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New pH-Sensitive Vesicles. Release Control of Trapped Materials from the Inner Aqueous Phase of Vesicles Made from Triple-Chain Amphiphiles Bearing Two Carboxylate Groups Yasushi Sumida,† Araki Masuyama,‡ Mayuko Takasu,‡ Toshiyuki Kida,‡ Yohji Nakatsuji,‡ Isao Ikeda,*,‡ and Masatomo Nojima‡ Cosmetic Laboratory, Kanebo Corporation, Kotobuki-cho 5-3-28, Odawara, Kanagawa 250-0002, Japan, and Department of Applied Chemistry, Faculty of Engineering, Osaka University, Yamadaoka 2-1, Suita, Osaka 565-0871, Japan Received June 26, 2000. In Final Form: November 21, 2000 The pH-induced release of trapped materials from the inner aqueous phase of vesicles made from the synthetic triple-chain amphiphile 1 bearing two carboxyl groups was investigated. While a particular pH-dependence of the release of trapped materials from the inside of vesicles made from distearoylphosphatidylcholine (DSPC) was not observed, vesicles made from compound 1 showed unique pH-sensitive character toward the pH of the outer bulk phase as follows: it released less than 10% of 5(6)carboxyfluorescein (CF) as a water-soluble marker at pH 7.5 after 1 year. But the vesicular assemblies were broken below pH 3.5-4.0, resulting in the liberation of the trapped CF into the bulk phase. The latter result is associated with a full-protonation of the carboxylate groups of compound 1. In the case of vesicles made from a mixture of DSPC and compound 1, the addition of cholesterol into the vesicle membranes was effective in providing clear pH-sensitive character for this type of vesicle. The ζ potentials of a series of vesicles containing compound 1 at various pH synchronized well with the pKa1 value of vesicles made from compound 1 and the pH-dependence of the release of trapped CF from these vesicles.
Introduction Self-organized molecular assemblies of phospholipids in water, liposomes, or vesicles have been attracting attention as biomembrane model systems or carriers of great promise in drug delivery systems (DDSs). Many investigations have been performed on the pharmacological and pharmaceutical aspects of liposomes in vivo or in vitro.1,2 For the practical use of liposomes or vesicles as DDSs, the following two substantial subjects have to be considered. One is the suppression of leakage of the entrapped materials during storage. The other is the design of methodology for release control of the entrapped materials under the desired conditions. Our approach to the former subject was a strengthening of the bilayer structure by enhanced hydrophobic interaction between amphiphiles, that is, close packing of hydrophobic chains of the amphiphile that constitute the vesicle. We have previously reported that the vesicular system made from the triple-chain amphiphiles 1 bearing two carboxylate groups, which had been found to form a highly packed monolayer at the air-water interface,3 showed much higher stability toward the leakage of a marker entrapped inside the vesicles and less sensitivity toward leakage induced by temperature change in comparison with vesicles made from distearoylphosphatidylcholine (DSPC).4,5 In particular, the vesicles made from * To whom correspondence may be addressed. Fax: +81-6-68797359. Tel: +81-6-6879-7356. E-mail:
[email protected]. † Kanebo Corporation. ‡ Osaka University. (1) Crowe, J. H.; Crowe, L. M.; Carpenter, J. F.; Rudolph, A. S.; Wistram, C. S.; Spargo, B. J.; Anchordoguy, T. J. Biochim. Biophys. Acta 1988, 947, 367. (2) Heath, T. D. Methods Enzymol. 1987, 149, 135. (3) Sumida, Y.; Oki, T.; Masuyama, A.; Maekawa, H.; Nishiura, M.; Kida, T.; Nakatsuji, Y.; Ikeda, I.; Nojima, M. Langmuir 1998, 14, 7450.
