plasma (50 #l) were applied t spin clumns (5 clumns/muse) and immediately centrifuged (Juan Centrifuge G4.11; 1000 rpm, l rain, 4 C). Clumn fractins were cllected in glass culture tubes by applying 50 /.tl f VBS t the spin clumns and centrifuging (1000 rpm, 1 min, 4 C). The first tw clumn fractins cntaining radiactivity (typically fractins 5 and 6, r 6 and 7) were cllected, pled and cncentrated using Centricn 30 micrcncentratrs (Amicn, Danvers, MA) at 4 C. The ra-, diactivity recveries frm the Centricn 30 micrcncentratrs were typically greater than 95%. The samples were stred at -20 C. Cmpetitive ELiSA fr C3. C3 cmpetitive ELISA was perfrmed n serially diluted islated iipsmes using the prcedure described by Mld [18]. Human C3 was purified accrding t the methd f Tack and Prahl [19]. Hrse radish perxidase-cnjugated gat anti-human C3 (CalBichem) was used at a 1/10000 dilutin. SDS-plyacrilamide gel electrphretic analysis f prtebts assciated with lipsmes. Prtein separatin was perfrmed by SDS-plyacrylamide gel electrphresis (SDS-PAGE) using the Mini Prtean-ll eleetmphretic apparatus (Bi-Rad) n precast 4-20% gradient Mini Prtean-ll gels (Bi-Rad) under nnreducing cnditins. Prestained SDS-PAGE mlecular weight standards (Diversified Bitech, Newtn, MA) were used t estimate the mlecular weights f the prteins. Prteins were detected using an ptimized silver stain prcedure [20]. Results lslathm f large unilameilar lipsmes frm plasma ushlg Bi-Gel A- 15m spin clumns Initial studies indicated that cventinai Bi Gcl A-15m, mesh, gel filtratin chrmatgraphy was effective in islating LUVs (extruded thrugh 100 nm pre sized filters)'fi,am human serum cmpnents (Fig. 1). Hwever, this prcedure, requiring lengthy prcessing ~imes (typically 3-4 h per separatin) and relatively large sample vlumes, was net practical fr ur studies. Thereire, several factrs including use f a finer mesh ( mesh) t increase the numbc, f theretical plates; use f shrter clumns, thereby reducing the sample vlume requirement; and increasing perating pressures, t reduce the separatin time were cnsidered in rder t imprve this islatin prcedure. A rapid methd fr the islatin f LUVs u3,r,,,, O.< i 2 r '10 n Clumn Fractin # C Fig. I. Elutin prfile f EPC/Clt/CL LUV/human serum incubatin mixture:; chrmatgraphed n cnventinal clumns. EPC/CH/CL (35:45: 10) LUV/human serum incubatin mixtures were chrmatgraphed n a cm Bi-Gel A-15m, I(X)=200 mesh, clumn as described in Methds and Materials. The phsphrus cntent (e) r the prtein cntent (ll) f the clumn fractins was determined as in Methds and Materials.

