Liposomes from polymerizable phospholipids

Chemistry and Physics of Lipids, 33 (1983) 355-374 Elsevier Scientific Publishers Ireland Ltd.

355

LIPOSOMES FROM POLYMERIZABLE PHOSPHOLIPIDS

B E R N D H U P F E R * , H E L M U T RINGSDORF* and HANS SCHUPP**

Institut fiir Organische Chemic, Johannes Gutenberg-Universitiit, J.-J.-Becher-Weg 18-20, D-6500 Mainz (Federal Republic of Germany) Received February 25th, 1983

accepted June 18th, 1983

The synthesis and characterization of a great variety of single and double chain phospholipids containing the diacetylene and butadiene moiety is described. These substances can be dispersed in water by ultrasonication and the resulting vesicles can be photopolymerized with the retention of their original structure. Absorption spectra of the polymerized diacetylenic lipids show significant differences depending on the molecular structure of the monomers. By the polymerization reaction, the gel to liquid crystalline phase transition is suppressed, which does not correspond to the properties of biological membranes. Evidence for enhanced stability of polymerized vesicles is given by treatment with ethanol and detergents showing that trapped markers are released to a much smaller extent than in the case of unpolymerized vesicles. Diacetylenic lipids show a pronounced hysteresis of the phase transition. If the membrane of supercooled vesicles crystallizes, all trapped marker is released within seconds. Possibilities for overcoming this extreme rigidity of the membranes are discussed.

Keywords: phospholipid polymer; polymeric membrane; liposome stability; membrane tightness; phase transition.

Introduction

For many reasons there is increasing interest in the synthesis of new stable analogs for biomembranes. One of the more important motivations for these studies is that most of the conventional membrane models (mono- and multilayers, black lipid membranes (BLM), and liposomes) lack in long-term stability [11. Mono- and multilayers cannot exist without an aqueous or solid support, BLM usually do not survive longer than minutes or hours, and liposomes (vesicles) on prolonged standing undergo fusion [1[. Approaches to stabilization of model membranes have been undertaken, e.g. by incorporating cholesterol into the membrane of vesicles [2] or by using phospholipids carrying photoactivable groups in the hydrophobic

*Present address: Kalle Niederlassung der Hoechst AG, D-6200 Wiesbaden. **Present address: BASF AG, Kunststofflaboratorium, D-6700 Ludwigshafen. tTo whom reprint requests should be addressed. 0(X)9-3084/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

356 chains [3, 4]. Another approach that has recently been developed is the stabilization of model membranes by polymerization reactions. It has long been known that single chain amphiphiles bearing polymerizable groups-mainly carboxylic acids and their esters---can be polymerized in mono- and multilayer form [5-7]. Only recently attempts were successful to build up polymeric BLM [8] and vesicles [9-15] from a variety of single and double chain amphiphiles. Diacetylenic [9,10,13,42], acryloylic [12,14], methacryloylic [11,12,15], butadienic [12], and vinylic [43] moieties were used as polymerizable units. Besides their enhanced stability, in some cases a drastically decreased leakage rate of entrapped substances was reported for polymerized vesicles [15,16], making them useful as controllable drug carrier systems in vivo. In addition, first approaches to the potential use of partially polymerized vesicles as cell models [16] have recently been described. The FoF1-ATP synthetase complex can be incorporated in partially polymerized liposomes from a synthetic sulfolipid under complete retention of its enzymatic activity [17[. Furthermore, mixed monolayers and vesicles built up from polymerizable and natural lipids also exhibit an improved stability but show an increased flexibility of their membrane [18] compared to fully polymerized model membranes [17]. Here, we describe the synthesis, vesicle formation and polymerization of amphiphiles carrying naturally occurring (2-11) and synthetic (1) head groups with the diacetylene (3-11) and butadiene (la, 2a) moiety as the polymerizable units. Furthermore, initial results on phase transitions and leakage behavior of monomeric and polymerized vesicles will also be reported. An additional paper [19] deals with the spreading and polymerization properties of the synthetic lipids at the gas/water interface. Materials and methods

Materials

Unless otherwise stated all reagents and chemicals were obtained commercially and used without further purification. Pyridine, chloroform and carbon tetrachloride (reagent grade) were liberated from water and alcohol by percolation through an alumina column [21]. Light petroleum, b.p. 40-70°C, was distilled before use. The following chemicals or materials used in this work are listed along with their commercial source. Methyl bis(2hydroxyethyl)amine, N,N'-dicyclohexylcarbodiimide, silica gel (Kieselgel 60; 70-230 mesh), 1,2-isopropylideneglycerol, alumina (Aluminiumoxid 90, basisch, 70-230 mesh), D-glucose, Triton X-100, sodium dodecylsulfate, tris(hydroxymethyl)aminomethane, aluminum TLC-sheets precoated with silica gel (Merck); 4-dimethylaminopyridine, palmitic acid, 2,4,6-tri-

357

isopropylbenzene sulfonylchloride, D-mannitol (Aldrich); Florisil (100-200 mesh), ATP, N A D P +, hexokinase from Yeast, Type C-130 (EC 2.7.1.1), glucose-6-phosphate dehydrogenase from Bakers Yeast, Type VII as suspension in 3.2M (NH4)2504, Sephadex G-50 (Sigma); 6-carboxyfluorescein (Eastman). Fractions on TLC plates were detected with the Haynes-Isherwood reagent [24]. The synthesis of lipids 1-11 is outlined in Scheme 1. HO~(CH2)2\.N-'-CH3

R~OOH

HO~(CH2)2/

)

(DCC/DMAP)

R-~CO~(CH2)2~ / N'~CH3 R--CO~O'~(CH2)2

CH3Br> R ~ O - - O - - ( C H 2 ) 2 \ +/CH3 N R~O~O--(CH2)2 / \CH3

Br

12a, 12b

(la) R = CH~------CH---CH~-----CH~(CH2)12~CH3 ( l b ) R = (CH2)14--CH3

CH2--OH

I

R~OCl/pyridine or R--COOH/DCC,DMAP

CH----OH I CHz---O--CO---O--CH2CCI3

CH2--O~CO~R I CH~O~R CHz~O~CO~O--CH2CCI3 16a, 16b, 16(:

CH2~O---CO~R Zn/HAc

I

(a) R = CH==CH~CH~--mCH--(CH2)~2~.CH3 (b) R = (CH2h4---CH3 (¢) R = (CH2)~-43~C---C~(CH2h2~CH3

> CH~O--R

I

CH2~-OH 13a, 13b, 13(:

