1937
Langmuir 1993,9,1937-1939
Counterion Control over the Barrier Properties of Bilayers Derived from Double-Chain Ionic Surfactants'
they addressthe questionof whether or not increaeedlevele of ionic association within the bilayer can be used to modulate membrane permeability. In order to place our permeability data into perspective, we have chosen as a frame of reference, bilayers made from the extensively investigated phospholipid, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC).
Yong-Chan Chung and Steven L. Regen' Department of Chemistry and Zettlemoyer Center for Surface Studies, Lehigh University, Bethlehem, Pennsylvania 18015 Received February 4,1993. In Final Form: April 20,1993
Early pioneering studies by Kunitake and co-workers demonstrated the feasibilityof assemblingbilayer vesicles from totally synthetic, double-chain ionic ~urfactants.~*3 Since that time, a wide variety of analogous amphiphiles and vesicles have been prepared.' Although the barrier properties of such bilayers are of considerabletheoretical as well as practical interest (e.g., for use in fundamental transportstudies, time-releaw devices,etc.),relativelylittle effort has focused on defining and controlling their permeability characteristics. In this paper we show that the counterion of a double-chain anionic(A) and a doublechain cationic (C) surfactant has a major influence on the barrier properties of assembled bilayers. We further show that an amphiphilic dianion can be used to "patch up" leaky membranes that are prepared from C.
I
I1
111
Materials and Methods General Methods. Unless stated otherwise, all chemicalsand reagents were obtained commercially and used without further purification. Dihexadecylphosphoricacid (Sigma) was converted into its sodium salt (I) by titration with sodium methoxide in methanol, followed by solvent evaporation and recrystallization in CHsOH/CHCb. Hexadecylmalonic acid was prepared via alkylation of diethylmalonate followed by saponification." [W]Sucrose (250 mCi/mmol, 20% ethanol solution) was obtained from ICN Laboratories. Water was purified using a Milli-Q Specific counterions that were selected for A-type system consisting of one carbon, two ion exchange, and one surfactants were Na+, (CH3)4N+, and CH~(CH~)ISN+- Organex-Q cartridges. Analytical methods that were used for (CH3)3. Those counterions that were chosen for C were nitrogen analysis, dynamic light scattering, liquid scintillation B r , CH3(CHhC02-, and CH~(CH~)ISCH(CO~-)~; i.e., counting, differentialscanning calorimetry (DSC),and membrane permeability were similar to those previously employed.8 An surfactants I-VI. Replacement of a sodium ion with a ion-exchange resin (Bio-Rad, AGl-X8, hydroxide form, 3.2 more hydrophobic cation was expected to result in mequiv/g) was extracted with methanol (Soxhlet) for 12 h under increased ion-pairing within the Stem layer due to van nitrogen and then packed into a column (2 X 7 cm) prior to use. der Waals interactions with the alkyl segments of A. In Tetramethylammonium Dihemadeaylphospbate(11). Tetprinciple, an increase in ion-pairing should lead to a ramethylammonium bromide (0.085 g, 0.55 "01) in 10 mL of reduction in electrostatic repulsion between the head methanol was converted into ita hydroxide by pawage through groups, an increase in chain packing, and a decrease in an AG1-X8resin. To theresultingsolutionwasthenadded0.300 permeability. Whether