Polymerizable phospholipids with lipoic acid as head group: synthesis and phase properties

Chemistry and Physics of LIPIDS ELSEVIER SCIENTIFIC PUBLISHERS IRELAND

Chemistry and Physics of Lipids 66 (1993) 63-74

Polymerizable phospholipids with lipoic acid as head group: synthesis and phase properties Horst Pax, Alfred Blume* Fachbereich Chemie der Universti~t Kaiserslautern, Erwin Schr6dinger Strafle, D-67653 Kaiserslautern. Germany'

(Received 29 December, 1992; accepted 24 February, 1993)

Abstract

Lipoic acid-substituted phospholipids were synthesized from phosphatidyl cholines by exchanging the choline residue with a glycol group using phospholipase D, forming phosphatidylglycols, and coupling of lipoic acid (l,2dithiolane-3-pentanoic acid) to the residual glycol hydroxyl group by esterification. The thermotropic properties of aqueous dispersions of the phosphatidylglycols and the phosphatidylglycolipoates with different chain lengths were studied by differential scanning calorimetry (DSC) and differential scanning densitometry (DSD). The exchange of choline with glycol increases the phase transition temperature Tm for the gel to liquid-crystalline phase transition by - 5°, independent of the chain length. Consecutive esterification of the glycol residue with lipoic acid causes a decrease of the transition temperature Tm by - 2 2 ° compared with the phosphatidylglycols. Polymerization of vesicles prepared from phosphatidylglycolipoates was induced by the addition of dithiothreitol and followed by DSC and DSD. Polymerization has only slight influence on the transition behavior, indicating that the polymer chain in the head group is effectively decoupled from the hydrocarbon region. Polymerized vesicles show increased stability towards aggregation and fusion. Key words: Differential scanning calorimetry; Differential scanning densitometry; Lipoic acid; Phase transition;

Polymerizable phospholipids

1. Introduction

In biomembranes the chemical structure of the head group of phospholipids is limited to a few residues such as choline, serine, glycerol, ethanolamine and inositol. The influence of chemical modifications of the head-group structure on the * Corresponding author at, Fachbereich Chemic, Universt.~t Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany.

phase transition properties of model membranes has been a subject of continued interest over the last 20 years and was investigated in detail using various physical-chemical methods (for reviews see Refs. 1-5). Polymerizable phospholipids have attracted interest because polymerization of lipids in vesicles can improve the stability of lipid vesicles towards aggregation a n d fusion. Polymerizable groups can be introduced into the hydrophobic chains as well as into the head group. The concept of coupling

0009-3084/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0009-3084(93)02169-R

64

the polymerizable group via a spacer to the side chain has been applied in the field of liquidcrystalline polymers and is also useful for polymerizable phospholipids. This spacer concept reduces the perturbation of the polymer backbone on the packing characteristics of the lipid hydrocarbon chains. Potential applications of these polymerizable lipids are in the field of drug delivery systems (for reviews see Refs. 6-8). Lipoic acid, with its disulfide bond in a fivemembered ring, is a naturally occurring molecule and functions as a co-factor to pyruvate dehydrogenase. It is covalently linked to a lysine residue of the E2 sub-unit via an amide bond. Lipoic acid was introduced by Regen and coworkers as an interesting moiety for coupling to phospholipids, because the disulfide bond should allow polymerization of the lipid molecules under mild physiological conditions, yielding stable vesicles that can also be depolymerized [9-11]. Such polymerized vesicles should be well suited for use as drug carriers in vivo, because of their potential biodegradability. Regen and coworkers described the introduction of lipoic acid into phospholipids, the lipoic acid residue being always coupled to the hydrophobic chains [9-11]. In this paper we describe phospholipids in which the lipoic acid is coupled to the head group and report on its influence on the phase transition properties of lipid vesicles. First experiments on the effects of polymerization on the phase transition properties are also reported. 2. Materials and methods

2.1. Chemicals 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) were obtained from Sigma (Deisenhofen). Epikuron 200 H, a product of Lukas Meyer (Hamburg), is a hydrogenated soy bean phosphatidylcholine with an average composition of 83.2% stearic acid and 16.8% palmitic acid. The lipids were checked for purity by TLC and used without further purification. Glycol (Riedel de HaEn, Seeize), ethylenediaminetetraacetic acid (EDTA) (Merck, Darmstadt), lipoic acid (DE-l,2-dithiolane-3-pentanoic acid, DL-

