Biochi~ic~a et Biophysica A~ta

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1 ELSEVIER Bichimica et Biphysica Acta 1195 (1994) BB Bichi~ic~a et Biphysica A~ta Factrs influencing the retentin and chemical stability f ply(ethylene glycl)-lipid cnjugates incrprated int large unilamellar vesicles Michael J. Parr *, Steven M. Ansell, Lewis S. Chi, Pieter R. Cullis The University f British Clumbia, Department f Bichemistry and Mlecular Bilgy, Faculty f Medicine, 2146 Health Sciences Mall, Vancuver, B.C., Canada V6T 1Z3 Received 15 December 1993; revised 5 May 1994 Abstract Ply(ethylene glycl)(peg)-lipid anchr cnjugates can prlng the circulatin lifetimes f lipsmes fllwing intravenus injectin. In this wrk we investigate the influence f the lipid anchr and the nature f the chemical link between the PEG and lipid mieties n circulatin lifetime. It is shwn that incrpratin f N-(mnmethxyply(ethylene glycl)20-succinyl)-l-palmityl-2-leylphsphatidylethanlamide (MePEG200-S-POPE) int large unilamellar vesicles (LUVs) cmpsed f distearylphsphatidylchline (DSPC) and chlesterl (DSPC/chlesterl/MePEG200-S-POPE, 50 : 45 : 5, ml/ml) results in nly small increases in the circulatin lifetimes as bserved in mice. This is shwn t be due t rapid remval f the hydrphilic cating in viv, which likely arises frm exchange f the entire PEG-lipid cnjugate frm the lipsmal membrane, althugh chemical breakdwn f the PEG-lipid cnjugate is als pssible. The chemical stability f fur different linkages was tested, including succinate, carbamate and amide linkages between MePEG derivatives and the amin head grup f PE, as well as a direct link t the phsphate head grup f phsphatidic acid (PA). The succinate linkage was fund t be the mst labile. The anchring capability f DSPE as cmpared t POPE in PEG-PE cnjugates was als examined. It is shwn that incrpratin f MePEG200-S-DSPE cnjugates int DSPC/chlesterl LUVs results in little lss f the PEG cating in viv, lng circulatin lifetimes and reduced chemical breakdwn f the PEG-lipid cnjugate. This wrk establishes that DSPE is a cnsiderably mre effective anchr fr PEG20 than POPE and that the chemical stability f PEG-PE cnjugates is sensitive t the nature f the linkage and exchangeability f the PEG-PE cmplex. We suggest that retentin f the PEG cating is f paramunt imprtance fr prlnged circulatin lifetimes. Keywrds: Lipsme; Ply(ethylene glycl); Drug delivery system; Stability; Exchange; Bidistributin 1. Intrductin The use f lipsmes as drug delivery vehicles in viv requires lng circulatin lifetimes. Fr example, increased circulatin times result in enhanced tumr uptake and enhanced efficacy when the lipsmes cntain anticancer drugs [1-6]. It has been shwn that the incrpratin f gangliside GM~ int the lipsmes can imprve circulatin lngevity [7]. Alternatively, incrpratin f mnmethxyply(ethylene glycl)- phsphatidylethanlamine (MePEG-PE) cnjugates int lipsmal systems can als significantly extend the circulatin lifetimes f intravenusly injected lip- * Crrespnding authr. Fax: + 1 (604) smes [8-12], ften t an extent greater than that prvided by GM1. It has been prpsed that MePEG-PE prvides a 'steric stabilizatin' f the surface by virtue f the hydrphilic brush cat prvided by the MePEG plymer [13-15]. This cat may inhibit serum prtein binding t the lipsmal surface [16,17] which wuld therwise prmte uptake by the reticulendthelial system (RES), the circulating phagcytic cell ppulatins r fixed ppulatins such as thse fund in the liver and spleen. The extent t which the PEG cating remains assciated with the lipsme after injectin int the circulatin has nt yet been adequately addressed. The lipid miety f the mlecule must bviusly be sufficiently lipphilic t firmly anchr the hydrphilic cat t the surface. In this regard, lipsmally incrprated PEGchlesterl r PEG-mnstearate are relatively inef /94/$ Elsevier Science B.V. All rights reserved SSDI (94)00156-J

2 22 M.J. Parr et al / Bichimica et Biphysica Acta 1195 (1994) fective at imprving the circulatin lifetimes f intravenusly injected lipsmes [11]. Presumably, the hydrphbic miety in these cmpunds is an ineffective anchr and thus the hydrphilic cat is rapidly lst frm injected LUVs. Reprts n the anchring prperties f diacylphsphatidylethanlamine anchrs have been cnflicting. It has been suggested that the lipid miety has little effect n 'stealth' characteristics f LUVs [18], while thers indicate that the lipid anchr is a imprtant factr [11]. The chemical stability f MePEG-PEs in viv has nt received detailed attentin. In this wrk we examine the influence f the lipid anchr and linkage chemistry n the ability f MePEG- PE t imprve circulatin lifetimes f LUV systems. We shw that the anchring capacity f PE anchrs is extremely sensitive t the acyl chain cmpsitin, where distearyl PE species are cnsiderably mre effective anchrs than palmitylleyl species. Secnd, depending n the type f linkage between the PEG and the PE, breakdwn can ccur either n the LUV surface r after release f PEG-PE frm the LUV. These factrs shuld be cnsidered when discussing the usefulness r mechanisms f PEG-PEs incrprated int lipsmes. 