0

II

CH2~O~CO~R

I

13 1: CI2P----O(CH2)z~B[ CH---O~CO~R 2, H20

C Hz--O---CO--R

I

1. NX3 CH---O.~CO.~R > I 2. ---R--~r ] + CH2~O~P~(CH2)2Br CH2~O.~PO.~O(CH2)2---NXs

I

OH

I

O_ (2a) R - CH~H---CH~---CH--(CH2)12~.CH3 X = CH3 (2b) R = (CH2)14-~CH3 X = CH3 (9) R = (CH2)~C--C=~=~C---(CH2)12--CH3 X=H (10) R = (CH2)~C~C.-~C==C~(CH2)12~CH3 X = OH3

358

H3C~-(CH2)~~(CH2)9~H

1. POCI3 2. H20

14

H3C--(C H2)12~C~-~-C~C~C~(C H2)g---O--PO(OH)2 3 O 14

1•

II

CI2PO--O(CH2)z~Br > H3C~(CH2)12.~C=~=~C~(CH2)g__O~P__O__(CH2)2~B r 2. H20 I

0a

15

1. NR1R2R3 2. -HBr O H3C~(C H2)1 2 - - C ~ - - - - C ~ ( C

11

H2)9--O.~P~(C

H2)2.~1~R 1R2R3

I

O (4) R 1 = R2

R3 - H (5) R I = R 2 = H , R3=CH3 (6) R 1 H, R2 - R3 OH3 (7) R 1 = R2 - R3 OH3

HO--CH2---CH--CH2

o×o [

(TPS/pyridine)

I

O

II

• H3C~(C H2) ~z - - C ~ C ~ - - ( C

H2)9~

p - - O ~ C H 2 - - C H - - C H2

I

I

o

I (HCI)

O

H

H3C---(CH2)I2 - - C ~ C ~ C - - ( C H2)9--O---P--O--CH2~ H - - C H 2

I

I

OH

OH

i

OH

8

CH2~-OH

CH2---O--CO--R 1

I

R'--COCI.

CH---OH

(pyridine) ;~ CH-OH

I

I

I CH~,---I

I

CH2--I

CH2~O~CO--R 1 R2--COCI I ) CH---O--CO~R 2 (pyridine)

CH2--I

17

o

II

AgO---P[OC(CH3)3]2 -Ag I

CH2~O~CO--R 1

I

L

CH--O--CO~R21 C H 2-"O--PO~'OC ( C H 3)3

I

OC(CH3)3 S c h e m e 1.

CH2---O~CO~R ~ (HCl) > CI H ~ O - - C O ~ R CH2---O---PO(OH)2 11 R ~= (CH2) lo--CH3 R2= (C H2)8--C~-~'C~C~-~-C--(C H2)12---CH3

359 Dimethyl bis[ 2-( octadeca-2, 4-trans, trans-dienoyl )oxyethyl ] a m m o n i u m bromide (la), dimethyl bis(2-palmitoyl)oxyethylammonium bromide (lb) (a) To a solution of 14.05 g (50 mmol) octadeca-2,4-trans, trans-dienoic acid [22], 2.98g (25mmol) methyl bis(2-hydroxyethyl)amine, and 100mg 4dimethylaminopyridine in 100 ml chloroform, 13.28 g (65 mmol) of dicyclohexyl carbodiimide dissolved in 20 ml chloroform was added dropwise at 0°C. The solution was stirred overnight at ambient temperature followed by filtration from N,N-dicyclohexylurea upon which the filtrate was washed with 1 N HC1, saturated NaHCO3 and water. After drying over Na~SO4 the solvent was evaporated in vacuo with the residue dissolved in 10 ml CHCI3 and percolated through a short silica gel column with CHC13 as eluent. Fractions containing methyl bis[2-(octadeca-2,4-trans, trans-dienoyl)oxyethyl] amine were pooled, the solvent evaporated in vacuo, and the resulting product recrystallized from petroleum ether. 12a, m.p.: 36°C, 65% yield. Catc. for C41H73NO4 (644.04): C 76.46; H 11.43; N 2.17%. Found: C 76.4; H 11.6; N 2.5%. IR (KBr): 1690 (C=O); 16411, 1610 (C=C); 1090 (C-O); 1000 (=C-H, 6oov.) cm-~In a similar manner the dipalmitoyl ester of methyl bis(2-hydroxyethyl) amine was prepared. 12b, m.p.: 38°C, 82% yield. Calc. for C37H73NO4 (596.00): C 74.57, H 12.35; N 2.35%. Found: C 74.8; H 12.5; N 3.2%. IR (KBr): 1720 (C=O); 1180 (C-O) cm -I. (b) Five millimoles of the above tertiary amines was dissolved in 50 ml acetone and 2 ml methyl bromide was added at 0°C. After stirring for 2 h at room temperature the solvent was evaporated and the residue recrystallized from acetone. la, m.p.: 93°C, 73% yield. Calc. for CazH76BrNO4 (738.98): C 68.26; H 10.37; N 1.90%. Found: C 68.6; H 10.4; N 2.2%. lb, m.p.: 88°C, 83% yield. Calc. for C38H76BrNO4 (690.94): C 66.06; H 11.09; N 2.03%. Found: C 66.06; H 11.2; N 2.0%. rac-l,2-Bis(octadeca-2,4-trans, trans-dienoyl)glycero-3-phosphoryl choline (2a), rac-l,2-dipalmitoyl glycero-3-phosphoryl choline (2b) (a) Glycerol-l-(fl,/3,/3-trichloroethyl)carbonate [23] was esterified twice with octadeca-2,4-trans, trans-dienoic acid [22] and palmitic acid, respectively, as described in l a and lb yielding rac - 1,2 - bis(octadeca - 2,4-trans, trans - dienoyl)glycerol - 3 - fl,fl,~ - trichloroethyl carbonate (70%), and rac - 1,2 dipalmitoylglycerol - 3 - fl,fl,~ - trichloroethyl carbonate (75%). The final purification of these compounds was carried out as described in Ref. 23 by Florisil chromatography using petroleum ether/ether mixtures as eluents. Small fractions (20 ml) were examined by T L C (petroleum ether/ethyl acetate, 8:1). The protecting trichloroethyl carbonate groups were split off with zinc in