H. Pax, A. Blume / Chem. Phys. Lipids 66 (1993) 63-74

thioctic acid) (Sigma) and dithiothreitol (DL-I,4dimercapto-2,3-butandiol) (Sigma) were used as obtained. Dicyclohexylcarbodiimide (DCC) (Merck-Schuchardt, Hohenbrunn) and 4-dimethylaminopyridin (4-DMAP) (Aldrich, Steinheim) were recrystallized from toluene before use. Chloroform, dichloromethane, methanol and acetone (Riedel de Hahn) were of p.a. grade. Dichloromethane was dried over phosphorus pentoxide.

2.2. Synthesis The reactions are described below for DMPC as starting compound. The reactions with other phosphatidylcholines are completely analogous. 2.3. 1,2-Dimyristoyl-sn-glycero-3-phosphoglycol (DMPGlycol, la) Phospholipase D was isolated from cabbage, as previously reported by Eibl and Kovatchev [12]. Storage of the enzyme solution at -18°C is possible for several months without a significant loss of activity. The enzymatic exchange of choline with glycol is also described by Eibl and Kovatchev [12]. The reaction was started with 1.5 mmol DMPC, and the yield of isolated la was 90%. TLC: Rf = 0.64. IH-NMR (CDCI3), 6(ppm): 0.89 (t, 6H, C_H3), 1.28 is, 40H, CO,), 1.59 is, 4H, OCOCH2CH2), 2.30 (q, 4H, OCOC_H2) 3.64-3.76 (m, 4H, POC_H2C_H2), 3.88-4.02 (m, 2H, CHC_HzOP), 4.12-4.22 (m, 1H, CH2CHCH2OP), 4.35-4.45 [C_H2CHCH2OP), 5.19-5.28 (m, 1H, C_HCH2OP). For storage the lipid should be kept at -5°C in the dark. 2.4. Lipoic acid anhydride The anhydride was synthesized and characterized as reported, using DCC as condensing agent [13]. IR reaction control showed the presence of the lipoic acid anhydride carbonyl (1745 and 1817 cm -I) and the absence of the acid carbonyl band (1700 cm-I). The reaction was started with 5 mmol lipoic acid and yielded 88% of the anhydride. 2.5. 1,2-Dimyristoyl-sn-glycero-3-phosphoglycolipoate ( D M PGlylipoate, 2a) A mixture of 580 mg (0.89 mmol) la, 870 mg

H. Pax, A. Blume/ Chem. Phys. Lipids 66 (1993) 63-74 (2.2 mmol) lipoic acid anhydride and 110 mg (0.89 mmol) 4-DMAP was dissolved in 20 ml of dry dichloromethane and stirred for 24 h under argon. The reaction was controlled by TLC. After removing the solvent the residue was dissolved in 15 ml chloroform. The lipid was then precipitated by the addition of 80 ml acetone. The yellow product was filtered by suction. Chromatographic purification on a silica gel column (1.9 x 140 mm) with a chloroform/methanol gradient yielded 303 mg (0.36 mmol) 2a (40%). TLC: Rf = 0.84, IH-NMR (CDCi3), ~5(ppm): 0.88 (t, 6H, C_H3), 1.28 (s, 40H, CH2), 1.42-1.51 (m, 2H, SCHCH2C_H2CH2), 1.53-1.80 (m, 8H, SCHCH2CH2CH 2 and OCOCH2CH2), 1.86-1.97 (m, 2H, SSCH2CH2), 2.27-2.51 (m, 6H, POCH2CH2OCOC_H2 and OCOC_H2), 3.07-3.22 (m, 2H, SSCH2), 3.51-3.61 (m, 1H, SSC_H), 3.88-3.95 (m, 2H, POC_H:CH2), 3.96-4.17 (m, 2H CHC_H2OP), 4.18-4.23 (m, 1H, C_H2CHCH2OP), 4.24-4.33 (m, 2H, POCH2C_H2), 4.35-4.43 (m, 1H, C_H2 CHCH2OP), 5.18-5.27 (m, 1H, CHCH2OP) [14]. For storage the lipid should be kept at -5°C in the dark.