2. Materials and methds 2.1. Mnmethxyply(ethylene glycl)-lipid (MePEGlipid) synthesis The verall chemical structures f the f the varius MePEG-lipids synthesized are shwn in Fig. 1, which include MePEG linked t phsphatidylethanlamine via succinate (MePEG-S-PE), carbamate (MePEG-C- 8 b OR. 1 _L, IVT'. r l H 0 OR d ~-]v ~,. Fig. 1. Summary f PEG-lipid cnjugate chemical structures. (a), succinate linkage: MePEG-S-PE; (b), carbamate linkage: MePEG-C- PE; (c), amide linkage: MePEG-A-PE; (d), direct linkage: MePEG- PA. (See Materials and methds fr detailed descriptins f the cmpunds synthesized.) R PE) and amide (MePEG-A-PE) linkages, and directly t phsphatidic acid (MePEG-PA). All PEG-lipids were islated as a single cmpnent n TLC, with similar Rf values, and shwed 1H NMR resnances characteristic f the MePEG and lipid grups. MePEG2000-S-POPE and MePEG2000-S-DSPE were synthesized as fllws. Mnmethxyply(ethylene glycl) (MePEG200-OH) was treated in pyridine with ten equivalents f succinic anhydride at rm temperature fr tw days. The slutin was diluted with water, acidified, extracted with methylene chlride, and the rganic extracts were dried ver magnesium sulfate, filtered, and the slvent remved. The resulting residue was subjected t silica gel clumn chrmatgraphy in methylene chlride/methanl (96:4, v/v)and Me- PEG20-Succinate islated. The dry MePEG2000-S, DCC, and NHS were disslved in chlrfrm, stirred fr 1 h and filtered. Dry 1-palmityl-2-leylphsphatidylethanlamine (POPE) r 1,2-distearyl phsphatidylethanlamine (DSPE) was disslved in warm chlrfrm and added t the filtrate, then triethylamine was added and the reactin mixture stirred fr half an hur. Cmbined rganic extracts were dried ver magnesium sulphate, filtered and the slvent remved under reduced pressure. The residue was subjected t silica gel clumn chrmatgraphy. Fractins cllected were analyzed by TLC using methanl/chlrfrm as the slvent (15 : 85, v/v) and visualized by expsure t idine. Fractins cntaining pure MePEG200-S-POPE r MePEG200-S-DSPE were cmbined, taken up int distilled water, centrifuged at 1500 x g fr 30 min and the supernatants dialyzed against distilled water vernight. The resultant slutins were lyphilized t yield a white pwder. The synthesis f MePEG200-[t4c]s-POPE was carried ut as described fr MePEG200-S-POPE with the exceptin that [1,4-]4C]succinic anhydride was reacted first befre additin f an excess f unlabelled succinic anhydride. The prduct was islated by preparative TLC using tw successive plates run in methanl/chlrfrm (15 : 85, v/v). The MePEG2000-S-POPE cmpnent was extracted frm the apprpriate scraped bands with methanl and then methanl/water (1 : 1, v/v) t yield MePEG200-[14C]S-POPE with specific activity 1.74 mci/mml. The extract was dispersed in water, centrifuged at 1500 x g fr 30 min, frzen and lyphilized. The synthesis f MePEG200-S-[3H]DSPE first required making MePEG200-S-DOPE similarly t the prcedure described fr MePEG200-S-POPE. The Me- PEG20-S-DOPE and Pd-C were then added with methanl t a vial which was sealed and flushed with nitrgen. Sdium brtritide (490 mci/mml) was injected and the mixture stirred fr 1 h. Sdium brhydride was added and the slutin stirred fr 3 h. After carefully pening in a fume hd, the suspensin was

3 M.J. Parr et al. / Bichimica et Biphysica Acta 1195 (1994) acidified with a drp f hydrchlric acid (10%), mre methanl added, and then centrifuged. The supernatant was filtered thrugh celite, diluted with water and extracted with methylene chlride. The rganic fractins were dried ver magnesium sulfate, filtered, and the MePEG2-S-[3H]DSPE was purified by preparative TLC as described abve (99% f the remaining radiactivity was lcated in the MePEG2-S- DSPE cmpnent, specific activity 140 mci/mml). T synthesize [3H]MePEG2-S-DSPE, a sample f [3H]MePEG2-OH (Amersham, custm synthesis) was used as the starting material with the remainder f the synthetic prcedure fllwed that f MePEG2-S- DSPE. Purificatin was carried ut using preparative TLC plates as described abve t yield [3H]Me- PEG2-S-DSPE (specific activity 44 mci/mml). [3H]MePEGs0-S-POPE (specific activity 156 mci/ mml) was synthesized similarly t [3H]MePEG2-S- DSPE. MePEG2-S-(1-palmityl-2-(4-pyrenyl)-butyryl)PE was made by first synthesizing MePEG2-S-(1-palmityl-2-hydrxy)-phsphatidylethanlamine as the starting species using the prcedure described fr Me- PEG2-S-POPE. A slutin f pyrenebutyric acid and DCC in alchl free chlrfrm was allwed t stir at rm temperature fr 1 h, filtered and then added t the reactin mixture. After wrk up, the prduct was islated by clumn chrmatgraphy and preparative thin-layer chrmatgraphy as befre t yield a pure flurescent cmpund, MePEG20-S-(1-palmityl-2- (4-pyrenyl)-butyryl)PE (MePEG20-S-PPBPE). MePEG2-C-POPE was made accrding t the fllwing prcedure. Dry MePEG2-OH was disslved in chlrfrm/tluene (50:2, v/v), reacted with triphsgene and the prduct precipitated by additin f ether. The precipitate, MePEG2000-chlrfrmate (MePEG2000-C-CI), was filtered and dried under vacuum. The MePEG2000-C-CI and dry POPE were disslved in chlrfrm and treated with triethylamine. The slvent was remved under vacuum and the residue