Langmuir 1994,10, 1735-1740
1735
Single-Tail Phosphates Containing Branched Alkyl Chains. Synthesis and Aggregation in Water of a Novel Class of Vesicle-Forming Surfactants Bart Jan Ravoo and Jan B. F. N. Engberts' Department of Organic and Molecular Inorganic Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Received November 5,1993. I n Final Form: February 28,1994" Four disodium monoalkyl phosphates containing branched undecyl and dodecyl groups have been synthesized. The aggregation properties of these surfactants were characterizedby transmissionelectron microscopy of uranyl acetate-stained samples and freeze-fracturereplicas. As predicted from a packing parameter analysis, the branching of the alkyl substituents induces aggregation into bilayers. Thermally stable unilamellar vesicles with diameters between 25 and 60 nm were prepared by vigorous stirring in water at room temperature. Lowering of the pH leads to partial protonation of the phosphate headgroup and to less electrostaticrepulsion between headgroups and bilayers. This results in larger vesicle diameters (50-100nm), a higher tendency to aggregate,and reduced thermal stability. Phase penetration experiments show a lamellar boundary phase upon dissolving the crystalline surfactant in water and growth of myelin structures upon increasing the temperature or decreasing the pH. Upon addition of Ca2+,the monoalkyl phosphate vesicles aggregate at [Ca2+] > 0.5 mM and undergo bilayer fusion, leading to stable vesicles (diameters between 100 and 250 nm) above a Ca2+ threshold concentration of 1.0mM. Introduction Attempts to predict the morphology of a surfactant aggregate on the basis of the molecular structure of the amphiphilic monomer have been successfully couched in terms of geometric packing constraints.' To this end, a critical packing parameter P has been introduced
P = v/a,l,
aqueous compartment of vesicles of di-n-dodecyl phosphate.6 Ca2+ions induce aggregation and fusion of such vesicles! Under appropriate conditions vesicles of di-ndodecyl phosphate can undergo specificasymmetricfusion with target membranes like phospholipid vesicles: erythrocyte ghosts? and Sendai virus.B The discovery that monoalkyl surfactants can associate to form vesicles if the alkyl substituent is branched,2J and the apparent versatility of di-n-dodecyl phosphate vesicles as membrane mimetic systems? directed our attention toward the synthesis of a series of structurally related monoalkyl phosphates with a branched alkyl chain and a study of their amphiphilic properties in aqueous solution. Although much is known about mono-n-alkyl phosphates which associate into micelles in aqueous solution,10-12the properties of phosphates with a branched alkyl chain have not been examined. In fact, to the best of our knowledge, only one such compound has been reported.l3 Herein we report the synthesis and aggregation behavior of three different disodium dodecyl phosphates, Le., 6-propylnonyl phosphate (PNP), 4-butyloctyl phosphate (BOP),and 2-pentylheptyl phosphate (PHP). Convenient procedures for the synthesis of these compounds were available.3J4J6 A precursor to disodium 6-undecyl phosphate (6UP) was available in our laboratory. The structures of these new surfactants are presented in Figure 1. The aggregation behavior of these surfactant molecules can be predicted using the packing parameter analysis. The chain volume v and the chain length 1, of an alkyl
in which v is the volume of the hydrocarbon chain(s), 1, is the critical chain length, and a, is the optimal crosssectional surface area per monomer in the aggregate.Under conditions of thermodynamic equilibrium, the approach has remarkable predictive power. Cone- or truncatedcone-shapedsurfactants possess Pvalues less than 0.5 and preferentially form micelles. For surfactant molecules with P values in the range of 0.5-1.0, the molecule is more cylindricallyshaped and the formation of bilayers or closed bilayers (vesicles) is favored. An increase of P from 0.5 to 1.0implies that the bilayer curvature tends to decrease and, concomitantly, that the average vesicle diameter will increase. A comparison of the aggregation behavior of 4-dodecyl-l(N)-methylpyridinium iodide surfactants in aqueous solution2*3showed that branching of the dodecyl chain favors aggregation into rodlike micelles and vesicles instead of into spherical micelles. Branching decreases the length of the chain at constant volume and headgroup size. Thus, P will increase. The critical rod or critical vesicle concentration decreases upon bran~hing.~ Di-n-alkyl phosphates with alkyl chains containing 10(6) Fonteijn, T. A. A. Ph.D. Thesis, University of Groningen, 1992. 18 carbon atoms form metastable vesicles in ~ a t e r . ~ > ~ (7) Fonteijn,T. A. A.;Hoekatra,D.;Engberta, J. B. F. N. J. Am. Chem. SOC.1990,112,8870. Various solutes can be entrapped in the bilayer or in the (8)Fonteijn,T. A. A.;Engberta, J. B. F. N.;Hoekstra, D. Biochemistry
* To whom correspondence should be addressed. Abstract published in Advance ACS Abstracts, May 1, 1994. (1) Israelachvili,J. N.;Marcelja, S.;Horn, R. G.Q. Reu. Biophys. 1980,
@
13, 121. (2) Engberta, J. B. F. N.; Nusselder, J. J. H. Pure Appl. Chem. 1990, 62, 47. (3) Nuaselder, J. J. H.; Engberta, J. B. F. N. Langmuir 1991,7,2089. (4) Rupert, L. A. M.; Engberta, J. B. F. N. J . Colloid Interface Sci. 1987,120, 125. (5) Wagenaar, A.; Rupert, L. A. M.; Engberta, J. B. F. N.; Hoekstra, D.J . Org. Chem. 1989,54, 2638.
