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Photochemically triggered reaction of glucose oxidase in a fused vesicle containing Malachite Green leuconitrile derivative
Colloids and Surfaces B: Biointerfaces 87 (2011) 510–513
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Short communication
Photochemically triggered reaction of glucose oxidase in a fused vesicle containing Malachite Green leuconitrile derivative Ryoko M. Uda ∗ , Keisuke Mori, Takuya Sugioka Department of Chemical Engineering, Nara National College of Technology, Yata 22, Yamato-koriyama, Nara 639-1080, Japan
a r t i c l e
i n f o
Article history: Received 6 April 2011 Received in revised form 12 May 2011 Accepted 25 May 2011 Available online 1 June 2011 Keywords: Vesicle fusion Enzyme Photoresponsive Malachite Green
a b s t r a c t Glucose oxidase (GOD) was encapsulated in vesicles containing a photoionizable Malachite Green leuconitrile derivative (MGL). Subsequent UV irradiation of MGL afforded the fusion of GOD- and glucose-encapsulating vesicles and thus decreased the concentration of glucose in the vesicles. The time dependence of the vesicle fusion was studied using fluorescent probe molecules. This phototriggered fusion could be instrumental in the development of a system for the production of nanometer-sized bioreactors. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Vesicles are spherical structures having an aqueous interior enclosed by a bilayer membrane of amphiphiles. Because vesicles are stable entities that do not undergo exchange of their water content, they have been widely used as encapsulating systems. Therefore, they are potential agents for promoting controlled reactions [1–6]. In particular, the encapsulation of water-soluble enzymes can produce a functional bioreactor. Substantial research has been conducted in relation to the enzyme-encapsulating vesicles in which the permeability properties of vesicle bilayers play a key role; a substrate in bulk phase penetrates through the bilayers to converge with an enzyme in the vesicle’s aqueous interior by increasing the substrate permeability [7–10]. Walde and coworkers have reported the uptake of adenosine 5 -diphosphate (ADP) into vesicles encapsulating an enzyme that catalyzes ADP polymerization [7]. The percentage of uptake was less than 1%, and consequently, a small amount of the polymerized ADP product was obtained. An alternative phenomenon in relation to the change in permeability is vesicle fusion [11]. The fusion between substrate- and enzyme-encapsulating vesicles is expected to afford effective conversion of substrate into products. Although the use of vesicle fusion in a bioreactor is attractive, there have been few studies concerning enzyme reactions in fused vesicles [12].
∗ Corresponding author. Tel.: +81 743 55 6164; fax: +81 743 55 6169. E-mail address: [email protected] (R.M. Uda). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.05.049
A controlled reaction of vesicle-encapsulated compounds represents a promising system for designing nanometer-sized reactors. Ideally, the vesicles would act as robust containers that would become destabilized in response to a variety of physical and chemical stimuli, including temperature, light, pH, and ions. Among the various methods reported to stimulate vesicles, irradiation offers attractive possibilities because it provides both temporal and spatial control. However, only a few studies have analyzed photocontrolled enzyme reactions in the vesicle interior. This study develops novel nanometer-sized bioreactors based on vesicle fusion activated by a photoresponsive compound. We selected a Malachite Green leuconitrile derivative (MGL) as the photoresponsive compound to trigger vesicle fusion (Scheme 1). MGL undergoes photoionization, exhibiting both hydrophilicity and hydrophobicity through its triphenylmethyl cation and long alkyl chain, respectively, thereby resulting in photogenerated amphiphilicity which is expected to drastically affect the vesicle bilayer [13]. Under dark conditions, the head group of MGL remains sufficiently nonpolar and the MGL solubilization in the vesicle membrane was revealed by exciplex formation with pyrene [14]. Once irradiated by UV light, MGL has an amphiphilicity and the hydrophilic head group is directed to the aqueous solution. The membrane packing is affected by the migration of ionized MGL to result in various events. Vesicle fusion is one of the phenomena induced by the photoionization of MGL incorporated in a vesicle membrane [14]. Therefore, an enzyme reaction in the vesicle’s interior can be triggered by irradiation, if this photoresponsive vesicle encapsulates both enzyme and its substrate separately (Fig. 1). Here, we report the phototriggered reaction of glucose oxidase (GOD) that occurs in fused vesicles.
