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Photochemically triggered transfer of bovine serum albumin by reverse micelle containing a Malachite Green leuconitrile derivative
Colloids and Surfaces A: Physicochem. Eng. Aspects 337 (2009) 180–184
Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Photochemically triggered transfer of bovine serum albumin by reverse micelle containing a Malachite Green leuconitrile derivative Ryoko M. Uda a , Takashi Hirai a , Takafumi Koshida a , Keiichi Kimura b,∗ a b
Department of Chemical Engineering, Nara National College of Technology, Yata 22, Yamato-koriyama, Nara 639-1080, Japan Department of Applied Chemistry, Faculty of Systems Engineering, Wakayama University, Sakae-dani 930, Wakayama 640-8510, Japan
a r t i c l e
i n f o
Article history: Received 24 June 2008 Received in revised form 2 December 2008 Accepted 11 December 2008 Available online 24 December 2008 Keywords: Reverse micelle Photoionization Protein extraction Malachite Green
a b s t r a c t UV irradiation of a Malachite Green leuconitrile derivative, solubilized in the organic phase of a chloroform/1-hexanol/aqueous mixture, affords a cationic surfactant. These surfactant molecules associate more readily than do the parent molecules to form reverse micelles, which have been investigated by water solubilization and transmission electron microscopy. The photo-induced formation of reverse micelle is shown to internalize bovine serum albumin originally present in the bulk aqueous phase. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Reverse micelles are nanometer-sized aqueous droplets coated with a monolayer of an amphiphilic surfactant and dispersed in a nonpolar solvent. The reverse micelle’s polar aqueous core is named the “water pool”. This water pool is a unique microenvironment that differs from that of the bulk polar medium [1,2]. The physical and chemical properties of reverse micelles have been studied extensively because their interiors provide microenvironments that are conducive to certain processes, including solubilizing proteins, enzymatic reactions [3], and synthesis of inorganic materials with submicron dimensions [4] (nanoparticles). Previous studies have examined the effects of water concentration [5,6], temperature [7], and ionic strength [8] on reverse micelle construction and water pool collisions. Such studies are necessary if the goal of building reverse micelles, acting as microreactors, is to be achieved. Alternatively, light could also be used to control the construction of reverse micelles. Molecules containing a photosensitive chromophore whose electrostatic state controls the molecules’ hydrophilic/hydrophobic balance exist. While there are many reports describing photoresponsive molecular assemblies, such as normal micelles [9], vesicles [10,11], and mono- [12,13] and multi-layers [14], only a few studies have targeted the photochemical control of reverse micelles’ physical and chemical properties. Such studies used photoresponsive molecules as additives to con-
∗ Corresponding author. Tel: +81 73 457 8255; fax: +81 73 457 8254. E-mail addresses: [email protected] (R.M. Uda), [email protected] (K. Kimura). 0927-7757/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2008.12.020
trol the disruption [15] of or percolation [16,17] of conventional reverse micelles. The photochemical control of an emulsion state, i.e. water-in-oil or oil-in-water, has been investigated using an azobenzene derivative grafted to poly(sodium acrylate) [18]. Zhang and co-workers [19] characterized a mixture of an azobenzenecontaining amphiphile and a block copolymer micelle that after exposure to light had increased amounts of the amphiphile in a reverse micelle-like state. While such studies have shown that a photoresponsive additive, such as an azobenzene derivative, can affect the state of an assemblage, a change in the net charge of the structure’s building blocks, i.e. the (potential) surfactants, should be more effective because their amphiphilic nature will be directly affected. With this expectation in mind, we synthesized a photoionizable Malachite Green leuconitrile derivative carrying a long alkyl chain (1) (Scheme 1) [20,21]. The head group of 1 is sufficiently nonpolar that, in the absence of light (dark conditions), 1 is best described as lipophilic. However, when 1 is exposed to UV light, one of the products is a significantly more amphiphilic compound, 1+ , with a hydrophilic triphenylmethyl cationic head group and a hydrophobic alkyl tail (Scheme 1). The photochemically generated electrical charge on the head group should drastically affect the amount and type of the aggregated state. Indeed, we have already shown that, in a system where 1 forms micelles, the electrostatic state of the head group controls the critical micelle concentration [20] and controls the extent to which an oily substance can be dissolved in the micelle solution [21]. Herein, we report a novel means of photochemically inducing reverse micelle formation. A general description of this method is illustrated in Fig. 1 with 1 as the photoresponsive compound. Compound 1 is dissolved in the organic phase of an organic/aqueous
R.M. Uda et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 337 (2009) 180–184
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2.2. Reverse micelle preparation A defined amount of 1 was dissolved in the organic phase of a two-phase mixture composed of 5.0 cm3 chloroform/20% (v/v) 1-hexanol and 0.5 cm3 aqueous solution (see below for the compositions of the aqueous solutions). Although reverse micelles composed of single-tailed surfactants usually contain only small amount of water, the water pool can be significantly enlarged when a short-chain aliphatic alcohol cosurfactant, such as 1-hexanol, is present [8]. The sample was irradiated or left undisturbed in the dark for 30 min after which it was agitated for 60 min so that the partitioning reached an equilibrium. Then the mixture was centrifuged for 30 min to cleanly separate the two phases at the interface. For the transmission electron microscopy (TEM) experiments, the aqueous solution was 0.52 mmol dm−3 12-tungstophosphoric acid. For the BSA extraction experiments, the aqueous solution was 0.15 mmol dm−3 BSA in 0.1 mmol dm−3 phosphate buffer (pH 7.0). A xenon lamp (500 W) that was equipped with a photoguide tube and a Toshiba UV-D33S filter was the UV light source (<330 nm). Reverse micelle samples were prepared at 25 ◦ C in a thermostated water bath. 2.3. Extraction of BSA into reverse micelles Scheme 1. Photochemically generated amphiphilicity of the Malachite Green leuconitrile derivative carrying a long alkyl chain.