4 218 A E. " ~ > tu.m " ee n A -7 i O to < v r- a) t- O 0 l. G) Q p! n" A 4 O,qtO <: 3 *".. O t- O 2 O t-, u e) t.._ 1 Ix ~ Clumn Fractin # Clumn Fractin # Fig. 2, (A) Spin clumn prfiles f lilx~smcs r human serum prteins, Bi-Gel A-15m, mesh, 1.0 ml spin clumns were calibrated using 50 ttl 20 mm EPC/CH (55:45) LUVs radilabelled with [~i+l]chlesterylhexadecyl ether (, 4 clumns) r 50/~1 80% human serum (ra, 4 clumns) chmmatgraphed separately under identical cnditins. Clumn fractins represent the eluant recvered frm ne centrifugatin (1000 rpm, I rain). (B) Spin clumn prfiles f lipsme/human serum incubatins. EPC/CH/DOPA (35:45: 20) (13), EPC/CH/DOPS (35:45: 20) (~), r EPC/CH/P! (35:45:20) () LUVs cntaining trace amunts f ['~H]chlesterylhexadecyl ether were incubated with human serum at 37 C fr 30 min, The incubatin mixtures were then chrmatgraphed using the Bi-Gel A-15m spin clumns as described in Methds and Materials. The pen symbls are the radiactive cntent and the filled symbls are the prtein cntent f the clumn fractins. frm plasma cmpnents was thus develped using a 'spin clumn' prcedure described in Methds. The efficiency f separatin using thi~ technique is depicted in Fig. 2, Fig. 2A shws the c/amn prfiles f EPC/CH (55:45) LUVs r f 80% human serum hrmatgraphed n Bi-Gel A-15m 1.0 ml spin clumns, Fig, 2B shws the clumn prfiles f varius LUV/80% human serum incubatin mixtures. As shwn in Fig, 2B, the lipsme cmpsitin des nt 1S affect the elutin prfile f the lipsmes. These prfiles are representative f hundreds f clumns using LUVs cmpsed f net neutral r aninic lipids. By centrifuging the clumn in a 13 x 100 mm glass culture tube carrier, the flw rate thrugh the clumn was cnsiderably increased. The prfiles shwn were btained using the ptimized spin cnditins f 1000 rpm fr 1 min. These ptimal cnditins were determined by varying the rate and duratin f centrifugatin. The cnditins el ~ 1000 rpm fr 1 rain gave the mst cnsistent elutin prfiles and fl actin vlumes (typically ~, 0, a "~ ,20 B: 0.10 A in,< a) c t,). u q) L_ O "-" m 200 koa ''~ S 10 ls 20 2S Clumn Fractin # Fig, 3, Separatin f 100 nm LUVs frm VLDL and LDL. Bi-Gel A-15m, Z mesh, 1,0 ml spin clumns were calibrated using 50 pl 20 mm EPC/CH (55:45) LUVs radiblabelled with [3Hk'hlesterylhexadecyi ether (, 4 clumns), 50 p.i purified VLDL (n, 4 clumns) r 50/tl purified LDL (zx, 4 clumns). VLDL and LDL cntent were detected using the BCA prtein assay. These were chrmatgraphed separately n similar spin clumns under identical cnditins. Fig. 4. SDS-plyacrylamide gel electrphretic analysis f the prtein cntent f the spin clumn fractins. The clumn fractins f chrmatgraphed 50 mi 80% human serum were analyzed fr their prtein cntent by SDS-plyacrylamide gel electrphresis fllwed by silver staining as described in Methds and Materials.

5 /~1). Spin times lnger than 2 min resulted in channel frmatin at the tp f the clumn. Clumn bed vlumes averaged 1.00 _ ml. The mean lipsme recvery in the first 9 fractins was 70%. Elutin prfiles btained with purified iipprteins shwed that the lipsmes were effectively reslved frm the very lw density and lw density lipprteins (Fig. 3) using the spin clumn prcedure. The clumn fractins were analyzed fr their prtein cntent by SDS-PAGE fllwed by silver staining (Fig. 4). When 80% serum in VBS was chrmatgraphed, the fractins where the lipsmes wuld elute (fractins 5-7) did nt cntain any detectable prtein. The fractin where the serum prteins started eluting (fractin 9) cntained very high mlecular weight prteins having mbilities crrespnding t mlecular masses greater than 200 kda. "['he prtein prfile f the lipsmes islated using the spin clumn