preparation and characterization of unsymmetrical “liposomes in a net”

Makromol. Chem. 189,299-315 (1988) 299

Preparation and characterization of unsymmetrical “liposomes in a net”

Helmut Ringsdorf *, Bernhard Schlarb

Institute for Organic Chemistry, University of Maim, J.J.-Becher-Weg 18 -20, D-6500 Maim, Federal Republic of Germany

(Date of receipt: April 15, 1987)

SUMMARY: Negatively charged lipid molecules were converted into polymerizable lipids by introduction

of polymerizable mono- or bifunctional counterions. As an attempt to mimic the cytoskeleton of biomembranes, unsymmetrical polymeric vesicles were prepared, where the polyelectrolyte is attached either only to the inner or to the outer bilayer surfaces. Polymerizable cations were introduced to the outer surface of preformed small unilamellar vesicles via ion-exchange. The outer counterions of vesicles bearing polymerizable counterions at both sides of the membrane were replaced by Na’ . Polymerization of these systems leads to unsymmetrical vesicles. The introduction, separation and polymerization of the organic counterions was monitored by UV- spectroscopy. Due to polymerization of the counterions, the phase transition temperature of the membranes is shifted remarkably to higher temperatures. For a comparison of ionically attached and covalently bound polymers, two cationic lipids were synthesized, which have a bifunctional polymerizable headgroup. In contrast to the lipids with “ionic” spacers, the polymerization of the covalently bound methacrylic units resulted in a decrease of the phase transition temperature. Thus, by application of ionically bound polymerizable units, unsymmetrical polymerized vesicle membranes are readily available. These systems may serve as models for mimicking the cytoskeleton of living cells.

Introduction: Attempts to mimic the cytoskeleton of biomembranes

Liposomes, i. e. spherically closed lipid bilayers, have become a very useful bio- membrane model for biochemical and biophysical investigations during the last two decades This model was simplified even more by the introduction of readily available synthetic amphiphiles3), which led to an enormous arsenal of new membrane forming compounds4). Within a short time, vesicles could be stabilized by covalent cross-linking of lipid molecules within the double layer, which resulted in “polymerized vesicles”, a field which has been reviewed already4-7.7as 7b).

Biomembranes are stabilized in an entirely different manner @: A polymer coating supports the membrane. The architecture of biomembranes is highly unsymmetric, and the supporting biopolymers usually occur only at one side of the double layer. For example, erythrocytes possess a spectrin net, which forms a kind of cytoskeleton attached to the inner membrane surface, while bacteria have a murein coating sur- rounding the membrane surface as a component of the cell wall.

Attempts to mimic this natural process of membrane stabilization by coating a vesicle membrane with polymers were described in the literature. Polymers have been fixed to vesicle membranes by ionic interactions y- 1 7 ) , via hydrophobic anchor groups 1 8 * lY), or by polymerization of charged, water-soluble monomers at membrane

0025-1 16)3/88/$03.00

300 H. Ringsdorf, B. Schlarb

surfaces. These ionic monomers were fixed to charged lipid molecules either via salt formation u)*21) or as counterions 22-24). Other approaches for the preparation of cytoskeleton models are based on compound membranes of polymerized macrolipids with phospholipids 25,26) or on the polymerization of liposome-encapsulated hydrophilic monomers2T. For the case of monolayers from cationic amphiphiles at air- water interfaces, a stabilization by polyion complex formation has been described recently (e. g. ref. z*)).

This paper describes some attempts to mimic the cytoskeleton: Using charged lipids with polymerizable counterions there is the chance to prepare unsymmetrical polymer membranes with synthetic polyelectrolyte chains or even a net only at one side of the vesicle membrane. The characterization of these systems is discussed and compared with polymerized vesicles from lipids, bearing a covalently bound bifunctional polymerizable headgroup.

Results and discussion

In formulas 1 - 5 are shown the lipids with polymerizable mono- or bifunctional counterions which were used in this study:

CH3 I

I CHI

H,C, + /CH~-CHZ-OOC-C=CH~ N

H3C/ \CHI-CH2-00C-C=CH2

H~C-(CH~),,-COO-CH~ I

4 H,c-(cH,~,,-COO-CH 0 I II

I 0- Na'

CHZ-O-P-0-

H3C-(CH2)12-COO-CH2 I

5 H~C-(CH~)~I-COO-CH 0 I I I

C H I - 0 - P a - I 0- Na'

7 H 3

H3C\ + /CHZ-CH2-OOC-C=CH2 N

H,C/ \CH~-CH~-OOC-C=CH~ I

CH3

Preparation and characterization of unsymmetrical “liposomes in a net” 301

The structure and synthesis of bifunctional lipids, with covalently bound polymerizable units in the headgroup region, is shown in the following reaction Scheme 1.