0743-7463/94/2410-1735$04.50/0
1991,30, 5319.
(9) Fonteijn, T. A. A.; Engberts, J. B. F. N.; Nir, S.; Hoekstra, D. Biochim. Biophys. Acta 1992,1110, 185. (10) Chevalier, Y.; Chachaty, C. Colloid Polym. Sci: 1984, 262, 489. (11) Brackman, J. C.; Engberta, J. B. F. N. J. Collord Interface Sci. 1989,132,250. (12) Romsted, L. S.; Yoon, C. 0. J. Am. Chem. SOC.1993, 115, 989. (13) Hardy, C. J.; Greenfield, B. F.; Scargill, D. J . Org. Chem. 1961, 26, 174. (14) Nelson, A. K.; Toy, A. D. F. Inorg. Chem. 1963,2,775. (15) Meakin,B. J.; Mumford, F. R.;Ward, E. R. J. Pharm. Pharmacol. 1959, 11, 540.
0 1994 American Chemical Society
Ravoo and Engberts
1736 Langmuir, Vol. 10, No. 6, 1994
PNPNa2 (20
PHPNa2 (3g)
BOPNa2 (10
6UPNa2 (4c)
Figure 1. Structures of (PNPINaZ (20, (BOPINaz (10,(PHPINaz (3g),and (6UP)Naz (40).
substituent containing n, carbon atoms can be estimated from empirical e uations,16yielding volumes of 350 A3for dodecyl and 323 3 for undecyl Substituents and 1, values of 12.92, 11.66, 10.40, and 9.13 A for nonyl, octyl, heptyl, and hexyl substituents, respectively. The effective crosssectionalheadgroup area a. for monoalkyl phosphates was estimated to be 54 A2,the value calculated for micelles of n-CsH170P03NaH.10 The a. value is strongly dependent on pH. This approximation yieldsPvalues for PNP (0.501, BOP (0.56),PHP (0.62), and 6UP (0.66). For comparison, the value calculated for di-n-alkyl phosphates is approximately 0.78. Thus, it was expected that all four surfactants would form small unilamellar vesicles in aqueous solution. This prediction was borne out in practice. The properties of these vesicles were studied using several microscopic techniques (transmission electron microscopy (TEM) of stained samples and freeze-fracture replicas,phase penetration experimenta). The morphology of the vesicles strongly depends on pH. In order to study the fusogenic properties of the vesicles, the response to the addition of Ca2+ was also examined.
1
Experimental Section General Procedures. Bromoalkanes, acetic acid, acetic anhydride, trifluoroacetic acid, deuterated chloroform,benzene, LiAlH,, and Pd/C were obtained from Merck. Magnesium, the lactones, and diethyl malonate were purchased from Janssen, and pyrophosphate was purchased from Fluka. The lactones were purified by distillation prior to use. Benzene and diethyl ether were distilled from PzOs, and water was distilled twice in an all-quartz distillation unit. Products were distilled in a Kugelrohr unit. NMR spectra were recorded on a Varian VXR 300 spectrometer at 300 MHz unless indicated otherwise. Elemental analyses were performed in the analytical department in our laboratory. Synthetic Procedures. 4-Butyloctanol (Id). Id was prepared as described in the literature.3J"J* 1H NMR (CDC13): 60.88 (6H, t), 1.24 (15H, br s), 1.53 (2H, m), 1.99 (lH, br s), 3.60 (2H, t) ppm. l9c NMR (CDCls): 6 14.0 (CH3), 23.0 (CHz), 28.8 (CH,), 29.4 (CH2), 29.8 (CHz), 33.1 (CHz),37.0 (CH),63.3 (CHz0) PPm. 4-Butyloctyl Dihydrogen Phosphate (le). The conversion A of Id to le was carried out as described in the 1iterat~re.l~ 1.86-g (10-mmol) sample of Id was dissolved in 30 mL of dry benzene, and 3.56 g (20 "01) of dry pyrophosphate was added. 40%) of pure Id could be From the mixture, 0.75 g (4.0 "01, recycled. A 1.33-g (5.0-mmol,50%) sample of le was isolated as a dark viscous oil. Attempts to crystallize the product were not successful. Pyrophosphate impurities (as checked by *lP NMR and elemental analysis) were removed by precipitation upon addition of 20% acetonitrile to a solution of crude le in (16)Tanford, C. J. Phys. Chem. 1972, 76,3020. (17)Mousseron, M.;Bolle, J. M6m. Seru. Chim. fitat 1957, 41, 293. (18)Vozza, J. F.J. Org. Chem. 1959,24, 720.
