Langmuir 1991, 7, 1045-1047
1045
Polymerizable Ion-Paired Amphiphiled Koji Hirano and Hiroyuki Fukuda* Nagoya Municipal Industrial Research Institute, 4-41, Rokuban 3-chome, Atsuta- ku, Nagoya 456, Japan
Steven L. Regen* Department of Chemistry and Zettlemoyer Center for Surface Studies, Lehigh University, Bethlehem, Pennsylvania 18015 Received February 19,1991. In Final Form: March 18,1991 Sonicdispersalof NJ-dimethyl-N-octadecyl-N- [3-(1-acrylamido)propyl]ammonium stearate (I)in water produces vesicles that range in diameter between 335 and 730A. Photopolymerizationresults in membranes that consist of linear polymers having polystyrene equivalent molecular weights up to M+ = 175 000 and M,= 40 000. Examination by transmission electron microscopy,dynamic light scattering,and differential scanning calorimetry establishes that the polymerization process does not significantly alter the vesicle’s overall supramolecular structure and melting behavior. Compared with their monomeric counterparts, polymerized dispersions of I show enhanced stability toward Trition X-100, NaC1, and NaI.
Introduction The geometry of a surfactant molecule is currently believed to play a major role in defining its aggregation behavior.2 In particular, those surfactants that possess a conical shape are expected to maximize intermolecular hydrophobic interactions by organizing into spherical micelles; those that are cylindrical form planar bilayers. Classic examples of the former are cetyltrimethylammonium bromide and sodium dodecyl sulfate; biologically relevant examples of the latter are the double-chain phosphatidylcholines. Recently we have begun to develop a new class of surfactants that consists of single-chaincations that are paired with single-chain anions.3~~ Unlike their micelle-forming precursors, “ion-paired amphiphiles” (IPAs) produce bilayer vesicles upon sonic dispersal in water. On the basis of such behavior, we have suggested that their effective head group size is somewhat reduced, due to electrostatic attraction between the counterions and a decrease in hydration (Scheme I). In principle, ionically paired singlechain surfactants should help to “bridge the supramolecular gap” between single- and double-chain amphiphiles. This fact, together with the ready availability and low cost of single-chain surfactants (as compared with double-chain counterparts),provides considerablestimulus for exploring and exploiting IPA’s as lamellar-forming material. Although it is now well-established that double-chain surfactant vesicles can be stabilized through polymerization: we sought to investigate whether IPA-based analogueswould behave similarly. Specifically,we wanted to establish (i) whether or not an IPA-based vesicle could be polymerized to a significant degree with overall retention of its supramolecular structure and (ii) if polymerization of only one of its ionic “partners” would lead to enhanced stability. Our primary motivation for an(1) Supported by Nagoya Municipal Government (Grant 1989-01, H. F.) and by the National ScienceFoundation (Grant CHE-87-00833,S.L.R.). (2) Reviews: Evans, D. F. Langmuir 1988,4, 3. Israelachvili, J. N.; Jarcelja, S.; Horn, R. G., Q. Rev. Biophys. 1980,13, 121. (3) Fukuda, H.; Kawata, K.; Okuda, H.; Regen, S. L. J. Am. Chem. SOC.1990,112, 1635. (4) Related vesicleshave alsobeen prepared,spontaneously,by mixing oppositelychargedsingle-chainsurfactants: Kaler,E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. N. Science 1989,245,1371. (5) See: Regen, S.L. In Liposomes: From Biophysics to Therapeutics; Ostro, M. J., Ed.; Marcel Dekker: New York; 1987; p 73, and references 10-26 cited therein.
0743-7463/91/ 2407-1045$02.50/0
Scheme I micelle-forming~-b surfactants
lamellar-forming & -. ion-pair amphiphiles
”cylinders”
I
swering these questions was to see if we could extend the utility of IPA’s for membrane modeling and device applications by creating more robust analogue^.^ For this purpose, we have investigated the aggregation and poly[3merization behavior of N,N-dimethyl-N-octadecyl-N(1-acrylamido)propyl]ammoniumstearate (I). Our principal results are described herein.