2 CH,=C-COOH I

CH3

1

CH2-CH2-OH H,N-(cH,),-N’

‘CH,-CH~-OH I 10

CH,Br 1

Br

12 d H ,


The preparation of unsymmetrical vesicle membranes from charged lipids was carried out by the exchange of counterions at the outer bilayer surface of preformed small unilamellar vesicles. The vesicle preparations used in this study had an average diameter of 50 - 80 nm, as determined by dynamic light scattering. Three methods for the preparation of vesicles, with polymer attached to the inner, outer, or to both bilayer surfaces, are shown in Fig. 1.

Preparation of vesicIes with polymer attached to the outer membrane surface

When vesicles from charged lipids are passed through a column which is filled with an ion-exchange resin, the counterions at the outer membrane surface can be ex- changed (e. g. refs. 29,30)). This method was used to replace sodium ions by polymerizable organic cations only at the outer membrane surface (Fig. 1, left route). The

0 0

/ \ 0 O - N a *

SONlCATlON / N a'

\ ION \ EXCHANGE

0 0 'P4 @-A 0' '0-

SON1 C AT ION I

l h u l h v 1 I h u

POLYMER OUTSIDE

POLYMER INSIDE POLYMER A N D OUTSIDE INSIDE

Fig. 1. Possible methods for the preparation of vesicles with charged polymers attached to the inner, outer, or both bilayer surfaces


introduction of these counterions can be observed by UV-spectroscopy. Both polymerizable counterions, the cross-linker as well as the monofunctional choline methacrylate, have an absorption maximum at 209 nm in vesicular solutions. As an example, Fig. 2 shows the UV-spectra of a vesicle solution of dimyristoylphosphatidic acid (DMPA) before and after the introduction of bifunctional cross-linker cations.

Fig. 2. UV-spectra of DMPA- vesicles before (A) and after (B) the introduction of bifunctional polymerizable counterions to the outer bilayer surface

A

x .= 1.50 C a, U - 0

a 0

u 1

0.75

C

-0OC

-0oc

190 210 230 250 270 290 Wavelength in nm

To check the amount of exchange, vesicular solutions of DMPA and dihexadecyl- phosphate (sodium salt, DHPNa) were lyophilized after the introduction of choline methacrylate counterions by the ion-exchange process. This material was used to record a NMR-spectrum in isotropic solution. From the integrated signals, the ratio of lipid anion to polymerizable choline methacrylate cations was determined (data not shown). For DMPA-vesicles with two negative charges this ratio was found to be one, for DHPNa with only one negative charge, it is two. Only one half of the negative charges of the vesicles participate in the exchange process, which indicates, that the exchange occurs only at the outer membrane surface. This can also be shown by GPC experiments: When these vesicles with UV-active counterions at the outer bilayer surface are passed through a GPC-column (Sephadex G25, 20 m~ NaCl as eluent), all polymerizable counterions are removed from the vesicle fraction as indicated by UV- spectroscopy. The vesicle fraction after GPC shows no absorption at 209 nm, as illustrated in Fig. 3.


Fig. 3. Separation of polymerizable counterions from the outer membrane surface by GPC: UV-spectra of DHP- vesicles before (A) and after (B) the replacement of choline methacrylate ions by Na+ at the outer bilayer surface

190 210 230 250 270 290 Wavelength in nm

Preparation of vesicles with polymer only inside

The anionic polymerizable lipids 1 - 3 were synthesized by ion-exchange in isotropic solution. While lipid 1 with two short alkyl chains is not able to form vesicles, lipids 2 and 3 form vesicles with polymerizable counterions at both bilayer surfaces very easily upon ultrasonication. Using GPC, the polymerizable counterions were removed only from the outer membrane surface (20 m NaCl as eluent). The remaining organic counterions at the inner bilayer surface were polymerized by UV-irradiation (Fig. 1, right route). For example, Fig. 4 shows the UV-spectra of DHP-vesicles with bifunctional counterions at both membrane surfaces before and after GPC. The UV-spectra shown in this paper were obtained by dilution of an aliquot of the original vesicle solutions to a final volume of 3 ml. The dilution of the vesicle solutions due to GPC was corrected before recording the UV-spectra by application of an appropriate aliquot. From these UV-spectroscopic data, the amount of remaining counterions at the inner membrane surface can be estimated to be 34%. This value is in good agreement with the theoretical value which is based on the different amounts of lipid molecules in the inner and outer bilayer half of a small unilamellar vesicle. E. g. for a 50 nm vesicle - as used in the experiments - only 39% of the lipid molecules are located in the inner half of the double layer.