R.M. Uda et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 510–513
511
The vesicles encapsulating ANTS, DPX, and glucose were prepared with buffer solutions containing 0.1 mM ANTS, 1.6 mM DPX, and 0.2 M glucose, respectively. To remove the unencapsulated contents, the vesicle dispersion was dialyzed against 0.1 M acetate buffer. The vesicles encapsulating GOD were prepared with buffer solutions containing 4.03 × 104 units L−1 GOD. Unencapsulated GOD was separated from vesicles by gel filtration (Sephacryl S-300). 2.2. Time dependence of vesicle fusion
Scheme 1. Photogenerated amphiphilicity in the Malachite Green leuconitrile derivative carrying a long alkyl chain (MGL).
2. Materials and methods MGL was synthesized in accordance with the literature [15]. Cetyltrimethylammonium chloride (CTAC) was recrystallized from tetrahydrofuran. Sodium octyl sulfate (SOS), 8-aminonaphthalene1,3,6-trisulfonic acid disodium salt (ANTS), and p-xylene-bispyridinium bromide (DPX) were used as received from Aldrich, Biochemika, and Sigma, respectively. GOD from Aspergillus niger was purchased from Wako Chemicals and used directly, and the deionized water was used in our experiments. All other materials were of analytical grade and were utilized without further purification. 2.1. Preparation of vesicle samples
The time dependence of vesicle fusion was examined using a dye/quencher pair [17]. The samples were obtained from a mixture of equal volumes of ANTS- and DPX-encapsulating vesicle dispersions. The quenching of ANTS was monitored by measuring the dequenching of ANTS. The excitation and emission wavelengths were set at 337 and 507 nm, respectively. To correct the bleaching of ANTS caused by the irradiation and ionization of MGL, the percentage of bleached ANTS was determined by comparing the fluorescence of only ANTS-encapsulating vesicles under dark conditions with that after UV irradiation. The measurements were performed at 25 ◦ C using a RF-5300PC spectrofluorometer (Shimadzu, Japan). The quenching of ANTS is derived by % quenching ANTS =
I0 − bIt , I0
where It represents the fluorescence intensity at time t, I0 indicates the fluorescence intensity under dark conditions, and b reflects the bleaching factor. Experimental errors in percentage quenching were within 5%. 2.3. Reaction of GOD with glucose in a fused vesicle
Vesicle samples containing MGL were prepared by mixing solutions of CTAC and SOS. Single-tailed amphiphiles, such as CTAC and SOS, can also form vesicles. In particular, simple mixtures of cationic and anionic surfactants, often referred to as “catanionic” systems, can spontaneously give rise to unilamellar vesicles in water [16]. The concentrations of CTAC and SOS were 25.0 and 51.7 mM, respectively, because we found this proportion desirable for fusion of catanionic surfactant vesicles induced by irradiation of MGL [14]. After a brief vortex mixing, the solutions were not subjected to any other form of mechanical agitation. An appropriate buffer solution was required, because MGL undergoes nonphotochemical ionization under conditions more acidic than pH 3.8. When the pH of the sample solution was raised above 4.2, the photoionized MGL was immediately hydroxylated and the positive charge on the Malachite Green moiety disappeared. Therefore, all samples were prepared using 0.1 M acetate buffer at pH 4.0, unless otherwise noted. The vesicle dispersion were irradiated for 15 min by UV light. The UV light source (<330 nm) consisted of a xenon lamp (500 W) equipped with a photoguide tube and a Toshiba UVD33S filter. The photoionization ratio of MGL was 0.25 [14].