disslved in water. The aqueus slutin was acidified and extracted with methylene chlride. The cmbined rganic fractins were dried ver magnesium sulphate, filtered, slvent remved and the residue subjected t clumn chrmatgraphy using silica gel and methanl/ methylene chlride t yield pure MePEG2000- carbamate-pope (MePEG2000-C-POPE). The prduct was dispersed in water, centrifuged at 1500 g fr 30 min and the supernatant lyphilized t yield a white pwder. MePEG2000-A-POPE and MePEG2000-A-DSPE were synthesized as fllws. MePEG2000-acetic acid was synthesized by an adaptin f the prcedure in Sessler et al. [19]. Briefly, MePEG2000-OH was added t a slutin f sdium dichrmate in dilute sulfuric acid (10%) and the slutin stirred at rm temperature vernight. The slutin was extracted with methylene chlride and the cmbined rganic extracts washed with sdium hydrxide slutin (1 M). The rganic fractin was dried ver magnesium sulphate, filtered and the slvent remved under reduced pressure. The residue was disslved in a minimum f chlrfrm and precipitated with ether. The precipitate was filtered and dried, yielding MePEG2000-acetic acid as a pale blue pwder (clur due t cmplexed chrmium). MePEG2000-A- POPE and MePEG200-A-DSPE were then prepared using the same prcedure as the succinate analgs, substituting MePEG2000-acetic acid fr MePEG2000- succinate. T synthesize MePEG2000-POPA, a mixture f 1-palmityl-2-leylphsphatidic acid (POPA), Me- PEG2000-OH and 2,6,6-triisprpylbenzenesulphrylchlride (TIPBSC) was suspended in pyridine. The reactin mixture was prtected frm light and allwed t stir vernight. Water was added and the mixture allwed t stir fr a further 3 h. The slutin was diluted with water, acidified and extracted with methylene chlride. After remval f the slvent, the residue was dispersed in water, filtered, centrifuged and the supernatant lyphilized. The resultant pwder was subjected t clumn chrmatgraphy using silica gel and methanl/chlrfrm. Pure fractins were cmbined, taken up in water, centrifuged and lyphilized t yield a white pwder. All lipids utilized in the abve synthetic prcedures were frm Avanti Plar Lipids. Unless indicated therwise, all ther materials were frm Sigma. Clumn chrmatgraphy was carried ut using silica gel 60 ( Mesh ASTM) (Merck). Analytical TLC emplyed aluminum backed silica gel 60-F254, 0.2 mm thick (Merck) and preparative TLC emplyed glass backed silica gel 60, 0.5 mm thick (Merck) Preparatin f large unilamellar vesicles (LUVs) The prductin f LUVs was carried ut as previusly described [20]. Briefly, lipid mixtures cmpsed f distearylphsphatidylchline (DSPC) and chlesterl (55:45, ml/ml), DSPC, chlesterl and MePEG-PE (50:45:5, ml/ml), r DSPC, chlesterl and GM1 (45:45:10, ml/ml), each with trace amunts f [14C]- r [3H]chlesteryl hexadecyl ether (CHE) as a nn-metablizable and nn-exchangeable lipsme marker [21] were freeze-dried frm benzene/methanl slutin and hydrated in physilgical sterile Hepes-buffered saline (HBS) (20 mm Hepes, 150 mm NaC1, ph 7.4). The sample was freeze-thawed five times and then extruded at 65 C ten times thrugh tw stacked 100 nm pre size plycarbnate filters (Cstar/Nuclepre, Canada) emplying an Extruder (Lipex Bimembranes, Canada). Lipsme size ranged frm 95 t 115 nm as determined by

4 24 M.J. Parr et al. / Bichimica et Biphysica Acta 1195 (1994) quasi-elastic light scattering n a NICOMP Mdel 270 submicrn particle sizer. The resultant LUVs were laded nt a cnventinal Bi-Gel A-15m ( mesh) (Bi-Rad, Canada) 10 x 1 cm clumn equilibrated with HBS t remve unincrprated MePEG- PE, and the pled lipsme peak diluted with HBS t an apprpriate cncentratin. All initial lipsme preparatins were checked fr cncentratin by determinatin f phsphrus [22] using a Shimadzu UVvisible recrding spectrphtmeter at 815 nm, and thereafter by scintillatin cunting using a Beckman LS3801 with Pic-Flur 40 scintillatin fluid (Packard). [14C]- and [3H]CHE was frm NEN/DuPnt, DSPC frm Avanti Plar Lipids, and chlesterl and ther chemicals were frm Sigma Exchange studies The MePEG-PE t lipsme radilabel ratis fr 5 mm (ttal lipid) preparatins incrprating either MePEGE000-[14C]S-POPE, [3H]MePEG2000-S-DSPE, r MePEGE000-s-[aH]DSPE were taken t determine the starting rati fr the exchange studies. Then 500 #l f the lipsme preparatin was diluted in 500 /.d f either HBS r nrmal muse serum (Cedar Lane, Canada). Fr MePEG2000-S-POPE, an additinal incubatin invlving 500 /xl f lipsmes with 200 /~l f muse serum and 300 /zl f HBS was carried ut. These mixtures were incubated at 37 C and at varius times, tw 50 /.d aliquts were remved and passed dwn 1 ml Bi-Gel A-15m spin clumns t separate lipsmes in the vid vlume frm serum and unincrprated MePEG-PE radilabel. (The use f these spin clumns t separate lipsmes frm serum cmpnents has been previusly described [23]). The peak tw lipsme fractins frm bth clumns were cunted and the MePEG-PE t lipsme radilabel ratis were determined by a crrected DPM dual label determinatin Chemical stability studies 5 mg f the MePEG-lipid indicated was disslved first in 200/.d f water, and then incubated in 1000/xl nrmal muse serum at 37 C. Additinal incubatins invlving 500 /~l f lipsmes (20 mm ttal lipid) cmpsed f DSPC/chlesterl/MePEG-lipid in 500 /zl serum at 37 C were als carried ut. At varius times, aliquts frm the micellar r lipsmal incubatins were remved and lipid cmpnents extracted by the fllwing prcedure. 