prcedure was cmpared t that btained using the cnventinal gel filtratin clumn prcedure by analyzing equal amunts f lipid n 4- TABLE! Amunt f C3 assciated with varius LUVs The LUVs recvered frm human serum incubatins using the spin clumn prcedure were analyzed fr C3 cntent using a C3 cmpetitive ELISA (see Methds and Materials). The results frm tw separate experiments are given. LUV cmpsitin EPC/CH (55 : 45) EPC/CH/PI (35:45 : 20) EPC/CH/PG (35:45:20) EPC/CH/DOPS (35:45:20) EPC/CH/DOPA (35 : 45.20) EPC/CH/CL (35 : 45:10) Amunt f C3 bund a (nml C3/mml ttal lipid) 3.15, , , , , , % gradient SDS-plyacrylamide gels (Fig. 5). Fig. 5 shws that the prtein prfiles are very similar in type and amunt f prteins assciated with EPC/CH/CL (35 : 45 : 10) LUVs. EPC/CH/CL LUVs were used because this cmpsitin bund the mst cmplex prtein prfile f all the aninic lipsmes investigated. kda "" "- 36 "- A B Measurement f C3 bund per lipsme recvered firm human serum incubatin mixtures using the spin clumns Previus studies have demnstrated that aninic lipsmes activate the cmplement system via the classical pathway leading t the lipsmal assciatin f C3b [14]. It was suggested that C3b was ne f the plasma prteins which marked the lipsmes as freign particles because C3b is a ptent psnin. As the ppulatin f lipsmes is essentially unilamellar fr all lipid cmpsitins emplyed here, we can estimate the amunt f C3b bund per lipid as a functin f lipid cmpsitin. Using a human C3 cmpetitive ELISA, the amunt f C3 assciated with the varius lipsmes was quantitated (Table I). The amunt f C3 assciated with EPC/CH/CL (35:45: 10) r EPC/CH/DOPA (35:45:20) LUVs is apprximately 4-10-times greater than fr EPC/CH (55:45), EPC/CH/EPG (35:45:20) r EPC/CH/Pi (35:45 : 20) LUVs. Fig. 5. Cmparisn f the prtein prfile assciated with EPC/CH/CL LUVs islated using the spin clumn prcedure r cnventinal chrmatgraphic prcedures. EPC/CH/CL (35:45:10) LUVs recvered using the Bi-Gel A-15m, mesh, cnventinal clumn (Lane A) r the Bi-Gel Al5m, mesh, spin clumn (Lane B) prcedures were analyzed fr the prteins assciated with their membranes by SDS-PAGE analysis as in Methds and Materials. Equal amunts f lipid (30 nml ttal lipid) were applied t each lane. Lane C is 25 p,! f 1/750 dilutin f pled nrmal human serum. In viv characterizatin f murine plasma prteins assciated with EPC / CH r EPC / CH / DOPS LUVs ver time The utility f this spin clumn methd in recvering LUVs frm bld f mice administered intravenusly with EPC/CH (55 : 45) r EPC/CH/DOPS (35:45 : 20) lipsmes was investigated. Fig. 6 shws the recvery f LUVs frm the plasma f CD1 mice ver a 1 h perid. Whereas EPC/CH/DOPS LUVs are cleared rapidly frm the plasma, EPC/CH LUVs are relatively lng-lived in the circulatin. The lipsmes were islated frm the bld samples in the absence f chelatrs r ther cagulatin inhibitrs by cling the

6 220 "6 O. > 0 U J 10 " I 1 0,1 I I '----~ Time (rain) Fig. 6, Recveries f EPC/CH and EP('/CII/DOPS LUVs ver time frm muse plasma, EPC/CH (55:45) (e) r EPC/CH: DOPS (35:45:20) ( A ) lir~)smes were administered intravenusly int CDI mice and ver time aliquts f plasma were cunted fr "all radiactivity I fllw the clearance f lipsmes frm the circulatin. Each muse received a dse f apprx. 