2.6. Differential scanning calorimetry ( DSC) DSC experiments were carried out with a Microcal MC2 calorimeter (MicroCal, Inc., Amherst, MA, USA). DSC data was analyzed using the ORIGIN program supplied by MicroCal. The lipid concentrations varied between 2 and 5 mg/ml. All samples were scanned several times at a rate of 20 K/h until reproducible curves were obtained. 2. 7. Differential scanning densitometry ( DSD) Densitometric data was accumulated on a DMA60 densitometer with two external measuring cells DMA 602H (Paar KG, Graz). The two cells were thermostatted using a Haake F3C circulating water bath. The temperature was measured inside the sample cell with a platinum resistance thermometer. Temperature scanning and data aquisition and evaluation was under computer control using an Atari ST microcomputer connected to a Rhomodul-BUS-adapter (Rhothron, Homburg). Lipid concentrations ranged between 9 and 13 mg/ml. Before the measurements the lipid dispersions were thoroughly degassed for at least 5 min

65 under water aspirator vacuum. The scan rate was 20 K/h.

2.8. Dynamic light scattering ( D L S ) Vesicle diameters were determined by dynamic light scattering using a Malvern Zetaziser 3 with an AZ10 cell and the Malvern program for size analysis. 2.9. Nuclear magnetic resonance spectroscopy The IH-NMR spectra were measured on an AMX400 NMR spectrometer (Bruker, Karlsruhe). 2.10. Thin layer chromatography ( T L C ) For TLC we used silica gel 60 plates with fluorescent indicator F254 (Merck). As eluting solvent a mixture of CHC13/MeOH/7M NH3, 230:90:15 (v:v:v), was used. Phospholipids were detected with molybdenum blue spray reagent (1.3% molybdenum oxide in 4.2 M sulfuric acid), other substances by charring. 2.11. Vesicle preparation For the vesicle preparation we used the ultrasonic method described by Saunders [15]. Phospholipid (4-26 mg) was dispersed in a 0.05 M KC! solution above the transition temperature of the lipid and mechanically shaken for several minutes. The dispersion was then homogenized in an ultrasonic bath (Branson 1200) at 60°C for 20 min. The vesicle diameters as determined by DLS were in the range 60-200 nm for freshly prepared suspensions. The pH of the lipid dispersions was 6. 2.12. Vesicle polymeri-ation The pH of the monomer lipid dispersion was raised to 8.5 by the addition of dilute NaOH. Polymerization was started by the addition of an aliquot of an aqueous 10 mM dithiothreitol solution to the vesicle suspension until a proportion of 10 mol"/,,dithiothreitol in relation to the phospholipid was reached. Polymerization was complete after 2 days, as determined from the DSC curves, which showed no further changes. As judged from a TLC analysis of the polymerized vesicles, - 90% of the material was polymerized, showing one major spot with an Rf value of 0.0.

66

H. Pax, A. Blume / Chem. Phys. Lipids 66 (1993) 63-74

dilute aqueous dispersions can be caused by modifications of the chains or the heads of the lipids. For a given chain Tm critically depends on the chemical nature and charge of the head group and its hydration properties [3-5]. Changes in transition temperature are combined effects of changes in intermolecular interactions between the lipid head groups modulated, for instance, by hydrogen bonding, by changes in hydration and changes in charge, the last-named being of minor importance [3]. It is well known that the exchange of the choline residue in PC by a glycerol moiety has almost no effect on the phase transition temperature, PGs thus having a very similar phase behaviour to PCs, as long as the ionic strength of the solution is high enough, i.e. larger than 10 mM [3-5]. In pure

3. Results and discussion

3.1. Synthesis The synthesis followed described procedures. A general outline of the steps involved in our synthesis and the chemical structure of the newly synthesized phospholipids is shown in Fig. 1.

