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Cosolvent Effects on the Stability of Catanionic Vesicles Formed from Ion-Pair Amphiphiles Shao-Jen Yeh, Yu-Min Yang,* and Chien-Hsiang Chang Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan 70101, Republic of China Received November 14, 2004. In Final Form: March 16, 2005 Four ion-pair amphiphiles (IPAs), derived from the pairing of alkyltrimethylammonium chlorides and sodium alkyl sulfates, were used to form catanionic vesicles in water upon the mechanical dispersion method. For the first time in the literature, short-chained alcohols (methanol, ethanol, 1-propanol, and 1-butanol) were added as cosolvents at a variety of concentrations and systematically studied for their effects on the stability of the ensuing vesicles. Dynamic light scattering measurements indicated that vesicles formed from one of the IPAs (i.e., dodecyltrimethylammonium dodecyl sulfate) could be efficiently and successfully stabilized by the addition of appropriate amounts of 1-propanol and 1-butanol. Maximum lifetimes of more than 1 year and 132 days for stable vesicles in 5% 1-butanol and 15% 1-propanol solutions, respectively, were observed, and this demonstrates that a novel method for the stabilization of catanionic vesicles formed from IPAs becomes available by means of cosolvent addition. Furthermore, the stability of catanionic vesicles was found to be strongly dependent on cosolvent concentration. In general, the vesicle stability increased with increasing the cosolvent concentration, reached a maximum at a specific concentration, and thereafter decreased with further increasing the concentration. The vesicles finally disintegrated into constituent molecules in solutions of high cosolvent concentrations. An explanation of cosolvent effects based on the medium dielectric constant was proposed.
Introduction Vesicles, which are involved in many biological processes, are usually constituted by double-chain amphiphiles such as phospholipids. However, a great deal of work has now been done which demonstrates the possibility of forming vesicles from single-chain surfactants, when specific conditions are fulfilled. This is the case for mixtures of anionic and cationic surfactants, whose association through the interactions of their polar heads can mimic the type of structures encountered in phospholipids. The word “catanionic” vesicle is now commonly accepted to qualify such types of structures. Vesicle formation in that case is usually described as a spontaneous process. A subtle distinction exists in the literature between simple anionic/cationic surfactant mixtures, where each surfactant is accompanied by its own counterion, and the so-called “ion-pair amphiphiles” (IPA), in which the preceding counterions were removed leaving two amphiphilic ions oppositely charged. Recent reviews on the current status of catanionic vesicles research are available.1,2 The most common IPAs (i.e., catanionic surfactants or catanionics) are formed by pairing two single-chained oppositely charged amphiphiles with an equimolar ratio. The resulting surfactants are, thus, uncharged and can be considered as pseudo-double-chained surfactants, in the sense that the two chains are not covalently bonded to the same headgroup.2 Typical catanionics consist of alkylammonium alkyl sulfates or sulfonates and alkylpyridinium alkyl sulfates or sulfonates. Catanionics with two symmetric alkyl chains and Cn ) Cm g 10 (where n and m represent the number of carbon atoms) or with * Corresponding author. Prof. Yu-Min Yang: tel., 886-6-2757575 ext. 62633; fax, 886-6-2344496; e-mail,
[email protected]. (1) Tondre, C.; Caillet, C. Adv. Colloid Interface Sci. 2001, 93, 115134. (2) Marques, E. F.; Regev, O.; Khan, A.; Lindman, B. Adv. Colloid Interface Sci. 2003, 100-102, 83-104.
asymmetric alkyl chainssone long alkyl chain, Cn g 11, and a shorter one, Cm g 9sare practically insoluble in water.3 Catanionics with shorter chains than the latter are soluble in water and yield micellar solutions. The geometry of a surfactant molecule is currently believed to play a major role in defining its aggregation behavior. It is well-known that the kind of aggregates forming in a system will depend on the value of critical packing parameter, Pc:
Pc ) v/a0lc
(1)
where a0 is the minimum interfacial area occupied by the headgroup; v and lc are the volume and maximum extended length, respectively, of the hydrophobic tail (or tails) in a fluid environment. According to Israelachvili,4 a surfactant with Pc e 0.33 will be able to form spherical micelles. Similar analyses for surfactants with 0.33 e Pc e 0.5 predict that cylindrical or disk-shaped micelles will result. For vesicle formation, the proper value of Pc is between 0.5 and 1, and for planar bilayers, the Pc value will be 1. Inverted micelles formed for surfactants with Pc > 1. On the basis of the critical packing parameter, the formation of catanionic vesicles in the case for mixtures of anionic and cationic surfactants can then be reasoned that the effective interfacial headgroup area of each partner of an IPA may be substantially smaller than that for each individual surfactant, due to the electrostatic attraction between headgroups and a reduction in hydration. Unlike their micelle-forming precursors, IPAs prefer to assemble into bilayer vesicles. This fact, together with the ready availability and low cost of single-chain surfactants as compared with double-chain phospholipids, (3) Khan, A.; Marques, E. F. In Specialists Surfactants; Robb, I. D., Ed.; Chapman & Hall: London, 1997; pp 37-80. (4) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: London, 1991; Chapter 17.