The photoinduced vesicle fusion and enzyme reaction were performed at 25 ◦ C. The samples were obtained by mixing equal volumes of glucose- and GOD-encapsulating vesicle dispersions. Trapping efficiency was determined by the relationship content amount encapsulated by vesicles × 100%. initial content amount before separation Trapping efficiency was found to be ca. 2.5% and 2.3% for glucoseand GOD-encapsulating vesicles, respectively. The concentration of GOD was determined by ellipticity at 210 nm using the calibration curves prepared for native GOD. The mixture was irradiated or left undisturbed under dark conditions for 15 min, after which it was incubated for 6 h. Ethanol was then added to the mixture to disrupt the vesicles. The amount of unreacted glucose was determined by the Schales method [18]. The oxidized glucose is given by % oxidized glucose =
C0 − Cr , C0
where Cr denotes the glucose concentration after incubation and C0 indicates the initial glucose concentration prior to mixing the glucose- and GOD-encapsulating vesicles.
Photoresponsive vesicle
Photoinduced fusion
GOD
Glucose
Reaction of GOD with glucose
Fig. 1. Conceptual representation of photoinduced vesicle fusion and the resulting reaction of GOD with glucose in fused vesicle.
3. Results and discussion 3.1. Time dependence of vesicle fusion Prior to examining the photoinduced enzyme reaction, it is important to investigate the time range of vesicle fusion. Fig. 2 shows the typical time dependence of quenching of ANTS fluorescence by DPX. ANTS was encapsulated in one group of vesicles and DPX was encapsulated in another. ANTS- and DPX-encapsulating vesicles were mixed and then UV irradiated. Under these experimental conditions, the simple dilution of DPX in the medium did not
512
R.M. Uda et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 510–513
40
60
40
Oxidized glucose/%
a
Quenching ANTS /%
50
40
30
Irradiation
30
b 30
UV
20
20
Dark 10
10
0
20
UV, Dark
0 0
0.1 [MGL]/mM
0.2
0
0.1
0.2
[MGL]/mM
10 Fig. 3. Oxidized glucose for a mixture of GOD- and glucose-encapsulating vesicles (a) and for a mixture of GOD-encapsulating vesicles and 5.0 mM glucose solution. (b) The average values for four measurements are plotted with error bars. Under dark conditions: closed circles; after UV irradiation: open circles.
Dark 0 -20
20
60
100
140
Time/min Fig. 2. Time dependence of the quenching of ANTS encapsulated in the vesicle containing 1.68 × 10−5 M (circles), 8.40 × 10−5 M (triangles), and 1.68 × 10−4 M (squares) MGL. The vesicles were subjected to UV irradiation for 15 min. Under dark conditions: closed symbols; after UV irradiation: open symbols.
cause the quenching of ANTS fluorescence outside of the vesicles [14]. Therefore, we concluded that the quenching of ANTS fluorescence occurs when the ANTS-encapsulating vesicles are fused with the DPX-encapsulating vesicles. Fig. 2 thereby shows the time dependence of photoinduced vesicle fusion; subsequent to irradiation, the percentage of quenching increased with time. A sharp rise was observed for the vesicles containing 1.68 × 10−4 M MGL, while vesicles containing a smaller concentration (1.68 × 10−5 M) of MGL showed a gradual increase in the quenching percentage after irradiation. This indicates that the ionized MGL accelerates vesicle fusion. The percentage of quenching increased with increase in the MGL concentration after irradiation, whereas this increase was negligible under dark conditions. Quenching was completed within 140 min of the cessation of irradiation in the MGL concentration ranging from 1.68 × 10−5 to 1.68 × 10−4 M. We also investigated the fluorescence intensity 24 h after terminating the irradiation, and found that it was almost identical to the intensity at 140 min. From Fig. 2 it can be concluded that photoinduced vesicle fusion is completed in 140 min, and that the incubation period for the GOD reaction is longer than 140 min. 3.2. Reaction of GOD with glucose in fused vesicle Photoinduced enzyme reaction was investigated by a mixture of GOD- and glucose-encapsulating vesicles. Fig. 3a shows