two-phase mixture in the dark. Upon UV irradiation, molecules of 1+ , cationic surfactants, associate within the organic phase forming reverse micelles with water pools in their cores. If the bulk aqueous phase contains a solute, then while exposed to the reverse micelles, the solute may be transferred into their water pools. Reverse micelles made of photoresponsive components might prove to be very useful containers for biomolecular microreactions. Although Seno and co-workers [22] studied the photochemically controlled extraction of amino acids from aqueous solution into reverse micelles, there are no such studies using biopolymers. Therefore, we aim at photochemically triggered transfer of protein into reverse micellar phase. To the best of our knowledge, this is a novel method for the extraction of a protein by a reverse micelle because it incorporates a photochemical trigger. This would be a step towards proof of function for photochemically induced reverse micelles as microreactors.
Because the strong UV absorbance of 1+ precludes using UV spectroscopy to determine the concentration of BSA within the reverse micelles, it was necessary to separate any BSA present from the organic phase. To isolate BSA solubilized in the water pools, 1+ and the organic components were removed from a 4.0 cm3 reverse micelle solution using a combination of evaporation and subsequent dialysis against 0.1 mmol dm−3 phosphate buffer (pH 7.0). After dialysis, the volume of the retentate was adjusted to 5.0 cm3 with the addition of phosphate buffer. To this end, BSA was isolated from the organic solution. The UV spectrum of the retentate had the absorption peak at 280 nm that is typical of proteins containing aromatic amino acids. Because there was no absorbance in the visible region of the retentate’s spectrum, the Malachite Green derivative was completely removed from the sample and cannot be responsible for the absorbance in the UV region. Therefore the concentration of BSA was determined by the absorbance at 280 nm of the retentate. The yield of BSA transfer was based on the percentage of BSA extracted into the organic phase relative to the initial amount of BSA in the aqueous phase.
2. Experimental 2.4. Analytical methods 2.1. Materials Malachite Green derivative 1 was synthesized according to the literature [21]. Bovine serum albumin (BSA) was purchased from Sigma. Other materials were of analytical grade and were used without further purification.
The water content of the organic phase was determined by a Karl–Fisher titration. TEM samples were prepared by placing a drop of the reverse micelle solution onto a TEM grid and evaporating the organic solvent. TEM measurements were performed using the HITACHI HF-2000 electron microscope. The circular dichromism
Fig. 1. Conceptual representation of a photochemically induced reverse micelle formation and protein extraction.
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Fig. 2. Absorption spectra of 0.1 mmol dm−3 1 in chloroform/20% (v/v) 1-hexanol before (dark) and after (UV) UV irradiation for 30 min.
(CD) spectra of the native and the isolated BSA in the 0.1 mmol dm−3 phosphate buffer (pH 7.0) were measured with a J-820 spectropolarimeter (Jasco Corporation, Tokyo, Japan). The sample of isolated BSA was the retentate which was prepared for the extraction measurement. 3. Results and discussion 3.1. Photochemical reaction of Malachite Green derivative 1 Fig. 2 shows typical absorption spectra for a sample of 1 dissolved in chloroform/20% (v/v) 1-hexanol that was or was not irradiated. The UV-irradiated samples have a new absorption peak with a maximum near 600 nm, which is assigned to a transition associated with 1+ . The cation is stable as the absorption peak around 600 nm persists for more than 5 h. The ionization ratio—which is defined as AMGL /AMGO , where AMGL and AMGO are respectively the molar absorptivities, measured at 600 nm, for a reverse micelle sample and a Malachite Green oxalate sample in chloroform/20% (v/v) 1-hexanol mixtures—is 0.11 and is independent of the pH value and ionic strength of the aqueous solution.