2-propanol. 1H NMR (CDCU: 6 0.89 (6H, t), 1.24 (16H, br s), 1.66 (lH, m), 4.02 (2H, dt, 'Jpn = 3.0 Hz) ppm. 13C NMR (CDCh): 6 14.1 (CHs), 23.1 (CHz), 27.4 (CHz), 28.8 (CHz), 29.2 (CHz), 33.2 (CHz), 37.0 (CH), 68.7 (CH20) ppm. 31P NMR (CDCl3): 6 1.59 (e) ppm. Anal. Calcd for ClzHnPOd: C, 54.14; H, 10.15; P, 11.65. Found: C, 54.23; H, 10.28; P, 11.28. Disodium 4-Butyloctyl Phosphate (If). A 460-mg (20mmol) sample of sodium was dissolved in 100 mL of ethanol. A 20-mL sample of this sodium ethanolate solution was added to a solution of 532 mg (2.0 mmol) of le in 10 mL of ethanol. After 30 min of equilibration, the ethanol was evaporated. lH NMR (DzO): 6 0.90 (6H, t), 1.25 (16H, br s), 1.57 (lH, m), 3.75 (2H, dt, 3 J p= ~ 6.0 Hz) ppm. "C NMR (DzO): 6 14.1 (CHg), 25.3 (CHz), 30.4 (CHz), 30.9 (CHz), 31.9 (CH2), 35.2 (CHz), 39.3 (CH), 65.7 (CH20,d, 3Jpc = 4.8 Hz) ppm. 31PNMR (DzO): 6 6.08 ( 8 ) ppm. 6-Propyl-l,6-nonanediol(2a).2a was prepared analogously to ld.3 Using 38.1 g (0.31 mol) of 1-bromopropane, 7.4 g (0.30 mol) of magnesium, and 16.8 g (0.15 mol) of c-caprolactone, 24.8 g (0.12 mol, 82%)of 2a was isolated as a colorless oil. lH NMR (60 MHz, CDC13): 6 0.95 (6H, m), 1.1-1.8 (16H, br e), 3.6 (2H, t) ppm. 6-Propylnonenyl Acetate (2b). 2b was prepared analogously to id.3 Starting from 0.10 mol of 2a in 100mL of an AcOH/AczO mixture, 2b could be isolated as a colorless oil (bp 120 OC, 0.05 mmHg) in 69% yield. 1H NMR (CDCh): 6 0.86 (6H, m), 1.33, 1.60, 1.93 (14H, all m), 2.01 (3H, s), 4.02 (2H, t), 5.08 (lH, m) ppm. 13C NMR (CDCU: 6 14.2 (CH3), 20.8 (CHs), 64.5 (CHzO), 171.0 (CO) ppm and several resonances between 20 and 40 ppm (alkyl CHz) and between 120and 140ppm (CH and CH2 alkene). 6-Propylnonyl Acetate (2c). The hydrogenation of 2b was carried out analogously to the synthesis of ld.3 Working on a 60 mM scale, a yield of 95% was obtained. 2c is a colorless oil. lH NMR (CDCL): 6 0.85 (6H, t), 1.20 (15H, m), 1.59 (2H, m), 2.02 (3H, s), 4.03 (2H, t) ppm. 13C NMR (CDCl3): 6 14.4 (CHs), 19.7 (CH2),20.9(CH3),26.2(CH2),26.3 (CHz),28.6 (CHz),33.4(CHz), 36.0 (CHz), 36.8 (CH), 64.6 (CHzO), 171.0 (CO) ppm. 6-Propylnonanol(2d). The reduction of 2c was carried out analogously to the synthesis of ld.9 Working on a 50 mM scale, a yield of 80% was obtained. 2d is a colorless oil with bp 80 OC, 0.01 mmHg. 1H NMR (CDCU: 6 0.87 (6H, t), 1.23 (15H, br s), 1.57 (3H, m), 3.63 (2H, t) ppm. 13C NMR (CDCb): 6 14.4 (CHs), 19.7 (CHz), 26.1 (CHz), 26.3 (CHz), 32.7 (CHz), 33.5 (CHz), 35.9 (CH2),36.8 (CH), 62.9 (CH20) ppm. 6-Propylnonyl Dihydrogen Phosphate (2s). A 9.00-g (48.4mmol) sample of 2d was converted into 2e as described for Id. A 7.50-g (28.1-"01, 58%) sample of 2e could be isolated, and 1.83 g (9.68 mmol, 20%) of 2d was recycled. Pyrophosphate impurities (as checked by 3lP NMR) were removed by precipitation upon addition of 15% acetonitrile to a solution of crude 28 in 2-propanol. Pure 2e was isolated as a dark viscous oil and could not be crystallized. lH NMR (CDCla): 6 0.93 (6H, t), 1.28 ~ 6.32 Hz) ppm. 13C (15H, br s), 1.73 (2H, m), 4.09 (2H, dt, 3 J p = NMR(CDCl& 6 14.5 (CH3),19.8 (CHz),25.9 (CHz), 26.3 (CHz), 30.1 (CH2),33.6 (CHa), 36.1 (CHz),37.0 (CH), 68.2 (CHzO, br s) ppm. 3lP NMR (CDCl3): 6 1.29 ( 8 ) ppm. Anal. Calcd for C12HnPOd: C, 54.14; H, 10.15; P, 11.65. Found: C, 53.95; H, 10.11; P, 11.45. Disodium 6-Propylnonyl Phosphate (2f). 