Fig. 4. Preparation of DHP-vesicles with bifunctional polymerizable counterions only at the inner membrane surface: UV- spectra before (A) and after (B) separation of the organic cations from the outer vesicle surface

$ 1.5- c a, U

0 0

a 0

- .- c

1.0 -

- 200 220 210 260 280 300

Wavelength in nm

100%

31%

Polymerization behavior

The ionically bound counterions can be polymerized on the vesicle surface by irradiation with UV-light. For example, the UV-spectra of vesicles from DHP anions with sodium counterions at the inner and choline methacrylate cations at the outer membrane surfaces, before and after one and two minutes of UV-irradiation, are shown in Fig. 5 . It is obvious, that there is a total conversion of the monomer already after two minutes of irradiation. The dynamic light scattering measurements revealed no changes in size of the vesicles after polymerization (data not shown).

Influence of the polymer coat on the phase transition temperature of vesicles

The influence of the introduction of polymerizable counterions and the resulting polyelectrolytes on the phase transition temperature (T,) of the vesicle systems was studied by light scattering measurements. Due to the introduction of polymerizable counterions, the phase transition temperature of large vesicles prepared from a mixture of the phosphatidic acid derivative 4 (95 mol-To) and the appropriate cross- linker 5 (5 mol-%) is shifted to lower temperatures: While the disodium salt of DMPA shows its transition at 49°C (lit.3’): 50°C), the transition of the vesicle preparation from this mixture of the polymerizable lipids 4 and 5 appears at 31 OC.


0.70 Ln C a, U

0 V

Q 0

- .- I

0.35

0

Fig. 5. Polymerization of choline methacrylate counterions attached to the outer vesicle surface of DHP-vesicles: UV-spectra before and after 1 and 2 min of UV-irradiation. (Polymerization method A)

190 210 230 250 270 290

Wavelength in nm

The transition of this system is shifted remarkably to higher temperatures after polymerization of the counterions (T, = 51 "C), as shown in Fig. 6.

Thus, the influence on T, of the ionically bound polyelectrolyte networks prepared by the methods described in this paper is comparable to the effect of poly(1ysine) bound to negatively charged bilayers. The addition of positively charged poly(1ysine) molecules to phosphatidic acid membranes increases the transition temperature of the DMPA bilayers from T, = 50°C to T, = 62"Cl4).

A similar effect of polymerization on the phase transition was found in the case of a polymerizable lipid, which has a long hydrophilic spacer group between the alkyl chains and the polymerizable unit 32). This behavior was explained by a decoupling of the motions of the unordered polymer chain and the ordered bilayer, together with an enhanced cooperativity of the polymerized lipids. This explanation can be used also for a polymer backbone, which is linked ionically to the lipid bilayer ("ionic spacer"), because each monomeric unit is not linked to a certain lipid molecule. Therefore, the polyelectrolyte chain is able to move laterally at the membrane surface, so that the decoupling effect should be even better than in the case of a hydrophilic spacer group.

Two lipids with a covalently bound bifunctional polymerizable headgroup (8 and 12) were synthesized to compare the effect of an ionically fixed network with a network fixed covalently to the vesicle surface. In contrast to lipid 8, the dimethacrylate