80/~l f sample was added t 920/zl f water. T this was added 2.1 ml f methanl and 1.0 ml f chlrfrm. After mixing, 1.0 ml f water was added, vrtexed, and then an additinal 2.0 ml f chlrfrm was added. After thrugh vrtexing, the sample was allwed t sit fr 10 min befre centrifuging at 1500 x g fr 1 h. The rganic layer was islated, cncentrated, and then sptted n 0.25 mm thick, silica gel 60, 5 x 10 cm TLC glass plates (Merck). The slvent system used t develp the plates was chlrfrm/methanl (85:15, v/v), and were visualized with idine vapr. Where [3H]MePEGs000-S-POPE was used, 0.5 cm sectins f each running lane were scraped and extracted with 3 x 1.0 ml chlrfrm/ methanl/water (50 : 40 : 10, v/v). The extract was placed in scintillatin vials, slvent remved, 5.0 ml scintillatin fluid added, and left vernight befre cunting. Where MePEG200-S-PPBPE was used, flurescence assciated with scraped sectins was extracted as abve, slvent remved, and resuspended in 4.0 ml f 0.5% (w/v) sdium chlate detergent. Flurescence was read n a Perkin Elmer LS50 luminescence spectrmeter perating at 600 V using an excitatin wavelength f 339 nm (2.5 mm slit width), emissin wavelength f 377 nm (2.5 mm slit width), and filter set at 350 nm. TLC standards used t aid identificatin included the apprpriate free MePEG-OHs, extracted serum, free 4-(1-pyrenyl)butyric acid, and the apprpriate MePEG-lipids Bidistributin and circulatin lngevity studies The LUV preparatins emplying trace [3H]- r [14C]CHE as lipsme markers were injected via lateral tail vein in a vlume f 200/zl (1/zml ttal lipid) int 25 g CD-1 mice (Charles River, Canada). At varius times, the mice were sacrificed and bld withdrawn by cardiac puncture and cllected in micrtainer tubes with EDTA (Bectn-Dickinsn, Canada). After centrifuging at 1500 x g fr 10 min, the plasma was islated and shwed n hemlysis. Tw 100/zl samples frm each muse were cunted directly in 5.0 ml scintillatin fluid. The percentage recvery f lipsmes remaining in circulatin was based n a plasma vlume f 4.55% f individual muse bdy weight. Liver and spleen tissue were hmgenized by Plytrn t 20% and 10% in saline, respectively. 200/zl f tissue hmgenate was slubilized with 500 Izl Slvable (NEN/DuPnt) fr 2 h at 60 C, after which the samples were cled and treated vernight with 200 /.d hydrgen perxide. 5 ml scintillatin fluid was then added befre cunting. Liver and spleen assciated lipsmes are expressed as percent injected dse per tissue (ttal rgan weight). Where in viv exchange studies were carried ut, tw 50/zl aliquts f plasma were passed dwn spin clumns as described abve and the MePEG-lipid t lipsme rati determined.

5 3. Results 3.1. PEG2-S-POPE is lst frm the lipsme surface bth in viv and in vitr M.J. Parr et al. / Bichimica et Biphysica Acta s 1195 (1994) : 25 / / The first series f experiments were designed t ascertain the influence f MePEG200-S-POPE n the circulatin lifetimes f 100 nm diameter DSPC/chlesterl (55:45 ml/ml) LUVs in mice. As shwn in Fig. 2, the incrpratin f 5 ml% MePEG2000-S- POPE results in a relatively mdest increase in LUV circulatin lifetimes. At 24 h; the LUV preparatin incrprating PEG-POPE is almst cmpletely cleared. This may be cntrasted with previus reprts [11] that incrpratin f 5 ml% PEG-PE can result in up t 30% f DSPC/chlesterl LUVs remaining in the circulatin at 24 h, and the third curve where lipsmes incrprating MePEG2000-S-DSPE have much greater circulatin levels (ver 20% at 24 h). In rder t determine whether this culd be due t the lss f the PEG cating arising frm interactins with serum prtein factrs, the DSPC/chlesterl LUVs (labelled with [3H]CHE as a nn-exchangeable lipsme marker) incrprating MePEG2000-[14C]S-POPE were incubated with nrmal muse serum at 37 C and the retentin f radilabel mnitred. As shwn in Fig. 3A, this incubatin results in rapid lss f the MePEG-PE radilabel. While the MePEG-PE cntent f the LUVs is relatively unaffected when incubated in HBS alne, the MePEG-PE radilabel rapidly drps when incubated in the presence f either 20% r 50% serum. After 24 h in 50% serum, the MePEG-PE t lipsme marker rati has drpped t nearly 50% f its initial value, suggesting that practically all f the MePEG-PE 100 e 80 ~.g_ g 60 'u " '~ 40 ~.