the percentage of oxidized glucose as a function of the MGL concentration: under dark conditions, the percentage of oxidized glucose in the measured samples was low and did not show any significant dependence on the MGL concentration. In contrast, under UV irradiation, the percentage of oxidized glucose was high, and it increased with the MGL concentration. This result is consistent with the conceptual representation of the phototriggered reaction of GOD in the fused vesicles (Fig. 1). However, there is still a possibility of disruption of vesicle encapsulating GOD that might otherwise potentially cause a reaction with glucose. To preclude the possibility of vesicle disruption, the reaction of encapsulated GOD with external glucose was also examined (Fig. 3b). The samples were obtained by mixing equal volumes of GOD-encapsulating vesicle dispersion and the buffer solution containing 5.0 mM glucose, which corresponded to
the concentration in the case where the encapsulated glucose had been leaked into the media. Fig. 3b illustrates the percentage of oxidized glucose obtained by the disruption of GOD-encapsulating vesicle. Irrespective of whether MGL was irradiated, the percentage of oxidized glucose remained small over the entire range of the measured MGL concentration. Accordingly, it can be stated that photoionized MGL hardly caused the reaction with external glucose by the disruption of GOD-encapsulating vesicle. Through a comparison with the results from Fig. 3b, it may be suggested that the increase in oxidized glucose due to irradiation observed in Fig. 3a must have resulted from photoinduced vesicle fusion. The percentage of oxidized glucose in the irradiated sample at 1.68 × 10−5 M MGL in Fig. 3a is negligible, although the vesicle fusion occurs at this concentration of MGL (Fig. 2). The reason for this is not apparent, and we plan to investigate the differences between the mixture of ANTS and DPX and that of GOD and glucose discovered in fused vesicles. At an MGL concentration above 1.68 × 10−5 M, the percentage of oxidized glucose under irradiated conditions corresponds to that of the quenching ANTS 140 min after irradiation. In order to investigate the effects of ionized MGL and UV irradiation on the GOD reaction, we examined the activity of GOD in the buffer solution containing Malachite Green oxalate under the irradiated condition and determined that neither Malachite Green oxalate nor irradiation caused any significant effect on the GOD reaction. 4. Conclusion Irradiation of MGL incorporated in a catanionic vesicle membrane induced vesicle fusion, which was completed within 140 min following irradiation. The photoresponsive vesicles encapsulated GOD and glucose separately, and the phototriggered vesicle fusion promoted the reaction of GOD with glucose. Because the vesicles are capable of encapsulating various enzymes and substrates, this system could be applied to a functional bioreactor. Although further investigation is required to elucidate the reaction occurring within the interior of the photoresponsive vesicles, this study may contribute to the design of a phototriggered reaction system. References [1] [2] [3] [4] [5]
F. Gauffre, D. Roux, Langmuir 15 (1999) 3738. S. Kazakov, M. Kaholek, I. Teraoka, K. Levon, Macromolecules 35 (2002) 1911. V. Noireaux, A. Libchaber, Proc. Natl. Acad. Sci. USA 101 (2004) 17669. K. Tsumoto, S.M. Nomura, Y. Nakatani, K. Yoshikawa, Langmuir 17 (2001) 7225. K. Hosoda, T. Sunami, Y. Kazuta, T. Matsuura, H. Suzuki, T. Yomo, Langmuir 24 (2008) 13540.
R.M. Uda et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 510–513 [6] J.S. Hong, W.N. Vreeland, S.H.D. Lacerda, L.E. Locascio, M. Gaitan, S.R. Raghavan, Langmuir 24 (2008) 4092. [7] M. Treyer, P. Walde, T. Oberholzer, Langmuir 18 (2002) 1043. [8] M. Naoi, M. Naoi, T. Shimizu, A.N. Malviya, K. Yagi, Biochim. Biophys. Acta 471 (1977) 305. [9] B. Solomon, I.R. Miller, Biochim. Biophys. Acta 455 (1976) 332. [10] A. Graff, M. Winterhalter, W. Meier, Langmuir 17 (2001) 919. [11] P. Walde, Curr. Opin. Colloid Interface Sci. 1 (1996) 638.
