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Photoinduced Increase in Vesicle Size and Role of Photoresponsive Malachite Green Leuconitrile Derivative in Vesicle Fusion Ryoko M. Uda,† Daisuke Yamashita,† Yoshiaki Sakurai,‡ and Keiichi Kimura*,§ Department of Chemical Engineering, Nara National College of Technology, Yata 22, Yamato-koriyama, Nara 639-1080, Japan, Technology Research Institute of Osaka Prefecture, Ayumino 2-7-1, Izumi, Osaka 594-1157, Japan, and Department of Applied Chemistry, Faculty of Systems Engineering, Wakayama UniVersity, Sakae-dani 930, Wakayama 640-8510, Japan ReceiVed March 21, 2007. In Final Form: May 16, 2007 The influence of photoirradiation on vesicles containing a Malachite Green leuconitrile derivative carrying a long alkyl chain, affording photogenerated amphiphilicity, was investigated. The photoresponsive Malachite Green leuconitrile derivative was embedded in the vesicle bilayer of two single-tailed amphiphiles with oppositely charged head groups consisting of cetyltrimethylammonium chloride (CTAC) and sodium octyl sulfate (SOS). Transmission electron microscopy, which was used for observing photoinduced structural change in the vesicles, demonstrated that photoirradiation of the vesicles containing the Malachite Green leuconitrile derivative increased the average size of the vesicle diameter from 116 to 243 nm in the [CTAC]/[SOS] ) 0.48 system. The mechanism for vesicle enlargement was studied with fluorescent probe molecules. The photoinduced change in the vesicle size can be explained by the destabilization of the vesicle bilayer, which is perturbed by photogenerated amphiphilicity. In addition, it was shown that the fusion process arising from the destabilized bilayer contributed to the increase in vesicle size.
Introduction Vesicles possess closed spherical structures with an aqueous interior and a bilayer consisting of amphiphiles. Therefore, they are widely used as model systems for cell membranes, reagent capsules, drug delivery,1,2 and microreactors for synthesis.3,4 Functionalized amphiphiles, which allow subsequent modification of bilayer properties, have attracted considerable interest. In particular, photoresponsive amphiphiles are of chemical and biochemical interest because of the possibility of mimicking biological processes for the conversion of solar energy and controlling vesicle formation or disruption. Much research has been conducted on the photochemical cis-trans isomerization of amphiphilic azobenzene derivatives, which leads to a change in the packing of the molecules and a disruption of the bilayer structure,5,6 resulting in the controlled release of entrapped reagents from the vesicle interior.7-10 Ringsdorf et al. have reported the photoreaction of pyridinioamidate amphiphiles, which exhibit a transformation of their polar head group to the corresponding nonpolar one.11,12 The photoirradiated vesicles * Corresponding author. E-mail:
[email protected]. Tel: +81-73-457-8254. Fax: +81-73-457-8255. † Nara National College of Technology. ‡ Technology Research Institute of Osaka Prefecture. § Wakayama University. (1) Papadhadjopoulos, D. Ann. N. Y. Acad. Sci. 1987, 1-462. (2) Ostro, M. J. Sci. Am. 1987, 256, 90. (3) Michel, M.; Winterhalter, M.; Darbois, L.; Hemmerle, J.; Voegel, J. C.; Schaaf, P.; Ball, V. Langmuir 2004, 20, 6127. (4) Kazakov, S.; Kaholek, M.; Teraoka, I.; Levon, K. Macromolecules 2002, 35, 1911. (5) Ichimura, K.; Suzuki, Y.; Seki, T.; Hosoki, A.; Aoki, K. Langmuir 1988, 4, 1214. (6) Mooney, W. F.; Brown, P. E.; Russell, J. C.; Costa, S. B.; Pedersen, L. G.; Whitten, D. G. J. Am. Chem. Soc. 1984, 106, 5659. (7) Kunitake, T.; Nakashima, N.; Shimomura, M.; Okahata, Y.; Kano, K.; Ogawa, T. J. Am. Chem. Soc. 1980, 102, 6642. (8) Song, X.; Perlstein, J.; Whitten, D. G. J. Am. Chem. Soc. 1997, 119, 9144. (9) Sakai, H.; Matsumura, A.; Yokoyama, S.; Saji, T.; Abe, M. J. Phys. Chem. B 1999, 103, 10737. (10) Morgan, C. G.; Thomas, E. W.; Sandhu, S. S.; Yianni, Y. P.; Mitchell, A. C. Biochim. Biophys. Acta 1987, 903, 504. (11) Haubs, M.; Ringsdorf, H. Angew. Chem., Int. Ed. Engl. 1985, 24, 882. (12) Haubs, M.; Ringsdorf, H. New J. Chem. 1987, 11, 151.