3.2. Differential scanning calorimetry (DSC) The thermotropic properties of the phospholipids were first investigated using differential scanning calorimetry (DSC). The DSC curves of the lipids l a - c and 2a-c are shown in Fig: 2. Their thermotropic data is summarized in Table I. Changes in lipid phase transition temperature Tm from the gel to the liquid-crystalline phase in O

II CH3-~CH2---~C--O--CH2 CH3-(CH2-}nj~-O--~H O

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Lipoic Acid Anhydride

O

II

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o

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~-c

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Fig. I. Scheme for synthetic procedure for the preparation of PGlycols (la-e) and PGlycolipoates (2a-c).

67

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Fig. 2. DSC curves for the phase transition of (A) DMPGlycol (top) and DMPGlylipoate (bottom); (B) DPPGIycol (top) and DPPGlylipoate (bottom) and (C) 'EpiGlycol' (top) and 'EpiGlylipoate' (bottom). water PGs tend to swell indefinitely, and small vesicles showing different phase behaviour are observed. This is very similar for the phosphatidylglycols. Only in 50 m M salt solution can reproducible D S C curves be obtained. The Tm values observed for the phosphatidylglycols are slightly higher than those o f the corresponding PCs. This Tm increase could be a simple size effect

caused by the smaller glycol head group as compared with the choline and also with the glycerol residue. With model compounds, such as the alkyl esters o f phosphatidic acids, a similar increase o f Tm with decreased head group size is observed, the ethyl ester o f phosphatidic acid (PA) having a higher Tm value than the propyl ester o f P A [16]. Increasing the size of the head group by coupl-

Table 1 Thermotropic data from DSC measurements for the phase transition of iipids la:2e Lipids

T m (°C)

ATI/2 (°C)

DMPC |5] DMPGlycoi (la) DMPGlylipoate (2a)

24.0 26.6 4.7

1.5 2.0

6.5 6.3 4.8 a

21.9 21.0 17.3a

DPPC [5] DPPGlycol (lb) DPPGlylipoate (2b)

41.5 45.9 22.9

2.4 1.5

8.7 8.6 8.4

27.7 26.9 28.4

Epikuron 200 H 'EpiGlycol' (le) 'EpiGlylipoate'(2e)

51.5 59. I 37.5

2.9 5.5 4.2

10.6 9.7 8.6

32.7 29.2 27.7

aEstimated by extrapolation to low temperature.

AHcaI (kcal/mol)

AScaI (cal/mol • K)

68

ing the lipoic acid to the glycol residue leads to a drastic decrease in Tm by -22°C. This decrease is again very similar to the effect of an increase in chain length in the alkyl esters of PA. The octyi ester of DPPA has a transition temperature similar to that of the dipalmitoyl compound 2h, namely -22°C [16]. For the dimyristoyl derivative the transition enthalpy AHca I decreases also, showing that the voluminous head group disturbs the packing of the lipids in the gel phase. This is also evident from the slight increase in the half width of the transition, i.e. the cooperativity of the transition is somewhat decreased. For the longer chain compounds the introduction of the lipoic acid residue in the head group increases the cooperativity somewhat without large changes in transition enthalpy (see Table I). Obviously the perturbation effects are chain-length-dependent, the influence of the head group being reduced with increased chain length. This is in agreement with the thermotropic behaviour of other phospholipid classes, where a reduction of the influence of different head-group structures on the phase transition was also observed [2-5], The low transition temperature, the reduction in transition enthalpy and cooperativity observed for DMPGlylipoate could be explained by the assumption that the dimyristoyl compound is close to a change to a different state of association, i.e. a micellar state. The van der Waals interactions between the chains seem just large enough to counterbalance the disturbing effect of the head group. With shorter chains a change into a micellar state could occur, as then the critical packing parameter v/ao.l~, describing the volume v of the molecule in relation to the product of the cross-sectional area of the head group a0 and the effective length lc of the molecule, becomes too small [17]. For phosphatidylcholines this occurs for compounds with chain lengths below twelve Catoms [3]. For the lipoic acid derivatives with their larger head group a change to a micellar state for the Ci2 derivative seems likely.