Fig. 3. Water concentration in an organic solution versus the corresponding concentration of 1. The inset shows the semi-logarithmic plot. The average values for five measurements are plotted with error bars. Under dark conditions, 䊉; after UV irradiation, .
organic phase and aqueous 0.52 mmol dm−3 12-tungstophosphoric acid, as the contrast agent. The images have a spherical shape that is consistent with the shape expected for reverse micelles. We did not observe images of aggregated reverse micelles as reported by others [24]. The particle diameters range from 30 nm to 70 nm; a plot of the diameter distribution is shown in the supporting material. The size of reverse micelle has been reported to depend on the molar ratio of [H2 O]/[surfactant] and there have been many investigations of the reverse micellar size in various systems
3.2. Photochemically induced reverse micelle formation Fig. 3 is a plot of the amount of water solubilized in the organic solution as a function of the initial concentration of 1 in the organic phase. Because, it is expected that the water present in the organic phase will be that of the water pool, its concentration can be used to determine the critical micelle concentration (cmc) of the surfactant [23]. For our system, under dark conditions, the water concentration was practically independent of the concentration of 1. On the other hand, after UV irradiation, there was an increase in the water concentration of the organic phase that correlates with the concentration of 1 and was particularly noticeable when the concentration of 1 was greater than 5.0 mmol dm−3 . This photochemically induced increase in the water concentration indicates that the reverse micelle formation is enhanced by UV irradiation. The cmc value for UV-irradiated 1 was 1.9 mmol dm−3 , which corresponds to the ‘break point’ in a semi-logarithmic plot of the data shown in Fig. 3. Under dark conditions, a cmc value was not obtained because there was no clearly abrupt increase for the slope of the corresponding semi-logarithmic plot. TEM imaging provides strong evidence for the existence of reverse micelles. Fig. 4 shows a typical portion of a micrograph of the irradiated sample that contained 10 mmol dm−3 1 in the
Fig. 4. A transmission electron micrograph of reverse micelles whose interiors are stained with 12-tungstophosphoric acid. This UV-irradiated system contained 1 at a 10 mmol dm−3 concentration.
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Fig. 5. Effect of the concentration of 1 on BSA transfer. The average values for three measurements are plotted. Under dark conditions, 䊉; after UV irradiation, .
[25–27]. For example, Naoe and co-workers investigated the size of reverse micelle containing cetyltrimethylammonium bromide and 1-haxanol and then they have obtained the empirical equation: diameter (nm) = 3.2 + 0.17 [H2 O]/[surfactant] [25]. Using their equation and the ratio of [H2 O]/[surfactant] for our system containing 10 mmol dm−3 of 1, the diameter of the reverse micelle in our system is calculated to be 51 nm which are consistent with the results of Fig. 4. The detailed calculation and the difference in the calculated diameter by using the other equations are discussed in the supporting material. Spherically shaped particles were also found in TEM micrographs of a sample containing 10 mmol dm−3 1 that was not irradiated. However, the number of particles was considerably less than those found for irradiated samples. Consequently, any tendency for size distribution could not be obtained. A TEM micrograph of a control sample that did not contain 1 was absent of spherically shaped particles; therefore, 1 and 1+ are responsible for the TEM images and can both form reverse micelles, although the latter is the more effective agent. 3.3. Photo-induced transfer of BSA to reverse micelles In general, the attractive electrostatic interactions between a biopolymer solubilized in the water pool and the head groups of
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the surfactants will be the driving forces for the phase transition [28,29], which is the reason why BSA (67 kDa; pI = 4.7) and an aqueous solution buffered at pH 7.0 (0.1 mmol dm−3 phosphate buffer) were chosen for the system of cationic 1+ . Fig. 5 shows the transfer yield of BSA as a function of the concentration of 1. Under dark conditions, the transfer yield was small in all the concentration of 1 measured. On the other hand, UV irradiation afforded an increase in the transfer yield with the increase of concentration of 1. The BSA transfer did not occur at the concentration of 1 lower than its cmc (1.9 mmol dm−3 ). Furthermore, the photochemically induced transfer was significantly promoted at the concentration of 1 greater than 5.0 mmol dm−3 , where the water concentration in the organic phase was enhanced by UV irradiation (Fig. 3). The results of Figs. 3 and 5 lead to the conclusion that BSA in the aqueous phase was transferred to the water pool of the photochemically generated reverse micelle. The CD spectrum of the BSA retentate isolated from an irradiated sample was recorded and compared to a spectrum of native BSA. These spectra are shown in Fig. 6. Although the spectrum of the retentate is noisy (Fig. 6b) because the amount of BSA present is small, the two spectra are otherwise identical. The shape and intensity of the far UV region of a protein’s CD spectrum is a consequence, in large part, of the protein’s secondary structure; therefore, native BSA is present in the reverse micelle. We confirmed in a separate experiment that the aqueous solution isolated from a sample under dark conditions did not show any significant CD signal in the UV region (Supporting material). 4. Conclusion We demonstrated that 1+ , which is a cationic form of 1, a Malachite Green derivative, more readily forms reverse micelles than does the lipophilic 1 itself and that the reverse micelles can internalize BSA. The photochemically induced transfer of BSA was attained by UV irradiation on 1 at the concentration greater than its cmc. We plan on testing the physical limits of the reverse micelle system by examining its ability to incorporate proteins of different charges, mass, structure, etc. into its water pool. We also intend to explore the suitability of the water pool and the external nonpolar environments to facilitate enzymatic reactions for which the substrates are water-insoluble, e.g. linoleic acid, which is a substrate for lipoxygenase. Finally, we also plan to investigate the reverse reaction illustrated in Scheme 1, i.e. 1+ → 1 by heating, as a way to release protein trapped in the water pools into aqueous solution,
Fig. 6. CD spectra of 0.15 mmol dm−3 native BSA (a) and BSA isolated from reverse micelles (b). Both samples were in 0.1 mmol dm−3 phosphate. The reverse micelle system contained 1 at an initial concentration of 10 mmol dm−3 .
