Biochimica et Biophysica Acta Received 21 May 1996; revised 18 September 1996; accepted 24 September 1996

Transcription

1 Ž. Biochimic et Biophysic Act Phse behvior of fluorocrbon di-o-lkyl-glycerophosphocholines nd glycerophosphoethnolmines nd long-term shelf stbility of fluorinted liposomes Veronique Rvily, Ctherine Sntell, Pierre Vierling,), Annette Gulik b Lbortoire de Chimie Moleculire, URA CNRS 426 nd Lbortoire de Chimie Bioorgnique, EP CNRS 104, Fculte des Sciences, UniÕersite de Nice-Sophi Antipolis, B.P. 71, Nice Cedex 2, Frnce b Centre de Genetique Moleculire, CNRS, Gif sur YÕette, Frnce Received 21 My 1996; revised 18 September 1996; ccepted 24 September 1996 Abstrct This pper is devoted to the morphologicl chrcteriztion, by freeze-frcture electron microscopy, nd to the thermotropic phse behvior, by differentil scnning clorimetry nd X-ry diffrction of the queous dispersions of vrious fluorocrbonrfluorocrbon or mixed fluorocrbonrhydrocrbon 1,2- or 1,3-di-O-lkyl-glycerophosphocholines Ž PC. nd 1,2-di-O-lkyl-glycerophosphoethnolmines Ž PE.. The fluorinted PCs form clssicl lmellr phses nd liposomes while n interdigitted lmellr phse hs been evidenced for hydrocrbon 1,3-nlog. The fluorinted PEs disply lmellr to hexgonl phse trnsition which occurs lmost simultneously with the gel-to-fluid lmellr phse trnsition. The impct of ech of the structurl fetures wether vs ester chemicl junction connecting the hydrophobic chins on glycerol, their position Ž 1,2- vs 1,3 isomers., number nd length of the perfluorolkylted chins, length of the fluorinted til nd hydrocrbon spcer, PC vs PE polr hedx of the fluorinted phospholipids on the phse trnsition thermodynmic prmeters Ž Tc, DH, DS. is discussed. Most of the liposomes formed from the fluorinted ether-pcs disply remrkble long-term shelf stbility: they cn be thermlly sterilized nd stored t room temperture for severl months without ny significnt modifiction of their size nd size distribution. Keywords: Perfluorolkylted phospholipid; Fluorinted liposome; Vesicle; Membrne; Polymorphism; Shelf-stbility 1. Introduction Liposomes Ž vesicles mde from phospholipids. provide chllenging in vivo delivery systems for enhncing the efficcy of vrious biologiclly ctive mterils w1,2 x, reducing the toxicity of vrious nti- ) Corresponding uthor. Fx: q E-mil: vierling@unice.fr tumor gents, fcilitting intrcellulr drug delivery, incresing the tumoricidl ctivity of mcrophges nd enhncing the immune response nd thus serve s rtificil vccines wx 1. Much effort hs been focused in more recent yers on liposomes with incresed residence in the bloodstrem Že.g., stelth liposomes. w2,3 x. Progress in this field hs llowed specific drug trgeting to cells nd orgns w1 3 x. The elbortion of liposoml systems with new or significntly improved properties implies the devel r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. Ž. PII S

2 2 V. RÕily et l.rbiochimic et Biophysic Act opment of components tht re substntilly different from those currently utilized. Highly fluorinted phospholipids Ž Fig. 1. re such components: they offer some of the specific fetures tht mke up the uniqueness of fluorinted mteril, e.g., their hywx 4. The fluori- drophobic nd lipophobic chrcter nted DFnCmPC phosphtidylcholines ŽFig. 1, wx. 5 were shown to form liposomes w6 8 x, their fluorinted tils creting inside the liposoml membrne highly hydrophobic nd lipophobic fluorocrbon lyer. This lyer modifies substntilly the phse behvior chrcteristics wx 7 nd dynmics wx 9, reduces membrne permebility w10 x, increses the stbility of the liposomes in biologicl medi Žin terms of relese Fig. 1. Chemicl structure nd code nme of the vrious fluorocrbon 1,2- nd 1,3-di-O-lkyl-Ž ether-connected. glycerophosphocholines nd glycerophosphoethnolmines together with those of the vrious fluorocrbon di-o-cyl-ž ester-connected. -glycerophosphocholines nd hydrocrbon nlogs used s references throughout this study.

3 V. RÕily et l.rbiochimic et Biophysic Act of encpsulted mteril. w10x nd, importntly, prow11 x. In order to further ct on nd modulte the hy- longs their in vivo blood circultion times drophobic-lipophobicrlipophilicrhydrophilic blnce, nd consequently, the physico-chemicl nd biologicl properties of fluorinted membrnes nd liposomes Žthermotropism, lipid mobility, order nd pcking, permebility, stbility in biologicl fluids, interctions with bio-compounds, in vivo fte., we hve recently extended the rnge of fluorinted phospholipids by performing the synthesis of vrious perfluorolkylted 1,2- nd 1,3-di-O-lkyl Žether-con- nected. glycerophospholipids Ž Fig. 1. w12 x. Their moleculr structures follow modulr design, which llows incrementl structurl vritions imed t the estblishment of structurerproperties reltionships. This design involves two hydrophobic chins, one Ž IIb nd IIIb. or both Ž I, IE, II, III nd IV. of which end in highly fluorinted til, in combintion with sturted Ž I, II, IIbE nd III IV. ndror unsturted Ž IE nd IIbE. liphtic chins connected through glycerol to phosphocholine Ž PC. ŽI III. or phosphoethnolmine Ž PE. Ž IV. polr hed groups. This pper is devoted to the polymorphic phse behvior of these perfluorolkylted di-o-lkylglycerophospholipids in wter. Such study is prerequisite when their use s liposoml components is intended. The morphologicl chrcteriztion of the suprmoleculr structures they form nd their thermotropic phse behvior ws performed by freeze-frcture electron microscopy Ž FFEM. nd by differentil scnning clorimetry Ž DSC. or X-ry diffrction, respectively. The impct of ech of the structurl fetures which composes the moleculr structure of the fluorinted phospholipids on the phse trnsition thermodynmic prmeters is furthermore discussed. In ll cses, the experimentl dt obtined re compred with those reported for their fluorinted ester Ž DFnCmPC., hydrocrbon ester nd ether nlogs Ž Fig. 1.. In ddition, the size nd size distribution of the liposomes they form nd their long-term shelf stbility with respect to prticle size evolution fter stndrd het-steriliztion process nd storge t room temperture Žnlyzed by light scttering spectroscopy, LSS. re lso described. These re key issues when the use of liposomes s drug crrier nd delivery devices is contemplted. 2. Mterils nd methods The synthesis nd chrcteriztion Želementl nl ysis, H, C, P, F NMR. of the fluorinted ether-connected glycerophospholipids Ž Fig. 1. used in this study re described in Ref. w12 x. Their purity Ž )99%. ws periodiclly checked by TLC. Rc-diplmitoylphosphtidylcholine Ž DPPC, 99%., egg phosphtidylcholines Ž EPC. nd cholesterol Ž CH. were supplied by Fluk, Lipoid nd Sigm, respectively, nd used without further purifiction. Dispersions of the lipids were prepred with Brnson B-30 sonifier. Alterntively, extrusion through polycrbonte membrne ŽLiposofst Milsch Equipment; size filter 100 nm. ws lso used to prepre liposomes. Prticle sizes nd size distributions were determined by LSS on Coulter N4SD Sub-Micron Prticle Anlyser Preprtion of the dispersions nd liposomes For the observtion by opticl microscopy with polrized light Ž Olympus BH2., the smples were prepred by dispersion Ž vortex. of the lipid in excess wter t room temperture, then plced onto the microscope between two glsses which were heted progressively. For the FFEM studies, unilmellr vesicles Žcon- centrtion of phospholipid 30 mm. were prepred, by soniction or by extrusion, in phosphte-buffered sline Ž PBS, BioMerieux. s described in Ref. w10x nd were ged for h t temperture 5 108C bove the phse trnsition temperture, Tc, detected by DSC, of the phospholipid under investigtion. For the stbility studies, 5-ml smples Ž 10 mm. were prepred by soniction or extrusion, then sterilized by utoclving the dispersions for 15 min t 1218C under pressure of 15 lbrsq.in in n oscillting utoclve Ž Subtil, Crepieux. nd stored t 25 Ž "1. 8C. Averge vesicle size mesurements were performed by LSS fter preprtion, fter het-steriliztion, then periodiclly during storge Freeze-frcture electron microscopy FFEM The dispersions were prepred s described bove nd stored t room temperture for three dys before nlysis. To prevent freeze dmge, the smples were