3.3. Differential scanning densitometry ( DSD) Whereas differential scanning calorimetry can only detect changes in enthalpy occurring at the phase transition temperature, differential scanning

H. Pax, A. Blume / Chem. Phys. Lipids 66 (1993) 63-74

densitometry (DSD) yields information on the change in molecular volume at the phase transition but also on the absolute values of molecular volume and thus on the packing properties of the molecules in the different phases [ 18,19]. The DSD curves obtained on heating vesicle suspensions of the iipids l a - e and 2a-c are shown in Fig. 3. The transition temperature Tm, the molecular volume Vm, the change in molecular volume at the phase transition AVm and the calculated volume of the lipoic acid Vlipoic residue are summarized in Table lI. The phase transition temperature Tm, as measured in the DSD experiments, shows the same effect due to the head group volume as observed before in the DSC measurements. Some transition temperatures derived from the DSD experiments are somewhat lower than from the DSC curves. This is caused by the different method of determination. The DSC temperatures were taken from the maxima of the Cdi~T graphs, wheras the DSD transition temperatures were obtained from the half height of the sigmoidal transition curve, i.e. at a degree of transition of 0.5. In the case of symmetric transitions both methods yield the same value for Tin. When the transitions are asymmetric the Tm values obtained from the DSC curves are somewhat higher. The change in molecular volume AVm at the transition and the transition enthalpy AHcaI are correlated. Changes in head group structure have similar effects on the AHcar and A Vm-values. The phosphatidylglycols with the small glycol head group show larger AH~al- and A Vm-values than the corresponding glycolipoate derivatives with their more voluminous head groups. The large head group apparently causes a less compact packing of the fatty acids and facilitates the formation of gauche conformers in the gel phase, thus reducing the differences of molecular volume between the gel and the liquid-crystalline phase. We will see below that the calculation of the volumes of the hydrophobic region of the different phospholipids supports this assumption. From the experimental data the molecular volumes of various parts of the lipid molecules, i.e. the head group, the backbone and the fatty acyl chains, can be calculated using certain assumptions. We will first assume that the hydrophobic

H. Pax, A. Blume / Chem. Phys. Lipids 66 (1993) 63.74

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Fig. 3. Molecularvolumesas a functionof temperaturefor (A) DMPGlycol(bottom)and DMPGlylipoate(top); (B) DPPGlycol (bottom) and DPPGlylipoate(top) and (C) 'EpiGlycol'(bottom)and 'EpiGlylipoate'(top).

region of the phospholipids at the same absolute temperature in the liquid-crystalline state is essentially identical. This assumption is justified by the finding that the difference between the hydrocarbon volumes of phosphatidylcholines and phosphatidylethanolamines is less than 0.5% [18,20]. Using this assumption, the volumes of the various head groups can be calculated. We will first use this approach to calculate the volume of the head group, which will then be subtracted from the molecular volumes in the gel phase to obtain the hydrocarbon volume for the ordered phase. Comparison of the molecular volume Vm of DMPGlycol (1129 A, 3) with that of DMPA (1011 A 3, averaged value from Refs. 19 and 21) at a temperature of 50°C yields for the volume of the glycol group a value of 118 A 3. This is a reasonable value compared with an estimation using values of - 3 0 A 3 for a CH 2 group and an OH group. Adding the average volume of the lipoic acid residue in the liquid-crystalline phase ( - 340 ,~ 3) gives a total volume for the glycolipoate head group without the glycero-phosphate backbone of - 458 ]k 3. Estimation of the molecular volume of glycol and lipoic residue using a volume of - 3 0

,~ 3 per methylene group and also for the sulfur atoms and the C--~-O moiety leads to a rough estimate of - 300 ,/~3 for the total volume of the lipoic acid residue. This compares well with the - 340 3 found from the difference between PGlycol and the PGlylipoate molecular volumes in the liquid-crystalline phase (see Table II). To obtain the total volume of the PGlycol and PGlylipoate head groups, two procedures are possible. The first one uses the difference between the hydrocarbon volume of known lipids and the molecular volume of DMPA in the gel phase to obtain the volume of the glycero-phosphate residue, which can then be added to obtain the total volume of the head group, including the backbone. The hydrocarbon volume of a bilayer in the gel phase can be calculated from the X-ray spacing yielding a value of 810 ~3 for DPPE with 16 carbon atoms, i.e. a volume of 25.3 A, 3 per methylene group [18]. For a 14-carbon atom chain this would give a value of 709 .~, 3. For DMPA in the gel phase, the average molecular volume is 935 A, 3 at 10°C, the difference of 226 A 3 thus being the volume of the glycero-phosphate backbone, including the sodium counter ion [19,21]. This has to

