Langmuir 1992,8, 1731-1735
1731
Comparative Dynamic Stabilities of Cyclopropyl and Olefinic Model Lipids in Liposomes. A Coordinated Kinetic and Spectroscopic Study Robert A.
Tsunehisa F u j i t a , I a Yukihisa Okumura,laZheng H u a , l a Richard M e n d e l s o h n , * J b and Lawrence Senaklb
Moss,*Ja
Department of Chemistry, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903, and Department of Chemistry, Rutgers, The State University of New Jersey, Newark, New Jersey 07102 Received March 5,1992. In Final Form: April 5,1992 Cyclopropanated dihydrosterculoyl(3-F,3-NF) and unsaturated oleoyl (cis-2-F,cis-2-NF) ammonium ion lipids were prepared. Li osomes were created by sonication of 1:lO functionallnonfunctional lipid blends. The small (300-450 liquid crystalline (T,< 15 "C) liposomes were surface differentiated by exovesicular glutathione in an endovesicularlexovesicdarpH gradient of 3.918. At 25 "C, the cyclopropyl liposomes of 3 reequilibrated by transbilayer lipid migration much more slowly (tip = 16 min) than the oleoyl liposomes of 2 (tip < 1 min). The origins of this difference were traced to variations in the lipids' acyl chain conformational states. Fourier transform IR studies revealed that the acyl chains of 3 contained fewer highly disruptive double gauche conformational sites than the chains of 2. This can be associated with better chain packing, increased chain order, and the observed enhanced resistance to lipid "flip-flop" in liposomes of 3.
1)
Introduction The precise role and significance of the cyclopropyl lipids are unclear. Their high local ordering and long Lipids that contain cyclopropanated fatty acid chains motional correlation times may fit them for a regulatory occur in certain bacteria and protozoa; representative is l-palmitoyl-2-dihydrosterculoyl-sn-glycero-3-phospho- role, for instance, control of the dynamic processes that affect membrane stability.3 Thus, the occurrence of cycholine, 1, where the cyclopropanated chain is derived clopropane-containingfatty acids in the stationary growth from dihydrosterculic acid.2 NMR studies of liposomes phase of E. coli produces membranes with enhanced constructed from specifically deuterated 1revealed liquid organizational stability.6 Moreover, the unsaturated lipcrystalline bilayers (above 0 "C)that were less fluid and ids are probably the biosynthetic precursors of their cymore ordered a t ambient temperatures than those of clopropyl analogues, so that the cyclopropanation step analogous lipid bilayers that featured sn-2 oleoyl chains; can be regarded as a final developmental stage in a bioi.e., a A9-cisdouble bond in place of the cyclopropyl group synthetic sequence that permits the membrane to attain of 1 3 3 maximal stability relative to external perturbations.3 In view of the potential importance of these structurally 0 II modified lipids, we initiated a study of the comparative dynamic stabilities of model cyclopropyl and olefinic lipids in synthetic liposomes. Recently, we described chemical methods for the differentiation of the inner and outer surfaces of bilayer liposomes that enabled us to monitor the subsequent decay of the imposed chemicalasymmetry brought about by transbilayer lipid migration (or flipflop).6 This, in turn, permitted a systematic study of the k 1 7 relation between a lipid's molecular structure and ita dynamic behavior within a liposomal aggregate.' We have 1 used this methodology to examine (inter alia) the effects of lipid chain unsaturation,8 and we apply the technique It was suggested that the cyclopropyl unit damped the here to a cyclopropanated lipid. propagation of motion along the acyl chain, rendering the Additionally, we have coordinatedthe kinetics/dynamica liposomal bilayer rather insensitive to large thermal study with a novel IR spectroscopic analysis that provides fluctuations. In another study, deuterated dihydrostercomparative, quantitative conformational information culic or oleic acid probes were biosynthetically incorporated about the lipidic hydrocarbon chains.+l1 This method is into the membrane lipids of A. laidlawii. The NMRbased on the observation12that the CH2 wagging modes measured ordering, in the liquid crystalline phase, was greater all along the chain with the cyclopropanated acid, (6)Sower, H. Biochim. Biophys. Acta 1982,691, 161. relative to ita olefinic analogue, suggesting that the cy(6) Mow, R. A,; Bhattacharya, S.; Chatterjee, S. J. Am. Chem. SOC. clopropyl function engendered a less "fluid" lipid m a t r i ~ . ~ 1989,111, 3680.
D
(1) (a) Rutgers, New Brunewick. (b) Rutgers, Newark. (2) Dufourc, E. J.; Smith, I. C. P.; Jarrell, H. C. Chem. Phy8. Lipids 1983, 33, 153. (3) Dufourc, E. J.; Smith, I. C. P.;Jarrell, H. C. Biochemistry 1984,23, 2300. (4) Jarrell, H. C.;Tulloch, A. P.; Smith, 1. C. P. Biochemistry 1983,22, 5611.
( 7 ) Moss, R. A.; Ganguli, S.;Okumura, Y.; Fujita, T. J . Am. Chem. soc. 1990,112,6391. (8) Moss, R. A.; Fujita, T.; Okumura, Y. Langmuir 1991, 7, 440. (9) Holler, F.; Callis, B. J. Phys. Chem. 1989,93, 2058. (IO)C a d , H. L.; McElhaney, R. N. Biochemistry 1990,29,5423. (11)Senak, L.; Davies, M. A.; Mendelsohn, R. J. Phys. Chem. 1991, 95, 2565. (12) Snyder, R. G. J. Chem. Phys. 1967, 47, 1316.