containing pyridinioamidate are transformed into their corresponding micelles or monolayers at the air-water interface. They also reported a benzylammonium salt amphiphile that undergoes photochemical cleavage of its head group and observed vesicle shrinking on photoirradiation.12 We designed another class of photoresponsive amphiphiles, Malachite Green leuconitrile derivative 1 (Scheme 1).13,14 Compound 1, when ionized photochemically, exhibits both hydrophilicity and hydrophobicity by its triphenylmethyl cation and its long alkyl chain, respectively, resulting in photogenerated amphiphilicity. Under dark conditions, 1 is less polar in the head group than after photoirradiation and behaves as a lipophilic compound. The photogenerated electrical charge on the head group is expected to affect molecular aggregation drastically. In fact, we observed significant changes induced by 1 in the critical micelle concentration13 and in the solubility of an oily substance in the micelle solution.14 Vesicles consisting of double-tailed amphiphiles are normally metastable, and mechanical energy (such as sonication or pressure filtration) or chemical treatment (detergent dialysis or reversephase evaporation) is essential for their preparation.15-17 The stable vesicle inhibits further modification in the vesicle bilayer. Kaler et al. have reported immediate vesicle formation by combining aqueous mixtures of two single-tailed amphiphiles with oppositely charged head groups.18-20 Vesicles formed in this manner allow gentle and efficient encapsulation without mechanical or chemical perturbation.18 Therefore, it may be possible to attain photochemical changes in bilayer properties, (13) Uda, R. M.; Oue, M.; Kimura, K. Chem. Lett. 2004, 33, 586. (14) Uda, R. M.; Kimura, K. Bull. Chem. Soc. Jpn. 2005, 78, 1862. (15) Fendler, J. Membrane Mimetic Chemistry; Wiley: New York, 1983. (16) Israelachvili, J. Intermolecular and Surface Forces; Academic Press: Orlando, FL 1985. (17) Gabriel, N. E.; Roberts, M. F. Biochemistry 1994, 23, 4011. (18) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. N. Science 1989, 245, 1371. (19) Herrington, K. L.; Kaler, E. W.; Miller, D. D.; Zasadzinski, J. A.; Chiruvolu, S. J. Phys. Chem. 1993, 97, 13792. (20) Yatcilla, M. T.; Herrington, K. L.; Brasher, L. L.; Kaler, E. W.; Chiruvolu, S.; Zasadzinski, J. A. J. Phys. Chem. 1996, 100, 5874.
10.1021/la700831z CCC: $37.00 © 2007 American Chemical Society Published on Web 06/23/2007
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Scheme 1. Photogenerated Amphiphilicity of the Malachite Green Leuconitrile Derivative Carrying a Long Alkyl Chain
affecting the vesicle shape, if photoionizable Malachite Green leuconitrile derivative 1 is applied to this kind of equilibrating vesicles. In particular, we aim at a system undergoing photoinduced vesicle fusion, which differs from the events caused by other types of photoresponsive surfactants (i.e., the disruption caused by amphiphilic azobenzene,8 shrinkage based on the benzylammonium salt,11,12 and the transformation of the pyridinoamidate vesicle into its corresponding micelle12). Yianni et al. have reported a vesicle fusion system containing azobenzene amphiphiles, although the detailed mechanism of the fusion was not understood.10 In this article, we report the photoinduced effects of 1 on the vesicles prepared by mixtures of cetyltrimethylammonium chloride (CTAC) and sodium octyl sulfate (SOS). Mixtures of cationic and anionic surfactants with different tail lengths were chosen to promote the formation of vesicles, which were neither of multilamellar structure nor crystalline precipitate.20 The photoinduced change in the vesicles was followed by glucose entrapment experiments and transmission electron microscopy (TEM). Furthermore, fluorescence analysis was performed using probe molecules of pyrene, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS), and p-xylene-bis-pyridinium bromide (DPX) to understand the mechanism for photoinduced change in the equilibrium vesicles. Experimental Section Materials. Malachite Green leuconitrile derivative 1 was synthesized according to the literature.14 CTAC was recrystallized from tetrahydrofuran. SOS, ANTS, and DPX were used as received from Aldrich, BioChemika, and Sigma, respectively. Pyrene was recrystallized twice from ethanol before use. Water was deionized. Other materials were of analytical grade and