4/zml ttal lipid in a vlume f 2111) ml HBS, Pla,~ma vlume f a muse was taken t be 5% f bdy weight. bld samples t 0 C t retard the cagulatin prcess, centrifuging t pellet the bld cells, and separated frm the plasma using the Bi-Gel A-15m spin clumns. The prtein prfiles were characterized using SDS- PAGE analysis (Fig. 7). Within a few minutes after administratin, EPC/CH LUVs are shwn t have an assciated prtein cmpsitin cnsisting mainly f albumin and very high mlecular weight prteins. Qualitatively, the verall prtein prfile assciated with circulating EPC/CH LUVs is nt markedly altered ver a I h perid. The rapidly cleared EPC/CH/DOPS LUVs, n the ther hand, have a mre cmplex prtein prfile, bth in amunt and type f assciated prteins, than the EPC/CH LUVs. N prtein was detected in these clumn fractins when muse serum alne was chrmatgraphed. (Fig. 1 in this reprt; Refs. 21 and 22), spin clumn prcessing times are extremely rapid. Frm the pint f sample applicatin t the cllectin f the lipsme fractins, the islatin prcedure takes apprx. 6-8 min. Other advantages f this spin clumn prcedure are that many clumns can be prcessed at the same time (up t 96 depending n the capacity f the centrifuge) and that small sample vlumes can be analyzed. The majr advantage in particular t the study f prtein-mediated iipsme clearance mechanisms, hwever, is that because prcessing times are rapid and the prcedure can be readily perfrmed at 4 C, the islatin prcedure can be dne in the absence f cagulatin inhibitrs which may affect prtein-lipsme interactins. Gd reprducibility in islating LUVs fl'm bld cmpnents using the spin clumn methd is bviusly necessary fr detecting the prteins assciated with circulating lipsmes. The rcsults presented here arc representative f hundreds f spin clumns; hwever, we have encuntered sme variatkm in the perfrmance f different batches f Bi-Gel A-15m gel. These variatins result in different flw rates and cl- kda A B C D E DL,,cussin The develpment f methds t rapidly islate LUVs frm bld cmpnents is imprtant t understanding the prtein/iipsme interactins which mediate lipsme leakage and clearance frm the circulatin. Until nw, there has been n satisfactry prcedure fr islating LUVs frm plasma. In this reprt, we describe a prcedure which is very cnvenient fr the study f prtein/lipsme interactins which ccur in viv. Cmpared t cnventinal clumn chrmatgraphic methds which take tbut 3-4 h per analysis..~,... Fig. 7. Prtein prfiles f EPC/CH (55:45) and EPC/CH/DOPS (35:45:20) LUVs recvered frm mice ver time. The prteins assciated with the recvered lipsmes were analyzed using 4-20% gradient SDS-plyacrylamide gel electrphresis and silver stained as in Methds and Materials. Lane A, EPC/CH, 2 rain; Lane B, EPC/CH, 30 min; Lane C, EPC/CH, 1 h, Lane D, EPC/CH/DOPS, 2 rain: EPC/CH/DOPS, 30 min. Lanes A-D represent the prteins assciated with 40 nml ttal lipid; Lane E represents the prteins assciated with 20 nml ttal lipid.

7 221 umn fractin vlun~es. The elutin prfiles are therefre smetimes different a~_d n ne ccasin resulted in serius tailing in the lipsme elutin prfile. This ptential prblem can be vercme by careful characterizatin f the elutin prfile: f the spin clumns using different batches f Bid-Gel A-15m gel. Within a particular batch, the elutin prfiles are very reprducible. Using this spin clumn prcedure a ppulatin f EPC/CH (55:45) LUVs which remained circulating ver a 1 h perid has been separated and the assciated prteins analyzed. We find that these net neutral lipsmes bund a number f high mlecular weight (> ) prteins which is similar t the in vitr findings f Julian and Lin [23] using human plasma. This similarity is ntewrthy since the earlier study invlved multihtmellar vesicles islated by ultracentrifugatin fllwed by multiple washes with buffer. This indicates that prteins which are assciated with the spin clumn-islated I.,UVs are tightly bund. The high mlecular weight prteins as well as albumin are assciated with EPC/CH LUVs within minutes f intravenus administratin, and d nt appear t enhance the clearance f lipsmes frm the