compound 1 released less than 10% of 5(6)-carboxyfluorescein (CF), as a water-soluble marker, after 1 year at 40 °C.4 This excellent stability of the vesicles has been explained from the standpoints of increasing surface charge of the vesicles and enhancement of hydrophobic interactions within the bilayer membrane of the vesicles.5 Concerning the release control of materials entrapped inside vesicles, many attempts have been made by designing liposomes or vesicles having perceiving characteristics toward a change in the surrounding conditions, such as pH,6-11 temperature,12-14 and UV light.15-17 Especially, the pH-sensitive liposomes or vesicles have been noteworthy as DDSs because, more often than is commonly realized, the pH value around any damaged tissue is different from that around other normal tissue.18-20 Up (4) Sumida, Y.; Masuyama, A.; Maekawa, H.; Takasu, M.; Kida, T.; Nakatsuji, Y.; Ikeda, I.; Nojima, M. Chem. Commun. 1998, 2385. (5) Sumida, Y.; Masuyama, A.; Takasu, M.; Kida, T.; Nakatsuji, Y.; Ikeda, I.; Nojima, M. Langmuir 2000, 21, 8005. (6) Yatvin, M. B.; Krentz, W.; Horwitz, M.; Shinitzky, M. Science 1980, 210, 1253. (7) Ellens, H.; Bentz, J.; Szoka, F. C. Biochemistry 1984, 23, 1532. (8) Leventis, R.; Diacovo, T.; Silvius, J. R. Biochemistry 1987, 26, 3267. (9) Connor, J.; Yatvin, M. B.; Huang, L. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 1715. (10) Brown, P. M.; Silvius, J. R. Biochim. Biophys. Acta 1989, 980, 181. (11) Collins, D.; Litzinger, D. C.; Huang, L. Biochim. Biophys. Acta 1990, 1025, 234. (12) Yatvin, M. B.; Weinstein, J. N.; Dennis, W. H.; Blumenthal, R. Science 1978, 202, 1290. (13) Tomita, T.; Watanabe, M.; Takahashi, T.; Kumai, K.; Tadakuma, T.; Yasuda, T. Biochim. Biophys. Acta 1989, 978, 185. (14) Iga, K.; Ogawa, Y.; Toguchi, H. Pharm. Res. 1992, 9, 658. (15) Ferritto, M. S.; Tirrell, D. A. Macromolecules 1988, 21, 3117. (16) You, H.; Tirrell, D. A. J. Am. Chem. Soc. 1991, 113, 4022. (17) Park, J. M.; Aoyama, S.; Zhang, W.; Nakatsuji, Y.; Ikeda, I. Chem. Commun. 2000, 231. (18) Wang, C. Y.; Huang, L. Biochem. Biophys. Res. Commun. 1987, 147, 980.
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Figure 2. The appearance of the vesicular systems made from compound 1 containing sulforhodamine B as a coloration reagent under various pH conditions. Figure 1. Release (%) of 5(6)-carboxyfluorescein (CF) trapped inside vesicles made from DSPC or the triple-chain amphiphile 1 after being kept at given pH for 1 h at 37 °C. Chart 1
to now, some pH-sensitive vesicular systems have been prepared, for example, by using a combination of unsaturated phosphatidylethanolamines and amphiphilic stabilizers.9-11 The stability of these pH-sensitive liposomes toward leakage, however, was reported to be relatively poor as compared to the normal phosphatidylcholine-based liposomes.21,22 In this study, we have investigated the pH-sensitive property of vesicles made from a synthetic triple-chain amphiphile bearing two carboxylate groups, 1, which already has been confirmed to construct very stable vesicles, because it is reasonably expected that the two carboxylate groups would provide a pH-sensitive character to the vesicles. Small unilamellar vesicles containing CF in an aqueous buffer solution (pH 7.5) were prepared by the conventional hydration-sonication method.4,5 The vesicles made from DSPC were used as a reference. The mixed vesicular systems of compound 1/DSPC and 1/DSPC/cholesterol were also explored. Structures of compound 1 and DSPC are illustrated in Chart 1. Results and Discussion The pH-Dependence of the Release of Trapped CF from the Inside of Vesicles Made from DSPC or the Triple-Chain Amphiphile 1. Figure 1 shows the released percentage of CF from the vesicle made from DSPC or the triple-chain amphiphile 1 after 1 h at the pH of the outer bulk phase (pH 2-6). The pH of the inner aqueous phase of vesicles was initially adjusted to 7.5 in all cases. In the case of the DSPC vesicles, the released percentage increased gradually with decrease of the pH value. A pH gradient and/or an ionic strength gradient (19) Torchilin, V. P.; Zhou, F.; Huang, L. J. Liposome Res. 1993, 3, 201. (20) Chu, C. J.; Szoka, F. C., Jr. J. Liposome Res. 1993, 4, 361. (21) Connor, J.; Norley, N.; Huang, L. Biochim. Biophys. Acta 1986, 884, 474. (22) Greidziak, M.; Bogdanov, A. A.; Torchilin, V. P.; Lasch, J. J. Controlled Release 1992, 20, 219.