were used without further purification. Preparation of Vesicle Samples. Vesicle samples were prepared by mixing solutions of CTAC ( 0.72 samples. From the results, it can be concluded that vesicle formation favors SOSrich mixtures in the range of [CTAC]/[SOS] ) 0.03-0.59, where the higher ratio of [CTAC]/[SOS] enhances vesicle formation, and that the precipitate is observed for samples in the range of [CTAC]/[SOS] ) 0.72-1.34, where vesicle formation is depressed. Photoinduced Change in Vesicle Formation. To attain the photoinduced structural change in the vesicles derived from mixtures of CTAC and SOS, photosensitive 1 was applied to the mixture. The total trapping volume of vesicles was estimated by measuring the amount of glucose encapsulated by the vesicles. Figure 2 shows the trapping efficiency for the vesicles containing 1.68 × 10-3 mol dm-3 1 as a function of the ratio of CTAC to SOS. Under dark conditions, the trapping efficiency increased with an increase in the ratio of [CTAC]/[SOS]. The increase in trapping efficiency corresponds to the turbidity in the range of [CTAC]/[SOS] ) 0.03-0.59 (Figure 2).
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Figure 2. Trapping efficiency dependence on the ratio of [CTAC]/ [SOS] in the system containing 1. The average values for five measurements are plotted, including error bars. Under dark conditions, b; after UV irradiation, O.
In the [CTAC]/[SOS] ) 0.59 system with 1, the trapping efficiency was decreased by photoirradiation. It is assumed that the photogenerated surfactant behaves as a cationic surfactant, which in turn brings about a precipitate-forming ratio of cationic to anionic surfactants. The precipitate formed when [CTAC]/ [SOS] was between 0.72 and 1.34 (Figure 1). The precipitate depressed vesicle formation, which resulted in a decrease in trapping efficiency. However, photoirradiation enhanced the trapping efficiency for the [CTAC]/[SOS] ) 0.48 system containing 1. Photoirradiation did not affect the trapping efficiency of the other systems. Thus, the following questions are raised: What kind of events take place in the [CTAC]/[SOS] ) 0.48 system as a result of photoirradiation of 1? Does photoirradiation affect samples in the range of [CTAC]/[SOS] ) 0.08-0.39? To understand the photoinduced structural change in the vesicle, TEM measurements were performed for the [CTAC]/[SOS] ) 0.48 system with an increased trapping efficiency and the [CTAC]/ [SOS] ) 0.18 system with an unchanged trapping efficiency. There are two factors that increase the trapping efficiency: increases in vesicle concentration and size. The concentration of the surfactants was not practically changed by photoirradiation because a small amount of Malachite Green surfactant can be neglected. Therefore, when the increase in vesicle size is observed, the enhanced trapping efficiency can be explained by the increased vesicle size. Typical transmission electron micrographs and size distributions of the vesicles obtained from the [CTAC]/[SOS] ) 0.48 system containing 1.68 × 10-3 mol dm-3 1 are shown in Figure 3. The vesicles under dark conditions have an average diameter of 116 nm. The average diameter was shifted to 243 nm by photoirradiation, and simultaneously, large vesicles with a diameter from 400 to 600 nm were obtained for the photoirradiated samples. It must be emphasized that photoirradiation did increase the vesicle size. The relation between trapping volume and diameter is obvious: larger vesicles trap larger amounts of glucose. Then it is revealed that the photoinduced increase in vesicle size is responsible for the photoinduced enhancement of the trapping efficiency in Figure 2. The results were compared with those for the [CTAC]/[SOS] ) 0.18 system containing 1.68 × 10-3 mol dm-3 1 (Figure 4). Though photoirradiation affords larger vesicles with a diameter of 250-300 nm, it also increases the frequency of small vesicles with a diameter of less than 50 nm (Figure 4B). Therefore, vesicle size was made polydisperse by photoirradiation, and the distinct change was not necessarily confirmed for the average diameter (81 nm under dark conditions and 70 nm after UV irradiation) in the [CTAC]/[SOS] ) 0.18 system. Consequently, the photogenerated surfactant led to an
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Figure 5. Schematic drawing of ANTS-encapsulating vesicles for bilayer destabilization experiments.