4 4 V. RÕily et l.rbiochimic et Biophysic Act diluted with n equivlent volume of n queous solution of glycerol Ž 50% wrw. nd incubted t room temperture for 4 h before cryofixtion. A smll drop of ech suspension Ž bout 0.5 ml. ws deposited on thin copper holder nd then quenched in liquid propne. The frozen smple ws frctured t Ž y7 y1258c in vcuo bout 10 Torr. with liquid nitrogen-cooled knife in Blzers 301 freeze-etching unit. The frctured smple ws replicted with pltinum-crbon Ž nm of metl deposit. bcked with bout 20 nm of crbon nd the replic ws clened with orgnic solvents, wshed with distilled wter nd observed with Philips 301 electron microscope. For ech smple, two or three independent freezings, frctures nd replics were relized Differentil scnning clorimetry DSC For DSC mesurements in the q4 to958cnd y25 to q48c temperture rnge, the smples were prepred by weighing the powdered phospholipid Ž 2 3 mg. into the DSC inox pn nd dding weighed mount of deionized wter or of n ethyleneglycolrwter Ž 70r30 wrw. solution Ž 4 6 mg., respectively. DSC mesurements were crried out with Perkin Elmer DSC-2 nd DSC-7 differentil scnning clorimeters. Ech smple ws heted nd cooled repetedly t rte of 2 58Crmin nd 108Crmin respectively. Trnsition enthlpies nd tempertures were determined fter severl hetingrcooling cycles. The reported vlues of the Fig. 2. A D: Freeze frcture electron microgrphs of 4% wrw queous liposoml dispersions of Ž A. wf8e11xwc16xopc, Ž B. 1,3-wF6C11xwC18xOPC, Ž C. wf4c11xwf8c11xopc nd Ž D. DF6C11OPC showing the simultneous presence of ribbon-like elements nd vesicles. Scle br is 200 nm.

5 V. RÕily et l.rbiochimic et Biophysic Act Tc represent the temperture t mximum excess het cpcity X-ry scttering The smples were prepred by mixing the lipid nd excess wter. In order to obtin the best hydrtion of the lipids, it ws performed by incubting the resulting suspensions t 708C for DF4C11OPE nd 908C for DF6C11OPE. The smples were then mounted in vcuum-tight cells with mic windows. The Debye-Scherrer spectr were recorded using Guinier-type focussing cmer with bent qurtz monochromtor. The spectr were recorded t selected tempertures or continuously s function of temperture. 3. Results nd discussion 3.1. FFEM nd LSS studies of the fluorinted phospholipid dispersions As shown by FFEM Ž Fig. 2A C., the queous dispersions of DF8C5OPC Ž not shown., wf4c11xwf8c11xopc, wf8e11xwc16xopc nd 1,3- wf6c11xwc18xopc performed by extrusion or soniction consist minly in unilmellr vesicles. Ech of these compounds being representtive of distinctive fmily of the fluorinted ether-connected PCs, it ppers tht formtion of liposomes is neither ffected by the number Ž one or two. of fluorinted chins nor by the connection in the 1,2- or 1,3-position on glycerol of the two hydrophobic chins. The sme dispersions were lso nlyzed by LSS. The vesicles of DF8C5OPC obtined by soniction, hd n verge prticle size of 50 nm nd were polydisperse. By contrst, the liposomes formed by ech of the other fluorinted lipids prepred by extrusion through 100-nm polycrbonte membrne, hd homogeneous prticle size distribution centered round 100 nm, in greement with FFEM Žsee Fig. 2A C.. By contrst to the bove mentioned dispersions, those of DF6C11OPC, DF8C11OPC, 1,3- DF6C11OPC or 1,3-DF8C11OPC were shown to consist in vesicles which slowly convert into ribbon-like phse t room temperture. The coexistence of vesicles with this phse is illustrted in Fig. 2D for DF6C11OPC. Such ribbon phse hs lredy been observed by FFEM nd confirmed by X-ry diffrction for the DF6C11PC nd DF8C11PC ester nlogs wx 7. It should be mentioned tht this phse is only observed for phosphocholines hving fluorinted til on both hydrophobic chins nd ech chin hving t lest 17 crbon toms. Indeed, ribbon phse is neither found for wf4c11xwf8c11xopc, wf8e11xwc16xopc nor for 1,3-wF6C11xwC18xOPC which hs two chins equl to or longer thn 17 crbon toms. Ž. Fig. 3. Freeze-frcture imge of 4% wrv viscoelstic gel viscous solution obtined by cooling to room temperture n queous liposoml dispersion of 1,3-DC18OPC. Scle br is 500 nm.