Preparation and characterization of unsymmetrical "liposomes in a net" 307

z1 c C al C

m C

Fig. 6. 90"-light v)

scattering intensity of L

large DMPA-vesicles .- with polymerizable counterions (A) and a al polymer net (B) c

attached to the membrane surface. L

(Heating curves. Mixture of lipids 4 and -+ 5 (5 mol-To). 0 Polvmerization ol

.- L

c

0 u ul

L m .-

B

Tm=3loC -\* rn o n o rn e r

method B) i 0 10 20 30 LO 50 60

T / O C

12 has a short hydrophilic spacer between the alkyl chains and the ammonium structure of the headgroup. Lipid 8 is not able to swell in water. The phase transition temperature of lipid 12 was determined in lamellar phases by DSC-measurements. It was found that the phase transition of the spacer-containing ammonium lipid 12 appears at T, = 40°C (see Fig. 8). Above its phase transition temperature, lipid 12 forms giant vesicles very easily by swelling a lipid film on a glass surface in water. For example, Fig. 7 presents a freeze fracture electron micrograph of a large vesicle, prepared from lipid 12 by this method. The influence of the cross-linking polymerization of the bismethacrylic headgroups of lipid 12 on the phase transition temperature was studied by DSC measurements (Fig. 8) and by light scattering (Fig. 9). The results from both methods correlate very well. In contrast to the ionically fixed polymer net, the covalently bound polymer net causes a decrease of the phase transition temperature: The initial value of 40°C for the monomeric vesicles from lipid 12 is decreased to 35 "C by polymerization. This behavior is comparable to that of vesicles from the

Fig. 7. Freeze fracture electron micrograph of a monomeric vesicle from the bifunctional polymerizable lipid 12. (Bar = 0,l pm)


0 290.00 300.00 310.00 320.00 330.00 310.00

Temp. in K

Fig. 8. DSC heating curves of monomeric and polymerized suspensions of lipid 12. (Polymeri- zation method c)

Fig. 9. 90°-light scattering intensity of monomeric and polymerized large vesicles from the cross- linker 12. (Polymerization method B)

10 20 30 40 50 60 T / O C

monofunctional polymerizable lipid (3-methacrylamidopropyl)methyldioctadecyl- ammonium bromide. The decrease of T, in this system was explained by a disorder- ing influence of the polymer chain on the head group packing5).

In summary, the results described in this paper show that unsymmetrical "liposomes in a net" are readily available by binding of polymerizable counterions to charged vesicle surfaces. The newly developed bifunctional lipid 12 is the first example of a polymerizable lipid, which can be cross-linked within the hydrophilic

Preparation and characterization of unsymmetrical "liposomes in a net" 309

headgroup region. This novel type of polymerizable lipid allows the formation of a two-dimensional covalently bound polymer network, coating the vesicle membrane.

By a combination of one of the ionic lipids 2 - 5 with lipid 12 it should be possible to create a polymeric net at the inner vesicle surface, which, in addition to ionic interactions, is attached to the membrane via the hydrophobic anchor groups of lipid 12. A structure of this type would be very similar to the red cell membrane skeleton, where the spectrin net at the inner membrane surface is coupled to an integral membrane protein (band 111) by ankyrin.

A study of the 3H-glucose permeation rate across monomeric and polymeric unsymmetric and symmetric vesicle membranes of the type described in this paper will be published in due course 33).

Experimental part

Preparation of lipids with polymerizable counterions

Preparation of choline methacrylate: This salt [ (2-methacryloyloxyethyl)trimethylammonium bromide] was prepared by quaternization of 2-dimethylaminoethyl methacrylate (Merck) with methyl bromide and purified by recrystallization from acetone; m. p.: 205 "C.

C9HI8BrNq (2522) Calc. C42,87 H7,20 N 5 3 5 Br 31,69 Found C 42,84 H 6,80 N 5,56 Br 3131

Preparation of bb(2-rnethacryloyloxyethyl)dimethylammonium bromide: a) Bis(2-methacry- 1oyloxyethyl)methylamine: 20 g (0,i 8 mol) of 2,2 '-(methy1imino)diethanol (Merck) and 30ml of pyridine were dissolved in 200 ml of CH2Cb. A solution of 40 g (0,38 mol) of methacryloyl chloride in 50 ml of CH2Cb was added to the stirred solution at 0 "C. Stirring was continued at room temperature for 24 h. Then the solvent was replaced by diethyl ether, and the solution was washed with sodium carbonate solution and water. After drying and removal of the solvent, the remaining product was stirred in high vacuum for several hours to remove pyridine. Yield: 26,4 g (57%) of a slightly yellow liquid. %I9: 1,4668.

IR (NaCl): 1730 (C=O), 1645 (C=C), 1 170 (C-0-C).