Figure 3. Release (%) of CF trapped inside vesicles made from the triple-chain amphiphile 1 at 37 °C as a function of elapsed time at pH 2.1.
between the inner phase and the outer bulk phase may promote the leakage of CF trapped inside the aqueous phase of the vesicles. In this instance, it was presumed that a pH gradient was a predominant factor for CF release because CF release was not observed at least for 2 h in all systems when the pH of the bulk phase was set for the same pH of the inner phase (pH 7.5). On the other hand, the vesicles made from compound 1 showed a clear sensitive character toward the pH of the bulk phase. The leakage of trapped CF was effectively suppressed above pH 4.0. The released percentage increased gradually from pH 4.0 to 3.2 and abruptly below pH 3.2. Alteration in the molecular assemblies composed of compound 1 by pH changes is visualized in Figure 2. The vesicle system containing sulforhodamine B, as a coloration reagent for the vesicles,23 was prepared according to similar procedures for the CF-trapped vesicle. While each system is translucent above pH 4.3, a purple insoluble mass of compound 1, including sulforhodamine B, is clearly observed below pH 4.0. Figure 3 shows the released percentage of CF from the inside of the vesicles made from compound 1 as a function of elapsed time at pH 2.1. A large portion of trapped CF was released to the outer bulk phase after 1 h. The pKa Values of the Triple-Chain Amphiphile Bearing Two Carboxylate Groups 1. The pKa values of the triple-chain amphiphile 1 were measured at 25 °C by the conventional titration method24 in two solvent systems. The results are listed in Table 1 along with the pKa values of sebacic acid, adipic acid, and acetic acid measured under the same conditions.25 In a water-ethanol (23) Torchilin, V. P.; Lukranov, A. N.; Klibanov, A. L.; Omelyanenko, V. G. FEBS 1992, 305, 185. (24) Parke, T. V.; Davis, W. W. Anal. Chem. 1954, 26, 642. (25) Simonetta, M.; Carra, S. General and Theoretical Aspects of the COOH and COOR Groups. In The Chemistry of Carboxylic Acids and Esters; Patai, S., Ed.; Interscience: London. 1969; pp 14-17.
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Table 1. The pKa Values of Compound 1 and Other Monobasic or Dibasic Acids at 25 °C compound
solvent systema
1 1 sebacic acid adipic acid acetic acid acetic acid
A B A B A B
a
pKa
6.87 4.76
pKa1
pKa2
origin
4.86 4.08 6.14 4.42
5.77 8.78 7.08 5.41
this work this work this work ref 25 this work ref 25
System A, water/ethanol (2/8, v/v); system B, water only.