or not a well-anchoredcounterion g (0.55 mmol) of dihexadecylphosphoric acid. After being stirred (one that is an integral part of the bilayer) would be more for 12 h at 23 OC, the solution was concentrated under reduced pressure, and the residue was recrystallized (2 times) from C&or lese effective than one that is expected to be localized OH/ether to afford 0.264 g (78%) of I1 as a colorlese solid having at the vesicular surface (i.e., I1 versus 111)was not obvious mp 165-167OC; 'H NMR (CDCl,, 500 MHz) 6 0.90 (t,6 H, CHs), and deemed worthy of investigation. Comparison of 1.2-1.4 (8, 52 H, CHz), 1.55 (9, 4 H, CHzCHzO), 3.45 (8, 12 H, bilayers produced from IV-VI was ale0of interest, because CHsN+), 3.8 (q, 4 H, CH20). Anal. Calcd for C&&JOplH20 C, 67.77 H, 12.63; N, 2.19. Found C, 67.91; H, 12.14; N, 2.32. (1) Supported by the National Science Foundation (Grant CHEN~~-Trimethyl-N-hexadecylammonium Dihexadec9022681) and by the U.S.Army Research Office (Grant DAALO3-91-Gylphosphate(111). N ~ ~ - T r i m e t h y l - N - h e x a d e c y ~ o ~ ~ 0081). (2) Kunitake,T.;Okahata,Y.;Tamaki,K.;Takayanagi,M.;Ku"mu,bromide (0.30 g, 0.82 mmol) was converted into its hydroxide F. Chem. Lett. 1977,387. form and reacted with dihexadecylphosphoric acid (0.45 g, 0.82 (3) Kunitake, T.; Okahata,Y.J. Am. Chem. SOC.1977,99,3860. mmol), using procedures similar to that described for the (4) Fender, J. H. Science 1984,223,888. O'Brien, D.F.; Ramawami, preparation of 11. Surfactant 111 (85% yield) was obtained as V. Encycl. Polym. Sci.Eng. 1989,17,108. Bader,H.;Dom, K.;Hashimoto, a colorless solid having mp 160-163 OC; 'H NMR (CDCb, 600 K.; Hupfer, B.; Petropouloa, J. H.; Ringsdorf, H.; Sumimoto, H. In Polymeric Membranes; Gordon, M., EX;Springer Verlag: Berlin, 198s; MHz) 6 0.90 (t, 9 H, CHs), 1.2-1.5 (8, 78 H, CHz), 1.6 (q, 4 H, p 1. Johnston,D.S.;Chapman,D.InLiposome Technology;Gregoriadia, CHzCHzO), 1.7 (br s,2 H, CHzCHzN+)3.4-3.6 (8, 11 H, CH&P G., Ed.; CRC hem, Inc.: Boca Raton, FL, 19M, Vol. 1, p 123. Regen, + CH2N+), 3.8 (9,4 H, CH20). Anal. Calcd for C6lHlW S. L. InLipoeomes: h m Biophysics To Therapeutics;Ostro, M.J.,Ed.; Marcel Dekker: New York, 1987; p 73. Kaler, E. W.; Murthy, A. K.; (5) Sharma, B. D.;Biswaa, A. B. A w l . Chem. 1968,30,1367. Rodriguez,B. E.; Zaaadziiki, J. A. N. Science 1989,246,1371. Fukuda, (6) Chung,Y. C.;Regen,S. L.; Fukuda, H.; Hirano, K. Langmuir 1992, H.; Kawata, K.;Okuda, H.; Regen, S. L. J. Am. Chem. SOC.1990,112, 8, 2843.
1636.
0743-7463/93/2409- 1937$04.Oo/O
0 1993 American Chemical Society
Notes
1938 Langmuir, Vol. 9, No. 7, 1993 N0&3H20: C, 69.25 H, 12.99; N, 1.58. Found C, 69.47; H, 12.41; N, 1.83. N , N - - D i m e t h y l - N J V d h d ~ y a OBromide ~m (IV). A mixture of 1-bromohexadecane (2.0 g, 6.55 "01) and N f l dimethyl-N-hexadecylamine(4.8Bg, 8.51 m o l ) in 50 mL of dry methanol (CaH2) was refluxed under a nitrogen atmosphere for 5 days. Subsequent cooling to room temperature, removal of solvent under reduced pressure, and recrystallization (3 times) of the oily residue from ethanol/ether afforded 3.06 g (81 %) of a colorless solid having mp 155-16OoC; lH NMR (CDCl3, 500 MHz) 13 0.90 (t, 6 H, CHd, 1.2-1.4 (8, 52 H, CH2), 1.7 (m, 4 H, CH2CH2N+),3.4 (8, 6 H, CHsN+), 3.5 (m, 4 H, CH2N+). Anal. Calcd for C&,2NBr.lH20: C, 68.87; H, 12.58; N, 2.36. Found C, 68.67; H, 12.42; N, 2.61. ~ p - D h ~ ~ t h y l - N p d h ~ a d e ~Palmitate ya~ni (V). m N f