IH NMR (CDCb): 6 = 6,09 (t, 2H, ROOC/ H3C'C=C/ ,H 1 , 5 3 5 (t. 2H, ROOC/C=C<G 1,

4,25 (t, 4H, CH2-OOC), 2,78 (t, 4H, N-CH2), 2,% (s, 3H, CH3-N), 1,93ppm (s, 6H,

H H3C\ H

CH3-C=CHz).

C I ~ H ~ I N O ~ (255,3) Calc. C 61,16 H 8,29 N5,49 Found C 61,35 H 7,92 N 5,45

b) Bis(2-methacryloyloxyethyl)dimethylammonium bromide: Bis(2-methacryloyloxyethy1)- methylamine was dissolved in acetone and quaternized with methyl bromide. The ammonium salt crystallized from the reaction mixture at - 18 "C and was purified by recrystallization from acetone: m. D.: 117 "C.

IR (KBr):-i 720 (C=O), 1635 (C=C), 1 170 (C-0-C). H,C\ H H3C' H

H 'H H NMR (CDCh): 6 = 6,09 (t, 2H, ROOC/C=C< ), 5,61 (t, 2H, RooC/C=C/-),


C,,H,BrNO, (350,3) Calc. C 4 7 9 H 6,91 N4,W Br 22,82 Found C 47,75 H 6,82 N 3,86 Br 22,93

Preparation of polymerizable lipids 1, 2 and 3

Didodecyl hydrogen phosphate was synthesized as described by Kunitake 34) and purified by recrystallization from acetone; m.p.: 60°C (lit.34): 51 -52°C). The free acid was converted into the sodium salt; m.p.: 70°C (DSC).

Dihexadecyl hydrogen phosphate was purchased from Sigma and converted into the sodium salt.

Lipids 1 - 3 were prepared by ion-exchange from the appropriate sodium salts of the dialkyl phosphates. For this purpose, a glass column (1 7 X 1 3 cm), filled with a cation-exchange resin (Merck Lewatit SP 1080), was loaded with the polymerizable counterion by treatment with an excess of a 10% solution of the appropriate bromide salt in water. Excess salt was then removed by washing the resin with water. After this, water was replaced by methanol. Then a solution of sodium dialkyl phosphate in methanol was passed through this column. The eluent fractions containing the lipid were collected and the solvent evaporated. The residue was recrystallized twice from acetone. (2-Methacryloyloxyethyl)trimethylammonium didodecyl phosphate (1); m. p.: 42 "C.

G3GN0(iP (m5,9) Calc. C65,42 H 11,31 N2,31 P 5,11 Found C 65,08 H 11,49 N 2,33 P 5,OO

(2-Methacryloyloxyethyl)trimethylammonium dihexadecyl phosphate (2); m. p. : 61 "C.

IR (KBr): 2910, 2840 (CH3,CH2), 1715 (C=O), 1630 (C=C), 1470 (CH3,CH2), 1225 (P=O), 1 180 (C-0-C), lo90 (P-0-C, P-0).

H H3C\ H ' H NMR (CDCb): 6 = 6,W (t, lH, Root/ H3C\C=C/ \H ), 5,62 (t, 1H, ROOC/C=C<k 1,

4,60 (m, 2H, CH2-OOC), 4,04 (m, 2H, 'N-CY), K75 (2t, 4H, CH2-0-P), 3,42 (s, 9H, CH3-N+), 1,91 (s, 3H, CH3-C=C), 1,54(m, 4H, CH2-CH2-O-P), 1,20-1,28(m,52H, (CH2)13), 0,84 ppm (t, 6H, cH3-(cH2)15).

C41 &NO6 P (7 1 891) Calc. C 6 8 3 H 11,79 N 1,95 P 4,31 Br - Found C 68,19 H 12,03 N 2,19 P 4,65 Br -

Bis(2-methacryloyloxyethyl)dimethylammonium dihexadecyl phosphate (3); m.p.: 59 - 60 "C.

H NMR (CDCb): same signals as lipid 2. Intensity ratio: 2 : 2 : 4: 4 : 4 : 6 : 6 : 4 : 52: 6.

C&I&J'QP (8162) Calc. C67,69 H 11,ll N 1,72 P3,79 Found C 66,73 H 11,02 N 1.53 P 3,60

Preparation of lipids 4 and 5

Small unilamellar vesicles were prepared from DMPA (disodium salt, Fluka) to achieve a final lipid concentration of 3 mg/ml water. This vesicular solution was passed through a cation exchange column (Merck Lewatit SP 1080 or Pharmacia CM-Sephadex C25), which had been loaded with the appropriate polymerizable cation, and the eluted vesicle fractions were collected and lypophihed. The NMR spectrum of this material indicated the presence of about one polymerizable counterion per lipid molecule.