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where the temperature on the reverse reaction should be carefully chosen to avoid the denaturalization of protein. Acknowledgements This work was supported in part by the Mukai Science and Technology Foundation. The authors thank Dr. K. Naoe for helpful discussions concerning the results. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.colsurfa.2008.12.020. References [1] P.L. Luisi, B. Straub, Reverse Micelles-Technological and Biological Relevance, Plenum, New York, 1984. [2] P. Neogi, in: S.E. Friberg, P. Bothorel (Eds.), Microemulsions: Structure and Dynamics, CRC Press, Boca Raton, FL, 1987. [3] P.L. Luisi, Enzymes hosted in reverse micelles in hydrocarbon solution, Angew. Chem. Int. Ed. Engl. 24 (1985) 439–528. [4] M. Boutonnet, J. Kizling, P. Stenius, G. Maire, The preparation of monodisperse colloidal metal particles from microemulsions, Colloids Surf. 5 (1982) 209–225. [5] I. Lisiecki, M.P. Pileni, Copper metallic particles synthesized “in situ” in reverse micelles: influence of various parameters on the size of the particles, J. Phys. Chem. 99 (1995) 5077–5082. [6] I. Lisiecki, M. Bjoerling, L. Motte, B. Ninham, M.P. Pileni, Synthesis of copper nanosize particles in anionic reverse micelles: effect of the addition of a cationic surfactant on the size of the crystallites, Langmuir 11 (1995) 2385–2392. [7] P.L. Luisi, M. Giomini, M.P. Pileni, B.H. Robinson, Reverse micelles as hosts for proteins and small molecules, Biochim. Biophys. Acta 947 (1988) 209–246. [8] S.H. Krishna, N.D. Srinivas, K.S.M.S. Raghavarao, N.G. Karanth, Reverse micellar extraction for downstream processing of proteins/enzymes, in: Advances in Biochemical Engineering/Biotechnology, Springer-Verlag, Berlin Heidelberg, 2002, pp. 119–183. [9] T. Kozlecki, A. Sokolowski, K.A. Wilk, Surface activity and micelle formation of anionic azobenzene-linked surfactants, Langmuir 13 (1997) 6889–6895. [10] T. Kunitake, Y. Okahata, M. Shimomura, S. Yasunami, K. Takarabe, Formation of stable bilayer assemblies in water from single-chain amphiphiles. Relationship between the amphiphile structure and the aggregate morphology, J. Am. Chem. Soc. 103 (1981) 5401–5413. [11] M. Haubs, H. Ringsdorf, Photoreactions of N-(1-pyridinio)amidates in monolayers and liposomes, Angew. Chem. Int. Ed. Engl. 24 (1985) 882–883. [12] D.A. Holden, H. Ringsdorf, Photosensitive monolayers. Studies of surface-active spiropyrans at the air-water interface, J. Phys. Chem. 88 (1984) 716–720.
