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Disruption of reverse micelles and release of trapped ribonuclease A photochemically induced by Malachite Green leuconitrile derivative
Journal of Colloid and Interface Science 355 (2011) 448–452
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Disruption of reverse micelles and release of trapped ribonuclease A photochemically induced by Malachite Green leuconitrile derivative Ryoko M. Uda ⇑, Tsuyoshi Nishikawa, Yoshitsugu Morita 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 5 August 2010 Accepted 16 December 2010 Available online 19 December 2010 Keywords: Reverse micelle Disruption Malachite Green Photochemical control Enzyme transfer
a b s t r a c t Photoinduced disruption of a sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelle is triggered by a Malachite Green leuconitrile derivative (MGL). UV irradiation of MGL solubilized in an AOT-waterchloroform mixture creates a cationic surfactant that interacts electrostatically with the anionic AOT. We investigated the disruption of the reverse micelle by using proton nuclear magnetic resonance spectroscopy and found that UV irradiation of MGL decreases the number of water molecules solubilized in the interior of the AOT reverse micelles. Furthermore, the photoinduced disruption of the reverse micelle is shown to release ribonuclease A, which is trapped in the water in the interior of the AOT reverse micelle. This photoinduced release may offer a desirable transport system of biopolymers. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Reverse micelles are aggregates of amphiphilic molecules wherein the polar headgroups are oriented toward the core of the reverse micelle and the hydrocarbon tails are extended outward toward a bulk nonaqueous solvent. Water contained in the core of the reverse micelles can accommodate a range of hydrophilic molecules such as amino acids [1,2], peptides [3,4], and proteins [5–7], which are thus protected from denaturation. Therefore, reverse micelles have attracted significant attention owing to their ability to carry biomolecules. Indeed, if reverse micelles perform as a functional transport system, then controlling the load and release of biomolecules is a crucial consideration. Therefore, several investigations on the effects of water concentration [8,9], temperature [10], and ionic strength [11] on reverse-micelle construction have been conducted. Alternatively, the use of light as a stimulus is promising, because it does not require any change in the system composition or thermodynamic conditions. Furthermore, light offers an attractive method to couple temporal and spatial control. These advantages of using light have created considerable interest in the control of colloidal materials. If a photoresponsive compound is incorporated into the reverse micellar carrier [12–17], selective photochemical transport of the contents can be induced by light [12].
⇑ Corresponding author. Address: Department of Chemical Engineering, Nara National College of Technology, Yata 22, Yamato-koriyama, Nara 639-1080, Japan. Fax: +81 743 55 6169. E-mail address: [email protected] (R.M. Uda). 0021-9797/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2010.12.047
Although photochemical control of the percolation of reverse micelles by using photoresponsive additives such as a stilbene derivative has been reported [15], an even more effective change in the molecular state can be achieved through charge generation on the amphiphilic molecules, which is attributed to the fact that their amphiphilic nature will be directly affected. Considering this possibility, we synthesized a photoionizable Malachite Green leuconitrile derivative carrying a long alkyl chain (MGL) [18,19]. The head group of MGL is sufficiently nonpolar under dark conditions. However, ionized MGL exhibits both hydrophilic and hydrophobic properties because of its triphenylmethyl cation and its long alkyl chain, respectively, resulting in photogenerated amphiphilicity (Scheme 1). The photogenerated electrical charge on the head group is expected to provide assemblies having a strong effect. The purpose of this study is to examine the MGL-triggered photoinduced disruption of the reverse micelles and the transfer of encapsulated enzymes into an aqueous phase. Our implementation of the disruption of the reverse micelle depends on investigations on the transfer of proteins encapsulated in sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles, which is performed by adding a counterionic surfactant [20,21]. The transfer mechanism is possibly caused by electrostatic interaction between oppositely charged surfactant molecules, which leads to the collapse of the reverse micelle. In the present system, MGL is mixed with anionic AOT reverse micelles. As illustrated in Fig. 1, MGL is solubilized into the organic solvent under dark conditions. After undergoing UV irradiation, MGL transforms into a cationic surfactant that interacts with the anionic AOT, which results in the disruption of the AOT reverse micelles. At this point the enzyme encapsulated in the reverse micelle is expected to be transferred into the
R.M. Uda et al. / Journal of Colloid and Interface Science 355 (2011) 448–452
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agitated in a vortex mixer for 60 s. The resulting phases were separated by centrifugation at 4000 rpm for 5 min. Agitation and centrifugation were performed subsequent to these procedures unless otherwise noted. The aqueous solution was sodium phosphate buffer (pH 5.0, 0.1 M) containing 10 g/L of RNase A. The reverse micelle encapsulating RNase A was obtained by removing the upper aqueous phase. The concentration of RNase A in the reverse micellar phase was determined by absorbance at 274 nm to be ca. 0.9 g/ L.