(2/8; v/v) system (system A in Table 1), compound 1 was unaggregated and soluble, in contrast to its vesicular aggregation in water (system B). In both solvent systems, compound 1 showed two pKa values (pKa1 and pKa2) like the other dibasic acids. These values of compound 1 in a water-ethanol system were considerably lower than those of sebacic acid in the same system. In water, compound 1 also showed two pKa values, but the order of magnitude of pKa2 was quite different from not only the pKa2 value of compound 1 measured in a water-ethanol system but also the pKa2 value of adipic acid measured in water.25 It already has been known that aggregated carboxylic acids have different pKa values from the corresponding unaggregated ones, due to the different manner of protonation of the carboxylate groups.26,27 An acid-anion dimer in which one proton is shared by two adjacent carboxylate groups is formed in aggregated carboxylic acids at a certain pH range, so that pKa values of aggregated carboxylic acids are generally higher than those of unaggregated ones. Taking these reported results into account, it is surmised that compound 1 aggregated in water is partly protonated between pH 4.1 and 8.8, forming intra- or intermolecular acid-anion pairs, and it is completely protonated below pH 4.08. From these results of the pKa values of vesicular aggregates of compound 1 in water, the pH dependence of the release of trapped CF from the inside of vesicles made from compound 1 (Figure 1) will be explained as follows: between pH 6 and 4, compound 1 will be half-protonated and form the intra- or intermolecular acid-anion pairs in vesicular aggregates. Below pH 4.0, vesicular aggregates will be destroyed by increasing lipophilicity of compound 1 through complete protonation of the carboxylate groups. This phenomenon causes the steep release of CF from the inner aqueous phase of the compound 1 vesicles to the outer bulk phase at low pH. In the Case of Vesicles Made from Mixtures of DSPC and the Triple-Chain Amphiphile. Figure 4 indicates the released percentage of CF from the vesicles made from a mixture of DSPC and compound 1 after being kept for 1 h under various pH conditions. With increasing the mixing ratios of DSPC to compound 1, the pH-sensitive characteristic of the system becomes vague. In addition, the vesicle made from the mixture of DSPC and compound 1 unexpectedly released trapped CF more than the DSPC vesicle in the pH region below 6. This result implies that protonation of the carboxylate groups of compound 1 significantly influenced the situation of the bilayer membranes composed of mixtures of DSPC and compound 1. Effect of the Addition of Cholesterol on the pHDependence of the Release of CF from Vesicles Made from Mixtures of DSPC and the Triple-Chain Amphiphile. To suppress the release of CF from vesicles (26) Hargreaves, W. R.; Deamer, D. W. Biochemistry 1978, 17, 3759. (27) Haines, T. H. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 160.
Figure 4. Release (%) of CF trapped inside vesicles made from the single and mixed components of DSPC and compound 1 after 1 h at 37 °C as a function of pH.
Figure 5. Release (%) of CF trapped inside vesicles made from the mixed systems of DSPC, compound 1, and cholesterol after 1 h at 37 °C as a function of pH.
made from mixtures of DSPC and compound 1 in the pH region of 4-6, the effect of the addition of cholesterol to the bilayer membranes was examined because cholesterol is well-known to stabilize bilayer membranes.28 Figure 5 illustrates the effect of the addition of cholesterol (0, 10, and 20 mol % of the total quantity of components in the membrane) on the release of trapped CF in the cases of DSPC/1 ) 75/25 and 50/50 systems. In both mixed systems, as the ratio of cholesterol increased, the release of CF decreased in the range above pH 4 and increased below pH 4. This clearly means that the pH dependence of the release of CF was improved dramatically by the addition of cholesterol. It is considered that the suppression of the release of CF at a higher pH range is attributed to enhancement of a hydrophobic interaction between the hydrophobic moieties of amphiphiles by the addition of cholesterol and that the promotion of release at a lower pH range is due to acceleration of coalescence of the vesicles (28) Seeling, A.; Seeling, J. Biochemistry 1974, 13, 4839.
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Figure 6. The ζ potential of vesicles composed of single and mixed components of DSPC, compound 1, and cholesterol at 37 °C as a function of pH.