2e was converted into 2f as described for le. The salt was isolated by evaporation of the ethanol. 1H NMR (DzO): 6 0.88 (6H, t), 1.31 (15H, br s), 1.61 (2H, m), 3.74 (2H, dt, 3 J p =~ 5.65 Hz) ppm. 13CNMR (DzO): 6 14.3 (CHs), 19.6 (CHz), 26.1 (CHz), 26.3 (CHz),31.0 (CHz), 34.4 (CHz), 35.9 (CHz),36.4 (CH), 65.1 (CHzO, br s) ppm. NMR (DzO): 6 6.45 ( 8 ) ppm. 2-Pentylheptanol (3e). 3e was obtained by a procedure described in the literature.15 The boiling point, MS, and analysis have been reported previously.19 lH NMR (CDClS): 6 0.89 (6H, dist t), 1.29 (17H, br s), 3.52 (2H, d) ppm. '9c NMR (CDCla): 6 14.0 (CHs),22.6 (CHz),26.5 (CHz), 30.8 (CHz),32.3 (CHz),40.4 (CH), 65.5 (CH2O) ppm. 2-Pentylheptyl Dihydrogen Phosphate (30. A 5.00-g (26.8 mmol) sample of 3e was converted into 3f as described for Id. A 3.00-g (11.2-"01, 42%) sample of 3f was isolated, and 2.30 (19)Bestmann, H.J.; Roesel, P.; Vostrowsky, 0.Liebigs Ann. Chem.
1979, 1189.
Synthesis of Vesicle-Forming Surfactants g (12.4 mmol, 46%) of 30 could be recycled. 3f was obtained as a colored viscous oil and could not be crystallized. lH NMR (CDC13): 6 0.89 (6H, t), 1.28 (16H, br s), 1.62 (lH, br s),3.85 (2H, dd) ppm. 13CNMR (CDCb): 6 13.9 (CHs),22.5 (CH2),26.0 (CH2), 30.2 (CHt), 32.0 (CHz), 38.3 (CH, d, J 7 Hz), 69.8 (CH20, d, 2Jpc = 2.2 Hz) ppm. 3lP NMR (CDCh): 6 2.05 (s) ppm. Disodium 2-Pentylheptyl Phosphate (3g). 3f was converted into its disodium salt 3g as described for le. 3g was isolated by evaporation of the ethanol and crystalliied from a hot, filtered, clear solution in 2-propanol upon addition of a few drops of acetonitrile. lH NMR (D20): 6 0.89 (6H, t),1.31 (16H, br s), 1.51 (lH, br s), 3.64 (2H, dt, 3 J p = ~ 1.3 Hz) ppm. 13CNMR (DzO): 6 13.4 (CH,), 21.9 (CHz), 25.5 (CHz), 30.0 (CHa), 31.6 (CHz), 38.2 (CH,d,J=6.4H~),67.7(CH20,d,~Jp~=4.8Hz)ppm. 81PNMR (D2O): 6 6.51 ( 8 ) ppm. Anal. Calcd for C12HdOINa2: C, 46.45; H, 8.06;P, 10.00; Na, 14.84. Found C, 46.44; H, 8.00; P, 9.85; Na, 14.71. Di-tert-butyl6-undecyl phosphate (4a) waspreparedusing a literature proceduremand kindly provided by Anno Wagenaar. lH NMR (CDCl3): d 0.95 (6H, t), 1.25 (12H, m), 1.40 (18H, s), 1.65 (4H, m), 4.25 (lH, m) ppm. 13C NMR (CDCl3): 6 14.2 (CHs), 23.0 (CHz), 25.0 (CHz), 30.0 (CHs, d, 'Jpc = 4.0 Hz), 32.2 (CHz), 35.3 (CH2, d, 3Jpc = 5.0 Hz), 78.2 (CH, d, 'Jpc = 6.0 Hz), 80.9 (C, d, 2 J p = ~ 7.0 Hz) ppm. 6-Undecyl Dihydrogen Phosphate (4b). To 77 mg (0.21 mmol) of 4a in 1.2 mL of c&3 was added one drop of CF&02H. The mixutre was stirred at 50 OC, and the reaction was followed with NMR. The reaction was complete in 30 min. Evaporation of the solvent and CF&OzH yielded crude 4b in quantitative yield. lH NMR (C&): 6 0.85 (6H, t), 1.25 (4H, m), 1.50 (12H, m),4.20(1H,m)ppm. 1 3 C N M R ( C ~ & 614.2(CHa),22.9(CH2), 24.9 (CH2), 32.0 (CHz), 35.3 (CH2, d, 'Jpc = 4.0 Hz), 81.5 (CH, ~ 7.0 Hz) ppm. 3lP NMR (C&): 6 1.388 (s) ppm. d, 2 J p = Disodium 6-Undecyl Phosphate (4c). A 53-mg (0.21-"01) sample of 4b was dissolved in 2 