UV
2.3. Release of RNase A from AOT reverse micelle triggered by MGL
Hydrophobic group Hydrophilic group Scheme 1. Photogenerated amphiphilicity in Malachite Green derivative carrying a long alkyl chain.
Aqueous phase
2.4. Preparation of reverse micelle for 1H NMR measurements -
-
+
-
-
+
-
+ +
-
+
- - -
-
-
-
- - -
-
+
-
-
+ +
- - -
UV
-
- - -
- - -
-
Aqueous solution
- - -
-
MGL
- - -
RNase A
- - -
A predefined amount of MGL was dissolved in the reverse micellar phase, trapping RNase A. The resulting solution was then mixed with an equal volume (typically 2.0 mL) of sodium phosphate buffer (pH 8.0, 0.1 M). The aqueous solution into which RNase A is released has a higher pH than that of the encapsulated RNase A because the reverse micellar phase containing MGL underwent non-photochemical ionization under acidic conditions. The sample was irradiated or left undisturbed in the dark conditions for 5 min, after which it was agitated and centrifuged. The UV light source (k < 300 nm) was a 500-W xenon lamp equipped with a photoguide tube and a Toshiba UV-D33S filter. The upper aqueous phase was used for CD spectroscopy.
Organic phase
AOT reverse micelle
MGL
Ionized MGL
Fig. 1. Conceptual representation of disruption of AOT reverse micelles and release of RNase A triggered by photoionized Malachite Green leuconitrile derivative (MGL).
aqueous phase. Ribonuclease A (RNase A) was selected as the enzyme for this study because the attractive electrostatic interactions between the head groups of AOT and RNase A in the interior water at pH < pI is the driving force for the encapsulation [22]. Furthermore, the interest in ribonucleases has recently increased because of their reported cytotoxic activity against tumor cells, which may have potential in medical applications such as anticancer therapy [23,24]. Thus, we report herein the release of RNase A from AOT reverse micelles by MGL triggered photochemically. Proton nuclear magnetic resonance (1H NMR) spectroscopy was used to characterize the effect of MGL on reverse micelle disruption. The structure of the released RNase A was investigated using circular dichroism (CD) spectroscopy. 2. Materials and methods 2.1. Materials MGL was synthesized according to the literature [19]. Cetyltrimethylammonium chloride (CTAC) was recrystallized from tetrahydrofuran. RNase A (Type I-A) was purchased from Sigma (St. Louis, MO, USA). Other materials were of analytical grade and were used without further purification. 2.2. Preparation of reverse micelle encapsulating RNase A Chloroform solution containing 1.0 mM AOT and an equal volume (typically 4.5 mL) of an aqueous solution were mixed and
The reverse micellar solution was prepared in CDCl3 (0.03 volume% tetramethylsilane) solution containing various amounts of AOT. An equal volume (typically 1.0 mL) of sodium phosphate buffer (pH 5.0, 0.1 M) was added to the solution and the mixture was agitated and centrifuged. After removing the upper aqueous phase, the lower organic phase (CDCl3 solution) was used to determine the critical micelle concentration (cmc). To investigate the effect of the AOT-cationic surfactant complex, a reverse micellar phase prepared from 1.0 mM of AOT was used. MGL was then added and the reverse micellar phase was mixed with an equal volume of sodium phosphate buffer (pH 8.0, 0.1 M). Irradiation was performed as described for releasing RNase A, followed by agitation and centrifugation. For the samples of AOT–CTAC, a micellar phase prepared from 1.0 mM of AOT was mixed with an equal volume of sodium phosphate buffer (pH 8.0, 0.1 M) containing CTAC at various concentrations. The resulting bottom phase was used after removing the upper aqueous phase. 2.5. Analytical methods The experiment was conducted at 25 °C. 1H NMR spectra were acquired using a JNM-270 instrument (JOEL, Japan) operating at 270.05 MHz. The peaks were referenced with respect to tetramethylsilane (d = 0.000 ppm), which was used as an internal standard. The CD spectra of the native and the released RNase A in phosphate buffer (pH 8.0, 0.1 M) were measured with a J-820 spectropolarimeter (Jasco Corporation, Japan). 3. Results and discussion 3.1. Photoionization of MGL Fig. 2 shows typical absorption spectra for MGL dissolved in chloroform saturated with sodium phosphate buffer (pH 5.0, 0.1 M). The irradiated samples exhibit a new absorption peak with a maximum near 600 nm, which is assigned to an ionized MGL. The cation is considered stable because the absorption peak at around 600 nm persists for over more than 3 h. The ionization ratio is
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1.76
0.4
δobs (H2O)/ppm
Absorbance
1.74
UV 0.2
1.72 1.7 1.68
1.66
Dark 0 400
500
1.64
600
700
800
Fig. 2. Absorption spectra of 5.0-mM MGL in chloroform saturated with sodium phosphate buffer (pH 5.0, 0.1 M) before (dark) and after (UV) UV irradiation for 5 min. The light path length is 1 mm.