6 6 V. RÕily et l.rbiochimic et Biophysic Act The fluorinted 1,3-di-O-lkylglycero-2-phosphocholines which form vesicles ndror ribbon phse disply behvior which differs from tht observed for their hydrocrbon nlog 1,3-DC18OPC. After soniction t temperture bove its phse trnsition temperture, 1,3-DC18OPC forms trnslucent dispersion consisting in liposomes which leds to viscoelstic gel Ž viscous solution. fter cooling to room temperture Ž replic of this dispersion is shown in Fig. 3.. The formtion of gel nd its ppernce on the microgrphs seems to be the result of some fusion of the liposomes. This fusion my be relted to the low stbility of the rther smll-sized vesicles ndror to the trnsition from conventionl lmellr ŽL b or L b X. to n interdigitted lmellr phse Ž L b i.. Upon cooling, wheres prt of the chins remins non-interdigitted, nother prt interdigittes s evidenced by the FFEM imges which show both usul frctures long the mid-plne of the membrne Ž convex nd concve liposomes. nd cross-frctures, respectively. The formtion of gel interdigitted lmellr L b i phse in excess of wter for 1,3-DC18OPC hs been further confirmed by X-ry diffrction w13x nd is in line with the phse behvior of other ether-linked glycerophosphow14 x, or of the 1,3-iso- cholines, such s DC16OPC mer of the ester-linked DPPC w15 x. The bsence of n interdigitted lmellr L b i phse for the fluorinted ether-pcs thus constitutes the min difference with their hydrocrbon 1,3- DC18OPC or DC16OPC nlogs. Indeed, none of our FFEM microgrphs recorded for the fluorinted ether-pcs hs shown the presence of cross-frctures which re typicl of n interdigitted lmellr phse. The formtion of clssicl gel lmellr phse for severl of the fluorinted ether-pcs investigted here hs been further confirmed by X-ry diffrction study w16 x Thermotropic phse behõior of the fluorocrbon di-o-lkyglycerophosphocholine ( PC) deriõtiões The thermotropic phse behvior of smples consisting of hydrted powders of the fluorinted PCs ws studied by DSC. Thus, when smples of the Tble 1 Thermodynmic prmeters of the phse trnsitions for the perfluorolkylted 1,2-di-O-lkylglycero-3-phosphocholines nd their hydrocrbon DCmOPC nlogs Compound Pre-trnsition Min phse trnsition L X Ž or P X. b b tp L T Ž "18C. DH Ž "5%. DS Tc Ž "18C. DH Ž "5%. DS Ž. e Ž. Ž. Ž. e DT kjrmol Jrmol.K DT Ž kjrmol. Ž Jrmol.K. 1r2 DF8C5OPC 60 Ž DF8E5OPC b b DF4C11OPC 17 Ž DF6C11OPC 43 Ž Ž ,c DF8C11OPC 88 Ž w xw x F8C5 F4C11 OPC 10 Ž lrge. not mesurble w xw x,c F4C11 F8C11 OPC 33 Ž Ž Ž w xw x F8E5 C14 OPC -y25 w xw x F8E11 C16 OPC 5 Ž w xw x F8C11 C16 OPC 32 Ž Ž d DC14OPC d DC16OPC d DC18OPC DSC mesurements were performed in wter Ž 60% wrw.; for DF4C11OPC, the content of wter ws 70% wrw. b No trnsition detected between y25 nd q958c. c Ech trnsition is split in two fter severl hetingrcooling cycles. d From Ref. w17x nd refs. therein. e DT is the trnsition width t hlf-mximl excess specific het cpcity. 1r2 1r2

7 V. RÕily et l.rbiochimic et Biophysic Act vrious compounds shown in Fig. 1 re heted from y258c Ž or from 48C. up, in most cses min phse trnsition nd, in some cses, pretrnsition of lower energy re observed reproducibly, even fter the smple ws tken repetedly through severl hetingcooling cycles. Tbles 1 nd 2 collect the thermodynmic prmeters phse trnsition temperture, Tp Ž pretrnsition. nd Tc Žgel to fluid min phse trnsition, vide infr., nd their ssocited enthlpy, DH, nd entropy, DS, for the fluorinted di-o-lkyglycerophosphocholines together with those corresponding to their most direct hydrocrbon ether Ž DCmOPC. w17 x, fluorinted Ž DFnCmPC. wx 7 nd hydrocrbon ester phosphtidylcholine ŽDMPC, DPPC nd DSPC w18x. nlogs Ž see Fig. 1. which re tken s references for the discussion in Section 3.3. As supported by X-ry diffrction w16 x, FFEM Ž vide supr. nd opticl microscopy with polrized light, nd by nlogy with the thermotropic phse behvior of their DFnCmPC nlogs wx 7, the vrious fluorinted ether-connected PCs form lmellr phses, when dispersed in n queous phse. Thus, opticl microscopy performed on the hydrted powders, when heting the smples through the highest phse trnsition tempertures listed in Tbles 1 nd 2, showed the ppernce of the chrcteristic opticl pttern of Mltese crosses, which indictes tht the fluorinted PCs re typiclly orgnized in liquid-crystlline or fluid Ž L. lmellr phse bove these tempertures nd tht phse trnsition from lmellr L bž b X. or rippled PbŽ b X. gel phse hs most likely occurred. When two phse trnsitions re detected by DSC, the less energetic pretrnsition nd the higher energetic trnsition cn thus be ssigned to trnsition from lmellr L bž b X. to rippled gel PbŽ b X. phse nd from rippled gel to lmellr L trnsition, respectively. The unique phse trnsition observed for severl PCs is trnsition from LbŽ b X. phse to L phse Žor PbŽ b X. to L phse trnsition, if the L to P pretrnsition is energeticlly too low to be detected.. This nlysis is in full greement with our X-ry diffrction study w16x which showed, t 238C, gel lmellr L b X phse for wf8c11xwc16xopc, 1,3-DF6C11OPC, 1,3- wf8c11xwc16xopc, or 1,3-wF8C11xwC18xOPC, nd fluid lmellr phse for wf8e11xwc16xopc. In the cse of the phospholipids which lso form ribbonlike phse, one of the trnsitions detected by DSC could rise from ribbon-like to lmellr phse trnsition. However, this trnsition is only slowly reversible Žseverl dys wx. 7. Furthermore, the prend min phse trnsitions re lwys mesured by DSC, even fter severl hetingrcooling cycles. This most likely indictes tht none of these trnsitions cn be ssigned to ribbon-likerlmellr phse trnsition. Tble 2 Thermodynmic prmeters of the phse trnsitions for the perfluorolkylted 1,3-di-O-lkylglycero-2-phosphocholines nd their hydrocrbon nlogs 1,3-DCmOPC Compound Pre-trnsition Min phse trnsition L X Ž or P X. b b to L T Ž "18C. DH Ž kjrmol. DS T Ž "18C. DH Ž kjrmol. DS Ž DT. Ž "5%. Ž Jrmol.k. Ž DT. Ž "5%. Ž Jrmol.k. 1r2,b 1,3-DF6C11OPC 46 Ž Ž ,3-DF8C11OPC 87 Ž w xw x 1,3- F6C11 C18 OPC 39 Ž w xw x 1,3- F8C11 C18 OPC 44 Ž Ž c 1,3-DC14OPC c 1,3-DC16OPC ,3-DC18OPC 59 Ž c 59 c 46.8 DSC mesurements were performed in wter Ž 60% wrw.. b Ech trnsition is split in two fter severl hetingrcooling cycles. c From Ref. w17 x. d DT is the trnsition width t hlf-mximl excess specific het cpcity. 1r2 1r2

