Langmuir 1995,11, 3633-3635
3633
pH-Induced Rapid Vesicle-to-MicelleConversion of a Phase-Separated Bilayer Vesicle Naoyuki Nishikawa," Masao Arai, Mitsunori Ono, and Isamu Itoh Ashigara Research Laboratories, Fuji Photo Film Co., Ltd., Nakanuma, Minamiashigara, Kanagawa 250-01, Japan Received August 21, 1995@ A new pH-sensitive bilayer vesicle composed of dipalmitoylphosphatidylcholine and monophytanyl phosphate (3(RS),7(R),lUR),15-tetramethylhexadecylphosphate)having an isoprenoid chain was prepared. This vesicle immediately released entrapped 5- or 6-carboxyfluorescein with rapid vesicle-to-micelle conversion above pH 8.5. The evidence of the pH-induced vesicle-to-micelleconversion was demonstrated by transmission electron microscopy.
Much attention has been paid to pH-sensitive vesicles in various fie1ds.l Some unique vesicles which release their contents against a n external stimulus have been realized by using partially polymeric bilayers.2 pHresponsive permeation of nylon capsules coated by synthetic bilayers was also r e p ~ r t e d .When ~ one applies these vesicles for sensing, imaging, o r drug delivery systems (DDS),vesicles whichvanish from the system after release are desired to facilitate subsequent treatments. We would like to describe a new pH-sensitive bilayer vesicle which immediately releases its contents with rapid vesicle-tomicelle conversion of the vesicle above pH 8.5. The evidence of the pH-dependent conversion of the vesicle was demonstrated by transmission electron microscopy (TEM). Use of such a vesicle is particularly important in controlled release of materials. We designed a dipalmitoylphosphatidylcholine(DPPC) vesicle having pH-sensitive domains which act as a trigger for the conversion. Monophytanyl phosphate (3(RS),7(R),11(R),15-tetramethylhexadecyl phosphate) 1was selected as a trigger m ~ l e c u l e .The ~ bulky structure of the phytanyl chain of 1contributes to the formation of stable bilayers under neutral condition^.^ (See Figure 1.) Vesicles were prepared by sonic dispersal of a mixture of DPPC and 1 (8/2 molar ratio) in a 200 mM 5- or 6-carboxyfluorescein (CF) solution of 20 mM Tris-HC1 buffer (pH 7.0). Subsequently, they were sized by a n extruder through a 0.2-pm pore membrane and separated from unentrapped CF by gel permeation chromatography.6 As shown in Figure 2, the DPPC/1 vesicle immediately released the entrapped CF above pH 8.5 (0 in Figure 2). In contrast, the DPPC vesicle showed no pH-dependent release of CF in the range of pH 6.0 to 10.0 (A). Straightchain monoalkyl phosphate 2 (0)did not exhibit the high sensitivity shown by the DPPC/l vesicle. On the basis of Abstract published inAcluance ACSAbstracts, October 1,1995. (1) (a)Yatvin, M. B.; Kreutz, K.; Honvitz, B. A,; Shinitzky, M. Science 1980, 210, 1253. (b) Maeda, M.; Kumano, A.; Tirrell, D. A. J.Am. Chem. SOC.1988,110, 7455. (c) Nayar, R.; Schroit, A. J . Biochemistry 1985, 24, 5967. (d) Liu, D.; Huang, L. Biochim. Biophys. Acta 1990, 1022, 348. (e) Collins, D.; Connor, J.; Ting-Beall, H.