513
[12] S. Ichikawa, P. Walde, Langmuir 20 (2004) 941. [13] R.M. Uda, M. Oue, K. Kimura, Chem. Lett. 33 (2004) 586. [14] R.M. Uda, D. Yamashita, Y. Sakurai, K. Kimura, Langmuir 23 (2007) 7936. [15] R.M. Uda, K. Kimura, Bull. Chem. Soc. Jpn. 78 (2005) 1862. [16] A. Khan, Curr. Opin. Colloid Interface Sci. 1 (1996) 614. [17] H. Ellens, J. Bentz, F.C. Szoka, Biochemistry 23 (1984) 1532. [18] T. lmoto, K. Yamashita, Agric. Biol. Chem. 35 (1971) 1154.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Short communication
Photochemically triggered reaction of glucose oxidase in a fused vesicle containing Malachite Green leuconitrile derivative Ryoko M. Uda ∗ , Keisuke Mori, Takuya Sugioka Department of Chemical Engineering, Nara National College of Technology, Yata 22, Yamato-koriyama, Nara 639-1080, Japan
a r t i c l e
i n f o
Article history: Received 6 April 2011 Received in revised form 12 May 2011 Accepted 25 May 2011 Available online 1 June 2011 Keywords: Vesicle fusion Enzyme Photoresponsive Malachite Green
a b s t r a c t Glucose oxidase (GOD) was encapsulated in vesicles containing a photoionizable Malachite Green leuconitrile derivative (MGL). Subsequent UV irradiation of MGL afforded the fusion of GOD- and glucose-encapsulating vesicles and thus decreased the concentration of glucose in the vesicles. The time dependence of the vesicle fusion was studied using fluorescent probe molecules. This phototriggered fusion could be instrumental in the development of a system for the production of nanometer-sized bioreactors. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Vesicles are spherical structures having an aqueous interior enclosed by a bilayer membrane of amphiphiles. Because vesicles are stable entities that do not undergo exchange of their water content, they have been widely used as encapsulating systems. Therefore, they are potential agents for promoting controlled reactions [1–6]. In particular, the encapsulation of water-soluble enzymes can produce a functional bioreactor. Substantial research has been conducted in relation to the enzyme-encapsulating vesicles in which the permeability properties of vesicle bilayers play a key role; a substrate in bulk phase penetrates through the bilayers to converge with an enzyme in the vesicle’s aqueous interior by increasing the substrate permeability [7–10]. Walde and coworkers have reported the uptake of adenosine 5 -diphosphate (ADP) into vesicles encapsulating an enzyme that catalyzes ADP polymerization [7]. The percentage of uptake was less than 1%, and consequently, a small amount of the polymerized ADP product was obtained. An alternative phenomenon in relation to the change in permeability is vesicle fusion [11]. The fusion between substrate- and enzyme-encapsulating vesicles is expected to afford effective conversion of substrate into products. Although the use of vesicle fusion in a bioreactor is attractive, there have been few studies concerning enzyme reactions in fused vesicles [12].
∗ Corresponding author. Tel.: +81 743 55 6164; fax: +81 743 55 6169. E-mail address: [email protected] (R.M. Uda). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.05.049
A controlled reaction of vesicle-encapsulated compounds represents a promising system for designing nanometer-sized reactors. Ideally, the vesicles would act as robust containers that would become destabilized in response to a variety of physical and chemical stimuli, including temperature, light, pH, and ions. Among the various methods reported to stimulate vesicles, irradiation offers attractive possibilities because it provides both temporal and spatial control. However, only a few studies have analyzed photocontrolled enzyme reactions in the vesicle interior. This study develops novel nanometer-sized bioreactors based on vesicle fusion activated by a photoresponsive compound. We selected a Malachite Green leuconitrile derivative (MGL) as the photoresponsive compound to trigger vesicle fusion (Scheme 1). MGL undergoes photoionization, exhibiting both hydrophilicity and hydrophobicity through its triphenylmethyl cation and long alkyl chain, respectively, thereby resulting in photogenerated amphiphilicity which is expected to drastically affect the vesicle bilayer [13]. Under dark conditions, the head group of MGL remains sufficiently nonpolar and the MGL solubilization in the vesicle membrane was revealed by exciplex formation with pyrene [14]. Once irradiated by UV light, MGL has an amphiphilicity and the hydrophilic head group is directed to the aqueous solution. The membrane packing is affected by the migration of ionized MGL to result in various events. Vesicle fusion is one of the phenomena induced by the photoionization of MGL incorporated in a vesicle membrane [14]. Therefore, an enzyme reaction in the vesicle’s interior can be triggered by irradiation, if this photoresponsive vesicle encapsulates both enzyme and its substrate separately (Fig. 1). Here, we report the phototriggered reaction of glucose oxidase (GOD) that occurs in fused vesicles.