mL of ethanol at 0 OC. A 2.22-g sample of a 0.2438 mmol/g solution of sodium ethanolate in ethanol was added (i.e., 0.42 mmol of Na). Most of the ethanol was evaporated, and then 1mL of acetonitrile was added. The product crystallized, and the remaining solvent mixture was evaporated. The yield was 60.0 mg (0.20 mmol, 95%)of 4c. The crude salt was washed with dry ether and crystallized from warm ethanol upon addition of 10-20% acetonitrile and cooling. lH NMR (CDaOD): 6 0.90 (6H, t), 1.32 (12H, m), 1.61 (4H, m), 4.20 (lH, m) ppm. SlP NMR (CDsOD): 6 8.96 (s)ppm. Anal. Calcd for CIlH~P04Na2:C, 44.60; H, 7.77; Na, 15.54. Found C, 44.00, H, 7.82; Na, 15.40. Preparation of Vesicle Solutions. The surfactants were weighed to make 15-30 mM solutions in 1 or 2 mL of freshly distilled water. The suspensions were either stirred vigorously or sonicated for 15 min at room temperature. The pH of these solutions was 10-11. Solutions at lower pH were prepared in a NaAc/HOAc buffer (pH 5.5) or by the addition of aliquots of 0.1 N HC1 solution. The pH was measured with an Orion SA 720 pH electrode. Transmission Electron Microscopy. Negatively stained samples were obtained by staining aliquots of the surfactant solutions on carbon-coated formvar grids (300 mesh) with a 1.0 % or a 0.1% (w/v) aqueous solution of uranyl acetate. Freezefracture preparations were obtained by the method described by KoehleP on a Balzers EVM 052 A with evaporation head EK 552. The samples were fractured at -165 "C, shadowed with 20 Aof platinum/carbon under an angle of 45O, and coated with 200 A of carbon. The replicas were cleaned in concentrated chromic acid and water. All samples were examined in a Philips EM 201 electron microscope operating at 60-kV accelerating voltage. Conductivity Measurements. Conductivities were measured as described previously.11 Critical vesicle concentration (CVC) values were taken from the intersection of the tangents drawn before and after the first break in the conductivity versus concentration plot. (20) Zwierzak, A.; Kluba, M. Tetrahedron 1971,27, 3163.
(21)Koehler, J. H. In Principles and Techniques ,of Electron Microscopy; Hayat, M. A,, Ed.; Van Nostrand Reinhold Co.: New York, 1972;Vol. 2, p 53.
Langmuir, Vol. 10, No. 6, 1994 1737 Optical Polarization Microscopy. A Zeiss Axioskop polarizing microscope with phase contrast or a Nikon polarization microscope equipped with a hot stage was used. Disodium alkyl phosphates were squeezed between a microscopeslide and a cover slip and brought into contact with water or dilute acetic acid (pH 5.5). Fusion Experiments and Resonance Energy Transfer (RET)Assays. CaClt (0.10 M solution) was injected into 15-20 mM disodium alkyl phosphates solutions at room temperature to final Caa+concentrations of 0.2-5.0 mM. Sampleswere studied by TEM. RET assays were carried out as described in the and NliteratureM using N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)(lissamine Rhodamine B sulfonyl)-labeledphosphatidylethanolaminein 0.8 mol % Fluorescencemeasurementswere performed on a SLM Aminco SPF-SOOCspectrofluorometer and turbidities were measured at 400 nm on a Perkin-Elmer Lambda 5 UV-vis spectrophotometer.