Materials and Methods General Methods. Unless stated otherwise, all reagents and chemicals were obtained from Wako Pure Chemical Industries, Ltd., and used without further purification. Stearic acid was purified by recrystallization (3 times) from ethanol. Specific experimental procedures that were used throughout this work for vesicle preparationand characterization were similar to those previously described.3~6973-(N,N-Dimethylamino)propylacrylamide was purchased from Tokyo Kasei, Ltd. NJV-Dimethyl-N-octadecyl-N-[3-(1-acrylamido)propyl]ammonium Bromide. A mixture of 3.33 g (0.01 mol) of 1-bromooctadecane,1.56g (0.01mol) of 3-(N,N-dimethylamino)and propylacrylamide,0.05 g of 2,6-di-tert-butyl-4-methylphenol, (6) Fukuda, H.; Diem, T.;Stefely, J.; Kezdy, F. J.; Regen, S. L. J. Am. Chem. SOC.1986,108,2321. (7) Stefely, J.; Markowitz,M. A.; Regen, S. L. J. Am. Chem. SOC.1988, 110,7463.
0 1991 American Chemical Society
Letters
1046 Langmuir, VoZ. 7,No.6,1991 20 mL of acetone was refluxed for 20 h. After removal of solvent under reduced pressue, the residue was recrystallized from ethyl acetate/ethanol (9/1 (v/v)) to afford 4.72 g (96%) of Nfl-dimethyl-Nedecyl-N-[ 3-( 1-acrylamido)propyl]a"onium bromide. Further purification was carried out by recrystallization (2 times) from this same solvent mixture: mp 73.7 "C; IR (KBr) vm 3300, V1670, v w 1630 cm-l; lH NMR (CDCl3) 6 0.88 (t, 3 H), 1.27 (s,30 H), 1.59 (s, 2 H), 2.15 (m, 2 H), 3.18 (s,6 H), 3.45 (m, 4 H), 3.85 (m, 2 H), 5.55 (m, 1H), 6.35 (m, 2 H), 8.25 (br t, 1H). Anal. Calcd for CwHaN20BrJ/2H20: C, 62.61; H, 10.94; N, 5.62; Br, 16.02. Found: C, 62.37; H, 11.02; N, 5.58; Br, 16.15. N,N-Dimethyl-N-octadecyl-N-[3-(1-acrylamido)propyl]ammonium Stearate (I). A 12 mm diameter glass column was filled with 22 mL of wet Bio-Rad AG 1X-8 (hydroxide form, 1.2 mequiv/mL). After the resin was washed with 200 mL of l-propanol, 0.245 g (0.5 mmol) of Nfl-dimethyl-N-octadecyl-N- [3(1-acrylamido)propyl]ammoniumbromide in 50 mL of 1-propanol was then passed through the column. The eluant was mixed with 0.143 g (0.5 mmol) of stearic acid. The solvent was then removed under reduced pressure, and the residue was recrystallized twice from ethyl acetate/ethanol (95/5 (v/v)) to give 0.31 g (89%) of I, having mp 96.3 "C: IR(KBr) VNH 3450, v1670, v w 1630 cm-l; lH NMR (CDCl3) 6 0.88 (t, 6 H), 1.28 (s, 62 H), 1.53 (9, 2 H), 2.15 (m, 4 H), 3.19 (s, 6 H), 3.45 (m, 4 H), 3.95 (m, 2 H), 5.55 (m, 1H), 6.35 (m,2 H), 8.45 (br t, 1H). Anal. Calcd for C~HSJ&O~~/~H~O: C, 75.24; H, 12.80;N, 3.99. Found: C, 75.32; H, 12.71; N, 3.82. Vesicle Formation and Polymerization. Typically, 50 mg (0.072 mmol) of I was dissolved in a minimum volume of chloroform and placed in a flat-bottomed test tube (40 X 130 mm). After the chloroform was evaporated by passing a stream of argon through the test tube, the thin lipid film was dried under reduced pressure (25 "C, 12h (0.05 mmHg)). Distilled water (50 mL of "Milli-Q" reagent) was then added to the tube and the lipid dispersed via vortex mixing at room temperature. The tube was sealed by use of a No-Air stopper and the dispersion purged with argon. The dispersion was then sonicated a t 65 "C to constant turbidity (ca. 10 min, Branson Sonifier 450, Cup horn). Photopolymerizationof vesicle dispersions (10mL) was carried out in quartz tubes equipped with No-Air stoppers. Prior to irradiation, each sample was purged with a stream of argon for 30 min, and the tubes were