[13] K. Ichimura, Y. Suzuki, T. Seki, A. Hosoki, K. Aoki, Reversible change in alignment mode of nematic liquid crystals regulated photochemically by command surfaces modified with an azobenzene monolayer, Langmuir 4 (1988) 1214– 1216. [14] W.F. Mooney, P.E. Brown, J.C. Russell, S.B. Costa, L.G. Pedersen, D.G. Whitten, Photochemical reactions in organized assemblies. 37. Photochemistry and photophysics of surfactant trans-stilbenes in supported multilayers and films at the air-water interface, J. Am. Chem. Soc. 106 (1984) 5659–5667. [15] J. Eastoe, M.S. Dominguez, H. Cumber, P. Wyatt, R.K. Heenan, Light-sensitive microemulsions, Langmuir 20 (2004) 1120–1125. [16] T. Wolff, H. Hegewald, Light induced switching of conductivity in AOTisooctane-water microemulsions via photoreactions of solubilizates, Colloids Surf. A: Physicochem. Eng. Aspects 164 (2000) 279–285. [17] T. Wolff, D. Nees, Influence of solubilized stilbazolium salts in trans and cis configuration on the percolation equilibrium in AOT-isooctane-water microemulsions: light induced switching of conductivity, Prog. Colloid Polym. Sci. 111 (1998) 113–116. [18] I. Porcar, P. Perrin, C. Tribet, UV-visible light: a novel route to tune the type of an emulsion, Langmuir 17 (2001) 6905–6909. [19] N. Ma, Y. Wang, B. Wang, Z. Wang, X. Zhang, Interaction between block copolymer micelles and azobenzene-containing surfactants: from coassembly in water to layer-by-layer assembly at the interface, Langmuir 23 (2007) 2874–2878. [20] R.M. Uda, M. Oue, K. Kimura, Effective photocontrol of micelle formation by Malachite Green derivative carrying a long alkyl chain, Chem. Lett. 33 (2004) 586–587. [21] R.M. Uda, K. Kimura, Photocontrol of micelle triggered by Malachite Green carrying a long alkyl chain, Bull. Chem. Soc. Jpn. 78 (2005) 1862–1867. [22] M. Ino, H. Tanaka, J. Otsuki, K. Arai, M. Seno, Photo-controlled extraction and active transport of amino acids by functional reversed micelles containing spiropyran derivatives, Colloid Polym. Sci. 272 (1994) 151–158. [23] P. Alexandridis, K. Andersson, Reverse micelle formation and water solubilization by polyoxyalkylene block copolymers in organic solvent, J. Phys. Chem. B 101 (1997) 8103–8111. [24] H.M. Jung, K.E. Price, D.T. McQuade, Synthesis and characterization of crosslinked reverse micelles, J. Am. Chem. Soc. 125 (2003) 5351–5355. [25] K. Naoe, C. Takeuchi, M. Kawagoe, K. Nagayama, M. Imai, Higher order structure of Mucor miehei lipase and micelle size in cetyltrimethylammonium bromide reverse micellar system, J. Chromatogr. B 850 (2007) 277–284. [26] M. Giustini, G. Palazzo, G. Colafemmina, M.D. Monica, M. Giomini, A. Ceglie, Microstructure and dynamics of the water-in-oil CTAB/n-pentanol/nhexane/water microemulsion: a spectroscopic and conductivity study, J. Phys. Chem. 100 (1996) 3190–3198. [27] K. Vos, C. Laane, S.R. Weijers, A. Van Hoek, C. Vegger, J.W. Visser, Time-resolved fluorescence and circular dichroism of porphyrin cytochrome c and Zn-porpyrin cytochrome c incorporated in reversed micelles, Eur. J. Biochem. 169 (1987) 259. [28] M. Dekker, K.V. Riet, B.H. Bijsterbosch, P. Fijneman, R. Hilhorst, Mass transfer rate of protein extraction with reversed micelles, Chem. Eng. Sci. 45 (1990) 2949–2957. [29] G.A. Krei, H. Hustedt, Extraction of enzymes by reverse micelles, Chem. Eng. Sci. 47 (1992) 99–111.
Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Photochemically triggered transfer of bovine serum albumin by reverse micelle containing a Malachite Green leuconitrile derivative Ryoko M. Uda a , Takashi Hirai a , Takafumi Koshida a , Keiichi Kimura b,∗ a b
Department of Chemical Engineering, Nara National College of Technology, Yata 22, Yamato-koriyama, Nara 639-1080, Japan Department of Applied Chemistry, Faculty of Systems Engineering, Wakayama University, Sakae-dani 930, Wakayama 640-8510, Japan
a r t i c l e
i n f o
Article history: Received 24 June 2008 Received in revised form 2 December 2008 Accepted 11 December 2008 Available online 24 December 2008 Keywords: Reverse micelle Photoionization Protein extraction Malachite Green
a b s t r a c t UV irradiation of a Malachite Green leuconitrile derivative, solubilized in the organic phase of a chloroform/1-hexanol/aqueous mixture, affords a cationic surfactant. These surfactant molecules associate more readily than do the parent molecules to form reverse micelles, which have been investigated by water solubilization and transmission electron microscopy. The photo-induced formation of reverse micelle is shown to internalize bovine serum albumin originally present in the bulk aqueous phase. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Reverse micelles are nanometer-sized aqueous droplets coated with a monolayer of an amphiphilic surfactant and dispersed in a nonpolar solvent. The reverse micelle’s polar aqueous core is named the “water pool”. This water pool is a unique microenvironment that differs from that of the bulk polar medium [1,2]. The physical and chemical properties of reverse micelles have been studied extensively because their interiors provide microenvironments that are conducive to certain processes, including solubilizing proteins, enzymatic reactions [3], and synthesis of inorganic materials with submicron dimensions [4] (nanoparticles). Previous studies have examined the effects of water concentration [5,6], temperature [7], and ionic strength [8] on reverse micelle construction and water pool collisions. Such studies are necessary if the goal of building reverse micelles, acting as microreactors, is to be achieved. Alternatively, light could also be used to control the construction of reverse micelles. Molecules containing a photosensitive chromophore whose electrostatic state controls the molecules’ hydrophilic/hydrophobic balance exist. While there are many reports describing photoresponsive molecular assemblies, such as normal micelles [9], vesicles [10,11], and mono- [12,13] and multi-layers [14], only a few studies have targeted the photochemical control of reverse micelles’ physical and chemical properties. Such studies used photoresponsive molecules as additives to con-
∗ Corresponding author. Tel: +81 73 457 8255; fax: +81 73 457 8254. E-mail addresses: [email protected] (R.M. Uda), [email protected] (K. Kimura). 0927-7757/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2008.12.020
trol the disruption [15] of or percolation [16,17] of conventional reverse micelles. The photochemical control of an emulsion state, i.e. water-in-oil or oil-in-water, has been investigated using an azobenzene derivative grafted to poly(sodium acrylate) [18]. Zhang and co-workers [19] characterized a mixture of an azobenzenecontaining amphiphile and a block copolymer micelle that after exposure to light had increased amounts of the amphiphile in a reverse micelle-like state. While such studies have shown that a photoresponsive additive, such as an azobenzene derivative, can affect the state of an assemblage, a change in the net charge of the structure’s building blocks, i.e. the (potential) surfactants, should be more effective because their amphiphilic nature will be directly affected. With this expectation in mind, we synthesized a photoionizable Malachite Green leuconitrile derivative carrying a long alkyl chain (1) (Scheme 1) [20,21]. The head group of 1 is sufficiently nonpolar that, in the absence of light (dark conditions), 1 is best described as lipophilic. However, when 1 is exposed to UV light, one of the products is a significantly more amphiphilic compound, 1+ , with a hydrophilic triphenylmethyl cationic head group and a hydrophobic alkyl tail (Scheme 1). The photochemically generated electrical charge on the head group should drastically affect the amount and type of the aggregated state. Indeed, we have already shown that, in a system where 1 forms micelles, the electrostatic state of the head group controls the critical micelle concentration [20] and controls the extent to which an oily substance can be dissolved in the micelle solution [21]. Herein, we report a novel means of photochemically inducing reverse micelle formation. A general description of this method is illustrated in Fig. 1 with 1 as the photoresponsive compound. Compound 1 is dissolved in the organic phase of an organic/aqueous
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2.2. Reverse micelle preparation A defined amount of 1 was dissolved in the organic phase of a two-phase mixture composed of 5.0 cm3 chloroform/20% (v/v) 1-hexanol and 0.5 cm3 aqueous solution (see below for the compositions of the aqueous solutions). Although reverse micelles composed of single-tailed surfactants usually contain only small amount of water, the water pool can be significantly enlarged when a short-chain aliphatic alcohol cosurfactant, such as 1-hexanol, is present [8]. The sample was irradiated or left undisturbed in the dark for 30 min after which it was agitated for 60 min so that the partitioning reached an equilibrium. Then the mixture was centrifuged for 30 min to cleanly separate the two phases at the interface. For the transmission electron microscopy (TEM) experiments, the aqueous solution was 0.52 mmol dm−3 12-tungstophosphoric acid. For the BSA extraction experiments, the aqueous solution was 0.15 mmol dm−3 BSA in 0.1 mmol dm−3 phosphate buffer (pH 7.0). A xenon lamp (500 W) that was equipped with a photoguide tube and a Toshiba UV-D33S filter was the UV light source (<330 nm). Reverse micelle samples were prepared at 25 ◦ C in a thermostated water bath. 2.3. Extraction of BSA into reverse micelles Scheme 1. Photochemically generated amphiphilicity of the Malachite Green leuconitrile derivative carrying a long alkyl chain.
two-phase mixture in the dark. Upon UV irradiation, molecules of 1+ , cationic surfactants, associate within the organic phase forming reverse micelles with water pools in their cores. If the bulk aqueous phase contains a solute, then while exposed to the reverse micelles, the solute may be transferred into their water pools. Reverse micelles made of photoresponsive components might prove to be very useful containers for biomolecular microreactions. Although Seno and co-workers [22] studied the photochemically controlled extraction of amino acids from aqueous solution into reverse micelles, there are no such studies using biopolymers. Therefore, we aim at photochemically triggered transfer of protein into reverse micellar phase. To the best of our knowledge, this is a novel method for the extraction of a protein by a reverse micelle because it incorporates a photochemical trigger. This would be a step towards proof of function for photochemically induced reverse micelles as microreactors.