Preparation and characterization of unsymmetrical "liposomes in a net" 31 1

Preparation of lipi& with covalently bound polyrnerizable groups

Bis(2-hydroxyethyl)dioctadecylammonium bromide (6): 20 g (38 mmol) of dioctadecylamine (Fluka, recrystallized from ethyl acetate three times), 48 g (0,38 mol) of 2-bromoethanol and 6 g (46 mmol) of diisopropylethylamine in 60 ml of CHCI, were refluxed for three days. After washing of the resulting reaction mixture first with dilute HBr and then with water, the solvent was removed. The product was recrystallized twice from acetone. Yield: 22 g (840/0); m.p.: 71 "C (DSC).

IR (KBr): 3300 (OH), 2910, 2850, 1470 (CH2,CH3). ' H NMR (CDCI.,): 6 = 4,82 (s, 2H, OH), 4,08 (t, 4H, N-CH2--CH2-0), 3,67 (t, 4H,

CH2-0), 3,43 (m, 4H, (CH2),,-CH2-N+), 1,67 (m, 4H, (CH2),,-CH2-CH2-N+), 1,2- 1,4 (m, 60H, (CH2)15), 035 ppm (t, 6H, CH3-(CH2),,).

CN%,BrNq (691 ,O) Calc. C 69,53 H 12,25 N 2,03 Br 11,56 Found C69,23 H 12,15 N 1,75 Br 11,23

Bis(2-methacryloyloxyethyl)dioctadecylammonium bromide (8): 5 g (7,2 mmol) of the diol6, 2,5 g (29 mmol) of methacrylic acid and 200 mg of 4-dimethylaminopyridine (DMAP) were dissolved in 80 ml of CHCI,. Then 6 g (29 mmol) of dicyclohexylcarbodiimide (DCCI) in 20 ml of CHCI., were added at OOC, according to Neises et al. 35). After filtration and removal of the solvent, the product was recrystallized three times from acetone. Yield: 4,6 g (770/0); m.p.: 89°C (DSC).

IR (KBr): 2910, 2840 (CH3,CH2), 1720 (C=O), 1630 (C=C), 1 160 (C-0-C). H H3C\C=C/- H

1H NMR: 6 = 6,io (t, 2H, H 3 C \ ~ = ~ / ),5,63 (tt 2H, Root/ \H ),4JO (t, R O O 6 \H

4H, CH2-OOC), 4,lO (t, 4H, N-CH2-CH2-OOC), 3,48 (m, 4H, (CH2)16-CH2-N+), 1,90 (s, 6H, CH3-C= CH,), 1,74 (m, 4H, (CH2)15-CH2-CH2-N), 1,2-1,4 (m, 60H, (CH2)15). 035 PPm (t, C H ~ ~ C W I ~ ) .

C,q2BrNO, (827,2) Calc. C 69,70 H 11,21 N 1,69 Br 9,66 Found C 69,32 H 11,ll N 2,40 Br 10,36

N,N-Dioctadecylsuccinamic acid (7): 20 g (38 mmol) of dioctadecylamine and 5,4 g (54 mmol) of succinic anhydride were refluxed in 60 ml of CHCI., for 10 h. Then the solvent was removed and the product recrystallized two times from acetone. Yield: 23 g (970/0); m. p.: 59°C (lit.36): 59-61 "C).

C@H@O, (622,1) Calc. C 77,23 H 12,80 N 2,25 Found C 77,23 H 12,80 N 2,23

Succinimido N,N-dioctadecylsuccinamate (9): 5 g (8 mmol) of N,N-dioctadecylsuccinamic acid, 1,15 g (1 0 mmol) of N-hydroxysuccinimide and 100 mg DMAP were dissolved in a mixture of 30 ml of CHCI,,30 ml of CH2C& and 20 ml of acetone and cooled to 0°C. Then 2,l g (10 mmol) of DCCI, dissolved in 10 ml of CH2Cb, was added according to Neises et al. 35). After 2A h the precipitate and the solvents were removed and the product was recrystallized twice from acetone. Yield: 3,8 g (66Vo); m.p.: 61 "C (lit.37): 64-67OC).

