Langmuir 1991, 7, 440-441
440
Effect of Unsaturation on Lipid Dynamics within Synthetic Lipid Membranes Robert A. MOSS,*Tsunehisa Fujita, and Yukihisa Okumura Department of Chemistry, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903 Received October 29, 1990 Unsaturated oleoyl (cis-2-F,cis-2-NF)and elaidoyl (trans-2-F, trans-2-NF) ammonium ion lipids were synthesized. Liposomes were created by sonication of 1:lO functional/nonfunctionallipid blends. These small (300 8, liquid crystal phase ( T , < 25 "C) liposomes were surface differentiated by exovesicular glutathione in an exovesicular/endovesicularpH gradient of 8/3.9. At 25 "C, the liposomes were quite permeable to H+/OH- (tllz < 20 s) and reequilibrated rapidly by lipid flip-flop (tl/z(cis-2) < 1 min, tl/2(trans-2) 4 min). In contrast, liposomes created from analogous, saturated stearoyl lipids (1:lO 3-F/3-NF),where T , = 59 "C, were less permeable to H+/OH- (tip 21 min) and less prone to transbilayer lipid migration after surface differentiation (t1/2 > 30 min at 40 "C).
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The development of chemical methods for the differentiation of the inner and outer surfaces of synthetic bilayer liposomes enables us to readily monitor the subsequent decay of the imposed chemical asymmetry brought about by transbilayer lipid migration (or "flip-flop").l This, in turn, makes possible a systematic study of the relation between the molecular structure of lipid monomers and their dynamic behavior within liposomal aggregates. We could thus correlate the rate of flip-flop equilibration with lipid chain length in liposomes constructed of homologous alkylammonium ion lipids.2 In addition to chain length, other features of lipid structure (e.g.,functional group attachment of the chains, backbone architecture) affect the properties of liposomes; these could also be e ~ a m i n e d For . ~ example, strategically placed alkyl substituents disrupt chain packing, markedly altering bilayer phase transition temperatures, and ent h a l p i e ~ .Related ~ effects and associated changes in flipflop dynamics were observed in chemically differentiated liposomes derived from phytanyl lipid^.^ A particularly important structural modification is the introduction of unsaturation into the lipid chains. Relative to analogous, saturated lipid bilayers at ambient temperature, unsaturation leads to increasing fluidity and looser chain packing that may be expressed in enhanced liposomal permeability to water, small uncharged organic solutes, and ionophore-complexed metal ions.6 Unsaturated lipids are common components of biological memb r a n e s , ' ~that ~ it seemed important to compare liposomes derived from unsaturated and saturated lipids in the context of readily controlled model systems. Here, we describe the preparation of novel oleoyl (cis-9-octadecenoyl)and elaidoyl (trans-9-octadecenoyl)lipids, together with a study of their flip-flop dynamics within liposomes. The results are compared to those obtained with analogous, saturated stearoyl lipids, thereby highlighting the profound effects of lipid unsaturation. (1)Moss, R. A.; Bhattacharya, S.; Chatteriee, S. J. Am. Chem. SOC. 1989, 111, 3680. (2) Moss, R. A.; Fujita, T.; Ganguli, S. Langmuir 1990, 6, 1197. (3) Moss, R. A.: Ganauli, S.: Okumura, Y.: Fuiita, T. J.Am. Chem. SOC. 1990, 112, 6391. (4) Menger, F. M.; Wood, M. G.,Jr.; Richardson, S.; Zhou, Q.; Elrington, A. R.; Sherrod, M. J. J. Am. Chem. So. 1988,110,6797. Menger, F. M.: Wood, M. G.. Jr.: Zhou. Q.Z.: Houkins. H. P.: Fumero. J. J. Am. Chem. SOC.1988, 110,6804. (5) Moss, R. A,; Fujita, T. Tetrahedron Lett. 1990, 31, 7559. (6). Bittman, R.; Blau, L. Biochemistry 1977,11,4831, and references therein. See also Barton, P. G.; Gunstone, F. D. J.Biol. Chem. 1975,250, 4470. Carruthers, A.; Melchior,D. L. Biochemistry 1983,22,5797. Quinn, P. J.; Joo, F.; Vigl, L. Prog. Biophys. Mol. Biol. 1989, 53,71. (7) Jain, M. K. Introduction to BiologicalMembranes, 2nd ed.; Wiley: New York, 1988; Chapter 2.