R.M. Uda et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 510–513
511
The vesicles encapsulating ANTS, DPX, and glucose were prepared with buffer solutions containing 0.1 mM ANTS, 1.6 mM DPX, and 0.2 M glucose, respectively. To remove the unencapsulated contents, the vesicle dispersion was dialyzed against 0.1 M acetate buffer. The vesicles encapsulating GOD were prepared with buffer solutions containing 4.03 × 104 units L−1 GOD. Unencapsulated GOD was separated from vesicles by gel filtration (Sephacryl S-300). 2.2. Time dependence of vesicle fusion
Scheme 1. Photogenerated amphiphilicity in the Malachite Green leuconitrile derivative carrying a long alkyl chain (MGL).
2. Materials and methods MGL was synthesized in accordance with the literature [15]. Cetyltrimethylammonium chloride (CTAC) was recrystallized from tetrahydrofuran. Sodium octyl sulfate (SOS), 8-aminonaphthalene1,3,6-trisulfonic acid disodium salt (ANTS), and p-xylene-bispyridinium bromide (DPX) were used as received from Aldrich, Biochemika, and Sigma, respectively. GOD from Aspergillus niger was purchased from Wako Chemicals and used directly, and the deionized water was used in our experiments. All other materials were of analytical grade and were utilized without further purification. 2.1. Preparation of vesicle samples
The time dependence of vesicle fusion was examined using a dye/quencher pair [17]. The samples were obtained from a mixture of equal volumes of ANTS- and DPX-encapsulating vesicle dispersions. The quenching of ANTS was monitored by measuring the dequenching of ANTS. The excitation and emission wavelengths were set at 337 and 507 nm, respectively. To correct the bleaching of ANTS caused by the irradiation and ionization of MGL, the percentage of bleached ANTS was determined by comparing the fluorescence of only ANTS-encapsulating vesicles under dark conditions with that after UV irradiation. The measurements were performed at 25 ◦ C using a RF-5300PC spectrofluorometer (Shimadzu, Japan). The quenching of ANTS is derived by % quenching ANTS =
I0 − bIt , I0
where It represents the fluorescence intensity at time t, I0 indicates the fluorescence intensity under dark conditions, and b reflects the bleaching factor. Experimental errors in percentage quenching were within 5%. 2.3. Reaction of GOD with glucose in a fused vesicle
Vesicle samples containing MGL were prepared by mixing solutions of CTAC and SOS. Single-tailed amphiphiles, such as CTAC and SOS, can also form vesicles. In particular, simple mixtures of cationic and anionic surfactants, often referred to as “catanionic” systems, can spontaneously give rise to unilamellar vesicles in water [16]. The concentrations of CTAC and SOS were 25.0 and 51.7 mM, respectively, because we found this proportion desirable for fusion of catanionic surfactant vesicles induced by irradiation of MGL [14]. After a brief vortex mixing, the solutions were not subjected to any other form of mechanical agitation. An appropriate buffer solution was required, because MGL undergoes nonphotochemical ionization under conditions more acidic than pH 3.8. When the pH of the sample solution was raised above 4.2, the photoionized MGL was immediately hydroxylated and the positive charge on the Malachite Green moiety disappeared. Therefore, all samples were prepared using 0.1 M acetate buffer at pH 4.0, unless otherwise noted. The vesicle dispersion were irradiated for 15 min by UV light. The UV light source (<330 nm) consisted of a xenon lamp (500 W) equipped with a photoguide tube and a Toshiba UVD33S filter. The photoionization ratio of MGL was 0.25 [14].