70

H. Pax, A. Blume / Chem. Phys. Lipids 66 (1993) 63-74

Table 2 Molecular volumes and changes in volume at the phase transition for lipids l a - 2 c as determined by DSD Lipids

Tm (°C)

Vm ( ,~ 3/molecule)

A Vma ( A 3/molecule)

Gel phase ( 10)°C

Ic phase (50°C) b

Vlipoic residue ( ~" 3/molecule) Gel phase (10°C)

lc phase (50°C) b

DMPGlycol (la) DMPGlylipoate (2a)

26.7 3.9

1045 1425 c

1129 1488

44 27

380

359

DPPGlycol (Ib) DPPGlylipoate (2h)

44.1 22.6

1266 1600

1366 1691

65 55

334

325

'EpiGlycol' (Ic) 'EpiGlylipoate' (2e)

56.4 38.3

1333 1720

1445 c 1783

85 52

387

338

aAVm due to phase transition is calculated from the differences in Vm-values below and above Tm at temperatures where deviations from linearity are observed. bLiquid-crystalline phase. CExtrapolated with the coefficients of thermal expansion c~, belonging to the particular phase.

be added to the value of 118 ~, 3 of the glycol resi-' due to obtain the volume of the PGlycol head group of 344 ,~ 3 and to the value of 458 ,4, 3 of the glycolipoate residue to obtain 684 ,4, 3 as the total volume of the head group, including the backbone and the counter ion. These two values are thus lower limits for the total volumes of the head groups of the dimyristoyi compound. Using these values one would calculate hydrocarbon volumes for lipids in the gel phase that are too large compared with other lipids. The alternative approach leads to more reasonable data. The head group volume of PGlycol and PGlylipoate can be calculated from the differences in molecular volumes of PCs and the PGlycols and PGlylipoates, respectively, at temperatures in the liquid-crystalline phase. Taking a value of 344 ,~, 3 for the PC head group volume [18], this approach gives head group volumes of 363 ~3 for DMPGlycol and 722 ,~3 for DMPGlylipoate. They are thus larger than the estimates using the first procedure. A peculiar chain length effect is evident when the dimyristoyl compounds are compared with the dipalmitoyl derivative and the lipid with the mixed chains with predominantly stearic (83%) and palmitic acid (17%). Normally one observes an increase in molecular volume of - 2 5 A, 3 and

27.5-28 ~ . 3 per additional methylene group in the gel and the liquid-crystalline phase, respectively [18]. On going from the dimyristoyl to the dipalmitoyl compound the molecular volume should thus increase by -110 A, 3 in contrast to the observed increase of - 200-235 .~ 3 (see Table II). The difference between the dipalmitoyl compounds and the 'EpiGlycol' and 'EpiGiylipoate', however, is in the expected range of values, with 80 and 90 ]k 3. Thus the calculation of the head group volumes for the longer-chain derivatives leads to larger values of 490 ,~, 3 and 815 ~, 3 for DPPGlycol and DPPGlylipoate, respectively, and 480 A, 3 and 818 ,~,3 for 'EpiGlycol' and 'EpiGlylipoate'. These two pairs of values are very similar but considerably higher than the ones for the dimyristoyl compounds. We decided to use the estimated values of this second procedure to separately calculate the hydrocarbon volumes in the gel phase for the different compounds. Perturbations in chain packing in the gel phase should show up in the volumes calculated for the hydrocarbon region of the bilayers. When the volumes of the head group are subtracted from the total molecular volume for lipids in the gel phase, the volumes for the hydrocarbon chains in the ordered phase are obtained. Subtraction of the choline residue volume of 344 A~3 from DMPC -

72

H. Pax, A. Blume / Chem, Phys. Lipids 66 (1993) 63-74 80O0

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1750

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Fig. 4. Left: DSC curves of DPPGlylipoate vesicles before (top) and after (bottom) polymerization. Right: DSD curves of DPPGlylipoate vesicles before (top) and after (bottom) polymerization.