8 8 V. RÕily et l.rbiochimic et Biophysic Act It should be mentioned tht the min phse trnsitions of the fluorinted phospholipids re much broder thn those mesured for DMPC, DPPC or DSPC Žsee trnsition width t hlf-mximl excess specific het cpcity, DT1r2, vlues listed in Tbles 1 nd 2.. Furthermore, the longer the Fn til the lrger the phse trnsition, hence the lower its coopertivity. This hs lso been shown for the DFnCm- PCs for which it hs been estblished tht the phse trnsition is first induced by the melting of the Fn til nd tht ech portion of the Fn til nd the hydrocrbon Cm spcer experiences intrinsic chnges of moleculr motion with temperture wx 7. Tht the melting of the perfluorolkyl chins occurs over lrge temperture rnge Žup to 208C for F8Cm chin from the onset to completion. is thus in mrked contrst with the hydrocrbon phosphtidylcholine s behvior for which chin melting, due to high coopertivity, occurs within very short temperture rnge Ž 18C. t their phse trnsition temperture w19 x. By contrst, the heting curves of smples of DF8E5OPC or wf8e5xwc14xopc showed no observble phse trnsition in the y25 to 958C temperture rnge. Thus, the DSC experiments most likely indicte tht the gel to fluid phse trnsition occurs, for these compounds, t temperture either lower thn y258c or in the y25 to 958C rnge with n enthlpy too wek to llow its detection. Both compounds possess n ethylenic bond in ech or in one of their hydrophobic chins nd re mixtures of cisrtrns isomers Žcisrtrns rtio of 20r80 nd 10r90, respectively. w12 x. This is expected to broden the phse trnsition nd to lower the thermodynmic prmeters, s compred to those of their sturted nlogs w18,20x Žsee lso our discussion concerning this spect in Section The fct tht no phse trnsition ws observed for DF8E5OPC in the y25 to 958C temperture rnge could thus result from subsequent brodening of the phse trnsition of DF8C5OPC Ž which is lredy very lrge. nd from decrese in DH which mke the detection of the phse trnsition very difficult or even impossible. This ltter sitution is further supported by the thermotropic behvior of DF6E11PC Žcisrtrns rtio of 20r80. which displys much broder nd energeticlly much lower phse trnsition thn its sturted DF6C11PC nlog wx 6. When considering the fct tht decrese in chin length is generlly ccompw18,19 x, wf8e5xwc14xopc, whose hydrophobic chins re nied by decrese in Tc nd in DH shorter thn those of wf8e11xwc16xopc by six nd two methylenic crbon toms, is therefore expected to exhibit lower thermodynmic phse trnsition pwf8e11xwc16xopc ŽTc s rmeters thn those of 58C, Tble 3 Thermodynmic prmeters of the phse trnsitions for the fluorinted 1,2-di-O-lkyglycerophosphoethnolmines DFnCmOPE nd their hydrocrbon DCmOPE nlogs Compound L X b to L or H II Trnsition L to H II Trnsition Tc Ž "18C. DH Ž "5%. DS T Ž "18C. DH Ž "5%. DS Ž. g DT Ž kjrmol. Ž Jrmol.K. Ž kjrmol. Ž Jrmol.K. 1r2 DF4C11OPC 32 Ž d Ž. ) 31 2 DF6C11OPE 63 Ž e,b DF8C11OPE 94 Ž f DC14OPE f DC16OPE f DC18OPE DSC mesurements were performed in HepesrNCl 0.15 M Ž ph 7.3. buffer Ž 60% wrw.; ) in wter Ž 60% wrw.. b A pre-trnsition is detected t 858C Ž DHs0.9 kjrmol.. c The exct nture of this trnsition is not clerly estblished. d No trnsition detected bove 328C. e No trnsition detected bove 638C. f From Ref. w21 x. g DT is the trnsition width t hlf-mximl excess specific het cpcity. 1r2

9 V. RÕily et l.rbiochimic et Biophysic Act DHs7.7 kjrmol.. This indictes tht the min phse trnsition of wf8e5xwc14xopc most likely occurs t temperture below y258c Thermotropic phse behõior of the fluorocrbon di-o-lkyglycerophosphoethnolmine ( PE) deriõtiões The thermotropic phse behvior of smples consisting of hydrted powders of the fluorinted ether- PEs ws investigted by DSC nd X-ry scttering. Tble 3 collects the thermodynmic prmeters determined by DSC Tc Žgel to fluid min phse trnsition ndror lmellr to hexgonl phse trnsition, vide infr., nd their ssocited DH nd DS on these dispersions together with those reported for their most direct hydrocrbon ether DCmOPE nlogs Ž Fig. 1. w21x which re tken s references for the discussion in Section 3.4. Our X-ry diffrction study performed on hydrted DF4C11OPE or DF6C11OPE s function of temperture showed tht bove 288C nd t 708C, respectively, these compounds, in excess wter, form hexgonl phse. This is supported by the observtion of set of five reflections in the spcing rtio of 1r63r67r612r613 for DF4C11OPE nd of 1r63r64r67r613 for DF6C11OPE corresponding to cell dimension of 75.0 A nd 73.2 A, respectively. Below the temperture domin of the hexgonl phse, the low-ngle scttering spectr were more or less complex, depending on the history of the smple, nd exhibited either shrp reflections or, when recorded fter long storge period t room temperture, both shrp nd brod reflections. Among the shrp reflections, some indexed s orders of lmellr repet period, which we tke to indicte tht the fluorinted PEs form bilyer structure which coexists with Ž n. other phsež s.. The rther complex wide-ngle scttering regions for DF4C11OPE nd DF6C11OPE, with mixture of shrp nd brod reflections, could further indicte the presence of gel phse bilyer w22 x. Indeed, brod bnd extending from 5.4 to 4.5 A y1 with shrp inner edge t 5.4 A y1 nd superimposed reflection t 5.0 A y1, nd two brod bnds extending from 5.4 to 5.0 A y1 nd 4.6 to 4.3 A y1 together with shrp inner reflection t 5.4 A y1 were mesured for DF4C11OPE nd DF6C11OPE, respectively. The dependence of the X-ry diffrction ptterns on the smple history is most probbly relted to the mount of wter tht entersrremins within the different phses. This could lso ccount for the discrepncy with the phse trnsition temperture s detected by DSC. Furthermore, the presence of the chrcteristic opticl pttern of Mltese crosses by opticl microscopy with polrized light of their hydrted powders incubted t temperture close to their phse trnsition temperture indicted in Tble 3, suggests tht the fluorinted PEs re lso orgnized in fluid lmellr phse. All these results most likely indicte tht the unique nd brod phse trnsition Ž DT1r2 of4 58C. observed by DSC for the fluorinted PEs consists in superposition of trnsitions, mong which one cn recognize the lmellr gel to fluid phse trnsition followed, lmost simultneously, by trnsition to hexgonl phse. Their thermotropic phse behvior thus resembles, to some extent, tht of their hydrocrbon nlogs prt from the fct tht both the lmellr gel to fluid nd lmellr to hexgonl phse trnsitions occur for the fluorinted PEs t very close tempertures. In the hydrocrbon phosphtidylcholine series, replcing the phosphocholine polr hed by phosphoethnolmine one results in subsequent increse of the gel to fluid lmellr phse trnsition temperture nd in the ppernce of trnsition from lmellr to n inverted hexgonl H phse w18,19,22,23 x II. These two fcts re lso found when going from the fluorinted PC to the fluorinted PE series. Concerning more prticulrly Tc nd s illustrted in Fig. 4c, the DFnC11OPCs do indeed disply lower Tcs thn their respective DFnC11OPE nlogs. However, one cn observe tht the mgnitude in Tc increse when going from the PC to the PE series is less importnt for the fluorocrbon thn for the hydrocrbon derivtives. Formtion of lmellr phse or of hexgonl H II phse is fvored when the size of the polr hed is similr or smller to tht of the hydrophobic prt, respectively. The PE hed cross-section being smller thn tht of PC hed nd of the two hydrocrbon chins in the glycerophospholipids, nd this size disproportion becoming even lrger when hydrocrbon chins re replced by fluorocrbon ones Žcross-sectionl re of bout 30 A 2 for fully extended lkyl chin of CF groups compred to the 2