360 glacial acetic acid [23], and the resulting rac-l,2-diacylglycerols recrystallized from petroleum ether. rac-l,2-Bis(octadeca-2,4-trans, trans-dienoyl)glycerol (13a): m.p.: 59°C, 73% yield. Calc. for C39H6805 (616.97): C 75.92: H 11.11%. Found: C 76.2; H 11.2%. IR (KBr): 3500 (O-H), 1690 (C=O), 1640, 1610 (C=C), 1150 (C-O), 1000 (=C-H, 6o.o.p) cm -1. rac-l,2-Dipalmitoyl glycerol (13b), m.p.: 58°C, 70% yield. Calc. for C35H6805 (568.92): C 73.89, H 12.05%. Found: C 73.9, H 12.0%. (b) The reaction of the monofunctional alcohols with 2-bromoethylphosphoric dichloride and trimethylamine was carried out as described by Eibl [27]. The crude lecithins were purified by silica gel chromatography using CHCI3/CH3OH/NH3 mixtures as eluent. 2a, m.p.: 220-225°C, 43% yield. Analytical data for this compound have been given in a previous publication [20]. 2b, m.p.: 220-230°C (m.p. 220°C [28]), 60% yield. Calc. for C40Hs0NOsP'H20 (752.07): C 63.88, H 10.99, N 1.86%. Found: C 63.9, H 11.1, N 1.7%.

Hexacosa - 10,12- diyne- 1-phosph ate (3) (a) The coupling of 1-iodopentadecyne-1 and 10-undecyne-l-ol was carried out as described in the literature for a diacetylene carbonic acid [25] yielding hexacosa-10,12-diyne-l-ol (14) (m.p.: 59--60°C). (b) This alcohol (3.75 g; 10 mmol) was added to a solution of 2.30 g POCI3 in 25 ml CC14. The mixture was left at room temperature overnight, the flask being stoppered with a CaCI2 tube. After boiling under reflux for 6h the solvent was evaporated in vacuo and the residue heated with 10 ml H20 for 1 h. The product was dissolved in ether, washed with water and dried over Na2SO4. Pure 3 was obtained by recrystallization from petroleum ether. 3, m.p.: 79-82°C, 86% yield. Calc. for C26H4704P (454.63): C 68.69; H 10.42%. Found: C 69.6; H 10.8%. IR(KBr): 2700 (sh, P-OH); 1080 (P-OC); 1020 (P-OH); 2140, 2180 (C~-C) cm -1. NMR(CDCI3): 0.9 (q, CH3): 1.3 (m, H3C-(CH2)lo, and -CHz-(C_H2)6-CH2); 2.3 (t, CH_2-C=-C-C=--C--CH_2); 4.1 (t, C I-2Iz--OPO3H2) ppm.

Hexacosa-lO,12-diynophosphoryl-1-glycerol

(8) (a) 2,4,6-Triisopropylbenzenesulfonyl chloride (4.54 g, 15 mmol) and 1,2isopropylideneglyeerol (1.32 g, 10 mmol) were dissolved in 100 ml dry pyridine and 3 (2.28 g, 5 mmol) was added [26]. After stirring for 20 h at room temperature 60 ml chloroform was added while stirring was continued for another 60 rain. Thereafter 40 ml H20 was added with the solvents being evaporated in vacuo. To the residue, 200ml ether was added and after stirring for 2h, the precipitate was filtered off while the ether was

361 evaporated in vacuo. TLC (CHC13/CH3OH/HzO, 65:25:4) shows complete conversion of 3. (b) For hydrolysis of the acetal, the crude product was dissolved in 25 ml ether and after filtration 13 ml conc. HCI was added at 0°C. (No phosphate ester migration was reported under comparable reaction conditions [44]. Alternatively, the acetal may be cleaved under mild conditions by trimethylborate/boric acid treatment [45[.) After stirring for 30 min, 60 ml H20 was added with stirring continuing for an additional 60min. The product was filtered with suction, washed with water and dried over P205. Crude 8 was purified by silica gel chromatography using CHCI3/CH3OH/H20, 65:25:4 as eluent and finally precipitated with acetone from a concentrated chloroform solution. 8, m.p.: 130-135°C; 40% overall yield. Calc. for C29H5306P (528.71): C 65.88; H 10.10%. Found: C 65.32; H 10.52%.

Hexacosa-lO,12-diyno-l-phosphorylethanolamine (4), amine (5),-N,N-dimethylethanolamine (6),-choline (7)

-N-methylethanol-

(a) Hexacosa-10,12-diyne-l-ol (see above) was reacted with 2-bromoethylphosphoric dichloride [27] according to Eibl and Niksch [27] yielding the phosphoryl-(2-bromoethyl) ester of 3, which was recrystallized from petroleum ether. 15, m.p.: 64-67°C, 73% yield. (b) Compound 15 was directly aminated with ammonia, methyl-, dimethyland trimethylamine, respectively [27], yielding 4, 5, 6 and 7. The crude products were purified by silica gel chromatography as described in Ref. 27. 4, m.p.: 219-225°C; 70% yield. Calc. for C28H52NOaP (497.70): C 67.57; H 10.67; N 2.81%. Found: C 67.9; H 10.7; N 2.6%. 5, m.p.: 167°C, 74% yield. Calc. for C29H54NO4P (511.73): C 68.07; H 10.64; N 2.74% . Found: C 68.6; H 10.3; N 2.7%. 6, m.p.: 106°C, 73% yield. Calc. for CNH56NO4P (525.76): C 68.54; H 10.74; N 2.66% . Found: C 68.2; H 10.7; N 2.7% . 7, m.p.: 262-272°C, 67% yield. Calc. for C31H58NOaP(539.78): C 68.98: H 10.83; N 2.59%. Found: C 68.2; H 11.0; N 2.6%.

rac-l,2-Bis(hexacosa-lO,12-diynoyl)glycero-3-phosphorylethanolamine rac- l,2-bis(hexacosa-lO,12-diynoyl)glycero-3-phosphorylcholine (10)

(9),

(a) Hexacosa-10,12-diynoic acid (m.p. 68-69°C) was prepared from 1-iodo pentadecyne-1 and 10-undecynoic acid as described for homologous compounds [10,25]. For removal of polar and unpolar byproducts formed during asymmetric acetylene coupling the acid was purified by preparative HPLC (/~-Porasil, Waters) with petroleum ether/ethyl acetate/acetic acid, 90:10:0.01 (by vol.) as eluent.