l - D i m e t h y l - N ~ - d u m bromide (0.30g, 0.52 "01) was converted into its hydroxide form and reacted with palmitic acid (0.133 g, 0.52 mmol), using procedures similar to those described for the preparation of 11. Surfactant V (38% yield) was obtained as a colorless solid having mp 136-139 "C; 'H NMR (CDCb, 500 MHz) 6 0.90 (t,9 H, CHs), 1.2-1.5 (8, 76 H, CH2), 1.6-1.8 (m, 4 H, CHzCH2N+ + 2 H, CH&H&O), 2.2 (t, 2 H, CH2CO), 3.4-3.5 (m, 10 H, CH3N+CH2). Anal. Calcd for C d l ~ N O r l H 2 0 :C, 78.15 H, 13.77; N, 1.82. Found C, 78.28; H, 13.36; N, 1.83. Bie(N~~dimethyl-N~-dihe.adecyla"onium)Hexadecylmalonate (VI). Nfl-Dimethyl-Nfl-dihexadecylamme niumbromide (0.35g, 0.61 "01) was converted into its hydroxide form and reacted with hexadecylmalonic acid (0.100 g, 0.304 mmol), using procedures similar to that described for the preparation of 11. Surfactant VI (89% yield) was obtained as a colorless solid having mp 100-103 "C; 'H NMR (CDCb, 500 MHz) 6 0.90 (t, 15 H, CHd, 1.1-1.5 (8, 132 H, CH2), 1.6-1.8 (br 8, 8 H, CHaCHN), 1.9 (m, 2 H, CH2CH(C02M13.0 (t, 1 H, CH(C02)2), 3.2-3.4 (m, 20 H, CHsN+CH2). Anal. Calcd for C ~ H ~ & J ~ O CC,~76.24; H ~ H, O 13.53;N, 2.04. Found C, 76.31; H, 13.12; N, 2.07. Membrane Permeability. Typically,a vesicle dispersionwas prepared by vortex mixing 3.0 mg of surfactant (a dried thin f i i , from the evaporation of a chloroform solution) in 1 mL of pure water, containing 1 rCi of [l~lsucrose. The resulting multilamellar dispersion was then subjected to five successive freezethaw cycles with vortex mixing and extruded, sequentially, throughtw0stacked0.4~0.2,andO.lpmNucleporepolycarbonate membranes (five passes in each case) to give ca. lo00 A diameter large vesicles (dynamic light scattering). The temperature of the dispersion was maintained at least 10 "C above the highest endothermthat was observed by hs-DSC analysis. After removal of nonentrapped sucrose by gel filtration (Sephadex G-SO),the vesicle dispersion was analyzed,simultaneously,for nitrogen and sucrose content. The percentage of recovery of I-VI in the void volume ranged between 40 % and 60% (nitrogen analysis); with the exception of 11, the percent capture ranged between 0.3% and 0.6%;for vesicles made from 11,no evidence of entrapment was found. Efflux measurements were made immediately after gel fdtrationbyplacingasampleinadialysisbag(Spectrapr1 cellulose tubing, MW cutoff 8OOO), and then dialyzed against 300 mL of water for 15 h. Periodic analysis for radioactivity inside of the bag indicated that 15 h was sufficient to remove residual nonentrapped sucrose, as judged by the complete disappearance of a rapid f i i t phase of the release kinetics. The bag was then placed in 300 mL of pure water and the efflux rate monitored by periodic analysis for radioactivity.
Results and Discussion In order to assess the barrier properties of bilayers of I-VI, we have measured the efflux rate of radioactive sucrose out of extruded vesiclesthat were made from these surfactants.s These rates can be readily determined by dialysismethods, provided that diffusion out of the vesicles is significantly slower than diffusion across the dialysis membrane. Under such conditions, the efflux kinetics obeys the relationship shown in eq 1. Here A1 = surface
Table I. Efflux Rates of [lqC]Sucro~ from Surfactant
Vesicles surfactant I I1 I11 IV
V VI VI
Wkl 0.8 0.5 no entrapment* 1.9 0.3 1.6 0.2 2.3 0.5
* ** *