10.1021/la047207g CCC: $30.25 © 2005 American Chemical Society Published on Web 05/28/2005
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provides considerable incentive for exploiting IPAs as a novel class of vesicle-forming materials. Unfortunately, catanionic vesicles formed from IPAs usually showed only a short-term stability.5,6 To overcome this shortcoming, various methods have been employed which may increase the stability of catanionic vesicles. For instance, multiple-chain IPAs have been designed to improve the membrane tightness7 or to mimic archaebacterial membranes, which can sustain extreme physiological conditions.8 Other multiple-chain structures are based on dimeric (or gemini) surfactants, in which two single-chain amphiphiles are covalently attached through a spacer at the level of the headgroups.9 The polymerization concept was also applied to IPAs, and a few polymerized IPA vesicles have been already reported.10,11 Furthermore, cholesterol has been added to improve the stability of the ensuing vesicles.6,11,12 Huang and co-workers13-17 proposed a different approach to enhance the vesicle formability of mixed cationic and anionic surfactants by adding short-chained alcohols in water. It is worthy to note that the vesicle formation studied by Huang and co-workers was the one described as a spontaneous process. In contrast with their investigations, vesicle formation in this work is made possible by the conventional mechanical dispersion method, which is also the one for making traditional liposomes. For the first time in the literature, this paper aims to enhance the stability of catanionic vesicles formed from IPAs by means of adding short-chained alcohols in water. Materials and Methods The anionic surfactants used were 99% sodium dodecyl sulfate and sodium tetradecyl sulfate purchased from Sigma. The cationic surfactants used were 97% dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, and hexadecyltrimethylammonium chloride purchased from TCI. Methanol (99.8% pure), ethanol (99.8% pure), 1-propanol (99.5% pure), and 1-butanol (99.5% pure) used as cosolvents were purchased from Aldrich. All chemicals mentioned above were used as received without further purification. All experiments were conducted with pure water that was passed through a Milli-Q plus purification system (Millipore, U.S.A.) with a resistivity of 18.2 MΩ‚cm. IPAs as precipitates will come out when cationic and anionic surfactants in aqueous solutions of sufficiently high concentrations are allowed to react with each other. In this work, such precipitates were prepared by mixing equal volumes (500 mL) of 20 mM solutions of them. Twenty millimolar is a concentration beyond the critical micelle concentrations of these surfactants. After standing for 1 h, the precipitates were separated from the solutions by repeated centrifuging and washing. They were then (5) Fukuda, H.; Kawata, K.; Okuda, H. J. Am. Chem. Soc. 1990, 112, 1635-1637. (6) Chien, C. L.; Yeh, S. J.; Yang, Y. M.; Chang, C. H.; Maa, J. R. J. Chin. Colloid Interface Soc. 2002, 24, 31-45. (7) Chung, Y. C.; Lee, H. J.; Park, J. Y. Bull. Korean Chem. Soc. 1998, 19, 1249-1252. (8) Bhattacharya, S.; De, S.; Subramanian, M. J. Org. Chem. 1998, 63, 7640-7651. (9) Bhattacharya, S.; De, S. Langmuir 1999, 15, 3400-3410. (10) Hirano, K.; Fukuda, H.; Regen, S. L. Langmuir 1991, 7, 10451047. (11) Chung, M. H.; Chung, Y. C. Colloids Surf., B 2002, 24, 111-121. (12) Menger, F. M.; Binder, W. H.; Keiper, J. S. Langmuir 1997, 13, 3247-3250. (13) Huang, J. B.; Zhao, G. X. Colloid Polym. Sci. 1995, 273, 156164. (14) Huang, J. B.; Zhu, B. Y.; Zhao, G. X.; Zhang, Z. Y. Langmuir 1997, 13, 5759-5761. (15) Huang, J. B.; Zhu, B. Y.; Mao, M.; He, P.; Wang, J.; He, X. Colloid Polym. Sci. 1999, 277, 354-360. (16) Zhang, X. R.; Huang, J. B.; Mao, M.; Tang, S. H.; Zhu, B. Y. Colloid Polym. Sci. 2001, 279, 1245-1249. (17) Wang, C. Z.; Tang, S. H.; Huang, J. B.; Zhang, X. R.; Fu, H. L. Colloid Polym. Sci. 2002, 280, 770-774.