362 This acid was converted to the acyt chloride by oxalyl chloride treatment according to Mattson and Volpenhein [29]. (b) As described for a variety of other 1,2-diacylglycerols [23], 1,2bis(hexacosa-10,12-diynoyl)glycerol was prepared by acylation of glycerol/3,/3,/3-trichloroethyl carbonate with the acid chloride (see (a)) (1,2-bis(hexacosa-10,12-diynoyl)glycerol-/3,/3,/3-trichloroethyl carbonate (16c) has m.p. 50°C) and subsequent removal of the protecting group with zinc in glacial acetic acid. 13c, m.p.: 65-66°C, 53% yield (based on glyceroltrichloroethyt carbonate). (c) For phosphorylation of the diacyl glycerol, Eibl's method [27] was employed using 2-bromoethylphosphoric dichloride and direct amination with ammonia and trimethylamine. 9 was also prepared by a different method described previously [20]. 9, m.p.: 180°C, 58% yield. NMR(CDCI3): 8.4-8.7 (m, 3H, -NH~) ppm. FD-MS(24mA): m/e 958 (M+ H ÷, 100%); 833 (M+-PO(OH)-O-(CH2),. NH2, 83%). All other analytical data have been given in a previous publication [20]. 10, m.p.: 215°C, 65% yield. Calc. for Cs~HI~4NO~P-H20 (1016.48): C 70.90; H 10.51; N 1.38%. Found: C 70.6: H 10.3: N 1.5%. IR(KBr): 3420 (H20); 2180, 214(I (C=--C); 1730 (C=O); 1245 (P=O); 1180 (C-O); 1100 ( P - O ) ; 1065 (P-O-C) cm 1. NMR(CDCI3): 3.4 (s, 9H, N+(CH3)3) ppm. FD-MS(18 mA): m/e+999 (M + H +, 90%): 940 (M+-N(CH3)3, 43%): 833 (M+-PO(O)-O(CH2)z-N(CH3)3, 100%); 817 (M+-O-P(O-) O(CH2)z-N(CH3)3, 94%).

rac-2- (Hexacosa - 10,12-diynoyl)- 1-stearoyl-3-phosphatidic acid (11) (a) The synthesis of rac-l-stearoyl glycerol-3-iodohydrine (m.p.: 54-56°C) was performed as described for the L-enantiomer in the literature [30]. This compound was acylated [30] with the chloride of hexacosa-10,12-diynoic acid (see above) giving rac-2-(hexacosa-lO,12-diynoyl)-l-stearoyl glycerol iodohydrine. 17, m.p.: 33-36°C, 93% yield. The diacylated iodohydrine was reacted with silver-di-tert.butyl phosphate [30,31,32] as described in [30] yielding the di-tert.butyl phosphoric triester as an oil (98% yield). The tert.butyl protecting groups were removed with gaseous HC1 in CHCI3 and the crude product purified (as the barium salt) by silica gel chromatography [30]. 11, m.p.: 37-38°C, 30% yield. Calc. for C47H85OsP (809.16): C 69.77; H 10.59%. Found: C 69.5; H 10.5%. IR(KBr): 2720 (P-OH); 2180, 2140 ( ~ C ) ; 1735 (C=O); 1235 (P=O); 1065 (P-O-C); 1010 (P-OH) cm -1. FD-MS(9 mA): m/e 809 (M+ H +, 100%). Methods IR spectra were recorded with a Beckman IR 4220. For NMR measure-

363 ments a WH-90-FT (Bruker) was used. Chemical shifts are given in ppm with respect to TMS as an internal standard. FD mass spectra were obtained with a M A T 711 (Varian) using a tantalium wire emitter activated with benzonitrile. Absorption spectra were recorded with a Beckman 25 using 1-cm standard cuvets. A fluorescence spectrometer MPF-2A (Hitachi Perkin Elmer) was used for the fluorimetric measurements with 6-carboxyfluorescein (excitation wavelength: 490 rim, maximal fluorescence: 520 nm). H P L C was performed with a Prep LC/System 500 A (Waters) using a ~-Porasil column. Phase transition temperatures and melting points were determined using a DSC-2C instrument (Perkin Elmer). For crystalline substances (approx. 1 mg) standard pans and a heating rate of 20°C/min were employed. Lipid dispersions (50 i~1; 10 mg lipid/ml water) were measured using large volume pans with 5°C/min. In some cases turbidity measurements were also used to indicate phase transitions [33]. For this purpose a 1-cm cuvet containing the vesicle dispersion was placed in the thermostated sample chamber of a Beckman Acta IV spectrometer whose temperature was raised and lowered stepwise while the corresponding extinction (turbidity) at 300rim was measured. Lipids were dispersed ultrasonically in either water or buffer under a nitrogen atmosphere (Branson sonifier B15P, 3 5 W output power) at temperatures above the ordered-fluid phase transition with concentrations varying from 0.05 to 10 mg lipid/ml. Heterogeneities were removed by filtration through a 8 ~m Millipore filter. For polymerization, monomeric vesicles are irradiated in thermostated cuvets (1 cm) with a mercury high pressure lamp (240 W; Philips). The distance between lamp and sample was 30 cm. For determining the leakage rate of unpolymerized and polymerized vesicles two methods were used [34,35]. (a) Lipid (approx. 7 m g ) was ultrasonically dispersed in 2mt of 0.1 M 6-carboxyfluorescein (6-CF) [34] in 0.1 M Tris/0.1 M NaOH. Free 6-CF was removed by passing 500 ~1 of the sonicated solution through a short column (13× 180mm) of Sephadex G-50 at 20°C, with 0 . 1 M Tris-HCl/0.1N KCI (pH 8.4) as eluent. The void volume fraction (Ve 7-10ml) contains the marked liposomes. For determination of total entrapped 6-CF 0.5 ml 1 M sodium dodecylsulfate solution was added to 0.5 ml of eluate and the fluorescence measured after 30 min. Another 0.5 ml was added to 0.5 ml Tris-HC1/KC1 and the time-dependent release of 6-CF was measured. Irradiation of non-polymerizable 2b vesicles revealed that photolysis of 10% of the trapped 6-CF occurred under the employed polymerization conditions used for 2a (10 min UV, 0°C). This was considered for the c0.1culation of the amount of released dye from polymeric 2a vesicles. (b) The description of the assay for release of vesicle-trapped glucose is given in detail in Ref. 35 and is based on the phosphorylation and subsequent oxidation of released glucose by the hexokinase/glucose-6-phos-

364 phate dehydrogenase system with ATP, N A D P ÷ and Mg 2÷ as cofactors. Vesicles were prepared by ultrasonication of 10 mg lipid in 2 ml 0.3M D-glucose with the untrapped glucose was removed by Sephadex G-50 gel filtration using degassed 0.3 M D-mannitol at 45°C as eluent. Portions (200 ~1) of the void volume fraction (approx. 3 ml) were diluted with 300 ~10.15 M NaCI and the amount of glucose released was determined by adding 500 ~1 assay reagent [35] and measuring the absorption of N A D P H at 340 nm. For estimating the total trapped glucose, a 200 ~1 vesicle preparation was added to 100/~1 10% Triton X-100 solution (in 0.1 M Tris-HCl (pH 7.5)). After 10 min 200 pA of water and 500 ~1 of assay reagent were added and the amount of N A D P H was determined [35].