-P.; Huaung, L. Chem. Phys. Lipids 1990, 55, 339. (fl Straubinger, R. M.; Duzgtmes, N.; Papahadjopoulos, D. FEBS 1985, 179, 148. (2) (a)Regen, S. L.; Shin, J. S. J . Am. Chem. SOC.1984, 106, 5756. (b) Subbarao, N. K.; Parente, R. A.; Szoka, F. C., Jr.; Nadasdi, L.; Pongracz, K. Biochemistry 1987,26, 2964. (c) Ohno, H.; Takeoka, S.; Tsuchida, E. Polym. Bull. 1985,14,487. (d) Bader, H.; Ringsdorf, H.; Schmidt, B. Angew. Makromol. Chem. 1984, 1231124, 457. (3) Okahata, Y.; Ariga, K.; Seki, T. J.A m . Chem. SOC.1988, 110, 2495. (4) Monophytanyl phosphate (1)was prepared by the method of Joo, C. N. et al. (JOO,C. N.; Park, C. E.; Kramer, K. G.; Kates, M. Can. J. Biochem. 1973, 51, 1527). (5) (a) Yamauchi, K.; Doi, K.; Yoshida, Y.; Kinoshita, M. Biochim. Bzophys. Acta 1994, 1193, 41. (b) Nishikawa, N.; Mori, H.; Ono, M. Chem. Lett. 1994, 767. @
-0-l-OH
1 : C20Ha04P = 378.53 -0-y-OH
-
2 : CteH3504P 322.42
OH
R OH
CH&H2)&00-?H2 CH3(CH2)14COO-?H 0 CH2-O-~-O-CH2CH2N'(CH3)3
0
DPPC Figure 1. Chemical structures of monophytanyl phosphate (l),cetyl phosphate (21, and DPPC.
the fact that the DPPC/l suspension shows a large decrease in turbidity in a small pH range 8.5-8.7,7 we believe that a significant structural change occurred a t this point. To obtain direct information on the morphological change of the DPPC/1 vesicle, TEM observations of the suspensions were carried out. Vesicular structures confirmed a t pH 7.0 (Figure 3a) changed to small stacked disks a t pH 9.0 (Figure 3b). The discoidal structures observed here closely resembled those observed in complexes of lecithin with apolipoproteins or lysolecithin^.^ A similar vesicular transition induced by pH-dependent structural reorganization of polyelectrolytes which were placed in an outer medium was also reported.1° To form (6) A solution of the mixture of 23.34 mg of DPPC and 3.00 mg of 1 in 12 mL of CHC13 was evaporated in round-bottomed flask. After it was dryed under vacuum for 1h , the resulting liquid film was hydrated with 3 mL of 20 mM Tris-HC1 buffer containing 200 mM CF and 200 mM NaCl adjusted to pH 7.0 with vortex agitation at 50 "C. After subsequent sonication with a bath type ultrasonicater (Branson 2000) a t 40 "C for 30 min, the vesicles were passed through a 0.2 pm pore membrane with an extruder at 55 "C. This operation was repeated six times. The resulting liposomes containing CF were purified and separated from the unentrapped CF by gel permeation chromatography (Sepharose 4B,eluent 20 mM Tris-HC1 buffer containing 200 mM NaCl adjusted t o pH 7.0). Othervesicles were preparedby the same procedure. (7) For the measurement of turbidity, the liposome was prepared by use of 10 mg of the mixture of DPPC and 1 (8/2 molar ratio) in 1 mL of 20 mM Tris-HC1 buffer containing 200 mM NaCl adjusted to pH 7.0. The suspension (200 pL) was added to 400 pL of the buffer adjusted to various pH values. After the pH of the resulting suspension was measured, the optical density relative to the value at pH 7.0 was measured a t 25 "C. The value was 1.0 a t pH 8.5 and 0.51 at pH 8.7. ( 8 ) Wlodawer, A,; Segrest, J. P.; Chung, B. H.; Chiovetti, R., Jr.; Weinstein, J. N. FEBS Lett. 1979, 104, 231. (9) Inoue, K.; Suzuki, K.; Nojima, S. J.Biochem. 1977, 81, 1097. (10) Borden, K. A.; Eum, K. M.; Langley, H.; Tan, J. S.; Tirrell, D. A.; Voycheck, C. L. Macromolecules 1988, 21, 2649.