circulatin ver a 1 h perid because the ppulatin f lipsmes bearing these prteins remain in the circulatin ver this perid. in cntrast t the EPC/CH (55:45) prtein prfiles, the prfile f prteins assciated with rapidly cleared EPC/CH/DOPS (35:45:20) LUVs is mre cmplex. The same prteins which are assciated with EPC/CH LUVs are als fund assciated with EPC/CH/DOPS LUVs; hwever, mre prteins, bth in terms f amunt and type, are assciated with the aninic EPC/CH/DOPS LUVs. Sme f these prteins may include cltting prteins and cmplement prteins as it has been reprted that PS-cntaining lipsmes acrid rate the cagulatin [23-26] and cmplement systems [14,27]. The identificatin f the prteins assciated with these lipsmes, especially thse prteins which are uniquely assciated with rapidly cleared lipsmes, shuld elucidate prteins which affect lipsme clearance frm the circulatin. Changes in the prtein fingerprints f the lipsmes ver time may prvide insight int the prteins affecting lipsme stability in the circulatin. The finding that specific assciated prteins disappcar frm the prtein prfiles ver time sug~,est that lipsmes bearing these prteins ale cleared mre readily. Fr example, EPC/CH LUVs at 2 min and 30 rain have a prnunced band, migrating similarly t the 200 kda mlecular mass standard, assciated with their membranes. This band ntably disappears at the 1 h time pint. Similarly, a prminent band, migrating with a mlecular weight crrespnding rughly t 80000, is ~ssciated with the EPC/CH/DOPS LUVs at the 2 min time pint but is less intense at 30 min, althugh ii shuld be nted that Lane D in Fig. 7 has twice the amunt f ttal lipid laded as in Lane E. Similar studies that characterize the prtein fingerprint f [ipsmes ver time, invlving mre quantitative methds, shuld be very useful in elucidating the prteins invlved in lipsme clearance. The EPC/CH/DOPS (35 : 45 : 20) LUVs recvered frm the circulatin f mice had a mre extensive prtein fingerprint (Fig. 7) than the EPC/CH/CL LUVs recvered frm in vitr incubatins with human serum (Fig. 5), especially with regard t the very high mlecular weight prteins (> ). This underlines the necessity t study the in viv plasma prtein interactins lipsmes experience in rder t fully understand the prtein-mediated clem'ance behavir f lipsmes. One f the plasma prteins which has been implicated in mediating lipsme uptake is cmplement cmpnent C3, specifically the psnic trrn C3b. 'ffc had previusly demnstrated that lipsmes bearing a net surtace charge ale ptent activatrs f the cmplement system resulting in the depsitin f C3b mlecules nt the lipsme membranes [14]. The results presented here clearly shw that lipsmes cntaining DOPA r CL exhibit cnsiderably mre assciated C3. If lipsme clearance behavir is similar in mice al,d humans, these results suggest a crrelatin between the amunt f C3 bund per lipsme and lipsme clearance behavir. CL- and DOPA-cntaining systems, which bind the mst C3 (Table 1), are cleared very rapidly frm the circr!atin [12,28]. EPC/CH, and EPG- and Pl-cntaining systems are cleared mre slwly [29,30] and bind much less C3. in summary, the spin clumn prcedure is a rapid and effective methd fr recvering LUVs frm incubatin mixtures with bld cmpnents. This prcedure requires little manipulatin f the system; n cagulatin inhibitrs are necessary and n multiple washes with buffer are required. Thus, the lipsmes can be readily recvered frm the bld f mice after intravenus administratin f iipsmes. This was demnstrated in the recvery f EPC/CH (55:45) LUVs ver a perid f 1 hr (Fig. 7). The amunt f lipsmes recvered frm this prcedure is adequate t analyze the prtein cntent f the lipsmes by SDS-plyacrylamide gel electrphresis fllwed by silver staining. As well, the lipsme prteins are readii7 analyzed using immunlgical methds such as imraunsrbent assays. Further, this spin clumn islatin prcedure allws the use f large unilamellar lipsmal systems as ppsed t multilamellar lipsreal systems in studies invlving prtein/membrane interactins. The advantage f using LUVs, where all the lamellae are expsed t the extravesicular envirnment, as ppsed t MLVs, where nly the utermst