Results

Vesicle preparation and physical properties The synthetic compounds 1-11 were dispersible in water by ultrasound treatment above their phase transition temperature. Monomeric vesicles from 4, 5 and 8 will, however, precipitate within minutes when cooled to room temperature, and preparations of the phosphatidylcholine 10 with concentrations greater than 0.5 mg lipid/ml will also precipitate very rapidly. All other dispersions were stable at least for several days. In order to ensure that no significant lipid decomposition occurred under the sonication conditions used, aqueous preparations of all lipids were extracted with chloroform. TLC of the extracts showed no contaminations. Spherical bilayer structures were clearly visible in freeze-fracture electron micrographs. Further characterization of shape and size of monomeric and polymerized vesicles were carried out using low angle laser light scattering, photocorrelation spectroscopy, and gel filtration. This work will be published in detail elsewhere. Phase transition temperatures of the polymerizable vesicles are summarized in Table I. In the case of the butadiene tipids la and 2a the phase transition (T,,) occurs at about 20°C lower than for the corresponding dipalmitoyl compounds lb and 2b. The same is true for the diacetylenes, where the C26 chain containing two conjugated triple bonds (cf. 9, 10) has approximately the same effect on Tc as a saturated Cls chain (DSPC, DSPE). As expected in the series of the single chain phospholipids 4-7, T~ decreases with increasing bulkiness of the head group. A striking difference in T,: is visible in the case of the mixed chain phosphatidic acid 11 if compared, e.g. to the fully saturated DPPA. Moreover, diacetylenic lipids showed a marked hysteresis of the phase transition, which does not occur with the saturated compounds. This is probably due to the molecular structure of the polymerizable substances.

365 TABLE I T R A N S I T I O N T E M P E R A T U R E S F O R D I A C E T Y L E N I C A N D S A T U R A T E D PHOSP H O L I P I D S M E A S U R E D BY D I F F E R E N T I A L S C A N N I N G C A L O R I M E T P Y A N D TURBIDITY CHANGE

Compound la lb 2a 21) 3 4 5

Transition temperature by DSC (°C) 26" 45 a 21" 41 a 55 a,b 22c 43 a 33 ~ 11c 2a

6

15 c

7

- 10"~

8

Transition temperature by turbidity change (°C) 27" 21.5 a 52 ~ 25 ¢ ----

4 5 ~'¢

--

74aJ

9 10

11

DSPC g DPPA h DSPE ~

48.5 ¢ 51.5" 35 ¢ 36 ~ 18¢ 58 ~ 54 ~ 71 [36] 71 [37]

51.5 ~ 37 ~

58 • 52¢

aHeating run. bpretransition at 49°C. cCooling run. dPretransition at -16°C (measured in ethyleneglycol/water, 1 : 1). eAt p H 2. Tretransition at 59°C. gDistearoylphosph atidylcholine. hDipalmitoyl phosphatidic acid. ~Distearoytphosphatidyl ethanolamine.

Vesicle polymerization By irradiation

with UV

light, diacetylenic

and

1,4-trans-butadienic

polymerize

to form poly(diacetylene)s

with a conjugated

18 [38] a n d

1,4-trans-poly(butadiene)s

19, r e s p e c t i v e l y .

ene-yne

lipids

backbone

366

CH3 L (CH2),2

CH3 I (CH2)12

!

CH3 J (CH2),2

C%c

c

I

%0

CH3 ' ((~H2)12

/

!

h ' , ' . /7 "\

\

\

/

c

"

c I

(CH2/9

(0H2)9 I OPOaH2

I

OPOaHe

%,/o (CH2)9 ~ OPO3H2

3

OH3 I (CH2)12

[

CH % CH

I

COOH

(CH2)e I OPO3H2 18

OH3 I (CH2)~2

CH3 I (CH2)12

OH3 I (CH2)12

I

CH ~

COOH

COOH

CH

I

COOH 19

For diacetylene monomers the reaction proceeds only if the monomers are arranged in crystals [22], condensed mono- [7] or multilayers [6] and liposomes [9,10,13], whereas for the butadienes polymerization occurs only in membrane systems [12,22] or channel complexes [39]. Because of the conjugated nature of the polymer backbone the poly(diacetylene)s 18 have strong absorptions in the visible spectrum and exhibit a blue or red color. This presents the possibility of following the polyreaction by absorption spectroscopy. As is already known for other simple structured amphiphiles [6,9], vesicles from 3, 4 and 8 upon UV irradiation first form the blue polymer (Am,x 640 nm), which upon heating above the phase transition temperature of the monomer (Table I) (or by further irradiation) is converted to the red form (,~-max500, 540 rim) (Fig. 1). Since the topochemical polymerization of diacetylenes only occurs below To, vesicles from 6 and 7 are not polymerizable under the present experimental conditions without water freezing. A different absorption behavior was observed for vesicles formed from the phospholipids 9-11. On irradiation they do not form the blue form of the polymer but instantly show a red-orange absorption, whose maxima are shifted to 485 nm and 525 nm, respectively (Fig. 2). Similar results have been

%7 I

1,5

c

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..~.

/,,,'~

10

7o <

I

•

//

.....

/ ..............• / 'i .... i ' , / ......... i •

O5

0 400

/\

~(~

/,

~./.....,:

.

.

~............> , . ~ . _

t. / . ./\',

/.....\ .......\

/ ....

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t

i

T

500

600

\

/ \ ~\ / I :\ • ili'~k'.\

700

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F i g . 1. A b s o r p t i o n

s p e c t r a o f v e s i c l e s f r o m 3 (c 1 0 4 M ; 1 5 m i n s o n i c a t e d )

), a n d a f t e r i r r a d i a t i o n

( (

) and

for 3 0 s ( . . . .