Because the strong UV absorbance of 1+ precludes using UV spectroscopy to determine the concentration of BSA within the reverse micelles, it was necessary to separate any BSA present from the organic phase. To isolate BSA solubilized in the water pools, 1+ and the organic components were removed from a 4.0 cm3 reverse micelle solution using a combination of evaporation and subsequent dialysis against 0.1 mmol dm−3 phosphate buffer (pH 7.0). After dialysis, the volume of the retentate was adjusted to 5.0 cm3 with the addition of phosphate buffer. To this end, BSA was isolated from the organic solution. The UV spectrum of the retentate had the absorption peak at 280 nm that is typical of proteins containing aromatic amino acids. Because there was no absorbance in the visible region of the retentate’s spectrum, the Malachite Green derivative was completely removed from the sample and cannot be responsible for the absorbance in the UV region. Therefore the concentration of BSA was determined by the absorbance at 280 nm of the retentate. The yield of BSA transfer was based on the percentage of BSA extracted into the organic phase relative to the initial amount of BSA in the aqueous phase.
2. Experimental 2.4. Analytical methods 2.1. Materials Malachite Green derivative 1 was synthesized according to the literature [21]. Bovine serum albumin (BSA) was purchased from Sigma. Other materials were of analytical grade and were used without further purification.
The water content of the organic phase was determined by a Karl–Fisher titration. TEM samples were prepared by placing a drop of the reverse micelle solution onto a TEM grid and evaporating the organic solvent. TEM measurements were performed using the HITACHI HF-2000 electron microscope. The circular dichromism
Fig. 1. Conceptual representation of a photochemically induced reverse micelle formation and protein extraction.
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Fig. 2. Absorption spectra of 0.1 mmol dm−3 1 in chloroform/20% (v/v) 1-hexanol before (dark) and after (UV) UV irradiation for 30 min.
(CD) spectra of the native and the isolated BSA in the 0.1 mmol dm−3 phosphate buffer (pH 7.0) were measured with a J-820 spectropolarimeter (Jasco Corporation, Tokyo, Japan). The sample of isolated BSA was the retentate which was prepared for the extraction measurement. 3. Results and discussion 3.1. Photochemical reaction of Malachite Green derivative 1 Fig. 2 shows typical absorption spectra for a sample of 1 dissolved in chloroform/20% (v/v) 1-hexanol that was or was not irradiated. The UV-irradiated samples have a new absorption peak with a maximum near 600 nm, which is assigned to a transition associated with 1+ . The cation is stable as the absorption peak around 600 nm persists for more than 5 h. The ionization ratio—which is defined as AMGL /AMGO , where AMGL and AMGO are respectively the molar absorptivities, measured at 600 nm, for a reverse micelle sample and a Malachite Green oxalate sample in chloroform/20% (v/v) 1-hexanol mixtures—is 0.11 and is independent of the pH value and ionic strength of the aqueous solution.
Fig. 3. Water concentration in an organic solution versus the corresponding concentration of 1. The inset shows the semi-logarithmic plot. The average values for five measurements are plotted with error bars. Under dark conditions, 䊉; after UV irradiation, .
organic phase and aqueous 0.52 mmol dm−3 12-tungstophosphoric acid, as the contrast agent. The images have a spherical shape that is consistent with the shape expected for reverse micelles. We did not observe images of aggregated reverse micelles as reported by others [24]. The particle diameters range from 30 nm to 70 nm; a plot of the diameter distribution is shown in the supporting material. The size of reverse micelle has been reported to depend on the molar ratio of [H2 O]/[surfactant] and there have been many investigations of the reverse micellar size in various systems
3.2. Photochemically induced reverse micelle formation Fig. 3 is a plot of the amount of water solubilized in the organic solution as a function of the initial concentration of 1 in the organic phase. Because, it is expected that the water present in the organic phase will be that of the water pool, its concentration can be used to determine the critical micelle concentration (cmc) of the surfactant [23]. For our system, under dark conditions, the water concentration was practically independent of the concentration of 1. On the other hand, after UV irradiation, there was an increase in the water concentration of the organic phase that correlates with the concentration of 1 and was particularly noticeable when the concentration of 1 was greater than 5.0 mmol dm−3 . This photochemically induced increase in the water concentration indicates that the reverse micelle formation is enhanced by UV irradiation. The cmc value for UV-irradiated 1 was 1.9 mmol dm−3 , which corresponds to the ‘break point’ in a semi-logarithmic plot of the data shown in Fig. 3. Under dark conditions, a cmc value was not obtained because there was no clearly abrupt increase for the slope of the corresponding semi-logarithmic plot. TEM imaging provides strong evidence for the existence of reverse micelles. Fig. 4 shows a typical portion of a micrograph of the irradiated sample that contained 10 mmol dm−3 1 in the
Fig. 4. A transmission electron micrograph of reverse micelles whose interiors are stained with 12-tungstophosphoric acid. This UV-irradiated system contained 1 at a 10 mmol dm−3 concentration.