Yeh et al.
Figure 1. Turbidity as an indication of the solubility of IPAs in aqueous solutions at a variety of alcohol concentrations. dried for 36 h under vacuum and ground into fine powders for further studies. Four IPAs, that is, DTMA‚DS (dodecyltrimethylammonium dodecyl sulfate), TTMA‚DS (tetradecyltrimethylammonium dodecyl sulfate), HTMA‚DS (hexadecyltrimethylammonium dodecyl sulfate), and TTMA‚TS (tetradecyltrimethylammonium tetradecyl sulfate), by the pairing of two oppositely charged surfactants were thereby obtained. The solubilities of IPAs in aqueous alcohol solutions were determined by a turbidimeter (model 2100N, Hach, U.S.A.). Catanionic vesicles were prepared by the mechanical dispersion method. An appropriate amount of IPA in 10 mL of chloroform was dried to form thin films on glass beads in a test vessel by nitrogen blowing. The resulting films were then hydrated with 60 mL of water or aqueous alcohol solution while vortexing for 10 min and sonicating for 1 h. The compositions of the mixed solvents were represented by cosolvent volume percentage. The translucent solution with a concentration of 1 mM was then extruded under nitrogen pressure through 0.4 µm and 0.2 µm nuclepore polycarbonate membranes (Osmonics, U.S.A.) sequentially at 60 °C. An extruder from Lipex Biomembranes, Inc., Canada, was used for the extrusion of vesicles. It is noteworthy that the variation of particle size is a critical test of stability. Therefore, the stability of vesicles was studied by measuring the particle size and the size distribution as a function of time following vesicle preparation using a computerized zetasizer (model 3000 HS, Malvern, U.K.). A transmission electron microscope (model JEOL-TEM-200EX) was used to obtain the vesicle images with the negative-staining technique. For the sample preparation, a few drops of vesicle solutions were applied to a carbon-coated Cu grid and dried. A drop of uranyl acetate-ethanol solution was then added as the staining agent.
Results and Discussion Identification of IPAs. The resulting pure IPAs were subjected to elemental analysis and determination of mass and infrared spectra, all of which have been described in detail elsewhere.6 There was enough evidence to prove them pseudo-double-chained amphiphilic compounds that contain amphiphilic cations and amphiphilic anions in an equimolar ratio. Solubility of IPAs in Aqueous Alcohol Solutions. The turbidity measurements were performed on solutions of IPAs dispersed in pure water and aqueous solutions at a variety of alcohol concentrations by vortexing with an IPA concentration of 1 mM. As shown in Figure 1, it was found that the turbidity always decreased with the increase of cosolvent concentration. The turbidity, which may be regarded as the inverse of solubility, decreased in the order C3OH > C2OH > C1OH for a given IPA. For the same cosolvent (ethanol), higher concentrations were needed for solubilizing the IPAs with a higher number of carbon atoms in the chains. The turbidity data indicated
Stability of Catanionic Vesicles Formed from IPAs
Figure 2. Initial size distributions of catanionic vesicles formed from DTMA‚DS.
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Figure 4. Lifetimes of catanionic vesicles formed from DTMA‚ DS in aqueous solutions of (a) 15% 1-propanol and (b) 5% 1-butanol. The arrow indicates the time beyond the upper limit of accurate size measurement. Table 1. Most Stable Conditions for the Formation of Catanionic Vesicles from DTMA‚DS in Aqueous Alcohol Solutions cosolvent
concn
by eyes
by size measurement
methanol ethanol 1-propanol 1-butanol
40% 20% 15% 5%
45 min 72 min 132 days >374 days
check baselinea 135 min 132 days >374 days
a Check baseline: particle size beyond the upper limit of accurate size measurement.