-~.E 20,T Time (hurs) Fig. 2. Circulatin lifetime f DSPC/chlesterl/MePEG2-S- POPE lipsmes. Large unilamellar vesicles cmpsed f DSPC/chlesterl (55:45) (), DSPC/chlesterl/MePEG200-S- POPE (50: 45:5) (e), (r DSPC/chlesterl/MePEGz000-S-DSPE (50: 45 : 5) ( ), dashed line, taken frm Fig. 6A) were injected via the lateral tail vein int 25 g CD-1 mice (5 mm ttal lipid in 200/.d, 1 /zml ttal lipid/muse). At varius times, the mice were sacrificed and plasma islated. The marker [3H]CHE was used t determine lipsme recvery ([14C]CHE fr ( )). Results shwn represent the mean + S.E. f fur animals v.0...,,,,... ~n~ s, is = =s 60 ~ s U.I a. I,iJ el C 100; FRACTION # 0 4 a (7d) C Time (hurs) Fig. 3. Lss f PEG cating frm the surface f the LUV. (A) In vitr incubatin f DSPC/chlesterl/MePEG2000-[t4C]S-POPE (50:45:5, ml/ml) large unilamellar vesicles in nrmal muse serum at 37 C. 500/zl f the LUV preparatin (5 mm ttal lipid) was incubated in the presence f 500 ~1 HBS (e), 200/~1 serum plus 300 /~1 HBS (A), r 500/~1 serum (1), representing 0%, 20%, and 50% serum cncentratins, respectively. At varius times, tw 50 /zl aliquts f the incubatin mixture were remved and lipsmes separated frm the free PEG-PE and serum cmpnents by lading nt a 1 ml Bi-Gel A-15m spin clumn. The 14C (PEG-PE)/3H (CHE, lipsme marker) ratis were btained frm the peak tw lipsme fractins frm bth clumns and expressed as a percentage (+S.D.) f the initial rati befre incubatin. (B) Bi-Gel A-15m ( mesh) spin clumn prfile f DSPC/chlesterl/Me- PEG200-[~4C]S-POPE LUVs in 50% serum at 37 C fr 24 h. ( ), the 3H DPM (lipsme) label, and (n), the 14C DPM (PEG-PE) label measured fr 30/zl f each cllected fractin. (C) The in viv lss f the PEG-PE cating frm injected lipsmes. The 14C (PEG-PE)/aH (CHE) rati was determined fr lipsmes recvered frm mice injected with DSPC/chl/PEG-PE lipsmes bth befre ( ) and after ( ) separatin f lipsmes frm plasma cmpnents via spin clumn. Results represent the means f 14C (PEG-PE)/aH (CHE) ratis btained frm the fur mice befre, r after the peak tw lipsme fractins frm tw clumns fr each muse plasma sample, and expressed as a percentage (+S.D.) f the initial rati befre injectin. radilabel in the uter mnlayer has been remved. This is supprted by the results f Fig. 3B, which shws the spin clumn prfile fr the 24 h time pint. There is an exact verlap f the MePEG-PE and lipsmal peaks, and gd separatin frm the MePEG-PE n lnger assciated with the lipsmes. Integratin f the tw peaks yields a 52:48 rati fr lipsme assciated t free MePEG-PE. After an additinal 6 days at rm temperature in the 20% incubatin, this rati was als

6 26 M.J. Parr et al / Bichimica et Biphysica Acta 1195 (1994) nearly 50%. Given ur data, it is nt unreasnable t assume that little mre than 50% f the label is available fr exchange. In additin, studies with Me- PEG1900-carbamate-DSPE at 4 ml% indicate that the PEG extends utward frm the surface in a brush frmatin appximately 5 nm [14]. Althugh direct evidence is lacking, it has been suggested that the distributin shuld be apprximately equal between the tw leaflets f the bilayer as lng as the radius f the vesicle is large (greater than a factr f 10) relative t the length f the plymer [15] as is the case here. The in viv exchange results presented in Fig. 3C cnfirm that the MePEG-PE is lst frm the surface f the injected LUVs. Here, plasma samples were taken at varius times frm mice which had received [3H]CHE labelled lipsmes incrprating Me- PEG2000-[~4C]S-POPE and were cunted t cmpare the MePEG-PE t lipsme ratis bth befre and after separatin f the lipsmes frm the plasma. In agreement with the in vitr data, this rati drps significantly with time indicating rapid lss f the Me- PEG-PE. The differences in the ratis fr unseparated and separated lipsmes can be attributed t Me- PEG-PE which is n lnger assciated with the lipsmes but which cntinues t circulate fr a shrt perid f time and t the shrt time required t islate plasma and elute the spin clumns. The level f Me- PEG-PE still assciated with the lipsme appraches 50% by 8 h, and by 24 h has cmpletely leveled ff. The faster rate f lss f this cmpnent frm the uter mnlayer in viv versus in vitr is expected given the greater plasma/serum t lipsme rati in viv. It wuld als appear that the rate f lss f MePEG-PE is faster than the clearance rate f the LUVs initially cntaining this cmpnent. If nly apprximately 50% f the label is available fr exchange, label lss leads clearance by a significant amunt. Fr example, at 1 h pst injectin 76% f the vesicles remain in the bld whereas, after serum remval, 75% f the label remains. This indicates that 50% f the available exterir label, then 50% f the uter label has been lst and 24% f the vesicles have been cleared. At 4 h, by the same arguments, 75% f the uter label has been remved while 45% f lipsmes have been cleared. Thus, at these early time pints uter label remval leads clearance by 25-30%. It is als f interest t cmpare the clearance rate t the cntrl. One can see that when at least sme prprtin f the PEG cating remains, these lipsmes have a slwer clearance rate than the cntrl. Hwever, beynd sme critical value f PEG lss between 4 and 8 h after which there is very little PEG remaining n the surface, the clearance f these lipsmes quickly appraches that f the cntrl Chemical stability f varius linker grups in the MePEG-PE cnjugate There are tw pssible mechanisms fr the lss f the MePEG-PE radilabel frm the LUVs in vitr and in viv. These are cleavage f the MePEG miety frm the lipid anchr r exchange f the entire MePEG-PE ut f the LUV. Chemical breakdwn was mnitred by thin-layer chrmatgraphy (TLC) after incubatin f MePEG-PE micelles in nrmal muse serum. The data f Fig. 4 shws the effect f different chemical linkages between MePEG and the lipid anchr n the chemical stability f MePEG-PE. All fur versins a b c d Fig. 4. Thin-layer chrmatgraphy f the results f incubatin f micellar PEG-PE in serum at 37 C. (a), MePEG200-S-POPE; (b), MePEG2-C-POPE; (c), MePEGE00-A-POPE; (d), MePEG2000-POPA. 5.0 mg f the PEG-PE indicated was first disslved in 200/zl f water, then incubated with an additinal 1000/zl f nrmal muse serum at 37 C. At varius times, 80/zl aliquts were withdrawn, extracted, and cncentrated. The samples were run n 0.25 mm silica plates develped with chlrfrm/methanl (85 : 15, v/v) and spts visualized with idine vapr. Lanes were: (1), befre incubatin; (2), 5 min incubatin; (3), 1 h; (4), 4 h; (5), 8 h; (6), 24 h. (e) shws TLC standards nrmal muse serum extract (7) and free MePEGz0-OH (8). 78 e