defined as AMGL/AMGO, where AMGL and AMGO are the molar absorptivities at 600 nm of an MGL sample after irradiation and that of a Malachite Green oxalate sample in chloroform saturated with sodium phosphate buffer (pH 5.0, 0.1 M), respectively. The ionization ratio is 0.004, which is independent of the MGL concentration in the range 5–70 mM. 3.2. Effect of AOT–CTAC complex on reverse micelle disruption Prior to irradiation of AOT reverse micelles containing MGL, it is important to understand the effect of the AOT complex with cationic surfactant on the disruption of reverse micelles. This effect was investigated using CTAC, which has a hydrophobic chain having the same length as ionized MGL does, and is thus a satisfactory model of ionized MGL. 1H NMR was used to characterize the effect of the AOT–CTAC complex on reverse micelle disruption. Fig. 3 shows representative 1H NMR spectrum for the AOT reverse micellar system. The magnetic resonance of water protons exhibits a single peak, indicating a rapid exchange between water associated with monomer surfactant molecules (free water) and water molecules solubilized into the micelle interior. At low AOT concentrations, the observed chemical shift is similar to that of free water. As the concentration increases, the water begins to solubilize into the micelle core leading to a downfield chemical shift of water protons because of which their resonance approaches the value of bulk water at higher AOT concentrations (see Supplementary material). Fig. 4 shows the chemical shift of the water protons
H2O
AOT AOT (CH2) (CH3)
4.5
4
3.5
3
2.5
2
1.5
0
0.2
0.4
0.6
0.8
1
1.2
[CTAC]/mM
Wavelength/nm
1
0.5 ppm
Fig. 3. 1H NMR spectrum of reverse micelles formed by 2.0 mM AOT. Sodium phosphate buffer (pH 5.0, 0.1 M) was used for sample preparation.
Fig. 4. Chemical shift of H2O protons as a function of CTAC concentration in CDCl3 solution containing 1.0 mM AOT. Experimental errors in the chemical shift were within 0.01 ppm.
in CDCl3 solution containing AOT (1.0 mM) and CTAC at various concentrations. The AOT concentration of 1.0 mM is over the cmc (0.8 mM: determination of the cmc is included in the Supplementary material), and the chemical shift corresponds to the reverse micelle carrying core water at 0 mM CTAC. The addition of CTAC to the AOT reverse micellar solution causes an upfield chemical shift of the water protons, which results from the decrease in the number of water molecules solubilized into the micelle interior. Therefore, from Fig. 4 it can be understood that the reverse micelle is disrupted by the formation of the AOT–CTAC complex. The chemical shift shows an abrupt decrease up to 0.2 mM of CTAC, above which it decreases gradually. The abrupt decrease agrees with a 1:1 complex of AOT and CTAC. AOT is neutralized by adding CTAC to the reverse micellar solution, and accordingly the reverse micelle concentration decreases with increasing CTAC concentration. When the CTAC concentration exceeds 0.2 mM, noncomplexing AOT is considered to be less than 0.8 mM, which corresponds to the cmc. Below this critical value, AOT remains in the monomeric form with which water is associated. Therefore, the chemical shift gradually decreases with increasing CTAC concentration.
3.3. Disruption of AOT reverse micelles caused by photoionization of MGL The effect of MGL on AOT reverse micelles is also assessed via the magnetic resonance peak of the water protons. Fig. 5 shows the chemical shift of water protons in CDCl3 solution containing AOT reverse micelles (1.0 mM) and MGL at various concentrations. Irradiation of MGL in AOT reverse micelles decreases the chemical shift with increasing MGL concentration. Because MGL alone causes the downfield chemical shift of the water protons (see Supplementary material), the upfield chemical shift must result from the decrease in the number of water molecules solubilized into the micelle interior. Moreover, the upfield chemical shift of the AOT-ionized MGL system agrees with that of the AOT–CTAC system (Fig. 4). In other words, irradiation of MGL disrupts the reverse micelles owing to the formation of the AOT-ionized MGL complex, as is the case for the AOT–CTAC complex. By comparing Figs. 5 with 4 we can evaluate the effect of ionized MGL on the disruption of AOT reverse micelles. For example, the chemical shift of water protons was observed at 1.70 ppm for the irradiated sample containing 50 mM of MGL (Fig. 5). This chemical shift corresponds to that obtained upon adding 0.1 mM CTAC (Fig. 4). Because the MGL photoionization ratio is 0.004, the
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20
1.76
Normalized ellipticity/mdeg
Dark 1.74
1.7
1.76
δ (H2O)/ppm
H 2O)/ppm
1.72
1.68
1.66
UV
1.74
1.72
0
0.1
0
20
-20
-40 -60
-80 -100 200
10
[MGL]/mM
1.64
0
210
220
230
240
250
Wavelength/nm 40
60
Fig. 6. CD spectra of 0.11 mg L1 native RNase A (thick solid line) and released RNase A from AOT reverse micelles triggered by irradiation of 50-mM MGL (broken line). Both samples were in sodium phosphate buffer (pH 8.0, 0.1 M).