10 10 V. RÕily et l.rbiochimic et Biophysic Act A 2 for chin of CH w25,26 x. 2, one expects therefore tht the formtion of n inverted hexgonl phse is much more fvored for PEs hving fluorocrbon chins. This is wht we observe. Indeed, the lmellr to hexgonl phse trnsition occurs t much lower tempertures when replcing, in the fluorinted series, the PC hed for PE one Structurer phse trnsition thermodynmic prmeter reltionships Fig. 4. c: Evolution of the phse trnsition temperture, Tc, with the length, L, of the fluorocrbon Ž filled symbols. or hydrocrbon Ž open symbols. hydrophobic chins: Ž. in the 1,2- di-o-lkyl- Ž ',^. nd 1,2-di-O-cyl-glycero-3-phosphocholine Ž v, `. series, Ž b. in the 1,3-di-O-lkyl- Ž B, I. nd 1,3-di-Ocyl- Ž `. glycero-2-phosphocholine series, Ž c. in the 1,2-di-O-lkylglycero-3-phosphoethnolmine Ž ', ^. nd -choline Ž B, I. series. Dt of the fluorocrbon Ž DFnCmPC., hydrocrbon Ž DMPC, DPPC nd DSPC. 1,2-di-O-cyl-glycero-3-phosphocholines nd hydrocrbon 1,3-di-O-cyl-glycero-2-phosphocholines ) re tken from Refs. wx 7 nd w18 x, respectively. L represents the number of crbon toms of the fluorinted til Ž. n nd hydrocrbon spcer Ž m. or of the hydrocrbon Ž m. lkyl or cyl chin. X It is well known tht the L b ŽL b or PbŽ b X. to L phse trnsition of the lipid bilyer detected by DSC is n energetic event reflecting coopertive trnsition of the bilyers from n ordered gel stte to disordered fluid stte t Tc. The reltive energetic contents nd hence the reltive pcking modes of the lipid chins between two lipid bilyer systems cn thus be compred on the bsis of their thermodynmic prmeters ssocited with the min phse trnsitions. The pcking modes of the chins cn be conveniently described in terms of the conformtionl sttistics Ž trnsrguche rtio., lterl chinchin nd hydrophobic interctions. Consequently the observed chnges in Tc, DH nd DS vlues ssocited with the min phse trnsition my be ttributed primrily to differences in the conformtionl sttistics of the chins, lterl chin-chin nd hydrophobic interctions of the respective PCs or PEs in the gel stte. One of the min objectives of this study ws to determine the impct on the phse trnsition thermodynmic prmeters Ž Tc, DH, DS. of severl structurl elements nture of the chemicl junction Ž ester vs ether. nd polr hed ŽPC vs PE, which hs lredy been discussed bove in Section 3.3.., position of the hydrophobic chins on glycerol Ž1,2- vs 1,3 isomers., number nd length of the perfluorolkylted chins, length of the fluorinted til nd hydrocrbon spcer. The following discussion is orgnized ccording to these criteri Impct of the ester r ether junction Fig. 4 which represents the vritions of Tc in the ester nd ether series shows tht no mjor difference exists for the DF4C11PCrDF4C11OPC pir. However, more importnt difference cn be noted for compounds whose hydrophobic chins re ended by

11 V. RÕily et l.rbiochimic et Biophysic Act F6 or F8 til. Thus, it ws found tht the DF6C11OPC, DF8C5OPC nd DF8C11OPC ethers disply lower Tc vlues thn their corresponding ester nlogs Žrespectively by 6, 98C nd even more for the DF8C11OPCrDF8C11PC couple.. This tendency is thus opposite to tht which is observed in the hydrocrbon ester nd ether series: the ltter disply slightly higher Tc vlues Ž ; 0to58C. thn their ester nlogs due to tighter chin chin pckw24 x. The closer chin chin pcking, when going from ing owing to the bsence of the crbonyl groups the ester to the ether series, more thn outweighs the loss of inter- nd intrmoleculr hydrogen bonding interctions which involves the crbonyl groups nd wter molecules. For the fluorocrbon series, the greter steric hindrnce of the F6 nd F8 tils, s Fig. 5. f: Evolution of the phse trnsition enthlpy, DH Ž c., nd entropy, DS Ž d f., with the length, L, of the fluorocrbon Žfilled symbols. or hydrocrbon Ž open symbols. hydrophobic chins: Ž,d. in the 1,2-di-O-lkyl- Ž ', ^. nd 1,2-di-O-cyl-glycero-3-phosphocholine Ž v, `. series; Ž b,e. in the 1,3-di-O-lkyl- Ž B, I. nd 1,3-di-O-cyl-glycero-2-phosphocholine Ž `. series; Ž c,f. in the 1,2-di-O-lkylglycero-3-phosphoethnolmine Ž ', ^. series. Dt of the fluorocrbon Ž DFnCmPC. nd hydrocrbon ŽDMPC, DPPC nd DSPC. 1,2-di-O-cyl-glycero-3-phosphocholines nd 1,3-di-O-cyl-glycero-2-phosphocholines re tken from Refs. wx 7 nd w18 x, respec- ) tively. L represents the number of crbon toms of the fluorinted til Ž. n nd hydrocrbon spcer Ž m. or of the hydrocrbon Ž m. lkyl or cyl chin.