0743-746319512411-3633$09.0010 0 1995 American Chemical Society
3634 Langmuir, Vol. 11, No. 10, 1995
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Figure 2. pH-dependent release of entrapped CF from vesicles of DPPC/l (01,DPPC/2 (O), or DPPC (A)in 30 s at pH 7.0 at 25 "C. A suspension of the vesicles (1OOpL)was added to 2 mL of 20 mM Tris-HC1 buffer containing 200 mM NaC1, adjusted to various pH values. Then, the fluorescence intensity of CF in the aqueous phase (excitation at 492 nm, emission at 520 nm) was measured in 30 s. Percent release of CF from the
liposome was estimated by the fluorescence intensity, where 100% was the intensity measured after the addition of 20 pL of Triton X-100.
Figure 3. Transmission electron micrographs of DPPC/1 suspensions at pH 7.0 (a) and at pH 9.0 (b). The suspension (1 mg/mL) was stained by phosphotungstic acid.
the discoidal structure, some edge stabilization is needed for the mixed micelles. The composition of the discoidal micelle was estimated by 'H-NMR analysis after dialysis experiments. As 76%of 1 was retained after the vesicleto-micelle conversion, we consider that the ionized 1 probably serves to stabilize the edges of the disks.
The surface tension of a 0.1 mM suspension of 1 in 20 mM Tris-HC1 buffer increased with the decrease of hydrogen ion concentration of the suspension above pH 8.5 at 25 "C. As the secondary dissociation constant of 1 is 8.4,11 its solubility was enhanced accordingto the change in dissociation conditions. The mixtures of DPPC and 1 formed stable monolayers on 20 mM Tris-HC1 buffer containing 200 mM NaC1, adjusted to pH 7.0. The miscibility of the mixed membrane was estimated by surface pressure-area isotherms at 25 "C, considering the two-dimensional phase rule of Defay and Crisp.12Plots of the mean film area against the monolayer composition at a surface pressure of 40 mN0m-l were fitted with ideal line (Figure 4). The isotherms also showed two independent destruction points corresponding to those of DPPC and 1. These results clearly indicate that the mixture forms a complete immiscible membrane under pH-neutral conditions.13 Evaluated by the area of DPPC and that of 1, approximately one-third of the area of the mixed membrane was occupied by the domains of 1. Because the solubility of 1 increases at the secondary dissociation condition, the domains must shift into the outer aqueous phase from the bilayer when the pH of the medium rises above 8.5. As a result, the vesicles immediately ruptured accompanied by the rapid release of their contents. Then, the resulting DPPC fragments of the vesicles formed the discoidal lipid micelles with the resulting ionized phytanyl phoshate. In conclusion, a new pH-sensitive system was achived by use of the phase-separated bilayer vesicle composed of DPPC and phytanyl phoshate (1). To obtain stable mixed membranes under neutral conditions and rapid pHdependent release of their contents, an alkyl phosphate having a unique isoprenoid chain was required as a trigger molecule. In addition, i t is convenient for practical applications, because the vesicles transformed to small (11)The secondary dissociation constant of 1 was measured in a mixture of H.40 and CH30H ( l / l , v/v). (12) (a) Cnsp, D. J. In Surface Chemistry; Butterwoths: London, 1949; p 17. (b)Defay, R.; Prigogion, I.; Bellemans, A.; Everett, D. H. Surface Tension and Adsorption; Longmans: London, 1966; Chapter 6. (13)Bernd, H.; Ringsdorf, H.; Schupp, H. Makromol. Chem. 1981, 182, 247.
Langmuir, Vol. 11, No. 10, 1995 3635
Letters stacked disks which facilitate subsequent treatments after release of their contents. Our system would be useful for industry, biotechnology, diagnosis, and drug delivery systems.
Acknowledgment. The authors thank Ms. Haruko Shimizu and Ms. Akiko Ito for their helpful technical assistance.
Supporting Information Available: Optical density of DPPC/1 suspension at 650 nm (relative to the value at pH 7.0) as a function of pH and plots of surface tension of 0.1 mM suspension of 1in 20 mM Tris-HC1 buffer as a function of pH (2 pages). Orderinginformation is givenon any current masthead page. LA9506988