.
Results and Discussion Synthesis. Alkyl-substituted 1-hydroxyalkanes were prepared from the reaction between lactones and 2 equiv of Grignard reagent.3J7JB The tertiary alcohol function of the diol thus obtained was dehydrated to the alkene, while the primary alcohol group was protected with an acetate ester to inhibit irreversible formation of cyclic ether^.^ It was found necessary to isolate the diol, since no stable alkene acetate was obtained if the Grignard reaction, the dehydration, and the protection were carried out in one pot. The alcohol was obtained after catalytic hydrogenation and reduction of the ester. 4-Butyloctanol (Id) was prepared from ybutyrolactone and n-butylmagnesium bromide, and 6-propylnonanol(2d) was prepared from E-caprolactone and n-propylmagnesium bromide. A third branched-chain alcohol was prepared by the conventionalalkylation of diethyl malonate.lS Alkylation with 2 equiv of 1-bromopentane, hydrolysis of the ethyl ester functions, and decarboxylation and reduction of the carboxylic acid to the alcohol yielded 5-pentylheptanolin satisfying yield. Di-tert-butyl6-undecyl phosphate was prepared via a literature procedure.20 The tert-butyl substituents were removed selectively in quantitative yield. The primary alcohols were converted into alkyl dihydrogen phosphates using pyrophosphate, H9207, as the phosphorylation agent." In order to obtain only monoalkylated product, 2 equiv of the pyrophosphate was used. 31P NMR confirmed the formation of only alkyl dihydrogen phosphate. Pyrophosphate impurities could be removed by precipitation. The alkyl dihydrogen phosphates were converted to their disodium salts by addition of 2 equiv of NaOEt in ethanol (Scheme 1). Scheme 1. Conversion of 1-Hydroxyalkanes to Disodium Alkyl Phosphates
-
HqP2Q7benzene
ROH
-
NaOEtntOH
ROP03H2
ROP03Naz
Electron Microscopy. The formation of small vesicles from the disodium alkyl phosphates was confirmed by a TEM study. Stained TEM samples of (PNP)Na2,(BOP)Na2, (PHP)Na2, and (6UP)Naz showed spherical vesicles with diameters between 25 and 50 nm for (PNP)Na2, (BOP)Na2,and (PHP)Na2 and between 30 and 60 nm for (6UP)Naz. Smaller diameters (15-25 nm) are observed for vesicle dispersions prepared by sonication. In view of the small diameters, we assume that these vesicles are unilamellar. There is literature precedent that surfactants with highly charged headgroups favor formation of small (22) 4093.
Struck, D. K.; Hoekstra, D.; Pagano, R. E. Biochemistry 1981,20,
1738 Langmuir, Vol. 10, No. 6, 1994
Ravoo and Engberts
Figure2. Micrographsofvesicle solutions: (A,topleft) (6UP)Na2,16.9mM,pH 11,1.0% UAc, prepared by stirringat room temperature; (B, top right) (BOP)Na2,16.1 mM, pH 11, 1.0% UAc, prepared by sonication at room temperature; (C, bottom left) (PHP)Na2, pH 5.5,0.1% UAc, prepared by stirring at room temperature; (D,bottom right) (PHP)Naz, 30 mM, freeze-fracture replica, prepared by stirring at room temperature. Bars represent 200 nm.
vesicles.23 Since there have been reports in the literature of the fusogenicproperties of uranyl acetatetoward vesicles of di-n-alkyl phosphates,5 freeze-fracture replicas were prepared to eliminate the influence of the staining agent. Again, vesicular aggregates of similar diameters were observed. Micrographs of several vesicle solutions are presented in Figure 2. Critical Vesicle Concentration. A conductometric experiment at 25 “Cand pH 11yielded a sharp transition a t 10 mM for (BOP)Na2 and a less cooperative transition a t 4.5 mM for (PHP)Na2. Critical micelle concentrations of mono-n-alkyl phosphates have been reported in the literature and are strongly dependent on pH (determining headgroup charge and size), the alkyl chain length of the (23) Winterhalter, M.; Helfrich, W. J. Phys. Chem. 1992,sS, 327.
surfactant, and the technique employed.10JlJ2tu Moreover, it has been shown that critical vesicle concentrations decrease upon branching in the alkyl chain.3 The CVC’s of (BOP)Na2 and (PHP)Na2 in the dianionic form are consistent with the available literature data.lOJ Influence of pH on Vesicle Morphology. Solutions (20 mM) of the disodium alkyl phosphates prepared in water possess a pH of around 11due to the basic character of the headgroup. The dianionic disodium surfacant can be protonated to form its neutral dihydrogen analogue in two steps (Scheme 2). The two pKa’s are approximately 7 and 2 at room temperature for micellar solutions of n-dodecyl, n-decyl, and n-octyl phosphate.*O Acid titration revealed that the PKa values of the branched monoalkyl 1~12924
(24) Tahara, T.; Satake, I.; Matuura, R. Bull. SOC.Chem. Jpn. 1969, 42, 1201.