Figure 3. TEM image of the DTMA‚DS vesicles formed in a 15% 1-propanol aqueous solution.
the absence of surfactant aggregates or precipitates in the solutions with higher cosolvent concentrations that were investigated. This gives strong evidence that IPAs in the molecular dispersion state may be assumed under these conditions. Vesicle Formation in Water and Mixed Solvents. It is noteworthy that none of the catanionic vesicles formed in pure water from the four IPAs studied in this work were stable enough for complete measurements. At first the liquid appeared bluish to the eye, but insoluble precipitate usually came out within 30 min. Figure 2 shows three initial vesicle size distributions, which were measured immediately following the vesicle preparation, for DTMA‚DS. Initial mean size values of 117, 205, and 101 nm were observed for vesicles in pure water, 15% 1-propanol, and 5% 1-butanol solutions, correspondingly. The transmission electron microscopy (TEM) photograph shown in Figure 3 illustrates the DTMA‚DS vesicles formed in a 15% 1-propanol aqueous solution. In contrast with the instability of vesicles in pure water, significant
enhancement of stability was found for vesicles in some mixed solvents. The data in Figure 4 show that 15% 1-propanol and 5% 1-butanol may provide lifetimes of 132 days and more than 1 year, respectively. Furthermore, the vesicle size in the 5% 1-butanol solution nearly remained constant for more than 1 year. It should be noted that the upper and lower limits of accurate size measurement by the zetasizer used in this work are 3000 and 3 nm, respectively. The arrow in Figure 4, hence, indicates the time beyond accurate size measurement and, therefore, represents the lifetime of the vesicles. Table 1 summarizes the most stable conditions and lifetimes for catanionic vesicles formed from DTMA‚DS in aqueous solutions at a variety of alcohol concentrations. Methanol and ethanol were found to be not so effective in enhancing the stability of vesicles as compared with 1-propanol and 1-butanol. It is concluded that the effectiveness of vesicle stability enhancement increases in the order C4OH > C3OH > C2OH > C1OH. Figure 5 shows the lifetimes of catanionic vesicles formed from TTMA‚DS, HTMA‚DS, and TTMA‚TS in aqueous solution with 15% 1-propanol, by which the formation of vesicles from DTMA‚DS is significantly promoted. Obviously the same approach is unable to induce a noticeable increment in stability for IPAs other than DTMA‚DS. It should be noted that experiments with other concentrations of 1-propanol and using other alcohols at a variety of concentrations were also conducted in this work for their effects on vesicle formability of TTMA‚DS, HTMA‚DS, and TTMA‚TS. Unfortunately, none of the approaches may enhance noticeably the vesicle stability. A meaningful observation for these IPAs is, therefore, the appreciable dependence of the effectiveness of vesicle stability enhancement by a cosolvent on the alkyl chain lengths of IPAs: the cosolvents fail to enhance the vesicle stability when the alkyl chain lengths increase from 24
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Figure 5. Lifetimes of catanionic vesicles formed from (a) TTMA‚DS, (b) HTMA‚DS, and (c) TTMA‚TS in an aqueous 15% 1-propanol solution. The arrows indicate the times beyond the upper limit of accurate size measurement.
(DTMA‚DS) to 26 (TTMA‚DS) and 28 (HTMA‚DS, and TTMA‚TS) carbon atoms. Furthermore, symmetry in the alkyl chain length seems to be a minor factor, as compared with total carbon atoms in alkyl chains, that influences the effectiveness of vesicle stability enhancement by the addition of cosolvents. Dependence of Vesicle Stability on Cosolvent Concentration. We are now in a position to show how the variations in cosolvent concentration in the solution affect the stability of vesicles formed from IPAs. Figure 6 shows the measured results of initial size, initial count rate, and lifetime of catanionic vesicles formed from DTMA‚DS in aqueous solutions at a variety of ethanol concentrations. Turbidity data, which have been shown in Figure 1, are shown here again accompanied with count rate data in this figure for further examination. Although the stability enhancement by the addition of ethanol is not significant practically, there are certain noteworthy observations on the effects of ethanol concentration. First, the initial size measurement results indicated that three regimes (A-C in Figure 6) concerning ethanol concentration can be identified. In the presence of