[MGL]/mM Fig. 5. Chemical shift of H2O protons as a function of MGL concentration in CDCl3 solution containing 1.0 mM AOT. The inset shows a semilogarithmic plot for MGL concentration <10 mM. Experimental errors in the chemical shift were within 0.01 ppm. Key: under dark conditions, closed squares; after UV irradiation, open squares.
concentration of ionized MGL is considered to be 0.2 mM for a system containing 50 mM of MGL. This means that the concentration of ionized MGL effective in disrupting the reverse micelles is less than the actual concentration. The reduced concentration is possibly due to the neutral MGL, which can act as a co-surfactant contributing toward the formation of the AOT reverse micelles. The AOT reverse micelles consequently persist under irradiated conditions, assisted by neutral MGL, which does not undergo photoionization. No chemical shift of water protons for MGL concentrations exceeding 40 mM was obtained under dark conditions because the magnetic resonance peaks of OCCH2C of the MGL alkyl chain, which was observed at 1.71–1.81 ppm, overlaps those of water, effectively hiding it in the spectrum. Although the intensity of the MGL peaks was strong for concentrations ranging from 40 to 60 mM, the water peak was distinguished from the MGL peaks under irradiated conditions because the water peak exhibited sufficient upfield shift, and thus separated itself from the MGL peaks.
Fig. 7 shows the percentage of released RNase A as a function of MGL concentration. Under dark conditions, the percentage released is low for all the measured MGL concentrations. However, UV irradiation results in an increase in the percentage released with increasing MGL concentration. We also examined the effect of CTAC on the release of encapsulated RNase A. By adding the equimolar cationic surfactant, the percentage released was 84%, which agrees with the reported value for RNase A released from AOT reverse micelles using counterionic surfactants [20]. In contrast to these systems, wherein AOT is completely neutralized by cationic surfactants, the MGL system exhibits a maximum release of 32% (Fig. 7). Therefore, the unreleased RNase A remains in the reverse micellar solution in the MGL system; otherwise, it is denatured and precipitates in between the organic and aqueous solutions so that it avoids detection by ellipticity at 210 nm. The percentage released at 60 mM has the maximum concentration of MGL, above which the percentage released decreases. Although the reason for the maximum percentage is not obvious, we anticipate that the denaturation of RNase A is the cause of this phenomenon. Ionized MGL plays a role in the disruption of AOT micelles, and it can affect RNase A to denature itself.
3.4. Photoinduced release of RNase A from AOT reverse micelles
%release ¼
RNase A released to aqueous phase RNase A encapsulated in reverse micelle
ð1Þ
RNase A release/%
40
CD spectra in the far-UV region of the released RNase A from AOT reverse micelles were measured and compared to the native RNase A. The CD spectra of all samples were the same as that of native RNase A: a representative spectrum is shown in Fig. 6. Because the CD spectrum in the far-UV region reflects the secondary structure of a protein, we conclude that the secondary structure of the released RNase A remains unchanged after the procedures of encapsulation and release. We also examined the reaction of the released RNase A with RNA, and found that the released RNase A exhibits enzyme activity (see Supplementary material). Therefore, the activity of RNase A suggests that the cyanide ion, which is released after irradiation of MGL (Scheme 1), does not inhibit the enzyme reaction of RNase A. The concentration of the released RNase A was determined by ellipticity at 210 nm by using the calibration curves prepared for native RNase A. The percentage released was calculated using the following equation.
30
20
UV
10
Dark 0
0
20
40
60
80
[MGL]/mM Fig. 7. Release of encapsulated RNase A plotted as a function of MGL concentration. Experimental errors in the release percentage were within 3%. Key: under dark conditions, closed squares; after UV irradiation, open squares.