then placed into a constant temperature quartz vessel. Irradiation was carried out by use of a Toshiba high-pressure mercury UV lamp, SHL-100 at a distance of 10 cm from the light source. Thermal polymerization reactions were carried out by use of 3 mol 96 of azobis(2-amidinopropane) dihydrochloride as an initiator at 60 "C for 20 h. Gel Permeation Chromatography. The isocratic liquid chromatograph which was used for all GPC measurements was a modular system consisting of a Shodex Degas, a Jasco Model 880-PU solvent delivery system, a Shodex Model AO-50 injector and column oven system, and a Shodex Model RI SE-61 differential refractometer, which was interfaced with a System Instrument Chromatocorder 12. The columns (AC-803 and AC80M) and polystyrene standards (MW 2.75 X lo6, 1.75 X lo6,4.50 x 105,1.70 x io6,3.45 x 104,9.00 x 103,3.25 x 103,1.20 x i v , and 5.80 X lo2) that were used were purchased from Shouwa Denko K.K. The following conditions were employed: solvent, chloroform containing 5 mM tetrabutylammonium bromide; column temperature, 30 "C; flow rate, 1 mL/min; sample concentration, 1mg/mL; sample injection volume, 0.5 mL. All molecular weights that are reported are polystyrene-equivalent molecular weights. Samples were prepared for GPC measurements by (a) freezedrying the polymerized vesicle dispersion (2 mL), (b) dissolving the polymer in 2 mL of chloroform containing 5 mM tetrabutylammonium bromide, and (c)filteringthe resultingsample through a 0.45-pm Chromatodisc (Kurabo). Vesicle Stability. The stability of the vesicle dispersions were evaluated by adding 25-pL aliquots of aqueous solutions of Triton X-100 (22 mM), sodium chloride (43.2 mM), or sodium iodide (7.21 mM) to 0.5 mL of a vesicle dispersion (1mg/mL, 1.44 mM), followed by gentle mixing and measurement of the resulting turbidity at 500 nm. The apparent absorbance ratio is defined as the corrected absorbance/initial absorbance, where
Scheme I1 0
or, Cn;
I
I
Figure 1. Transmission electron micrographs of nonpolymerized vesicles of I (2% uranyl acetate stain). Bar represents 500
A.
the corrected absorbance equals the observed absorbance X (volume of initial vesicle dispersion + volume of aqueous agent that is added)/volume of initial vesicle dispersion.
Results and Discussion Quaternization of 3-(N,N-dimethylamino)propylacrylamide with 1-bromooctadecane, followed by passage through an anion-exchange resin (hydroxide form) and reaction with a stoichiometric quantity of stearic acid produced the requisite IPA, NJV-dimethyl-N-octadecylN-[ 3-(1-acrylamido)propyl]ammonium stearate (I) (Scheme 11). Sonication of I in pure water resulted in a translucent dispersion that consisted of microspheres ranging between 335and 730 A diameter, as judged by transmissionelectron microscopy (Figure 1);dynamic light scattering indicated particles having a mean diameter of 593 A. In order to confirm the presence of a lamellar phase, this dispersion was examined by differentialscanningcalorimetry. A welldefined gel to liquid-crystalline phase-transition temperature (T,)was readily apparent at 53.6 "C,with AH = 9.5 kcal/mol. It is noteworthy that these values are very similar to those which have been observed for the zwitterionicphosphatidylcholinehaving the exact same fatty acid
Langmuir, Vol. 7, No. 6,1991 1047
Letters Standard (Polrstyrene) 10'
20
25
30
lo6
105
104
103
35
Figure 2. Gel permeation chromatograms of polymers formed via UV irradiation of vesicular I.