Figure 3. Size distributions and transmission electron micrographs of vesicles. [CTAC]/[SOS] ) 0.48; [1] ) 1.63 × 10-3 mol dm-3. Under dark conditions, A; after UV irradiation, B.
Figure 4. Size distributions and transmission electron micrographs of vesicles. [CTAC]/[SOS] ) 0.18; [1] ) 1.63 × 10-3 mol dm-3. Under dark conditions, A; after UV irradiation, B.
increase in vesicle size in the [CTAC]/[SOS] ) 0.48 system whereas the photogenerated surfactant caused the vesicle size to be polydisperse for [CTAC]/[SOS] ) 0.18. 1 underwent photoionization with an ionization ratio of 0.25, which was independent of the ratio of CTAC to SOS (Supporting Information). Therefore, the difference in the photoinduced events observed in the [CTAC]/[SOS] ) 0.18 and 0.48 systems is not explained in terms of the ionization ratio of the Malachite Green derivative. Mechanism of Photoinduced Change in Vesicle Structure. Though the glucose entrapment experiments and TEM measure-
Figure 6. Fluorescence intensity of ANTS encapsulated in vesicles containing 1. The quencher for ANTS is 3.2 × 10-3 mol dm-3 DPX. Fluorescence (100%) was set with 3.0 × 10-4 mol dm-3 ANTS at 507 nm in acetate buffer (pH 4.0). UV irradiation was carried out for 15 min. Under dark conditions, 9; after UV irradiation, 0.
ments showed the photoinduced structural change in the vesicles containing Malachite Green surfactant 1, the mechanism for the increase and polydispersion of the vesicle size remains unclear. To elucidate the mechanism, we investigate the photoinduced release of vesicle-encapsulated fluorescent probes, which may indicate the ability of photogenerated surfactant to induce the destabilization of the vesicle bilayer. The analysis is based on the quenching of ANTS fluorescence by DPX.22,23 ANTS in the vesicles is not self-quenched, but highly water-soluble DPX is able to quench ANTS fluorescence by collisional transfer.22 ANTS was encapsulated in the vesicles, and then DPX was mixed with the vesicle. Fluorophore (ANTS) and quencher (DPX) were therefore separated by the vesicle bilayer at the initial stage. Once the vesicle bilayer was destabilized to release ANTS from the vesicles, the fluorescence of ANTS was quenched by DPX as illustrated in Figure 5. Figure 6 shows the fluorescence of ANTS encapsulated in vesicles containing 1. The graph demonstrates that photoirradiation causes a decrease in the intensity of the fluorescence, which seems to be due to the quenching of ANTS released from vesicles by DPX. In a control test on photoirradiation (15 min) using a solution containing only ANTS, we did not observe any significant decomposition of ANTS. A possibility for the quenching of ANTS fluorescence by the ionized Malachite Green derivative was also excluded by comparing the fluorescence of ANTS solution with that of the solution containing ANTS and Malachite Green oxalate, which does not undergo such photoinduced ionization as does Malachite Green leuconitrile derivative 1. Foerster transfer was not considered to take place between ionized Malachite Green and ANTS because they were not confined in a small area with dimensions of several nanometers. The vesicles in this work have a diameter of 70-240 nm as shown in Figures 3 and 4, and ANTS is a water-soluble compound that is not localized at the vesicle bilayer. Therefore, the fluorescence quenching in Figure 6 is completely attributed to DPX, and the photogenerated (22) Ellens, H.; Bentz, J.; Szoka, F. C. Biochemistry 1984, 23, 1532. (23) Smolarsky, M.; Teitelbaum, D.; Sela, M.; Gilter, C. J. Immunol. Methods 1977, 15, 255.