7 M.J. Parr et al. / Bichimica et Biphysica Acta 1195 (1994) degrade significantly after expsure t serum at 37 C. The succinate linked versin rapidly generates a cmpund which crrespnds t free MePEG, while the ther three (carbamate, amide and direct linked) versins shw very little f this prduct. The breakdwn f the succinate ester linkage is perhaps nt unexpected given a similar phenmenn bserved in the crrespnding MePEG-prtein cnjugates [24], hwever, this has nt been previusly reprted fr PEGlipid cnjugates. In additin, all fur versins shw the appearance f a prduct which is slightly mre plar than the starting MePEG-lipid, which in turn is brken dwn t yield a prduct with further increased plarity by 24 h. When these fur cmpunds were incrprated int lipsmes, incubatin in serum yielded the same patterns f breakdwn prducts, but the rates at which these prducts are frmed was significantly slwer (results nt shwn). Additinal experiments were perfrmed t characterize the breakdwn prducts. The [3H]MePEGs000- S-POPE was incubated in micellar frm in serum as described abve, and the TLC lanes were cunted fr radiactivity. The results (nt shwn) cnfirmed that the apparent free MePEG spt cntained [3H]Me- PEGs00-OH. When MePEG2000-S-(1-palmityl-2-(4- pyrenyl)-butyryl)pe (MePEG20-S-PPBPE) was incubated in serum, flurescence (due t the pyrene miety) was bserved in the mre plar spts as well as the spt crrespnding t free pyrene butyric acid (data nt shwn). This suggests that the mre plar spts are lys-mepeg-lipid cmpunds which have had ne r bth f the acyl chains cleaved frm the cnjugate. In additin, heat inactivatin f serum r additin f EGTA was fund t significantly reduce the rate f breakdwn. As cntrls, extractin efficiencies frm serum r buffer ver all time pints and cnditins were checked and fund t be cnsistently abve 90%. All versins f the MePEG-lipid cnjugates remain relatively stable in HBS (ph 7.4) ver 24 h, althugh the succinate versin did shw sme slw breakdwn. Lwer ph values (ph 2) resulted in increased appear- ill '= m ~ 0..,... i...,,,, Time (hurs) Fig. 5. In vitr incubatin in nrmal muse serum at 37 C f DSPC/chlesterl large unilamellar vesicles incrprating 5 ml% MePEG200-S-DSPE. 500/.d f the LUV preparatins (5 mm ttal lipid) incrprating [3H]MePEG200-S-DSPE (circles) r Me- PEG20-S-[3H]DSPE (squares) was incubated in the presence f 500 p.l HBS (pen symbls), r 500 ~1 serum (clsed symbls). At varius times, tw 50-/~1 aliquts f the incubatin mixture were remved and lipsmes separated frm free cmpnents by lading nt a 1 ml Bi-Gel A-15m spin clumn. The 3H (PEG-PE)/14C (CHE) ratis were btained frm the peak tw lipsme fractins frm bth clumns and expressed as a percentage (+ S.D.) f the initial rati befre incubatin ance f lys cmpunds, prbably due t acid catalyzed hydrlysis at the sn-1 and sn-2 psitins [21] PEG2-DSPE is retained in DSPC/chlesterl L UVs and exhibits enhanced chemical stability The chemical breakdwn f MePEG-lipids can ccur either n the surface f the lipsme r after exchange f the whle mlecule ut f the LUV. In rder t reduce the pssibility f exchange, Me- PEG200-PEs were synthesized with a DSPE anchr which may be expected t result in imprved retentin. The exchange f either [3H]MePEG200-S-DSPE r MePEG2000-S-[aH]DSPE frm [14C]CHE labelled LUVs in serum r HBS at 37 C is illustrated in Fig. 5. The acyl chain label remains assciated with the lipsme in bth HBS and in serum. Hwever, the MePEG ~ O Table 1 Bidistributin f DSPC/chlesterl large unilamellar vesicles incrprating GM1 r PEG-PE 24 h after injectin Lipsme cmpsitin (mlar rati) % f injected dse recvered per ttal tissue bld liver spleen DSPC/chl DSPC/chl/GM1 DSPC/chl/MePEG200 S-POPE DSPC/chl/MePEG2-A-POPE DSPC/chl/MePEG20S-DSPE DSPC / chl /MePEG 2-A-DSPE (55 : 45) : t (45 : 45 : 10) 11.3 _ :0.09 (50 : 45 : 5) (50 : 45 5) (50 : 45 : 5) 16.3:1: (50 : 45 : 5) The 5 mm 100 nm LUV preparatins were injected via lateral tail vein in a vlume f 200/~1 (1/zml ttal lipid) int 25 g CD-1 mice. At 24 h, the mice were killed and plasma, liver, and spleen islated. The percentage recvery f lipsmes remaining in circulatin was based n a plasma vlume f 4.55% f individual muse bdy weight Lipsmes assciated with liver and spleen tissues were determined based n ttal rgan weight Each preparatin emplyed trace [3H]CHE as a lipsme marker, and the results represent the mean f fur animals + S.E.