0743-7463192J2408-1731$03.00/0 0 1992 American Chemical Society
Moss et al.
1732 Langmuir, Vol. 8, No. 7, 1992 in disordered states absorb a t frequencies that are characteristic of specific two- and three-bond conformers. Together, the chemical and spectroscopic studies furnish a detailed view of the differing stabilities of unsaturated and cyclopropanated liquid crystalline liposomes, and of their underlying chain conformational origins.
Results and Discussion Synthesis. The model lipids that are the focus of this study are the pseudoglycerylammonium ions 2 and 3. The previously described! unsaturated, cis-A9-oleoyl lipids (2) were prepared from oleoyl chloride and rac-34dimethylamino)-l,Zpropanediol, followed by quaternization with either methyl bromide (affording 2-NF) or 3-(bromomethy1)-4-nitrophenyl benzoate6 (affording 2-F).
2 (oleoyll
I
OCOPh
nCiiH3&XXH
I
4 islearoyl)
d,b liposomeo nm
Tc: OC
2' 2'
31 C10 31 -30at 40 OC 4 a t 55 O C
See text for structures and conditions. The F/NF ratio was 1:lO. Hydrodynamic diameters from laser light scattering a t pH 4,O.Ol M KC1. Temperature of gel to liquid crystal transition, pH conditions as in footnote b. Reaction temperature. e Rate constant for glutathione cleavage of exoliposomalp-nitrophenyl benzoate moieties; see text. f Rate constant for endoiipoeomal cleavage reaction. 8 Ratio of fast (exolipoeomal)to slow (endoliposomal)kinetic phases from the corresponding absorbance changes a t 400 nm. Approximate half-time for the decay of surface differentiation; see text. From ref 8. j This work.
3 (dihydroslerculoyll
G7YsCOOCHz
CH2N*Me&H20. Br.
Table I. Dynamics in Liposomal Systems
NF Series, G
=
4
H; F Series. G =
I
NO2
The new cis-dihydrosterculoyl lipids (3) required the prior preparation of methyl dihydr~sterculate,~~ that was accomplished by Simmons-Smith methylenation of methyl oleate (CH212, Zn-Cu, refluxing ether, 5 days, 99%). The dihydrosterculoyl ester was then saponified; the resulting acid was converted to the acid chloride with SOC12, and the acid chloride was reacted with rac-34dimethylamino)-l,2-propanediol(Et3N, ether, 25 "C, 1 h, 67 % ) to afford the tert-amine precursor of 3 (i.e. 3 without "CH2G, Br-"). Quaternization of the amine with either methyl bromide (ether, sealed tube, 25 "C, 24 h, 91 9%) or 3-(bromomethyl)-4-nitrophenyl benzoate6 (THF, 25 "C, 6 days, 50%) gave the desired lipids, 3-NF or 3-F respectively, presumably as inseparable mixtures of diastereomers. The new lipids were characterized by NMR spectroscopy and elemental analyses. For reference purposes, the Cla-SatUrated, stearoyl lipids, 4-NF and 4-F, available from a previous study! were also employed. Liposomes. Covesicles or liposomes of 2-F/NF, 3-F/ NF, and 4-FINF were created by sonication of CHCl3-cast films of 1:lO F/NF surfactant blends in pH 3.9 aqueous HC1, p = 0.01 (KCl). Sonications of 2 and 3 were carried out with an immersion probe sonicator (61 W, 3 min, 25 "C), and the liposome solutions were filtered through 0.8pM Millex membrane filters. Sonication of 4-F/4-NF was performed above its gel-liquid crystalline phase transition temperature (59 Oca); the solution was cooled to 25 "C before filtration. Final surfactant concentrations were F = 5 x 10-5 M; NF = 5 x 10-4 M. Dynamic light scattering measurements (Ar laser, 488 nm, 90" scattering angle) gave the hydrodynamic diameters shown in Table I for the several liposome systems. The sizes are in accord with expectations for small unilamellar ~esic1es.l~ (13)Christie, W. W.; Gunstone, F. D.; Ismail, I. A.; Wade, L. Chem. Phys. Lipids 1968,2, 196. It was difficult to reproduce the literature yield without extensive modification. The procedure reported here is optimized. (14) Jain, M. K.lntroduction toBiologica1Membranes,2nded.;Wiley: New York, 1988; pp 86f.
The gel to liquid crystalline transition temperature (T,) of the 2 and 4 covesicles have been examined8 by monitoring the temperature dependence of the fluorescence polarization of covesicallized 1,6-diphenyl-1,3,5h e ~ a t r i e n c e ; ~they J ~ are recorded in Table I. The Ag unsaturations in the Cl8 oleoyl chains of 2 profoundly depress the liposomal T,in comparison with that of the saturated c 1 8 stearoyl liposomes, 4. The dihydrosterculoyl liposomes, 3, exhibit the onset of a discontinuity in the diphenylhexatriene fluorescence scan at -13 "C, but the companion discontinuity (reflecting the gel phase) is not observed by 8 "C, so that we estimate T,for 3 as