0743-7463/91/2407-0440$02.50/0
The requisite lipids were prepared as follows. Oleoyl chloride (Sigma)and rac-3-(dimethylamino)-1,2-propanediol (Aldrich) afforded the (dimethylamino) dioleoyl ester, cis-1, as the hydrochloride, in 65 % yield after 1h of stirring in CHC4 at 25 "C. Diester 1was purified by neutralization (NaOH), followed by chromatography over silica gel. Similarly, elaidoyl chloride (from elaidic acid, Sigma, and refluxing SOClz) was converted to trans-1 in 75 % yield by reaction with (dimethylamino)-1,2-propanediol (ether, 25 "C, 1h), followed by analogous purification. The tertiary amines, cis-1 or trans-1, were each quaternizeds with methyl bromide, affording nonfunctional (NF) surfactants cis-2-NF (76%)or trans-2-NF (89%). The tertiaryamines CHdCHz)$H=CH(CH2)7CWH2
CH3(CH97CH=CH(CH&CCKZH2
CHdCH&CH=CH(CH2)$COOH
CH~(CHZ)~CH=CH(CH~J~CCOOCH
I
I
I
I
CH2NMe2 cis-1 (oleoyl) trans.1 (elaidoyl)
CH2N*Me2CH2G,Br
1 1 '-,$I
cis-2-N F trans-2-N F
G-H
OCOPh
cis-2-F frans-2-F
NO2
n-C17H3sC00CiH2 nC17H3+XXXH
I
CH2N*Me2CH2G.Br-
3 (NF or F defined as in 2)
(1) were also quaternizedg with 3-(bromomethyl)-4-nitrophenylbenzoate,' affording functional (F)surfactants cis2-F (46%) or trans-2-F (43% ). For comparative purposes, we also prepared the nonfunctional and functional stearoyl lipids, 3-NF and 3-F. The synthetic methods and structural substantiation followed the procedures outlined for the unsaturated lipids.1° Covesicles of cis-a-F/NF, trans-2-F/NF, and 3-F/NF were generated by sonication of CHCla-cast films of 1:lO F/NF surfactant blends in pH 3.9 aqueous HC1, p = 0.01 (8) Conditions: Et20 saturated with MeBr, 25 OC, 15 h. The trimethylammonium salts were purified by recrystallization from ether: mp (cis2-NF) 75 "C (liquid crystal), 90 OC (melts); (trans-2-NF) 95 O C (liquid crystal), 145 "C (melts). Structurally consistent proton NMR spectra and satisfactory elemental analyses (C,H, N, Br) were obtained. (9)Conditions: Ethereal solutions of 50% excess 3-(bromomethyl)4-nitrophenylbenzoatee, 25 OC, 3 days; cis-2-F, purified by chromatography over silica gel was an oil a t room temperature; trans-2-F (recrystallized in acetone) had mp 68 OC (liquid crystal), 74 O C (melts). Structurally consistent proton NMR spectra and satisfactory elemental analyses (C, H, N, Br) were obtained. (10) The palmitoyl analogues of 3-NF and 3-F have been described; cf. ref 3.
0 1991 American Chemical Society
Letters
Langmuir, Vol. 7, No. 3, 1991 441 Table I. Dynamics of Liposomes
liposome'
cis-8-F/NF trans-t-F/NF 3-F/NF
Tn,b Tc; O C
"C
kf,d s-l
25