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Fig. 5. Effect of the concentration of 1 on BSA transfer. The average values for three measurements are plotted. Under dark conditions, 䊉; after UV irradiation, .
[25–27]. For example, Naoe and co-workers investigated the size of reverse micelle containing cetyltrimethylammonium bromide and 1-haxanol and then they have obtained the empirical equation: diameter (nm) = 3.2 + 0.17 [H2 O]/[surfactant] [25]. Using their equation and the ratio of [H2 O]/[surfactant] for our system containing 10 mmol dm−3 of 1, the diameter of the reverse micelle in our system is calculated to be 51 nm which are consistent with the results of Fig. 4. The detailed calculation and the difference in the calculated diameter by using the other equations are discussed in the supporting material. Spherically shaped particles were also found in TEM micrographs of a sample containing 10 mmol dm−3 1 that was not irradiated. However, the number of particles was considerably less than those found for irradiated samples. Consequently, any tendency for size distribution could not be obtained. A TEM micrograph of a control sample that did not contain 1 was absent of spherically shaped particles; therefore, 1 and 1+ are responsible for the TEM images and can both form reverse micelles, although the latter is the more effective agent. 3.3. Photo-induced transfer of BSA to reverse micelles In general, the attractive electrostatic interactions between a biopolymer solubilized in the water pool and the head groups of
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the surfactants will be the driving forces for the phase transition [28,29], which is the reason why BSA (67 kDa; pI = 4.7) and an aqueous solution buffered at pH 7.0 (0.1 mmol dm−3 phosphate buffer) were chosen for the system of cationic 1+ . Fig. 5 shows the transfer yield of BSA as a function of the concentration of 1. Under dark conditions, the transfer yield was small in all the concentration of 1 measured. On the other hand, UV irradiation afforded an increase in the transfer yield with the increase of concentration of 1. The BSA transfer did not occur at the concentration of 1 lower than its cmc (1.9 mmol dm−3 ). Furthermore, the photochemically induced transfer was significantly promoted at the concentration of 1 greater than 5.0 mmol dm−3 , where the water concentration in the organic phase was enhanced by UV irradiation (Fig. 3). The results of Figs. 3 and 5 lead to the conclusion that BSA in the aqueous phase was transferred to the water pool of the photochemically generated reverse micelle. The CD spectrum of the BSA retentate isolated from an irradiated sample was recorded and compared to a spectrum of native BSA. These spectra are shown in Fig. 6. Although the spectrum of the retentate is noisy (Fig. 6b) because the amount of BSA present is small, the two spectra are otherwise identical. The shape and intensity of the far UV region of a protein’s CD spectrum is a consequence, in large part, of the protein’s secondary structure; therefore, native BSA is present in the reverse micelle. We confirmed in a separate experiment that the aqueous solution isolated from a sample under dark conditions did not show any significant CD signal in the UV region (Supporting material). 4. Conclusion We demonstrated that 1+ , which is a cationic form of 1, a Malachite Green derivative, more readily forms reverse micelles than does the lipophilic 1 itself and that the reverse micelles can internalize BSA. The photochemically induced transfer of BSA was attained by UV irradiation on 1 at the concentration greater than its cmc. We plan on testing the physical limits of the reverse micelle system by examining its ability to incorporate proteins of different charges, mass, structure, etc. into its water pool. We also intend to explore the suitability of the water pool and the external nonpolar environments to facilitate enzymatic reactions for which the substrates are water-insoluble, e.g. linoleic acid, which is a substrate for lipoxygenase. Finally, we also plan to investigate the reverse reaction illustrated in Scheme 1, i.e. 1+ → 1 by heating, as a way to release protein trapped in the water pools into aqueous solution,
Fig. 6. CD spectra of 0.15 mmol dm−3 native BSA (a) and BSA isolated from reverse micelles (b). Both samples were in 0.1 mmol dm−3 phosphate. The reverse micelle system contained 1 at an initial concentration of 10 mmol dm−3 .
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