8 28 M.J. Parr et al. / Bichimica et Biphysica Acta 1195 (1994) leaves the lipsme t a measurable extent, resulting in apprximately 7% lss in HBS ver 24 h. In serum, the lss f this label is smewhat greater, up t apprximately 12% lss ver 24 h, althugh this rate is much reduced cmpared t MePEG2000-S-POPE. Thus, the results f Fig. 5 demnstrate that a primary factr fr retentin f the MePEG cating is the lipid anchr and that these cmpunds are relatively chemically stable if they remain assciated with the lipsme. Hwever, it als appears that slw hydrlysis f the succinate bnd can ccur n the lipsme surface leaving the lipid anchr behind. The ability f MePEG200-S-DSPE, when incrprated int DSPC/chlesterl LUVs, t prlng the circulatin lifetime is significantly imprved ver that bserved fr MePEG2000-S-POPE. Using [14C]CHE labeled lipsmes incrprating [3H]MePEG200-S-DSPE r MePEG2000-S-[3H]DSPE, apprximately 20% f the injected dse remains in the circulatin at 24 h as shwn in Fig. 6, with the tw preparatins exhibiting very similar clearance behavir. When the 3H//14C rati was checked at varius times bth befre and after separatin f lipsmes frm plasma cmpnents (Fig. 6B and 6C), bth the MePEG and acyl miety labels f the MePEG2000-S-DSPE are shwn t remain assciated with the lipsme. It may, hwever, be mre accurate t say that the lipsmes recvered, which are representative f thse still in circulatin, have retained mst f their PEG cating. It is prbable that lipsmes which have lst their prtective cating wuld have been rapidly cleared Bidistributins f DSPC/chlesterl LUVs cnmining different species f MePEG2-PE The final series f experiments were perfrmed t characterize the bidistributin at 24 h fr DSPC/ chlesterl LUVs incrprating varius species f MePEG200-PE varying in acyl chain cmpsitin r PEG-PE chemistry at a lipsme dse level f 1/~ml ttal lipid per muse. The results are shwn in Table 1. In the absence f a PEG-PE cating, DSPC/chlesterl LUVs are almst cmpletely remved frm the circulatin at 24 h (less than 1% f the injected dse remains), with high levels accumulated in the liver and spleen. The incrpratin f 10 ml% GM1 significantly increases the circulatin levels t apprximately 11% f the injected dse remaining while decreasing the amunt fund in the liver and spleen at 24 h by factrs f 2 and 4, respectively. Incrpratin f 5 ml% MePEG200-S-POPE and MePEG20-A-POPE have smaller effects than GM1 in altering the LUV bidistributin. Hwever, the presence f either MePEGz00- S-DSPE r MePEG20-A-DSPE greatly increased the circulatin levels present at 24 h t almst 20%, higher than achieved with GM1, while the accumulatin by the i -==i c ~ SO ~, 2O O 7 " ~ a 90 g ~. i * I... I i i * I * k * I... FII,~: B --I O C T....,..., Time (hunl) Fig. 6. Circulatin lifetime f DSPC/chlesterl/MePEG2-S- DSPE lipsmes and in viv exchange f MePEG200-S-DSPE frm injected lipsmes. (A) Circulatin lifetime. Large unilamellar vesicles cmpsed f DSPC/chlesterl/[aH]MePEGE-S-DSPE (50:45:5) ([:3) r DSPC/chlesterl/MePEG200-S-[aH]DSPE (50: 45:5) (Ill) were injected via lateral tail vein int 25 g CD-1 mice (5 mm ttal lipid in 200 /zl, 1 ~ml ttal lipid/muse). At varius times, the mice were sacrificed and plasma islated. The marker [IaH]CHE was used t determine lipsme recvery. Results shwn represent the mean 5: S.E. f fur animals. (B) The 3H/14C rati fr plasma islated frm A befre separatin f lipsmes frm plasma cmpnents via spin clumn. (O), [3H]MePEG2-S-DSPE, and (I), MePEGE-S-[3H]DSPE preparatins. Results represent the means f ratis btained frm the fur mice and expressed as a percentage (+S.D.) f the initial rati befre injectin. (C) The 3H/14C rati fr plasma islated frm (A) after separatin f lipsmes frm plasma cmpnents via spin clumn. (O), [3H]Me- PEG200-S-DSPE, and (ll), MePEGE-S-[3H]DSPE preparatins. Results represent the means f ratis f the peak tw lipsme fractins frm tw spin clumns frm fur mice, and expressed as a percentage (± S.D.) f the initial rati befre injectin. liver and spleen is reduced t an equal r better extent as GM1. While there is little difference in bidistributin behavir between the succinate and amide versins fr MePEG-POPE, the data fr DSPE anchred species suggest that the amide linkage may be slightly superir in bth imprved circulatin lifetimes and reduced liver and spleen uptake.