The photoinduced vesicle fusion and enzyme reaction were performed at 25 ◦ C. The samples were obtained by mixing equal volumes of glucose- and GOD-encapsulating vesicle dispersions. Trapping efficiency was determined by the relationship content amount encapsulated by vesicles × 100%. initial content amount before separation Trapping efficiency was found to be ca. 2.5% and 2.3% for glucoseand GOD-encapsulating vesicles, respectively. The concentration of GOD was determined by ellipticity at 210 nm using the calibration curves prepared for native GOD. The mixture was irradiated or left undisturbed under dark conditions for 15 min, after which it was incubated for 6 h. Ethanol was then added to the mixture to disrupt the vesicles. The amount of unreacted glucose was determined by the Schales method [18]. The oxidized glucose is given by % oxidized glucose =
C0 − Cr , C0
where Cr denotes the glucose concentration after incubation and C0 indicates the initial glucose concentration prior to mixing the glucose- and GOD-encapsulating vesicles.
Photoresponsive vesicle
Photoinduced fusion
GOD
Glucose
Reaction of GOD with glucose
Fig. 1. Conceptual representation of photoinduced vesicle fusion and the resulting reaction of GOD with glucose in fused vesicle.
3. Results and discussion 3.1. Time dependence of vesicle fusion Prior to examining the photoinduced enzyme reaction, it is important to investigate the time range of vesicle fusion. Fig. 2 shows the typical time dependence of quenching of ANTS fluorescence by DPX. ANTS was encapsulated in one group of vesicles and DPX was encapsulated in another. ANTS- and DPX-encapsulating vesicles were mixed and then UV irradiated. Under these experimental conditions, the simple dilution of DPX in the medium did not
512
R.M. Uda et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 510–513
40
60
40
Oxidized glucose/%
a
Quenching ANTS /%
50
40
30
Irradiation
30
b 30
UV
20
20
Dark 10
10
0
20
UV, Dark
0 0
0.1 [MGL]/mM
0.2
0
0.1
0.2
[MGL]/mM
10 Fig. 3. Oxidized glucose for a mixture of GOD- and glucose-encapsulating vesicles (a) and for a mixture of GOD-encapsulating vesicles and 5.0 mM glucose solution. (b) The average values for four measurements are plotted with error bars. Under dark conditions: closed circles; after UV irradiation: open circles.
Dark 0 -20
20
60
100
140
Time/min Fig. 2. Time dependence of the quenching of ANTS encapsulated in the vesicle containing 1.68 × 10−5 M (circles), 8.40 × 10−5 M (triangles), and 1.68 × 10−4 M (squares) MGL. The vesicles were subjected to UV irradiation for 15 min. Under dark conditions: closed symbols; after UV irradiation: open symbols.
cause the quenching of ANTS fluorescence outside of the vesicles [14]. Therefore, we concluded that the quenching of ANTS fluorescence occurs when the ANTS-encapsulating vesicles are fused with the DPX-encapsulating vesicles. Fig. 2 thereby shows the time dependence of photoinduced vesicle fusion; subsequent to irradiation, the percentage of quenching increased with time. A sharp rise was observed for the vesicles containing 1.68 × 10−4 M MGL, while vesicles containing a smaller concentration (1.68 × 10−5 M) of MGL showed a gradual increase in the quenching percentage after irradiation. This indicates that the ionized MGL accelerates vesicle fusion. The percentage of quenching increased with increase in the MGL concentration after irradiation, whereas this increase was negligible under dark conditions. Quenching was completed within 140 min of the cessation of irradiation in the MGL concentration ranging from 1.68 × 10−5 to 1.68 × 10−4 M. We also investigated the fluorescence intensity 24 h after terminating the irradiation, and found that it was almost identical to the intensity at 140 min. From Fig. 2 it can be concluded that photoinduced vesicle fusion is completed in 140 min, and that the incubation period for the GOD reaction is longer than 140 min. 3.2. Reaction of GOD with glucose in fused vesicle Photoinduced enzyme reaction was investigated by a mixture of GOD- and glucose-encapsulating vesicles. Fig. 3a shows the percentage of oxidized