1 3 5 0 s (-

), 1 5 0 s ( . . . . . . . .

before

UV

), 6 0 0 s ( . . . . .

irradiation ), 1 0 5 0 s

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obtained by other investigators for two diacetylenic phosphatidylcholines [10,13]: Polymerized vesicles from 9-11 also show the thermochromism described by C h a p m a n et al. [10] when heated a b o v e Tc of the m o n o m e r . In the case of 10 the long wavelength shoulder at 525 nm disappears at 51.5°C and returns if the vesicle solution is cooled below To. In all cases small vesicles obtained by prolonged sonication (1 h) did not polymerize. In the case of the butadienes (la, 2a) the polyreaction can qualitatively be followed by the decrease of the strong m o n o m e r absorption at 260 nm. Since the disappearance of the m o n o m e r absorbance must not necessarily be due to a polyreaction but can also be caused by, e.g. dimerization, additional proof for polymer formation was obtained by irradiation of the vesicles which were then freeze-dried and methylene chloride added to the residue. The product, on solvent addition, only swelled but did not dissolve. G P C of the extract s h o w e d only small amounts of oligomers and the absence of any m o n o m e r . (In contrast, uncrosslinked polymers from monofunctional butadienic lipids [22] are soluble in methylene chloride and are eluted from I ............'"" ........ " -.........

1.5 ....-

/ , / • ~ .~:.. /

1.0

I

<;.-..

<:'\

/ i

o <{

0.5

0 400

,

,

500

600

700

X (nm)

Fig. 2. Absorption spectra of vesicles from 10 (c 5 x 10 4 M; 15 min sonicated before (and after irradiation for 24min (. . . . ), 51 rain () and ll4min ( . . . . . . ).

368 a GPC column (exclusion molecular weight: 6000 (polystyrene) at the void volume.) Moreover, the IR spectrum of freeze-dried polymerized vesicles from l a was identical as that of an irradiated monolayer. This supports the formation of cross-linked 1,4-trans-poly(butadiene)s 19, which have also been observed for different amphiphiles in monolayers [22] and canal complexes [39]. Since the monomers are covalently linked by the polyreaction, the result is a restricted mobility of the alkyl chains. Figure 3 demonstrates how the intensity of the phase transition of 3 decreases with increasing conversion. Fully polymerized liposomes no longer show a phase transition. This is true for all investigated butadienic and diacetylenic monomers with similar results obtained by O'Brien et al. [13].

Stability and leakage behavior In former publications [9,16] the enhanced stability of polymerized vesicles was demonstrated by their resistance to osmotic shock or by obtaining a scanning electron micrograph. This was impossible in the case of unpolymerized vesicles. In addition, in all cases long-term storage (for months and years) of polymerized liposomes is possible. In the present study we investigated the effect of water soluble organic solvents on vesicles. The addition of ethanol to polymerized (blue) vesicles from 3 and 8 results in the formation of the red polymer, while the corresponding monomeric vesicles precipitate. Polymerized liposomes from 10 do not change their color on ethanol addition, but the influence of the organic solvent can be studied in the UV region by monitoring the turbidity change at 375 nm (Fig. 4). While monomeric vesicles already show a strong interaction with the addition of only small amounts of ethanol and finally precipitate with a considerable increase in turbidity if the solution contains more than 10% of ethanol, polymerized vesicles are not affected by the addition of up to 20% ethanol. If more alcohol was also added in this case the turbidity slowly increased but without precipitation of the polymerized vesicles. As marker molecules for the leakage tests, CF [34] and D-glucose [35] were used. CF at concentrations smaller than 0.01 M shows a fluorescence which is proportional to the CF concentration. At higher concentrations self quenching of the fluorescence occurs. Thus, after removal of untrapped CF from a vesicle dispersion in 0.1 M CF by gel filtration, the leakage rate of the liposomes could be determined by monitoring the increase of fluorescence. D-Glucose release from vesicles was measured by Kinsky's spectrophotometric method [35] based on enzymatic oxidation of the carbohydrate under formation of N A D P H , whose absorbance at 340nm could be measured.

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// / ]

0 64

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/

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100

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058 40

50

60 T { C}

Fig. 3

70

0

10

20 Vol

30

40

EtO~t

Fig. 4.

Fig. 3. Phase transitions of vesicles from 3 (c 0.02 M, 2 min sonicated) before ( after irradiation for 15 min (. . . . ) and 60 min (. . . . . . ).

), and

Fig. 4. Turbidity at 375 nm as a function of added ethanol for polymerized (&) and unpolymerized (0) vesicles from 10 (c 5 x 10-4 M).

In a previous work [16] it has already been shown that polymerization drastically decreases the leakage rate of vesicles from butadienic lecithin 2a. At 20°C, i.e. a b o v e Tc of 2a, unpolymerized vesicles release all trapped CF after approximately 50 h, whereas liposomes from D P P C (2b) (To 41°C) only release about 10% CF after the same time period. N o detectable release, h o w e v e r , could be observed for polymerized vesicles from 2a. The effect of ethanol on CF release from vesicles made from 2a and 2b is shown in Fig. 5. While unpolymerized liposomes at low concentrations already exhibit a c o m p l e t e release of fluorescent dye, polymeric 2a-vesicles lose only 15% trapped CF on addition of 30% ethanol. Similar results were obtained on addition of the surfactant, sodium dodecylsulfate (SDS), to different vesicle preparations (Fig. 6). Even a large excess of S D S failed to destroy all polymerized vesicles made from 2a. A s observed for 2b, m o n o m e r i c vesicles from the diacetylenic lecithin 10 (Tc 51.5°C) release 8% trapped CF after 50 h. Trapped glucose (Fig. 7) is freed at the same rate below Tc of these lipids, while a b o v e Tc the release occurs 1-2 orders of magnitude more rapidly. If vesicles from 10, however, were c o o l e d to 0°C, necessary for t o p • c h e m i c a l polymerization, all trapped glucose was released within seconds. This behavior was not observed with 2b

370

1O0

$

/e/e-

80

•//• /

6O

;

/

8o

/•

//•

J

/~

~ 60

40

./

20

ZI~

0 0

10

20

30

0

40

Vol. % EtOH

i

i-

--

0.5

10

CSD!g

(mrnol-k

15

2 0

] I

Fig. 5. Effect of ethanol on release of trapped CF from vesicles at 20°C. 2a, monomeric (O); 2b (A): 2a, polymeric (I). Fig. 6. Effect of SDS on release of trapped CF from vesicles. 2a, monomeric (O); 2b (A); 2a, polymeric (1).

]00

° / f ~ °

//~---m-

8O 6O t 40 2O 0 0

i 2

i 4

r 6

//.

i ,, 27

r 50

(h)

Fig. 7. Release of trapped D-glucose from vesicles. 2b, 24°C (&); 10, 24°C (O); 2b, 60°C (I); 10, 60°C (O); 10, 0°c (o). vesicles. A t t e m p t s to first p o l y m e r i z e and then r e m o v e untrapped marker failed: p o l y m e r i c vesicles from 10 were strongly adsorbed by S e p h a d e x gels and precipitated on dialysis or ultrafiltration against isotonic D-mannitol solution.