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4. Summary
Appendix A. Supplementary material
We demonstrated that MGL undergoes photoionization and induces the disruption of the AOT reverse micelle, thereby triggering the release of encapsulated RNase A. This is a novel method to achieve the photoinduced release of an enzyme encapsulated in a reverse micelle. Photoinduced destabilization of an AOT water-in-oil microemulsion was performed by the photolysis of azosulfonate surfactant [13,14]. The photochemical control of the macroemulsion was also been investigated using an azobenzene derivative grafted to poly(sodium acrylate) [16] and a mixture of an azobenzene containing amphiphile and a block-copolymer micelle [17]. Although such studies have indicated the importance of photoresponsive molecules, few studies have focused on the targeted delivery using a photoresponsive reverse micelle. Seno and colleagues studied the photochemically controlled extraction of amino acids from reverse micelles [12], but, there are no such studies that use biopolymers. By comparing with other published studies, we conclude that the reverse micellar system presented herein offers the important understanding that a change in the net charge of the surfactant is effective in disrupting reverse micelle and results in a triggered release of the encapsulated enzyme. Moreover, if electrostatic interaction is preferred between the head groups of the reverse micelle and biopolymer, this system can be applied to a variety of biopolymers. For wider application, future studies will investigate the molecular design of MGL thereby avoiding the release of the cyanide ion after irradiation.
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jcis.2010.12.047.
Acknowledgments This work was supported by Grants-in-Aid for Scientific Research from the Japan Society of the Promotion of Science (No. 22750135). The authors thank Dr. K. Kimura for helpful discussions concerning the results.
References [1] E.B. Leodidis, T.A. Hatton, J. Phys. Chem. 94 (1990) 6400. [2] M. Adachi, M. Harada, A. Shioi, Y. Sato, J. Phys. Chem. 95 (1991) 7925. [3] L.M. Gierasch, J.E. Lacy, K.F. Thompson, A.L. Rockwell, P.I. Watnick, Biophys. J. 37 (1982) 275. [4] K.F. Thompson, L.M. Gierasch, J. Am. Chem. Soc. 106 (1984) 3648. [5] P.L. Luisi, Angew. Chem., Int. Ed. Engl. 24 (1985) 439. [6] F.M. Menger, K. Yamada, J. Am. Chem. Soc. 101 (1979) 6731. [7] J.S. Thompson, H. Gehring, B.L. Vallee, Natl. Acad. Sci. USA 77 (1980) 132. [8] G.A. Krei, H. Hustedt, Chem. Eng. Sci. 47 (1992) 99. [9] R. Hilhorst, M. Sergeeva, D. Heering, P. Rietveld, P. Fijneman, R.B.G. Wolbert, M. Dekker, B.H. Bijsterbosch, Biotechnol. Bioeng. 46 (1995) 375. [10] P.L. Luisi, M. Giomini, M.P. Pileni, B.H. Robinson, Biochim. Biophys. Acta 947 (1988) 209. [11] B.A. Keiser, D. Varie, R.E. Barden, S.L. Holt, J. Phys. Chem. 83 (1979) 1276. [12] M. Ino, H. Tanaka, J. Otsuki, K. Araki, M. Seno, Colloid Polym. Sci. 272 (1994) 151. [13] J. Eastoe, M.S. Dominguez, H. Cumber, P. Wyatt, R.K. Heenan, Langmuir 20 (2004) 1120. [14] J. Eastoe, M.S. Dominguez, H. Cumber, G. Burnett, P. Wyatt, Langmuir 19 (2003) 6579. [15] T. Wolff, D. Nees, Prog. Colloid Polym. Sci. 111 (1998) 113. [16] I. Porcar, P. Perrin, C. Tribet, Langmuir 17 (2001) 6905. [17] N. Ma, Y. Wang, B. Wang, Z. Wang, X. Zhang, Langmuir 23 (2007) 2874. [18] R.M. Uda, M. Oue, K. Kimura, Chem. Lett. 33 (2004) 586. [19] R.M. Uda, K. Kimura, Bull. Chem. Soc. Jpn. 78 (2005) 1862. [20] S. Jarudilokkul, L.H. Poppenborg, D.C. Stuckey, Biotechnol. Bioeng. 62 (1999) 593. [21] K. Shiomori, Y. Kawano, R. Kuboi, I. Komasawa, J. Chem. Eng. Jpn. 32 (1999) 177. [22] M.J. Pires, M.R. Aires-Barros, J.M.S. Cabral, Biotechnol. Prog. 12 (1996) 290. [23] S.M. Mikulski, A. Viera, A. Deptala, Z. Darzynkiewicz, Int. J. Oncol. 13 (1998) 633. [24] P.A. Leland, R.T. Ranies, Chem. Biol. 8 (2001) 405.