12 12 V. RÕily et l.rbiochimic et Biophysic Act Fig. 6. Evolution of the phse trnsition temperture, Tc, with the length, L, of the fluorocrbon Ž filled symbols. or hydrocrbon Ž open symbols. hydrophobic chins in the 1,2-di-O-lkyl- Ž', ) ^. nd 1,3-di-O-lkyl-glycerophosphocholine Ž B, I. series. L represents the number of crbon toms of the fluorinted til Ž. n nd hydrocrbon spcer Ž m. or of the hydrocrbon Ž m. lkyl chin. compred to tht of the F4 ones, prevents such closer contct between the hydrophobic chins which is necessry to counterblnce the loss of hydrogen bonding. Where the phse trnsition DH nd DS prmeters re concerned, it ppers, s shown in Fig. 5,b, tht no significnt trend cn be evidenced when going from the ester to the ether series whether for the fluorinted or for the hydrocrbon derivtives Impct of the chin s position on glycerol ( 1,2- Õs 1,3-isomer) As shown in Fig. 6 for the Tc, no significnt differences re found for the DF6C11OPCr1,3- DF6C11OPC nd DF8C11OPCr1,3-DF8C11OPC pirs. This tendency prllels thus the trend estbw17 x. The 1,2-r1,3- lished for the hydrocrbon series isomerism lso hs no impct on the DH, DS prmeters Ž see Tbles 1 nd Impct of the presence of fluorinted chins nd of their number The effect of the introduction of fluorinted chin in glycerophospholipidic structure on the thermodynmic prmeters depends upon the number of chins Ž 1 or 2., their length, the reltive length of the fluorinted til vs the whole length, i.e., the fluorintion degree Ždefined s the rtio of the number of fluorinted crbons on the overll number of crbons constituting the hydrophobic chins.. Concerning Tc, s illustrted in Fig. 4,b, our results show tht, for comprble overll chin lengths L, the replcement of hydrocrbon til by its perfluorinted equivlent in both chins of the hydrocrbon glycerophosphocholines Žwhether they belong to the 1,2-, 1,3-ether or ester series wx. 7 induces n effect which is highly dependent upon the length of this til. Thus, nd in the 1,2-ether series, replcing C8 by F8 til results in lrge Tc increse: the Tc of DF8C5OPC Ž 608C for Ls13. is much higher thn tht of Tc of DC14OPC Ž 278C for Ls14. nd even higher thn tht of DC18OPC Ž 558C, for L s 18. which hs much longer chins. On the other hnd, no significnt effect on Tc is found when replcing C6 for F6 til: the Tc of DF6C11OPC Ž508C for L s 17. lies in between tht of DC16OPC nd DC18OPC. By contrst, Tc decrese is evidenced when replcing C4 for F4 til: thus nd despite longer chin length, DF4C11OPC Ž L s 15. displys lower Tc Ž 178C. thn DC14OPC. In the PE series nd s illustrted in Fig. 4c, the tendencies evidenced in the PC series re lso found for the F8 nd F4 tils but Tc decrese is observed for F6 til. However, the replcement of hydrocrbon til by its perfluorinted equivlent in only one chin of the hydrocrbon glycerophosphocholines results systemticlly in Tc decrese nd this even for the longest F8 til. This is illustrted in Fig. 7 which shows tht the three fluorocrbonrhydrocrbon mixed-chin derivtives possess Tc which is lower thn tht of their closest fully hydrocrbon nlog. Conversely, the replcement of one of the two perfluorolkylted chins in the fluorocrbonrfluorocrbon 1,3- DFnC11OPCs by long C16 or C18 hydrocrbon chin diminishes significntly the Tc Ž Fig. 7.; s n exmple, one cn notice tht the Tc of 1,3- wf8c11xwc18xopc lies fr below Ž by lmost 408C. tht of 1,3-DF8C11OPC. A similr result hs lso been evidenced for triple-chin fluorocrbon Žrespec- tively, hydrocrbon. mmonium mphiphiles where the replcement of one fluorocrbon Žresp. hydrocrbon. chin by hydrocrbon Ž resp. fluorocrbon. one resulted in decrese of Tc w27 x. Where the impct on Tc of the chin length increse is concerned, one cn see, s illustrted in Fig. 4 c nd 7, tht the Tc rises Ž 1. on incresing the Fn

13 V. RÕily et l.rbiochimic et Biophysic Act Fig. 7. Evolution of the phse trnsition temperture, Tc, with the length, L, of the hydrophobic chins in the fluorocrbonrfluorocrbon Ž filled symbols., hydrocrbonrhydrocrbon Žopen symbols. nd mixed fluorocrbonrhydrocrbon Ž htched symbols. di-o-lkyl-glycerophosphocholine series. ) For the two former series, L represents the number of crbon toms of the fluorinted til Ž. n nd hydrocrbon spcer Ž m. or of the hydrocrbon Ž m. lkyl chin. For the mixed series, L is the men number of X crbon toms Žsnqmqm r2.. til s length for given Cm spcer: Tc of DF8C11OPC Ž or OPE.) Tc of DF6C110PC Žor OPE.)Tc of DF4C11OPC Ž or OPE.; Ž 2. on incresing the Cm spcer s length for given Fn til ŽTc of DF8C11OPC) Tc of DF8C5OPC.. A similr correltion between Tc, which is closely connected to the melting of the hydrophobic chins, nd the lkylchin s length is observed for the hydrocrbon glycw18,19 x. In the ltter series, it ws erophosphocholines found tht monotonic chin lengthening of two methylenic crbon toms is ccompnied by nerly monotonic Tc increse of bout 208C Ž Fig. 4c.. By contrst, chin lengthening of two CF2s in the DFnC11OPC, DFnC11OPE Ž Fig. 4c. or 1,3- DFnC11OPC Ž Fig. 4b or Fig. 7. series results in lrger Tc increse Ž ;308C.. An elongtion by two CF2s on only one chin lso results in Tc increse but the mgnitude is less importnt: thus, the Tc s of 1,3-wF6C11xwC18x nd 1,3-wDF8C11xwC18xOPC differ by only 88C Ž Fig. 7.. A comprison between the Tc vlues of DF8C5OPC, DF4C11OPC nd DF6C11OPC shows tht the chin melting phse trnsition temperture is minly modulted by the length of the Fn segment rther thn by the overll chin length L Ž Fig. 4., hence by the fluorintion degree. It ws indeed expected tht, when considering only L nd by nlogy with the hydrocrbon series, the phse trnsition of DF8C5OPC Ž Ls13. should be lower thn tht of DF4C11OPC Ž L s 15. nd even of DF6C11OPC ŽL s 17.. In fct, of the three compounds, it is DF8C5OPC which hs the shortest chin length but the longest perfluorolkyl til, nd, consequently, the highest fluorintion degree, which displys the highest Tc. These results re in line with our previous study concerning their ester nlogs w6,7 x. Where the DH nd DS prmeters re concerned nd s illustrted in Fig. 5 f, it ppers tht the fluorinted Ž 1,3-. DFnCmOPC, DFnCmOPE, X wfncmxwfn Cm X xopc nd wfncmxwcm X xopc lmellr phses disply much lower DH nd DS vlues thn their corresponding hydrocrbon ones. This behvior is similr to tht evidenced for the fluorinted DFnCmPC phosphtidylcholines s compred to the hydrocrbon ones wx 7. One should notice tht the mixed fluorocrbonrhydrocrbon 1,3- wfnc11xwc18xopcs disply intermedite DH nd DS vlues to those mesured for the fluorocrbonrfluorocrbon 1,3-DFnC11OPCs nd fully hydrocrbon 1,3-DC18OPC. Furthermore, it is 1,3- wf6c11xwc18xopc which hs the lowest fluorintion degree mong ll the fluorinted compounds investigted here which displys the lrgest DH nd DS vlues. All these results indicte tht the trnsition is minly due to the reorgniztion of the hydrocrbon Cm prt of the FnCm chins nd is further in line with studies performed on other fluorocrbon mw28 x. The DH nd DS vlues in the hydrocrbon series, phiphiles excepted for the 1,3-diether series, re seen to increse nerly linerly with incresing chin length Ž Fig. 5 c nd Fig. 5d f., reflecting tht the pcking interctions re rising proportionlly to the chin length increse. This is lso the cse in the DFnCmŽ O. PCs series Ž Fig. 5,d., for which the DH nd DS vlues re rising on incresing the Fn til or the Cm spcer lengths. This tendency is not s evident in the 1,3- nd PE series ŽFig. 5b,e nd 5c,f, respectively.. It is therefore difficult to rtionlize our results in terms of DH nd DS increment per CF 2 nd per CH 2 groups. Owing to the geometric, electronic nd hydrophobic chrcteristics which differentite hydrocrbon from perfluorolkyl chin, one expects tht the