chain length, Le., distearoyl-sn-glycero-3-phosphocholine (54.9"C, AH = 10.6 kcal/mol).s Upon exposure to direct UV irradiation at 20 "C, vesicles of I were completely polymerized after 60 min (Figure 2). The resulting polymers had polystyrene equivalent molecular weights up to M p d = 175 OOO and Mn = 40 OOO. Similar photopolymerization reactions carried out at 50 "C for 3 h gave polymers having Mpea~= 374 0oO and Mn = 114 0o0.9 Transmission electron microscopy and dyamic light scattering established that both the particle size and the size distribution were unaltered after polymerization. Examination of the melting behavior of photopolymerized membranes of I by DSC revealed a T, = 53.6"C and AH = 9.9 kcal/mol. In order to judge the stability of polymerized and nonpolymerized dispersions of I, we have examined their lability toward added detergent (Triton X-loo),NaC1, and NaI. A plot of the apparent absorbance ratio (turbidity at 500 nm) as a function of the molar ratio of Triton X-1OO/Iis shown in Figure 3; similar plots are presented in Figure 4,using NaCl and NaI as the perturbing agents. From these data it is clear that polymerization significantly enhances the stability of the dispersion and that complete conversion to polymer is necessary in order to achieve maximum stabilization. The fact that iodide is significantly more effective in disrupting nonpolymerized dispersions of I, than is chloride, suggests that IPA's could find use as novel sensing devices that discriminate among ionic solutes. We presently believe that this ion selectivity derives, in large part, from the higher affinity of iodide ion toward the ammonium head groups. (8)Mabrey-Gaud, S. In Liposomes: From Physical Structure t o Therapeutic Applications; Knight, C. G., Ed.; Elsevier/North Holland Biomedical Press: Cambridge, 1981;p 114. (9)The precise reason why photopolymerization at elevated temperatures (50 "C) leads to a higher degree of polymerization is not presently clear. Further investigations into these two-dimensional reactions are currently in progress. It is noteworthy, however, that polymerization can also be accomplished by thermal initiation; i.e., addition of 2,2'-azobis(2-amidinopropane) dihydrochloride to a similar dispersion, and subsequent heating (60 "C) for 20 h resulted in polymerized vesicles having M. = 170000.
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Elution volume (ml)
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Figure 3. Apparent absorbance ratio as a function of molar ratio of Triton X-100/1: nonpolymerized (0);partially polymerized, 25-min irradiation at 20 O C (@);partially polymerized, 40-min irradiation at 20 O C (a); fully polymerized, 60-min Arrow indicates the onset of precipirradiation at 20 O C (0). itation. 1.5
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M o l a r r a t i o ( S o d i u m C h l o r i d e / IPA )
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M o l a r r a t i o ( S o d i u m I o d i d e / IPA )
Figure 4. Apparent absorbance ratio as a function of molar ratioof (a) NaCl/Iand (b)NaI/Ifornonpolymerized(O);partUy polymerized, 25-min irradiation at 20 O C ; ( 0 )fully polymerized, Arrow indicates the onset of 60-min irradiation at 20 OC (0). precipitation.
Studies that are currently in progress are aimed a t (i) exploiting the responsiveness of IPA-based vesicles toward ionic stimuli, (ii) modulating such responsiveness through polymerization, and (iii) expanding the scope of polymerizable and nonpolymerizable IPA's. Registry No. I, 133294-88-9;I bromide, 133294-86-7;I (homopolymer), 133294-89-0;NaCl, 7647-14-5;NaI, 7681-82-5;Triton X-100, 9002-93-1; stearic acid, 57-11-4.