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Disruption of reverse micelles and release of trapped ribonuclease A photochemically induced by Malachite Green leuconitrile derivative Ryoko M. Uda ⇑, Tsuyoshi Nishikawa, Yoshitsugu Morita 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 5 August 2010 Accepted 16 December 2010 Available online 19 December 2010 Keywords: Reverse micelle Disruption Malachite Green Photochemical control Enzyme transfer
a b s t r a c t Photoinduced disruption of a sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelle is triggered by a Malachite Green leuconitrile derivative (MGL). UV irradiation of MGL solubilized in an AOT-waterchloroform mixture creates a cationic surfactant that interacts electrostatically with the anionic AOT. We investigated the disruption of the reverse micelle by using proton nuclear magnetic resonance spectroscopy and found that UV irradiation of MGL decreases the number of water molecules solubilized in the interior of the AOT reverse micelles. Furthermore, the photoinduced disruption of the reverse micelle is shown to release ribonuclease A, which is trapped in the water in the interior of the AOT reverse micelle. This photoinduced release may offer a desirable transport system of biopolymers. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Reverse micelles are aggregates of amphiphilic molecules wherein the polar headgroups are oriented toward the core of the reverse micelle and the hydrocarbon tails are extended outward toward a bulk nonaqueous solvent. Water contained in the core of the reverse micelles can accommodate a range of hydrophilic molecules such as amino acids [1,2], peptides [3,4], and proteins [5–7], which are thus protected from denaturation. Therefore, reverse micelles have attracted significant attention owing to their ability to carry biomolecules. Indeed, if reverse micelles perform as a functional transport system, then controlling the load and release of biomolecules is a crucial consideration. Therefore, several investigations on the effects of water concentration [8,9], temperature [10], and ionic strength [11] on reverse-micelle construction have been conducted. Alternatively, the use of light as a stimulus is promising, because it does not require any change in the system composition or thermodynamic conditions. Furthermore, light offers an attractive method to couple temporal and spatial control. These advantages of using light have created considerable interest in the control of colloidal materials. If a photoresponsive compound is incorporated into the reverse micellar carrier [12–17], selective photochemical transport of the contents can be induced by light [12].
⇑ Corresponding author. Address: Department of Chemical Engineering, Nara National College of Technology, Yata 22, Yamato-koriyama, Nara 639-1080, Japan. Fax: +81 743 55 6169. E-mail address: [email protected] (R.M. Uda). 0021-9797/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2010.12.047
Although photochemical control of the percolation of reverse micelles by using photoresponsive additives such as a stilbene derivative has been reported [15], an even more effective change in the molecular state can be achieved through charge generation on the amphiphilic molecules, which is attributed to the fact that their amphiphilic nature will be directly affected. Considering this possibility, we synthesized a photoionizable Malachite Green leuconitrile derivative carrying a long alkyl chain (MGL) [18,19]. The head group of MGL is sufficiently nonpolar under dark conditions. However, ionized MGL exhibits both hydrophilic and hydrophobic properties because of its triphenylmethyl cation and its long alkyl chain, respectively, resulting in photogenerated amphiphilicity (Scheme 1). The photogenerated electrical charge on the head group is expected to provide assemblies having a strong effect. The purpose of this study is to examine the MGL-triggered photoinduced disruption of the reverse micelles and the transfer of encapsulated enzymes into an aqueous phase. Our implementation of the disruption of the reverse micelle depends on investigations on the transfer of proteins encapsulated in sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles, which is performed by adding a counterionic surfactant [20,21]. The transfer mechanism is possibly caused by electrostatic interaction between oppositely charged surfactant molecules, which leads to the collapse of the reverse micelle. In the present system, MGL is mixed with anionic AOT reverse micelles. As illustrated in Fig. 1, MGL is solubilized into the organic solvent under dark conditions. After undergoing UV irradiation, MGL transforms into a cationic surfactant that interacts with the anionic AOT, which results in the disruption of the AOT reverse micelles. At this point the enzyme encapsulated in the reverse micelle is expected to be transferred into the
R.M. Uda et al. / Journal of Colloid and Interface Science 355 (2011) 448–452
449
agitated in a vortex mixer for 60 s. The resulting phases were separated by centrifugation at 4000 rpm for 5 min. Agitation and centrifugation were performed subsequent to these procedures unless otherwise noted. The aqueous solution was sodium phosphate buffer (pH 5.0, 0.1 M) containing 10 g/L of RNase A. The reverse micelle encapsulating RNase A was obtained by removing the upper aqueous phase. The concentration of RNase A in the reverse micellar phase was determined by absorbance at 274 nm to be ca. 0.9 g/ L.
UV
2.3. Release of RNase A from AOT reverse micelle triggered by MGL
Hydrophobic group Hydrophilic group Scheme 1. Photogenerated amphiphilicity in Malachite Green derivative carrying a long alkyl chain.