14 14 V. RÕily et l.rbiochimic et Biophysic Act introduction in membrne forming double-chin mphiphile of one or two fluorocrbon chins induces two ntgonist effects on the thermodynmic Tc, DH nd DS prmeters of this membrne: Ž 1. enhncement of pcking disorder or fluidity, which results in lowering in the thermodynmic prmeter vlues, due minly to weker lterl inter- nd intrmoleculr interctions nd incresed steric repulsions between the fluorocrbon chins or between fluorocrbon nd hydrocrbon chin, s compred with those existing between hydrocrbon chins wx 4, nd Ž 2. increse in order or rigidity, which is only expressed here by n ugmenttion of Tc, relted to n increse in hydrophobic interctions w29 x. In the cse of the phospholipids hving in both of their chins short F4 Ž C F. 4 9perfluorolkyl til, the hydrophobic interctions re not likely to be sufficient to counterblnce the opposite effect. Thus, the replcement of prt of the hydrocrbon chins by F4 til ppers to induce membrne fluidifiction. A F6 Ž C F. 6 13til is sufficient in some cses to equilibrte the ntgonist contributions nd does not modify membrne fluidity. By contrst F8 Ž C F segment results in the predominnce of the hydrophobic interctions nd enhnces the membrne s rigidity. These results re found whtever the series Žester, 1,2- or 1,3-ether, PC or PE. to which the fluorinted phospholipids belong. However, in the cse of the mixed fluorocrbonrhydrocrbon phospholipids, the hydrophobic interctions induced by F8 til re not strong enough to compenste for weker intermoleculr fluorocrbonrfluorocrbon nd intr- nd intermoleculr fluorocrbonrhydrocrbon chin chin inwx 4 nd incresed steric repulsions w25,26 x. The presence of one fluorocrbon together with terctions hydrocrbon chin thus results in Tc decrese Impct of double bond in the hydrocrbon spcer It is well estblished tht membrnes formed from unsturted phospholipids hving double bond in one ndror in both chins, Žwhether of cis or trns configurtion. disply much lower Tcs, s compred to membrnes formed from their sturted nlogs w2,18 20 x. This is due to geometric perturbtions imposed by the double bond on the overll conformtion of liner long liphtic chin which re more importnt when the double bond is cis rther thn trns. In the cse of the fluorocrbon unsturted PCs ŽwF8E5xwC14xOPC, wf8e11xwc16xopc nd DF8E5OPC., these compounds consist in fct in mixture of cis nd, minly, trns isomers Žcisrtrns F 20r80.. The observed effects on the thermodynmic prmeters will thus lso depend upon the reltive proportion of cisrtrns isomers. The presence of double bond in only one chin, s in wf8e11xwc16xopc, which is the sole compound of the unsturted ones for which phse trnsition hs been detected, is found to be ccompnied by consequent decrese of Tc, from 448C for the stuwf8c11xwc16xopc to 58C for wf8e11xwc16xopc. This trend is thus similr to wht is usully found for rted the unsturtedrsturted phospholipids Symmetric Õs. mixed fluorocrbon r fluorocrbon phospholipids It is lso interesting to compre the Tcs of the m ixed fluorocrbonrfluorocrbon wf4c11xwf8c11xopc nd wf8c5xwf4c11xopc phospholipids w ith those of sym m etric fluorocrbonrfluorocrbon DFnCmOPC ones. The former mixed fluorocrbonrfluorocrbon wf4c11xwf8c11xopc possesses chins which differ by the length of their fluorinted tils nd by their overll chin length Ž 4 crbon toms. wf4c11xwf8c11xopc results formlly from the replcement of one of the F8 or F4 tils in the symmetric DF8C11OPC or DF4C11OPC, respectively. These replcements, which correspond to chin shortening or lengthening by F4 segment, respectively, re ccompnied, s expected, by Tc decrese Ž 488C. or increse Ž 238C., respectively. Although these results re in line with those lredy commented on bove concerning the impct of Fn til length decreserincrese on Tc Ž see Section , one cn furthermore notice tht two consecutive chin lengthenings by F4, hence when going from DF4C11OPC to wf4c11xwf8c11xopc then to DF8C11OPC, result in significnt difference in mgnitude of Tc increse, the second one Ž 488C. being much lrger thn the former one Ž 238C.. Such n increse in rigidity is mostly relted to the lrger hydrophobicity of the F8 compred to F4 segment nd, to lesser extent, to