8 222 lamellae is expsed t the extravesicular envirnment, is clearly demnstrated in the studies quantitating the amunt f lipsme-assciated C3 (Table I). The relatin between the amunt f assciated C3 (Table I) and lipsme clearance frm muse bld strngly suggests that C3 fragments play a rle in lipsme clearance. This prtein fingerprin[ing apprach has great ptential in elucidating the prtein/lipsme interactins which ccur in viv and in identifying prtein interactins which may affect lipsme clearance frm the circulatin. A detailed in viv study f the prteins that interact with varius lipsmes exhibiting markedly different clearance pharmackinetics, and identificatin f sme f the prteins mediating lipsme clearance are in prgress in this labratry. This spin clumn prcedure culd als have a general utility in studies invlving prtein-membrane interactins. Acknwledgements The authrs wuld like t thank Dr. Dana V. Devine fr helpful discussins. This research was supprted by the Medical Research Cuncil f Canada. References! Cullis, P.R,, Mayer, L.D., Baily, M.B., Madden T.D. and Hpe, MJ. (1989) Adv, Drug Deliv. Rev, 3, str, MJ. and Cullis. P.R, (1989) Am. J. Hsp. Pharm. 46, Senir, J. (1987) Crit. Rev. Ther. Drug Carrier Syst. 3, Bnte, F. and Julian. R.L. (1986) Chem. Phys. Lipids 40, , 5 Scherphf, G,L,, Damen J, and Wilschut. J. (1984) in Lipsme Technlgy, Vl, 3 (Oregriadis, G,, ed,), pp , CRC Press, Bca Ratn, FL. 6 Alvin8, C.R. and Richards. R.L. (1983) in Lipsmes (Ostr. M.. ed,), pp, , Marcel Dekker, New Yrk, 7 Derkesen, J.T.P., Mrseit, H.W.M., Kalicharan, D., Huistaert C.E. and Scherphf. G.L. (1987) Exp. Cell Res. 168, 105-!15. 8 Rerdink, F., Wassef, N.M., Richardsn E.C. and Alving, C.R. (1983) Bicbim. Biphys. Acta '.). 9 Hsu, M.J. and Julian, R.L. (1982) Bichim. Biphys. Acta 720, Senir, J. and G. Gregriadis (1984) in Lipsme Technlgy, Vl. 3. (Gregriadis, G., ed.), pp , CRC Press, Bca Ratn, FL. 11 Kirby, C,, Clarke J, and Gregriadis O. (1980) FEBS Le. 111, Jtdian, R.L. and ~'p, D. (1975) Bichem. Biphys. Res. C~mmun "i-658, 13 Schetnf, G. and Mrselt, H. (1984) Bichem. J. 221, Chnn, A., Ctdlis P.R. and Devine, D.V. (1991) J. lmmunl. 146, Hpe, MJ., Ba[ly, M.B., Webb G. and Cullis, P.R. (1985) Bichim. Biphys. Acta 812, Stein, Y., Halperin G. and Stein, O. (IOd0) FEBS Lett. III, I ()4-1 (}6. 17 Fiske, C.H. and Subbarw, Y. (1925) J. Bil. Chem. 66, Mld, C. (1989) J. lmmunl. 143, Tack, B.F. and Prahl, J,W, (1976) Bmchemistry 15, Heukeshven, J. and Dernick, R. (1988) Eiectrphresis 9, Huang, C.H. (1969)Bichemisl~'y 18, Reynlds, J.A., Nzaki Y. and Tanfrd, C. (1983) Anal. Bichem. 130, Julian, R.L. and Lin, G. (1980) in Lipsmes and Immunbilgy (Tm, B.H., Six, H.R., eds.), pp , Elsevier Nrth-Hlland, New Yrk. 24 Julian, R.L. (19~3) in Lipsmes (Ostr, M., ed.), pp , Marcel Dekker New Yrk. 25 Yshika, A. Peake, I.R., Furlng, B.L., FurlnlL A., Giddings J.C. and BKmm, A.L (1983) Br. J. Haematl. 5~, Andcrs~,fi, L.O. and Brwn, J.E. (1981) Bichcm. J. 200, Cmi,', A. and Easterbrk-Smith, S.B. (1986) FEBS Lett. 197, 32! ~ nir, J. and Gregriadis, G. (1982) Life Sci. 30, t~ Ka, TJ, and L, T.L. (1980) J. Pharm. Sci. 659, Gabizn, A. and Papahadjpuls, D. (1988) Prc. Natl. Acad. Sci. USA 85,