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Figure 7. Mechanism of bilayer destabilization induced by photoirradiation of the Malachite Green leuconitrile derivative.
surfactant is assumed to destabilize the vesicle bilayer. It should be noted that the quenching of ANTS fluorescence occurred in the systems over the entire [CTAC]/[SOS] range employed here. A plausible mechanism for the destabilization of vesicle bilayers by 1 is illustrated in Figure 7. Because 1 is a lipophilic compound under dark conditions, it is solubilized in the lipid bilayer of the vesicle. Once irradiated by UV light, 1 has an amphiphilicity, and thereby the hydrophilic head group is directed to the aqueous solution. The bilayer is destabilized by the migration of ionized 1, and the inner components of the vesicles are mixed with the outer components in aqueous solution. The fluorescence quenching shown in Figure 6 is explained by this process. The location of 1 under dark conditions was elucidated by exciplex formation with pyrene. Pyrene, a lipophilic compound, is located in the lipid bilayer of the vesicle and has been known to form an exciplex with N,N-dimethylaniline.24,25 Therefore, if 1 having an N,N-dimethylaniline moiety forms an exciplex with pyrene in the vesicle bilayer, then one can observe a new fluorescence peak assigned to the exciplex, which is distinguishable from that of the pyrene monomer. Also, exciplex formation of 1 with pyrene was observed in tetrahydrofuran solution (Supporting Information). Figure 8A shows fluorescence emission spectra of pyrene (10-6 mol dm-3) in the vesicle dispersion at a ratio of [CTAC]/[SOS] ) 0.48. The monomer emission of pyrene, where the fluorescence intensity ratio of the first and the third vibronic peaks of pyrene (I1/I3) is sensitive to medium (solvent) polarity,26,27 was observed. The I1/I3 ratio obtained from Figure 8A is 1.15, which is compatible with the reported value of 1.16 in the vesicle bilayer of 1,2-distearoylphosphatidylcholine.28 Therefore, the pyrene molecules are found to be located in the lipophilic bilayer in this case. It was noted that an excimer peak of pyrene around 470 nm29 was not observed under this experimental condition because the pyrene concentration is low enough. Figure 8B shows the fluorescence emission spectra of pyrene with 1 in the vesicle dispersion. The peak around 490 nm is assigned to the exciplex because 1 has no emission peak around 490 nm. The control test was carried out by vesicle dispersion at a ratio of [CTAC]/[SOS] ) 0.48 containing just 1 that was excited at the same wavelength as for pyrene samples, and no emission peak was observed at 490 nm in Figure 8C. Thus, 1 is revealed to be located under the same lipophilic conditions as pyrene (i.e., the vesicle bilayer). Fusion Process for Increasing Vesicle Size. As the destabilized bilayer proceeds through aggregate reconstruction, it may undergo a variety of processes, such as disruption, transformation into micelles, shrinking, and fusion. The resulting changes in the (24) Werner, U.; Staerk, H. J. Phys. Chem. 1995, 99, 248. (25) Sen, K.; Bandyopadhyay, S.; Bhattacharya, D.; Basu, S. J. Phys. Chem. A 2001, 105, 9077. (26) Kalyanasundaram, K. Langmuir 1988, 4, 942. (27) Dong, D. C.; Winnik, M. A. Can. J. Chem. 1984, 62, 2560. (28) Lissi, E. A.; Abuin, E.; Saez, M.; Zanocco, A.; Disalvo, A. Langmuir 1992, 8, 348. (29) Lianos, P.; Lang, J.; Strazielle, C.; Zana, R. J. Phys. Chem. 1982, 86, 1019.
Figure 8. Fluorescence emission spectrum of pyrene (A), pyrene with 1 (B), and 1 (C) in the [CTAC]/[SOS] ) 0.48 system under dark conditions. [Pyrene] ) 10-6 mol dm-3; [1] ) 1.63 × 10-3 mol dm-3. The excitation wavelength is 337 nm.