and a subsequent destruction of the vesicle structures by the addition of cholesterol. The pH-Dependence of the ζ Potential of Vesicles. For the purpose of exploring of the surface of a vesicle from the standpoint of electrochemical potential, the ζ potentials of vesicles made from DSPC, compound 1, and their mixed systems (molar ratio of DSPC/1 ) 50/50 and DSPC/1/cholesterol ) 40/40/20) were measured at various pH values. The results are shown in Figure 6. The DSPC vesicle had no apparent surface potential above pH 4 and had positive values below pH 4. On the other hand, the vesicles made from compound 1 showed high negative values above pH 4.0, indicating that its hydrophilic groups were carboxylate ions at this pH. The ζ potential of these vesicles sharply changed toward a neutral direction with decrease of pH value below pH 4, indicating that the carboxylate groups of compound 1 were fully protonated. As for the vesicles of the mixed systems of DSPC and compound 1, the curve of the ζ potential was similar to the case of compound 1 vesicle but was slightly shifted to a positive direction. Moreover, the addition of cholesterol to the mixed system of DSPC and compound 1 showed no further change in the ζ potential of the DSPC/compound 1 mixed vesicles. The pH dependence of the ζ potential of compound 1 vesicle synchronized with its pKa1 value of vesicular aggregates and the pH dependence of the release of CF from the vesicle made from compound 1. In conclusion, vesicles made from compound 1 showed a pH-sensitive character in the release of materials trapped inside the vesicles depending on the protonation of the carboxylate groups of compound 1. Concerning the vesicles made from mixtures of DSPC and compound 1, the addition of cholesterol improved the pH-sensitive character of the mixed vesicle system. The ζ potentials of these vesicles supported a series of findings regarding the pH dependence of the release of materials trapped inside the vesicles. Experimental Section Materials. Triple-chain amphiphile 1 was synthesized and purified by the previously reported method.4,29 DSPC was purchased from Nippon Fine Chemical Co., of 99.8% purity, and used without further purification. CF was purchased from
Sumida et al. Eastman Kodak and purified by the following process: dissolving in 1 M NaOH aqueous solution, treating with active carbon, acidifying the filtrate with 1 M hydrochloric acid, centrifuging, washing the resulting precipitate with distilled water, and finally drying in a desiccator. Sulforhodamine B was purchased from Aldrich and used without further purification. All other chemicals were commercial products of reagent grade and used without further purification. Methods. Preparation of vesicles containing CF was carried out as follows:5 A film of lipid and/or amphiphile with or without cholesterol (total 40 µmol) was prepared on the inside wall of a test tube by evaporation of its CHCl3 solution and stored in a desiccator overnight under reduced pressure. After addition of 4 mL of a Tris-HCl buffer solution (20 mM, pH 7.5) containing 100 mM of CF to the test tube, the mixture was vortex-mixed for 10 min and sonicated for 5 min at about 10 °C higher than its Tc (phase transition temperature between a gel state and a liquid crystal state, 59.5 °C for compound 1)5 using a probe-type sonicator (Ultrasonic Processor VC-50, the maximum power is 50 W) under a stream of nitrogen. Small unilamellar vesicles containing trapped CF were separated from untrapped CF by eluting the vesicle dispersion through a Sephadex G-50 gel column with 20 mM Tris-HCl buffer solution containing 100 mM of NaCl (pH 7.5). Then the vesicle fraction was diluted to 1 mM of the total constituents in the membrane with the same buffer solution used as eluent and subjected to measurement of the leakage of CF from vesicles. The vesicles containing sulforhodamine B were prepared by a similar procedure mentioned above except for using sulforhodamine B instead of CF. The mean size of vesicle in water was measured at 25 °C using a differential light scattering apparatus (Ohtsuka Electronics Co., Ltd. DLS70SAr, He-Ne laser light source). The mean sizes of these vesicles prepared in this work were estimated at 100-250 nm. The measurement of the pH-induced release of CF trapped inside the vesicles was carried out as follows: Forty microliters of the above vesicle suspension was added to 4 mL of 20 mM Tris-HCl buffer solution containing 100 mM of NaCl adjusted to a given pH value by aqueous HCl. After the suspension was held at 37 °C for a given period, an aqueous solution of NaOH was added to the dispersion until pH 7.5 was reached. Then, the amount of CF released (%) from the vesicles was calculated by means of eq 1
CF released % ) (Ix - I0)/(It - I0) × 100
(1)
where I0 is the fluorescence intensity of the vesicle suspension containing CF at the initial time, Ix is the fluorescence intensity of the final suspension, and It is the fluorescence intensity after addition of an aqueous solution of Triton X-100 (100 g L-1) to the suspension. The fluorescence intensity at 530 nm was measured at 25 °C using an excitation at 490 nm. The ζ potential of vesicles was measured using an Ohtsuka Electronics Co., Ltd. ELS-800, which is a laser doppler electrophoresis apparatus. For this measurement, vesicles without entrapped materials were prepared in 20 mM Tris-HCl buffer solution containing 10 mM of NaCl (pH 7.5) under the same conditions, mentioned above. After dilution of this suspension with 20 mM Tris-HCl buffer solution containing 10 mM of NaCl, which was adjusted to a given pH value by aqueous HCl, the measurement was carried out at 37 °C. All CF release experiments and the ζ potential measurements were carried out at least twice to confirm their reproducibility.
LA0008939 (29) Zhu, Y.-P.; Masuyama, A.; Kirito, Y.; Okahara, M.; Rosen, M. J. J. Am. Oil Chem. Soc. 1992, 69, 626.