Aqueous phase
2.4. Preparation of reverse micelle for 1H NMR measurements -
-
+
-
-
+
-
+ +
-
+
- - -
-
-
-
- - -
-
+
-
-
+ +
- - -
UV
-
- - -
- - -
-
Aqueous solution
- - -
-
MGL
- - -
RNase A
- - -
A predefined amount of MGL was dissolved in the reverse micellar phase, trapping RNase A. The resulting solution was then mixed with an equal volume (typically 2.0 mL) of sodium phosphate buffer (pH 8.0, 0.1 M). The aqueous solution into which RNase A is released has a higher pH than that of the encapsulated RNase A because the reverse micellar phase containing MGL underwent non-photochemical ionization under acidic conditions. The sample was irradiated or left undisturbed in the dark conditions for 5 min, after which it was agitated and centrifuged. The UV light source (k < 300 nm) was a 500-W xenon lamp equipped with a photoguide tube and a Toshiba UV-D33S filter. The upper aqueous phase was used for CD spectroscopy.
Organic phase
AOT reverse micelle
MGL
Ionized MGL
Fig. 1. Conceptual representation of disruption of AOT reverse micelles and release of RNase A triggered by photoionized Malachite Green leuconitrile derivative (MGL).
aqueous phase. Ribonuclease A (RNase A) was selected as the enzyme for this study because the attractive electrostatic interactions between the head groups of AOT and RNase A in the interior water at pH < pI is the driving force for the encapsulation [22]. Furthermore, the interest in ribonucleases has recently increased because of their reported cytotoxic activity against tumor cells, which may have potential in medical applications such as anticancer therapy [23,24]. Thus, we report herein the release of RNase A from AOT reverse micelles by MGL triggered photochemically. Proton nuclear magnetic resonance (1H NMR) spectroscopy was used to characterize the effect of MGL on reverse micelle disruption. The structure of the released RNase A was investigated using circular dichroism (CD) spectroscopy. 2. Materials and methods 2.1. Materials MGL was synthesized according to the literature [19]. Cetyltrimethylammonium chloride (CTAC) was recrystallized from tetrahydrofuran. RNase A (Type I-A) was purchased from Sigma (St. Louis, MO, USA). Other materials were of analytical grade and were used without further purification. 2.2. Preparation of reverse micelle encapsulating RNase A Chloroform solution containing 1.0 mM AOT and an equal volume (typically 4.5 mL) of an aqueous solution were mixed and
The reverse micellar solution was prepared in CDCl3 (0.03 volume% tetramethylsilane) solution containing various amounts of AOT. An equal volume (typically 1.0 mL) of sodium phosphate buffer (pH 5.0, 0.1 M) was added to the solution and the mixture was agitated and centrifuged. After removing the upper aqueous phase, the lower organic phase (CDCl3 solution) was used to determine the critical micelle concentration (cmc). To investigate the effect of the AOT-cationic surfactant complex, a reverse micellar phase prepared from 1.0 mM of AOT was used. MGL was then added and the reverse micellar phase was mixed with an equal volume of sodium phosphate buffer (pH 8.0, 0.1 M). Irradiation was performed as described for releasing RNase A, followed by agitation and centrifugation. For the samples of AOT–CTAC, a micellar phase prepared from 1.0 mM of AOT was mixed with an equal volume of sodium phosphate buffer (pH 8.0, 0.1 M) containing CTAC at various concentrations. The resulting bottom phase was used after removing the upper aqueous phase. 2.5. Analytical methods The experiment was conducted at 25 °C. 1H NMR spectra were acquired using a JNM-270 instrument (JOEL, Japan) operating at 270.05 MHz. The peaks were referenced with respect to tetramethylsilane (d = 0.000 ppm), which was used as an internal standard. The CD spectra of the native and the released RNase A in phosphate buffer (pH 8.0, 0.1 M) were measured with a J-820 spectropolarimeter (Jasco Corporation, Japan). 3. Results and discussion 3.1. Photoionization of MGL Fig. 2 shows typical absorption spectra for MGL dissolved in chloroform saturated with sodium phosphate buffer (pH 5.0, 0.1 M). The irradiated samples exhibit a new absorption peak with a maximum near 600 nm, which is assigned to an ionized MGL. The cation is considered stable because the absorption peak at around 600 nm persists for over more than 3 h. The ionization ratio is
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1.76
0.4
δobs (H2O)/ppm
Absorbance
1.74
UV 0.2
1.72 1.7 1.68
1.66
Dark 0 400
500
1.64
600
700
800
Fig. 2. Absorption spectra of 5.0-mM MGL in chloroform saturated with sodium phosphate buffer (pH 5.0, 0.1 M) before (dark) and after (UV) UV irradiation for 5 min. The light path length is 1 mm.