vesicle aggregation after bilayer destabilization are presented schematically in Figure 9. We focus on the photoinduced increase in vesicle size in the [CTAC]/[SOS] ) 0.48 system containing 1. There are two possible ways to increase the vesicle size as shown in Figure 9. One is fusion, allowing the membrane components of one vesicle to mix with another, and the other is the rearrangement of bilayer packing, allowing the membrane components of a vesicle to mix with the aqueous components. Figure 6 suggests that the rearrangement process of bilayer packing is caused by the destabilization of the vesicle bilayer. The vesicles in this work were obtained by mixing two singletailed surfactants with oppositely charged head groups, and the other aggregates coexist in the samples. Therefore, the destabilized bilayer accompanies the surfactants in aqueous solution, which do not participate in the bilayer arrangement. In particular, the photogenerated cationic Malachite Green surfactant takes the anionic SOS into the vesicle bilayer. As a consequence, the vesicle size and trapping efficiency of the vesicle were increased. However, it is difficult to explain how large vesicles with a diameter more than 400 nm (Figure 3) arise from the rearrangement of the bilayer packing of vesicles with an average diameter of 116 nm at [CTAC]/[SOS] ) 0.48. Thus, the vesicle fusion process seems to be a reasonable cause of large vesicles in the [CTAC]/[SOS] ) 0.48 system. The results in Figure 6 are not enough to support the fusion phenomena because the bilayer destabilization includes disruption, rearrangement of bilayer packing, and other processes. To support the fusion phenomena,
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Figure 11. Fluorescence intensity of ANTS encapsulated in vesicles containing 1. The quencher for ANTS is DPX encapsulated in vesicles containing 1. Fluorescence (100%) was set with 1.0 × 10-4 mol dm-3 ANTS at 507 nm in acetate buffer (pH 4.0). UV irradiation was carried out for 15 min. Under dark conditions, b; after UV irradiation, O.
[CTAC]/[SOS] range. Conversely, Figure 11 indicates that the photoinduced vesicle fusion depends on the ratio of [CTAC]/ [SOS]. In other words, in the [CTAC]/[SOS] ) 0.48 system, photoirradiation of 1 embedded in vesicles induces the destabilization of the vesicle bilayer, which in turn is responsible for vesicle fusion. In the [CTAC]/[SOS] ) 0.08 system, the destabilized bilayer produced by photoirradiation did not result in fusion; therefore, it may undergo other processes such as disruption and transformation to the smaller vesicles to reconstruct aggregation. Figure 9. Conceptual representation of bilayer destabilization followed by changes in vesicle aggregation.
Figure 10. Schematic drawing of ANTS- and DPX-encapsulating vesicles for fusion experiments.
the quenching of ANTS fluorescence by DPX was followed by using a method different from that shown in Figure 5. ANTS was encapsulated in a group of vesicles and DPX was encapsulated in another,30 and the two types of vesicles contained 1 as illustrated in Figure 10. We confirmed in a separate experiment that simple dilution of DPX in the medium does not cause the quenching of ANTS fluorescence outside the vesicles (Supporting Information). Therefore, the quenching of ANTS fluorescence may take place when the ANTS-encapsulating vesicles are fused together with the DPX-encapsulating vesicles. Figure 11 shows the quenching of ANTS fluorescence by encapsulated DPX. In the [CTAC]/[SOS] ) 0.48 system, the decrease in fluorescence intensity by photoirradiation indicates that the Malachite Green surfactant causes the fusion of vesicles. However, any significant change in the fluorescence intensity in the [CTAC]/[SOS] ) 0.08 system was not observed by photoirradiation. A comparison of Figures 6 and 11 leads to important results. Figure 6 indicates that the photoinduced destabilization of the vesicle bilayer occurs over the entire (30) Ellens, H.; Bentz, J.; Szoka, F. C. Biochemistry 1985, 24, 3099.
Conclusions We have shown that photoresponsive Malachite Green leuconitrile derivative 1 perturbs the vesicle bilayer consisting of CTAC and SOS and that the destabilized bilayer induces a remarkable change in vesicle aggregation. The photoinduced change depended on the mixing ratio of CTAC to SOS. At the mixing ratio of [CTAC]/[SOS] ) 0.48, the vesicle size was increased by photoirradiation. The mechanism for the photoinduced increase in vesicle size was investigated by fluorescence quenching analysis, which supported the processes of vesicle fusion. Photoinduced vesicle fusion triggered by functionalized compound 1 holds potential for the microreactor, which can afford clean, rapid control. We consider that the ratio of 1 to vesicles is important to photoinduced changes, so we are planning to study other types of photoinduced changes in vesicles at various ratios of 1 to vesicles and to conduct a kinetic analysis of vesicle fusion. Acknowledgment. This work was supported by Research for Promoting Technological Seed from the Japan Science and Technological Agency (no. 08-083). R.M.U. is also grateful for support from the Material Science Project of Nara National College of Technology. Supporting Information Available: Photoionization ratio of the Malachite Green leuconitrile derivative. Quenching of ANTS fluorescence by DPX in solution. Fluorescence emission spectrum of pyrene in tetrahydrofuran. This material is available free of charge via the Internet at http://pubs.acs.org. LA700831Z