glucose as a function of the MGL concentration: under dark conditions, the percentage of oxidized glucose in the measured samples was low and did not show any significant dependence on the MGL concentration. In contrast, under UV irradiation, the percentage of oxidized glucose was high, and it increased with the MGL concentration. This result is consistent with the conceptual representation of the phototriggered reaction of GOD in the fused vesicles (Fig. 1). However, there is still a possibility of disruption of vesicle encapsulating GOD that might otherwise potentially cause a reaction with glucose. To preclude the possibility of vesicle disruption, the reaction of encapsulated GOD with external glucose was also examined (Fig. 3b). The samples were obtained by mixing equal volumes of GOD-encapsulating vesicle dispersion and the buffer solution containing 5.0 mM glucose, which corresponded to
the concentration in the case where the encapsulated glucose had been leaked into the media. Fig. 3b illustrates the percentage of oxidized glucose obtained by the disruption of GOD-encapsulating vesicle. Irrespective of whether MGL was irradiated, the percentage of oxidized glucose remained small over the entire range of the measured MGL concentration. Accordingly, it can be stated that photoionized MGL hardly caused the reaction with external glucose by the disruption of GOD-encapsulating vesicle. Through a comparison with the results from Fig. 3b, it may be suggested that the increase in oxidized glucose due to irradiation observed in Fig. 3a must have resulted from photoinduced vesicle fusion. The percentage of oxidized glucose in the irradiated sample at 1.68 × 10−5 M MGL in Fig. 3a is negligible, although the vesicle fusion occurs at this concentration of MGL (Fig. 2). The reason for this is not apparent, and we plan to investigate the differences between the mixture of ANTS and DPX and that of GOD and glucose discovered in fused vesicles. At an MGL concentration above 1.68 × 10−5 M, the percentage of oxidized glucose under irradiated conditions corresponds to that of the quenching ANTS 140 min after irradiation. In order to investigate the effects of ionized MGL and UV irradiation on the GOD reaction, we examined the activity of GOD in the buffer solution containing Malachite Green oxalate under the irradiated condition and determined that neither Malachite Green oxalate nor irradiation caused any significant effect on the GOD reaction. 4. Conclusion Irradiation of MGL incorporated in a catanionic vesicle membrane induced vesicle fusion, which was completed within 140 min following irradiation. The photoresponsive vesicles encapsulated GOD and glucose separately, and the phototriggered vesicle fusion promoted the reaction of GOD with glucose. Because the vesicles are capable of encapsulating various enzymes and substrates, this system could be applied to a functional bioreactor. Although further investigation is required to elucidate the reaction occurring within the interior of the photoresponsive vesicles, this study may contribute to the design of a phototriggered reaction system. References [1] [2] [3] [4] [5]
F. Gauffre, D. Roux, Langmuir 15 (1999) 3738. S. Kazakov, M. Kaholek, I. Teraoka, K. Levon, Macromolecules 35 (2002) 1911. V. Noireaux, A. Libchaber, Proc. Natl. Acad. Sci. USA 101 (2004) 17669. K. Tsumoto, S.M. Nomura, Y. Nakatani, K. Yoshikawa, Langmuir 17 (2001) 7225. K. Hosoda, T. Sunami, Y. Kazuta, T. Matsuura, H. Suzuki, T. Yomo, Langmuir 24 (2008) 13540.
R.M. Uda et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 510–513 [6] J.S. Hong, W.N. Vreeland, S.H.D. Lacerda, L.E. Locascio, M. Gaitan, S.R. Raghavan, Langmuir 24 (2008) 4092. [7] M. Treyer, P. Walde, T. Oberholzer, Langmuir 18 (2002) 1043. [8] M. Naoi, M. Naoi, T. Shimizu, A.N. Malviya, K. Yagi, Biochim. Biophys. Acta 471 (1977) 305. [9] B. Solomon, I.R. Miller, Biochim. Biophys. Acta 455 (1976) 332. [10] A. Graff, M. Winterhalter, W. Meier, Langmuir 17 (2001) 919. [11] P. Walde, Curr. Opin. Colloid Interface Sci. 1 (1996) 638.
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