phase can easily be formed. When the ring o f the lipoic acid is opened the extended chain has a length o f - 7 - 8 ,~. This length is very similar to the average centre-to-centre distance of monomeric lipid molecules in lamellar phases. Polymerization, therefore, does not disturb the chain packing, as no additional strain is created. This is different with the methacryloyl group as

which the methacryloyl group is connected to the phosphate via an oligo-ethylene-glycol spacer, an increase in transition temperature is observed [6,7]. In the glycolipoate phospholipids the perturbation by polymerization seems to be minimal, because the average distance between the 'side chains', i.e. the lipid molecules, o f the polymer has such a value that an expanded liquid-crystalline

Table 3 Comparison of phase transition parameters of DMPGlylipoate (2a) and DPPGlylipoate (2b) before and after polymerization Lipids

Tm (DSC)

Tm (DSD)

AHcal (kcal/mol)

(C°)

Vm(A 3/molecule)

A Vma ( A 3/molecule)

Gel phase (10°C)

I-c phase (50°C)b

DMPGlylipoate Poly-DMPGlylipoate

4.7 7.4

3.9 5.5

4.8¢ 4.4

1425d 1313d

1488 1390

27 26

DPPGlylipoate Poly-DPPGlylipoate

22.9 26.5

22.6 25.7

8.4 8.6

1600 1542

1691 1633

55 52

aAVm values are determined as described in Table I1. bLiquid-crystallihe phase. CEstimated by extrapolation to low temperature. dExtrapolated with the coefficient of thermal expansion or, belonging to the particular phase.

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H. Pax, A. Blume / Chem. Phys. Lipids 66 (1993) 63-74

Table 4 Stability of DMPGlylipoate (2a) and DPPGlylipoate (2b) before and after polymerization. Number distribution for the diameter of the vesicles is given. The samples were stored at room temperature Lipids

Maximum (nm)

Range (nm)

Type of sample

DMPGlylipoate DMPGlylipoate Poly-DMPGlylipoate

102 472 122

Poly-DMPGIy!ipoate

155

60-137 Freshlysonicated 241-804 After20 days 89-195 Freshlysonicated and after 2 days of polymerization 89-220 After20 days

DPPGlylipoate DPPGlylipoate Poly-DPPGlylipoate

108 493 138

Poly-DPPGIylipoate

167

73-163 Freshlysonicated 222-1044 After 20 days 101-214 Freshlysonicated and after 2 days of polymerization 127-273 After20 days

polymerizable moiety, as in this case the 'side chains' are separated by only two carbon bonds. Polymerization thus induces additional strain on the packing, leading to a change in thermotropic properties. One of the potential advantages of polymerized vesicles is their greater stability towards aggregation and fusion. We have checked the size of our vesicles as a function of time to test this hypothesis. In Table IV the dynamic light-scattering DLS data of the polymerized and unpolymerized vesicles is summarized. Indeed the polymerized vesicles demonstrate a much higher stability to aggregation or fusion than the unpolymerized vesicles. After 20 days they show only a slight increase in vesicle size, whereas the vesicles made from monomers tend to fuse and aggregate, despite their net negative surface charge.

4. Summary and conclusions Phospholipids with glycolipoate head groups as a polymerizable moiety are potentially useful com-

pounds for the preparation of stable vesicles, which are increasingly discussed as drug delivery systems, particularly in dermatological applications [8,22]. The lipoic acid residue has the advantage of being a biological moiety, and is thus easily biodegradable [9-11]. The polymerization of the monomers in the vesicles occurs via an intermolecular disulfide formation. The S - - S bonds can be opened by mild cleavage, so that a depolymerization is possible resulting in vesicles consisting again of monomers. The phospholipids are easily synthesized in a two-step synthesis, the first step employing the phospholipase D catalyzed exchange of the choline group of PCs by glycol. In the second step the lipoic acid is coupled to the glycol residue by esterification. As starting compounds cheap hydrogenated or natural soy bean phosphatidylcholines can be used. Polymerization is initiated by addition of dithiothreitol. The polymerized vesicles show increased stability towards aggregation and fusion as compared with the vesicles prepared from the monomers. The phase transition properties of the polymeric vesicles are almost unchanged, indicating that the polymer backbone is effectively decoupled from the hydrocarbon region. A polymerizationdepolymerization cycle is in principle possible, which could be employed to influence the release of encapsulated molecules.

5. Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.

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