Discussion and conclusions T h e results d e m o n s t r a t e that vesicle formation is not restricted to naturally occurring two-chain p h o s p h o l i p i d s , but also occurs with lysolipid-like m o l e -

371

cules as 3-8, although the possibility of micelle formation cannot be completely ruled out. Also, much simpler structured amphiphiles with 19, 161 or without [ 11polymerizable groups have been shown to form bilayer assemblies. The phase transition temperatures of the polymerizable phospholipids 9 and 10 are comparable to distearoyl phosphohpids. Although the diacetylenic fatty acid used in this study contains 26 carbon atoms the disturbance of chain packing by the triple bonds-prevents proper crystallization in the gel state of the membranes. For fully saturated C,, chain phosphatidylcholines a T, of about 90°C was expected. The same behavior was observed for the butadienic phosphatidylcholine 2a (Cl8 fatty acid containing two conjugated double bonds), which exhibits a T, (21°C) similar to that of dimyristoyllecithin (23°C [lo]). A s expected the presence of double bonds near the hydrophilic head group does not affect T, to such a great extent while one double bond in the center of a Cl8 chain does (dioleoyllecithin, T, -23°C). In contrast, in the homologous series of the lysophospholipid analogs 4-7, the bulkiness of the head group is the limiting factor for chain crystallization. It was shown in monolayer experiments [19] that a pH decrease has a marked effect on T, of these compounds most likely attributable to changes in head group conformation. The low T, of the mixed chain phosphatidic acid 11cannot yet be properly explained. If the C, diacetylenic fatty acid had the same effect on T, as a stearoyl chain, a phase transition temperature similar to that of distearoyl phosphatidic acid (approx. 88°C) should be expected. Apparently the presence of the triple bonds in a mixed chain phospholipid more strongly prevents a regular crystallization than do the triple bonds in 9 and 10.In order to clarify this finding compounds are presently being prepared in which the positions of the alkyl chains as well as of the triple bonds are changed. In contrast to naturally occurring phospholipids the phase transitions of diacetylenic lipids show a marked hysteresis. As mentioned above the kink in the hydrocarbon chains prevents an instantaneous crystallizatian if vesicles are cooled only a few degrees below T,. The most pronounced effect is observed for 9 and 10,where two diacetylene chains are located in the same molecule. Since diacetylenes do only polymerize if they are perfectly ordered, i.e. in the crystalline state, their polymerization only occurs below T,. For freshly prepared vesicles from 10 (T, 51S”C) cooling to room temperature does not lead to crystallization and polymerization occurs only if the vesicles are cooled to 0°C. Evidence that this is a kinetic effect comes from the fact that vesicles from 10 after storage at room temperature for 2 weeks are photopolymerizable at all temperatures below T,. Similar results were obtained by O’Brien and coworkers [13,42] with another diacetylenic phosphatidylcholine. As discussed by these authors the reason for this finding could be attributed to the different distance of the polymerizable groups (of 10)from the hydrophilic head group. That this is, in fact, true is

372

shown by the fact that freshly prepared vesicles from symmetrically built double chain amphiphiles, where the diyne groups have the same distance from the hydrophilic head group, are already polymerizable at room temperature [9]. In similar observations by others [10,13,42], asymmetric compounds, such as 9, 10 and 11, on irradiation do not go through a blue intermediate phase before forming the final red-orange polymer. In addition, the absorption maxima of the red form were shifted to shorter wavelengths compared to single chain (e.g. 3) or symmetric double chain diacetylenic amphiphiles [9]. The reason for this is still unclear, although O’Brien [13,42] suggests that in molecules like 9 and 10 the diyne moieties are in a stereochemical arrangement that does not permit a simultaneous intramolecular and intermolecular reaction. If one diacetylene group of 9 or 10 has reacted, this probably decreases the reactivity of the remaining one leading to linear polymers which are soluble in a variety of organic solvents [lo], in strong contrast to the insolubility of most poly(diacetylene)s. This does, however, not explain the different absorbance behavior of symmetric and asymmetric lipids. An explanation for the shifted absorption maxima could be the formation of oligomers since it is known that, because of the short effective conjugation length, the absorption of non-planar poly(diacetylene) chains is shifted to smaller wavelengths [40]. Since the polymer from vesicle and monolayer polymerization is necessarily planar, the absence of a blue form and shifted absorptions of the red form can be explained by the presence of oligomers with 2-6 repeat units corresponding to short effective conjugation length of non-planar polymers [41]. Since the ‘polymer’ from 10 is soluble in a variety of solvents molecular weight determination is possible and this work is in progress. The restricted mobility of the alkyl chains caused by vesicle polymerization does not correspond to the smectic mesophase-like behavior of biological membranes. For this reason present attempts are being undertaken to synthesize phospholipids with one polymerizable and one nonpolymerizable chain. These can be handled more easily than 11 which decomposes rather quickly unless stored as sodium or potassium salt. In addition, polymerization in the head group should lead to enhanced chain mobility but may affect the head group properties. Thus another approach to more flexible polymerized membranes would be the build up of mixed monolayers and vesicles as discussed in Ref. 18. The butadienes la and 2a, in contrast to the diacetylenes, polymerized if the vesicles were either in the gel or in the liquid crystalline state. This reaction leading to 1,4-trans-poly(butadiene)s 19 is not topochemically, i.e. strictly lattice controlled. Because of the enhanced mobility of the poly(butadiene) backbone, also in polymerized vesicles of la and 2a, phase transitions were to be expected but not observed.