9 M.J. Parr et al. / Bichimica et Biphysica Acta 1195 (1994) Discussin The use f lipsmes as drug delivery vehicles depends upn their ability t remain in circulatin fr extended perids f time. The incrpratin f PEGlipids clearly allws extended circulatin lifetimes t be achieved. Hwever, the results presented here emphasize tw majr pints. First, relatively subtle changes in the acyl chain cmpsitin f the PE anchr can significantly influence retentin f the PEG-PE in the uter mnlayer f the lipsme. Secnd, significant chemical breakdwn f PEG-PE cnjugates may ccur, particularly after the PEG-PE is lst frm the LUV surface. The influence f acyl chain cmpsitin n PEG-PE retentin and related clearance behavir is particularly prfund. As shwn here, when MePEG2000-S-POPE is incrprated int DSPC/chlesterl LUVs, the circulatin lifetime is nly mdestly increased. This increase is less than that reprted fr MePEG200-DSPE [11,18], but cmparable t ther studies using MePEG200- DOPE [25]. The pr perfrmance f PEG-POPE is due t rapid remval f the exterir PEG cating, with a half-time f apprximately 2 h in vitr (50% muse serum at 37 C) and apprximately 1 h in viv. This may be cmpared with the rate f clearance f the injected LUVs, which exhibit a half-life in the circulatin f apprximately 5 h. The fact that lss f the hydrphilic cating precedes lipsme clearance suggests that lss f the PEG cating hastens clearance. As shwn here, the lss f the PEG-POPE cating is primarily due t exchange f the entire PEG-POPE mlecule ut f the external mnlayer. The use f DSPE as the lipid anchr in place f POPE results in a dramatic imprvement n the retentin f the PEG cating. When MePEG200-S-DSPE is incrprated int LUVs and incubated in 50% muse serum, apprximately 90% f the PEG-PE remains assciated with the LUVs after 24 h. The DSPE anchr als exhibits markedly superir prperties in viv. The circulatin half-life f LUVs incrprating MePEG2000-S-DSPE is apprximately 10 h, with ver 20% f the injected dse remaining in circulatin at 24 h. In additin, the LUVs recvered frm the circulatin even up t 24 h shw n exchange r breakdwn f the MePEG200-S-DSPE, althugh any lipsmes which have lst their PEG cating wuld likely have been cleared frm the circulatin. Previus wrk [26] examining the intervesicular exchangeability f several PEG-lipids in vitr shwed that transfer f saturated diacyl cnjugates f Me- PEG20 decreased expnentially with increasing chain length. In additin, transfer f POPE derivatives f MePEG20 and MePEGs00 was fund t be fld slwer than the crrespnding DMPE derivatives. Thus, the increase in LUV retentin in vitr and in viv between POPE and DSPE anchrs bserved here is nt unreasnable. The chemical stability studies n pure (micellar) PEG-lipids indicate that the succinate linkage is labile in muse serum, generating free MePEG-OH by 1 h with cmplete hydrlysis f this linkage within 24 h. A variety f ther linkages prved t be mre stable in this respect, including carbamate, amide, and direct linkages. The results presented here als indicate that the succinate bnd is prtected by retentin f the PEG-lipid in the LUV. Fr MePEG2000-S-DSPE, a slw lss f the PEG headgrup as cmpared t the lipid anchr (which is cmpletely retained) indicates that it is pssible t remve the PEG frm the LUV surface and leave the lipid anchr behind; hwever, this effect is small cmpared t lss f the POPE anchred versin which is almst cmpletely remved frm the uter mnlayer within several hurs. The ratinale fr the use f PEG-lipids is their ability t significantly reduce the rate f clearance f lipsmes frm the circulatin. At a practical level, the results presented here demnstrate that chemical stability and lipid anchring ability are determining factrs fr the ability f PEG-lipids t prvide imprved circulatin lifetimes fr LUVs in viv. While it has been reprted that the nature f the anchr f PEGlipids influences the circulatin lifetimes f injected lipsmes [11] and that the LUV lipid cmpsitin can als affect the circulatin lifetimes f lipsmes incrprating PEG-PEs [27,28], thers suggest that different PEG anchrs are equivalent and that the lipid cmpsitin may be varied with little effect if PEG-PEs are incrprated [18]. Our results cnclusively demnstrate the imprtance f the PEG-lipid anchr. In summary, the lipid anchr is a primary factr in the retentin f a PEG plymeric cating fr LUVs. Chemical breakdwn f the cnjugate largely ccurs after exchange ut f the LUV but can ccur n the LUV surface depending upn the PEG-lipid linkage. The use f a strng membrane anchr (DSPE) and chemically stable cnjugate bnd (amide) results in retentin f the plymeric cating and greatly enhanced circulatin lifetimes. We cnclude that since the majr ratinalizatin fr the use f PEG-lipids in lipsmes is the prlnged circulatin lifetimes and hence the greater chance f accumulatin in targets ther than the RES, by whatever detailed mechanisms PEG-PE is prpsed t wrk, this requires that the PEG cating is retained and shuld be a cnsideratin in any practical discussin f the use f PEG-lipids. Acknwledgments We wish t thank Dana Masin fr her assistance with the animal experiments. This wrk was supprted by The Natinal Cancer Institute f Canada.

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