IR (KBr): 2915, 2845 (CH3, CH2), 1820 (C=O, ester), 1 790, 1745 (C=O, imide), 1 645 (C=O, amide).

' H NMR (CDCI.,): 6 = 3,l- 3,4 (m, 4H, CH2-N-CO), 3,00 (t, 2H, CH2-COO-N), 2,80 (s, 4H, CH2 maleimide), 2,70 (t, 2H, N-CO-CH2), 1,l - 1,6 (m, 64H, (CH2),,), 0,86 ppm (t, 6H, CH3).

C44%2N205 (7192) Calc. C 73,49 H 11,49 N 3,90 Found C73,38 H 10,64 N 3,91


N-3-[Bis(2-hydroxyethyl)aminolpropyl-N'~dioctadecylsuccinamide (10): 4 g (5,6 mmol) of activated ester 9 were dissolved in 50 ml of CHCJ. Then 2 g (12,3 mmol) of 2,2'-(3-amino- propy1imino)diethanol (Aldrich), dissolved in 10 ml of CHCJ, were added at 0 "C. The mixture was stirred for 3 h at 0 "C and for 24 h at 20°C. Then the solvent was removed and the product recrystallized from acetone yielding 3,65 g (85%) of crude, waxy product. The product was used without further purification. Bis(2-hydroxyethyl)-[3-(N,N-di~tadecylsuccinamoylamino)propyflmethylammonium bro-

mide (11): 7 g (9,l mmol) of the tertiary amine 10 were dissolved in 80 ml of CHCJ and cooled to - 5 OC. Then 5 ml(88 mmol) of liquid C 4 B r was added and the flask sealed with a stopper. This mixture was stirred at 20 "C for 4 days. Then the solvent and excess CH3Br were removed and the residue was recrystallized twice from a mixture of 600 ml of acetone and 100 ml of CHCb. Yield: 3,9 g (50%); m.p.: 52°C (DSC).

IR (KBr): 3400 (OH), 2910,2850 (CH,, CH3), 1635 (C=O), 1545 (C=O, amide 11), 1470 (CH3, CY).

' H NMR (CDCb): 6 = 7,65 (t, lH, CO-N-H), 5,l (s, 2H, broad, OH), 4,09 (m, 4H, + N-CH,-CH,-OH), 3,68 (m, 6H, CHz-OH, NH-(CHz)z-CHz-N+), 3,35 (m, 2H, CO-NH-CH,), 3,30 (s, 3H, CH3-N+), 3,17 (m, 4H, (CHz),6-CHz-N), 2,62, 2,51 (2t, 4H, CO-CY-CHz-CO), 2,07 (m, 2H, CO-NH-CHz-CHz-CH,-N+), 134 (m, 2H, CH3-(CHz),5-CH,-C~-N-C0, cis), 1,42 (m, 2H, CH3-(CHz),5-CHz-C~-N-C0, trans), 123 (m, 6OH, (CY)15), 0,87 ppm (t,6H, CH3-(CH,)17).

C,q8BrN304 (861,2) Calc. C 66,94 H 11,46 N 4,88 Br 9,28 Found C 66,84 H 10,25 N 4,93 Br 9,63

Bis(2-methacryioy1oxyethyl)- [3-(N, N-dioctadecylsuccinamoylamino)propy fl methylammonium bromide (12): 3 g (3,9 mmol) of diol11, 1,3 g (1 5 mmol) of methacrylic acid and 100 mg DMAP were dissolved in 100 ml of CHCh. Then 3,l g (15 mmol) of DCCI, dissolved in 10 ml of CHC4, was added at 0 "C according to Neises et aI. 35) and the mixture was stirred at room temperature overnight. After filtration, the product was purified by adsorption chromatography using 300 g of silica gel (Merck, Silica Gel 60) and CHC&/CH30H = 9/1 (by vol.) as the elution medium. After removal of the solvents, the product was recrystallized from diethyl ether and then freeze dried from benzene solution. Yield: 657 mg (17%) of white crystals; m.p.: 41 "C (DSC).