373 E x p e r i m e n t s c o n c e r n i n g t h e stability of t h e vesicles i n d i c a t e d that p o l y m e r i z a t i o n i n c r e a s e d t h e r e s i s t a n c e of t h e vesicles to o r g a n i c s o l v e n t s a n d d e t e r g e n t s . M o r e o v e r , t h e l e a k a g e r a t e of t r a p p e d m a r k e r s was d r a s t i c a l l y d i m i n i s h e d [16]. U n p o l y m e r i z e d vesicles f r o m a d i a c e t y l e n i c p h o s p h a t i d y l c h o l i n e r e l e a s e all t r a p p e d m a r k e r s u p o n c o o l i n g to 0°C. A p p a r e n t l y t h e m e m b r a n e of s u p e r c o o l e d vesicles, if f o r c e d to crystallize, d i d so u n d e r r e o r g a n i z a t i o n a n d s u p e r i m p o s i n g of m o n o m e r islands which c r e a t e d h o l e s in t h e vesicle m e m b r a n e t h r o u g h which t r a p p e d m a r k e r e s c a p e d . V e s i c l e s r e m a i n e d intact d u r i n g this p r o c e s s as e v i d e n c e d b y e l e c t r o n m i c r o s c o p i c studies. F u r t h e r m o r e , t h e h o l e s in t h e vesicle m e m b r a n e a p p e a r to h e a l d u r i n g p o l y m e r i z a t i o n since n e g a t i v e l y s t a i n e d p o l y m e r i c vesicles d i d n o t s h o w any stain in t h e i r i n t e r i o r . U n f o r t u n a t e l y , p o l y m e r i z e d l i p o s o m e s f r o m 10 w e r e v e r y sensitive to c h a n g e s of t h e i r e n v i r o n m e n t a n d all a t t e m p t s to r e m o v e u n t r a p p e d m a r k e r r e s u l t e d in the d e s t r u c t i o n of t h e vesicles. O b v i o u s l y t h e r i g i d i t y of t h e u n p o l y m e r i z e d m e m b r a n e s c o u l d n o t b e o v e r c o m e by t h e p o l y r e a c t i o n . W h y p o l y m e r i z e d vesicles f r o m 10 a r e n o t d e s t r o y e d b y e t h a n o l is still u n c l e a r a n d w o r k o n this is in p r o g r e s s .

References 1 2 3 4

J.H. Fendler, Acc. Chem. Res., 13 (1980) 7. E. Oldfield and D. Chapman, FEBS Lett., 23 (1972) 285. P. Chakrabarti and H.G. Khorana, Biochemistry, 14 (1975) 5021. C.M. Gupta, C.E. Costello and H.G. Khorana, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 3139. 5 D. Naegele and H. Ringsdorf, in: H.G. Elias (Ed.), Polymerization of Organized Systems, Midland Macromolecular Monographs, Vol. 3, Gordon & Breach, New York, 1976, p. 79, and refs. cited therein. 6 B. Tieke, G. Lieser and G. Wegner, J. Polym. Sci., Polym. Chem. Ed., 17 (1979) 1631. 7 D. Day and H. Ringsdorf, J. Polyml Sci., Polym. Lett. Ed., 16 (1978) 205. 8 R. Benz, W. Pra8 and H. Ringsdorf, Angew. Chem., Int. Ed. Engl., 21 (1982) 368. 9 H.-H. Hub, B. Hupfer, H. Koch and H. Ringsdorf, Angew. Chem., Int. Ed. Engl., 19 (1980) 938. 10 D.S. Johnston, S. Sanghera, D. Pons and D. Chapman, Biochim. Biophys. Acta, 602 (1980) 57. 11 S.L. Regen, B. Czech and A. Singh, J. Am. Chem. Soc., 102 (1980) 6638. 12 A. Akimoto, K. Dorn, L. Gros, H. Ringsdorf and H. Schupp, Angew. Chem., Int. Ed. Engl., 20 (1981) 90. 13 D.F. O'Brien, T.H. Whitesides and R.T. Klingbiel, J. Polym. Sci., Polym. Lett. Ed., 19 (1981) 95. 14 T. Kunitake, N. Kakashima, K. Takarabe, M. Nagai, A. Tsuge and H. Yanagi, J. Am. Chem. Soc, 103 (1981) 5945. 15 S.L. Regen, A. Singh, G. Oehme and M. Singh, J. Am. Chem. Soc., 104 (1982) 791. 16 L. Gros, H. Ringsdorf and H. Schupp, Angew. Chem. Int. Ed. Engl., 20 (1981) 305. 17 N. Wagner, K. Dose, H. Koch and H. Ringsdorf, FEBS Lett., 132 (1981) 313. 18 R. Bfischl, B. Hupfer and H. Ringsdorf, Makromol. Chem. Rapid Commun., 3 (1982) 589.

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B. Hupfer and H. Ringsdorf, Chem. Phys. Lipids, 33 (1983) 263. B. Hupfer, H. Ringsdorf and H. Schupp, Makromol. Chem., 182 (1981) 247. G. Wohlleben, Angew. Chem., 68 (1956) 752. H. Ringsdorf and H. Schupp. J. Macromol. Sci.-Chem., 15 (1981) 1015. F.R. Pfeiffer, C.K. Miao and J.A. Weisbach, J. Org. Chem., 35 (1970) 221. C.S. Haynes and F.A. Isherwood, Nature, 164 (1949) 1107. B. Tieke, G. Wegner, D. Naegele and H. Ringsdorf, Angew. Chem., Int. Ed. Engl., 15 (1976) 764. R. Aneja, J.S. Chada and A.P. Davies, Biochim. Biophys. Acta, 218 (1970) 102. H. Eibl and A. Niksch, Chem. Phys. Lipids, 22 (1978) 1. R. Hirt and R. Berchtold, Pharm. Acta Helv., 33 (t958) 349. F.H. Mattson and R.A. Volpenhein, J. Lipid Res., 3 (1962) 281. A.F. Rosenthal, Methods Enzymol., 35 (1975) 429. H. Goldwhite and B.C. Saunders, J. Chem. Soc. (1957) 2409. A. Zwierzak and M. Kluba, Tetrahedron, 27 (1971) 3163. C.S. Chong and K. Colbow, Biochim. Biophys. Acta, 436 (1976) 260. J.N. Weinstein, S. Yoshikami, P. Henkart, R. Blumenthal and W.A. Hagins, Science, 195 (1977) 489. S.C. Kinsky, Methods Enzymol. 32 (1974) 501. H. Eibl and A. Blume, Biochim. Biophys. Acta, 553 (1979) 476. K. Harlos, Biochim. Biophys. Acta, 511 (1978)348. G. Wegner, Makromol. Chem., 154 (1972) 32. B. Tieke and G. Wegner, Angew. Chem., tnt. Ed. Engl., 20 (1981) 687. G.N. Patel, J.D. Witt and Y.P. Khanna, J. Polym. Sci., Polym. Phys. Ed., 18 (1980) 1383. G.N. Patel and G.G. Miller, J. Macromol. Sci.-Phys., (1983) in press. E. Lopez, D.F. O'Brien and T.H. Whitesides, J. Am. Chem. Soc., 104 (1982) 305. P. Tundo, D.J. Kippenberger, P.L. Klahn, N.E. Prieto, T.-C. Jao and J.H. Fendler, J. Am. Chem. Soc., 104 (1982) 456. E. Baer and D. Buchnea, J. Biol. Chem., 232 (1958) 895. P.P.M. Bonsen, G.H. de Haas and L.L.M. van Deenen, Chem. Phys. Lipids, l (1966) 33.