Synthesis of Vesicle-Forming Surfactants
Langmuir, Vol. 10, No.6,1994 1739
Scheme 2. Protonation of the Phosphate Headgroup H+
ROm3H2
H+
ROP03HPKa 2
I
ROF032' PKa 7
phosphates do not differ significantly from the literature data for mono-n-alkyl phosphates. Samples of the monoalkyl phosphates at various pH values were examined with TEM and compared to the solutions of small vesicles with diameters between 25 and 50nm observed at pH 11(Figure 2). Samples taken around or below the first pKa (pH 7.1, 6.4, 3.7) showed that reduction of the pH leads to (1) a steadily increasing number of much larger vesicles (diameters between 50 and 100nm) and (2)an increasing tendency to aggregation of the small (25-50 nm) vesicles. Upon further reduction of the pH (until below 3.5) the solutions become turbid, and below pH 3 the surfactants flocculate. We suggest that reduction of the pH of the vesicle solutions results in partial protonation of the phosphate headgroup, leading to substantial changes in headgroup electrostatic interactions and hydration. In the first place, the effective headgroup charge decreases, leading to less electrostatic repulsion between the headgroups and to an effectively smaller size (more diffuse character) of the hydration sphere. Consequently, there will be a smaller driving force for bilayer curvature, and vesicles of larger diameter are formed. This situation can be analyzed in terms of the packing parameter P: a reduction of the effective headgroup area a,, will lead to an increase of P, favoring aggregation in bilayers with less curvature. Secondly, protonation reduces the negative charge at the surface of the vesicles, and the intervesicular repulsion will be reduced concomitantly. Moreover, partial protonation favors hydrogen bond formation between the headgroups.25 Thus, the vesicles are expected to aggregate more extensively a t lower pH. Reduction of the intravesicular repulsion and possibilities for intervesicular H bond formation might also facilitate symmetric fusion. This is an explanation for the formation of large vesicles from very small ones and is in accord with observations concerning vesicles of di-n-dodecyl ph0sphate.~5 When the pH is lowered to below 3.5, an increasing fraction of the surfactant becomes fullyprotonated. The electrostatic interbilayer repulsion is reduced to a minimum. The vesicles can now approach closely, and the surfactant flocculates, which explains the increase in turbidity below pH 3.5. Diprotonated (neutral) alkyl phosphates are not soluble in water and precipitate from the solution. No birefringent lamellar phases were observed. Phase Penetration Experiments. Phase penetration experiments were carried out for (PHP)Naz and (6UP)Na2 to examine the transition from solid crystalline surfactant to vesicular bilayers in water. Different lyotropic phases could be identified as water penetrated into the crystal.26 The boundary layers between bulk crystal and aqueous solution represent a gradient of increasing water content, and the structure of the outer (most hydrated) phase is considered to be indicative of the aggregation form of the surfactant in water. Both (PHP)Na2 and (6UP)Naz show a lamellar phase a t the outer boundary with the bulk solution. A t higher temperature (>40 "C)or at lower pH (5.5)formation of myelin structures was observed. Both the lamellar phase and the myelins slowly dissolve in water. It has been proposed that this ~~
Figure 3. Micrographs of vescile solutions showing the effect of the addition of Ca2+: (A, top) (6UP)Naz (16.9 mM) and Ca2+ (2.0 mM) after 30 min, 0.1% ! UAc, vesicles prepared by stirring at room temperature;(B, bottom) (PHP)Na2(16.1 mM) and Ca2+ (3.5 mM) after 4 days, 0.1% UAc, vesicles prepared by stirring at room temperature. Bars represent 200 nm.
is a mechanism for the formation of vesicles from solid lipid film~.2~928 Stability of the Vesicle Solutions. One of the most remarkable features of the vesicle solutions of the present monoalkyl phosphates is their high stability. Contrary to most vescile solutions of synthetic surfactants, solutions of (BOP)Na2, (PHP)Na2, and (6UP)Na2 (prepared by stirring a t room temperature) that were stored a t room temperature for a period of over 3 days did not show any flocculation or appreciable morphological changes, as observed by TEM. The vesicles tend to become less neatly spherical, and the average vesicle size decreases (to a diameter of approximately 25 nm).