15 V. RÕily et l.rbiochimic et Biophysic Act incresed chin chin interctions when both chins re of similr length. It is lso interesting to note tht the mixed wf4c11xwf8c11xopc displys lower Tc Ž by 98C. thn the symmetric DF6C11OPC, lthough both compounds possess the sme number Ž 12. of fluorinted crbons nd the sme overll number of crbon toms Ž 34.. A similr difference in Tc hs lso been reported in the hydrocrbon series, where it ws found tht the Tc of the mixed wc15xwc19xpc Žn nlog of wf4c11xwf8c11x OPC; see structure in Fig. 1. is lower Ž by 58C. thn tht of the symmetric DC17PC Žn nlog of DF6C11OPC. w18 x. These differences in Tc re most likely relted to difference in length Ž4 crbon toms. between both chins in the mixed wf4c11xwf8c11xopc or wc15xwc19xpc which results in poorer chin chin overlp nd, consequently, in lower chin chin interctions compred to, respectively, DF6C11OPC or DC17PC where both chins re of identicl length. This is further supported by the non-idel mixing behvior of DF4C11PC nd DF6C11PC Žwhose chins differ by 2 fluorinted crbons. w30x or of DMPC nd DSPC w31x Žwhose chins differ by 4 crbons. or by the thermodynmic preference for DMPC to become in closer contct of nother DMPC in mixed DMPCrDSPC bilyer w32 x. The second mixed fluorocrbonrfluorocrbon wf8c5xwf4c11xopc possesses chins which differ by the length of both the fluorinted tils nd hydrocrwf8c5xwf4c11xopc Ž Tc; 108C. results bon spcers. formlly from the replcement of F8C5 chin in Tble 4 Vesicle size nd size distribution for vrious formultions fter preprtion, het-steriliztion nd storge t 25" 18C Liposome After preprtion b After steriliztion After storge t 258C Ž "18C. composition Dimeter Weight Dimeter Weight Dimeter Weight Storge Ž "S.D.. distribution Ž "S.D.. distribution Ž "S.D.. distribution Ž dys. Ž nm. Ž %. Ž nm. Ž %. Ž nm. Ž %. d,e DF4C11OPC 110 Ž Ž Ž DF6C11OPC 110 Ž Ž Ž deposit 30 wf8e11xwc16xopc 100 Ž Ž Ž wf4c11xwf8c11xopc 140 Ž Ž Ž Ž Ž Ž ,3-wF6C11xwC18xOPC 90 Ž Ž Ž Ž Ž Ž Ž Ž ,2-wF8C11xwC18xOPC 140 Ž Ž Ž Ž Ž ,3-DF6C11OPC 100 Ž Ž Ž Ž Ž deposit e DF4C11PC 130 Ž lrge Ž Ž Ž e DFFC 60 Ž Ž Ž Ž deposit EPCrCH 130 Ž deposit EPC 110 Ž Ž Ž mm of the phospholipid in Hepes buffer. b 1218C, 10 5 P, 15 min. c S.D. is the stndrd devition; 95% of the popultion is corresponding to "1.96 S.D. d not sterilized. e From Ref. wx 8.

16 16 V. RÕily et l.rbiochimic et Biophysic Act DF8C5OPC Ž Tcs608C. for F4C11 one, which is ccompnied by Tc decrese Ž 508C.. This Tc decrese ws expected in view of the lower hydrophobicity of F4Ž C11. chin compred to F8Ž C5. one Ž see Section It is therefore surprising tht, conversely, the replcement of F4C11 chin in DF4C11OPC Ž Tcs178C. for F8C5 one is ccompnied by Tc decrese Ž ;78C.. This is most probbly relted to difference in length between the two C5 nd C11 hydrocrbon spcers in the mixed wf8c5xwf4c11xopc which, when pcked in the bilyer, results in subsequent overlp of the F8 tils with the C11 spcer neighbours nd, consequently, in much lower F8C5rF4C11 chin chin interctions thn those existing between F4C11 chins. These lower F8C5rF4C11 chin chin interctions re not compensted by the lrger hydrophobic interctions developed by the F8Ž C5. chin, thus incresing membrne fluidity s compred to DF4C11OPC Long-term shelf stbility of the fluorinted liposomes The use of liposomes s drug crrier system requires the development of het-sterilizble nd long-term shelf-stble formultions in terms of prticle size nd prticle size distribution. Prticle size is indeed crucil prmeter, mong others, which governs the in vivo fte of the liposomes wx 1. Vesicles mde from pure monodispersed phospholipids hve low stbility. The development of more stble liposomes usully involves multicomponent systems nd elborte formultions. By contrst, we hve previously shown tht the fluorinted DFnCmPC phosphtidylcholines, s single components, form hetwx 8. sterilizble vesicles of exceptionl shelf stbility In this respect, it ws of prticulr interest to exmine the behvior of the liposomes formed from the new fluorinted di-o-lkylglycerophosphocholines, i.e., DF4C11OPC, DF6C11OPC, wf4c11xwf8c11xopc, wf8e11xwc16xopc, 1,3-wF6C11xwC18xOPC, 1,3- wf8c11xwc18xopc nd 1,3-DF6C11OPC, ech of these compounds being representtive of series of phospholipids which hve been synthesized. Tble 4 collects the men dimeters of the liposoml dispersions which hve been mesured by LSS, before nd fter het steriliztion Ž stndrd norms. nd fter storge t 25"18C for periods up to 600 dys. Most of the liposomes formed from the fluorinted ether-pcs cn be thermlly sterilized nd stored t room temperture for severl months without ny significnt modifiction of their men size nd size distribution. This is the cse whether their membrnes re in the fluid or gel stte t the storge temperture, nd formed from compounds hving one or two fluorocrbon chins connected to glycerol in 1,2 or 1,3. These results confirm the remrkble stbility of the fluorinted liposomes which contrsts strongly with tht of most of the conventionl liposomes Ž see Tble 4.. As evidenced by LSS, the fluorinted DF4C11OPC nd wf8e11xwc16xopc liposomes, whose membrnes re t 258C in the fluid stte, do indeed disply no significnt modifictions in size nd size distribution s those formed from their ester DF4C11PC nlog. This revels rther exceptionl stbility of these liposomes when submitted to such drstic steriliztion procedure ndror to storge period for up to 300 or 600 dys. This is lso the cse of the conventionl EPC liposomes which re, however, polydispersed system. For the fluorinted liposomes whose membrnes re in the gel stte t 258C, the therml steriliztion process implies tht they go through their phse trnsition. It ppers tht, for the fluorinted DF6C11OPC, wf4c11xwf8c11xopc, 1,3- wf6c11xwc18xopc nd 1,3-wF8C11xwC18xOPC liposomes, only minor chnges in terms of prticle size nd size distribution re detected. Most of these formultions cn lso be stored for severl months t room temperture, except DF6C11OPC which forms lrger ggregtes nd precipittes fter only onemonth storge period, nd 1,3-DF6C11OPC which displys even much lower stbility. The poorer stbility of these two ltter fluorinted formultions my be relted to the formtion of ribbon-like phse from the lmellr gel phse, s detected by FFEM Ž vide supr.. The much greter stbility of the former fluorinted liposomes contrsts strongly with tht of the conventionl gel DPPC or EPCrCH 1r1 liposomes for which precipitte is formed fter shorter period of storge or immeditely fter steriliztion, respectively Ž see Tble 4.. The stbility in terms of size nd size distribution of the fluorinted liposomes seems to be chrcter-