IR (KBr): 2920, 2850 (CH,, CH3), 1725 (C=O, ester), 1645 (CEO, amide), 1550 (C=O, amide 11), 1 470 (CH,, CH,). - -

H ' H NMR (CDCb): 6 = 7,86 (t, lH, CO-N-H), 6,lO (t, 2H, ROOC/ H3C\C=C/ 'I-J 1,5764 (t,

H 2H' ROOC/ 'H

H3 c\ c=c/- ), 4,67 (t, 4H, CHz-OOC), 4,02 (m, 4H, + N-CHz-CH,-OOC),

3,87 (m, 2H, NH--(CH,)z-CH,-N+), 3,40 (m, 5H, CH-,-N+, NH-CHz), 3,15 (2t, 4H,

2,17 (m, 2H, NH-CHz-CHz-CHz-N+), 1,91 (s, 6H, CH3-C=CY), 1,52 (m, 2H, CH3-(CI-€&-CHz-C&-N-C0, cis), 1,40 (m, 2H, C~-(CHz),5-CHz-CHz-N-C0, trans), 123 (m, 60H, (CY)15h 035 ppm 0, 6H, cH3-(c&)17).

(CHz),,-CHz-N-CO), 2,63 (t. 2H, CHZ-CO-NH), 2,50 (t, 2H, CHz-CO-N(alkylh),

G ~ H ~ M B ~ N ~ O ~ (997,4) CdC. C 67,44 H 10,71 N 4,21 Br 8,Ol Found C66,13 H 10,85 N3,86 Br 8,51

Methods

Water was purified by distillation and passed through a Mim-Q water purification system (Millipore Corp.). The purity of all lipids was checked by TLC. Melting points were determined


with a Bilchi melting point apparatus (uncorrected) or by differential scanning calorimetry (DSC) (onset). The infrared spectra were recorded with a Perkin-Elmer 457 IR spectrometer. NMR spectra were obtained with a Bruker AM 400 (400 MHz) spectrometer. The UV spectra were recorded with a Beckmann UV 6 spectrometer. Vesicle solutions were diluted before recording a UV-spectrum.

Determination of phase transition temperatures by DSC: The lipid (2 - 3 mg) and 60 p1 of water were placed into Al pans and hydrated at 60-70°C for three hours. The thermal behavior was investigated in a Perkin-Elmer differential scanning calorimeter (DCS2C) at a heating rate of 2,5 K/min.

The determination of phase transition temperatures by light scattering measurements and the preparation of freeze fracture replica was carried out as described in ref. 38). Dynamic light scattering measurements were carried out with a Coulter “Nano-Sizer”.

GPC: The vesicles were chromatographed on prepacked Sephadex G 25 columns (Pharmacia PD 10) at room temperature.

Preparation of symmetrical vesicles

The lipid-water mixture was sonicated above the phase transition temperature with a Branson sonifier B 15 P (microtip) for 15 min. The lipid concentration was 3 - 2 0 mg/ml. Titanium particles were removed by centrifugation.

Suspensions of thin-walled vesicles of some vm diameter for the light scattering measurements and for freeze fracture replica were prepared as described in ref. 38).

Preparation of unsymmetrical vesicles

Monomer and polymer only at the outer membrane surface: Symmetrical vesicles from DHPNa or DMPA were prepared by sonication, as described above. These small unilamellar vesicles were passed through a glass column filled with a cation-exchange resin (Merck Lewatit SP 1080 or Pharmacia CM-Sephadex C25), which had previously been loaded with a polymerizable cation. A peristaltic pump was used with a flow rate of 0,5 ml/min. The vesicle fraction, as indicated by a refractive index detector, was collected and used for further investigations, or for UV-polymerization.

Monomer and polymer at the inner membrane surface: Symmetrical vesicles from compounds 2 or 3 were prepared. The polymerizable counterions at the outer vesicle surface were removed by passing these vesicles through a GPC-column using 20 m~ NaCl solution as an eluent. Excess NaCl was then removed by a second GPC using pure water as an eluent.

Polymerization of vesicles

Method A: The vesicle solution was transferred into a 1 cm quartz cuvette with Teflon stopper and flushed with nitrogen for 5 min. The sample was irradiated at room temperature with a 200 W high pressure mercury lamp (Oriel) at a distance of 30 cm for 10 min.

Method B: The sample was irradiated at T = 6OoC with a low pressure mercury lamp (“Pen Ray”) at a distance of 4 cm for 15 min.

Method C: 5 mol-Yo of the azo-initiator 4,4‘-azobis(4-~yanovaleric acid) (ACVA) was added to the lipid suspension. This mixture was heated to 70 “C for 2 h.


We are grateful to Conny Fuhn (Technical University of Munich, Garching) for the preparation of the freeze fracture replica.

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