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(25) Rupert, L. A. M.; Van Breemen, J. F. L.; Hoekstra,D.; Engberta, J. B. F. N. J . Phys. Chem. 1988,92,4416. (26) Rosevear, F. B. J. SOC.Cosmet. Chem. 1968,19,581.
(27) Lasic, D. D. Biochem. J. 1988,256,l. (28)Fonteijn, T.A. A,; Hoekstra, D.; Engberts, J. B. F. N. Longmuir 1992,8,2437.
1740 Langmuir, Vol. 10, No. 6,1994 The high stability of the vesicles might be explained in terms of the dianionic nature of the headgroup. The surface of the vesicles will have an overall negative charge, and it is unfavorable for the vesicles to approach each other. Flocculation of the vesicles will occur only very slowly. At lower pH, the vesicle solutions clearly show a reduced stability. At pH 5.5 (monoanionic headgroups), (BOP)Na2, (PHP)Naz, and (6UP)Naz partially flocculate within 1day. However, TEM shows that vesicles are still present in solution after 3 days. The vesicles show an increasing tendency to aggregate, which is a consequence of the reduced intervesicular repulsion due to a lower surface charge and a higher degree of hydrogen bond formation at lower pH. A higher degree of aggregation leads to a more rapid flocculation of surfactant material. The vesicles have a reduced average diameter (50 versus 50100 nm) and a wrinkled spherical form as compared to a freshly prepared solution. Vesicles of all four monoalkyl phosphates studied also show a remarkable stability toward physiological salt concentrations: after 6 h in a 150 mM NaCl solution, no morphological changes (TEM) or increase of turbidity could be detected. Fusion Experiments. Upon addition of CaClz to 1520 mM solutions of (BOP)Naz, (PHP)Naz, and (6UP)Naz two approximatethreshold concentrations were identified. At Ca2+> 0.5 mM a sharp increase in turbidity of the solutions is observed. TEM showed a large increase in the tendency of the vesicles to aggregate (Figure 3A). At Ca2+ > 1.0 mM, some calcium alkyl phosphate salt precipitates (identified by elemental analysis), but TEM shows that large aggregates of vesicles persist and vesicles of 100-250 nm diameter have formed. Solutions of these vesicles in the presence of the calcium alkyl phosphate precipitates are stable for periods of over 3 days (Figure 3B). In order to obtain quantitative insight into the effect of Ca2+ on the monoalkyl phosphate vesicles, turbidity measurements to measure aggregation rates and RET assays%to measure rates and extents of fusion were carried out. However, we found it impossible to derive rates of
Rauoo and Engberts
aggregation from turbidity measurements, since this process occurs almost instantaneously upon addition of Ca2+and the measurement is hampered by the formation of the precipitate. Moreover,the RET assay technique is not applicable to this system: although we measured a large increase in NBD fluorescence in a mixture of labeled and nonlabeled (B0P)NaZ vesicles upon addition of Ca2+, this increase in fluorescence was completely annihilated upon addition of a 3-fold excess of EDTA, a strong calcium chelating agent. A control experiment revealed that upon addition of Ca2+to a NBD-PE-labeled solution of (BOP)Na2 an identical increase in fluorescence intensity occurred, although any dilution effect is impossible. Upon addition of EDTA, the fluroescence intensity decreased to the initial value. We believe that the high Ca2+ affinity of the dianionic headgroup of the monoalkyl phosphates induces morphological transformations in the bilayer that influence the NBD fluorescence intensity. Comparableobservations were made for vesicles of NDB-labeled dioleoylphosphatidic acidm Ca2+addition results in formation of patches in the bilayers, leading to high (local) fluorescence yields. A change in fluorescence intensity may also result from a reversible phase transition in the bilayer induced by Ca2+.30 At pH 5.5, when the phosphate headgroup is mono anionic, comparable observations were made. In summary we have shown that branched-chain monoalkylphosphates form vesicles even though the alkyl chains are short and the headgroup charge is high. Vesicles and vesicle formation were visualized with negative staining TEM, freeze-fracture TEM, and phase penetration experiments. The vesicles are small but show high thermal stability over periods of more than 3 days.
Acknowledgment. We are indebted to Mr. A. Wagenaar for valuable advice concerning the synthesis of the monoalkyl phosphates and to Dr. A. Sein for the preparation of the freeze-fracture replicas. (29)Haverstick, D.M.;Glaser, M. hoc. Natl. Acad. Sci. U.S.A. 1987, 84,4475. (30)Hong, K.;Baldwin, P. A.; Allen, T. M.; Papahadjopoulos, D. Biochemistry 1988,27, 3947.