defined as AMGL/AMGO, where AMGL and AMGO are the molar absorptivities at 600 nm of an MGL sample after irradiation and that of a Malachite Green oxalate sample in chloroform saturated with sodium phosphate buffer (pH 5.0, 0.1 M), respectively. The ionization ratio is 0.004, which is independent of the MGL concentration in the range 5–70 mM. 3.2. Effect of AOT–CTAC complex on reverse micelle disruption Prior to irradiation of AOT reverse micelles containing MGL, it is important to understand the effect of the AOT complex with cationic surfactant on the disruption of reverse micelles. This effect was investigated using CTAC, which has a hydrophobic chain having the same length as ionized MGL does, and is thus a satisfactory model of ionized MGL. 1H NMR was used to characterize the effect of the AOT–CTAC complex on reverse micelle disruption. Fig. 3 shows representative 1H NMR spectrum for the AOT reverse micellar system. The magnetic resonance of water protons exhibits a single peak, indicating a rapid exchange between water associated with monomer surfactant molecules (free water) and water molecules solubilized into the micelle interior. At low AOT concentrations, the observed chemical shift is similar to that of free water. As the concentration increases, the water begins to solubilize into the micelle core leading to a downfield chemical shift of water protons because of which their resonance approaches the value of bulk water at higher AOT concentrations (see Supplementary material). Fig. 4 shows the chemical shift of the water protons
H2O
AOT AOT (CH2) (CH3)
4.5
4
3.5
3
2.5
2
1.5
0
0.2
0.4
0.6
0.8
1
1.2
[CTAC]/mM
Wavelength/nm
1
0.5 ppm
Fig. 3. 1H NMR spectrum of reverse micelles formed by 2.0 mM AOT. Sodium phosphate buffer (pH 5.0, 0.1 M) was used for sample preparation.
Fig. 4. Chemical shift of H2O protons as a function of CTAC concentration in CDCl3 solution containing 1.0 mM AOT. Experimental errors in the chemical shift were within 0.01 ppm.
in CDCl3 solution containing AOT (1.0 mM) and CTAC at various concentrations. The AOT concentration of 1.0 mM is over the cmc (0.8 mM: determination of the cmc is included in the Supplementary material), and the chemical shift corresponds to the reverse micelle carrying core water at 0 mM CTAC. The addition of CTAC to the AOT reverse micellar solution causes an upfield chemical shift of the water protons, which results from the decrease in the number of water molecules solubilized into the micelle interior. Therefore, from Fig. 4 it can be understood that the reverse micelle is disrupted by the formation of the AOT–CTAC complex. The chemical shift shows an abrupt decrease up to 0.2 mM of CTAC, above which it decreases gradually. The abrupt decrease agrees with a 1:1 complex of AOT and CTAC. AOT is neutralized by adding CTAC to the reverse micellar solution, and accordingly the reverse micelle concentration decreases with increasing CTAC concentration. When the CTAC concentration exceeds 0.2 mM, noncomplexing AOT is considered to be less than 0.8 mM, which corresponds to the cmc. Below this critical value, AOT remains in the monomeric form with which water is associated. Therefore, the chemical shift gradually decreases with increasing CTAC concentration.
3.3. Disruption of AOT reverse micelles caused by photoionization of MGL The effect of MGL on AOT reverse micelles is also assessed via the magnetic resonance peak of the water protons. Fig. 5 shows the chemical shift of water protons in CDCl3 solution containing AOT reverse micelles (1.0 mM) and MGL at various concentrations. Irradiation of MGL in AOT reverse micelles decreases the chemical shift with increasing MGL concentration. Because MGL alone causes the downfield chemical shift of the water protons (see Supplementary material), the upfield chemical shift must result from the decrease in the number of water molecules solubilized into the micelle interior. Moreover, the upfield chemical shift of the AOT-ionized MGL system agrees with that of the AOT–CTAC system (Fig. 4). In other words, irradiation of MGL disrupts the reverse micelles owing to the formation of the AOT-ionized MGL complex, as is the case for the AOT–CTAC complex. By comparing Figs. 5 with 4 we can evaluate the effect of ionized MGL on the disruption of AOT reverse micelles. For example, the chemical shift of water protons was observed at 1.70 ppm for the irradiated sample containing 50 mM of MGL (Fig. 5). This chemical shift corresponds to that obtained upon adding 0.1 mM CTAC (Fig. 4). Because the MGL photoionization ratio is 0.004, the
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20
1.76
Normalized ellipticity/mdeg
Dark 1.74
1.7
1.76
δ (H2O)/ppm
H 2O)/ppm
1.72
1.68
1.66
UV
1.74
1.72
0
0.1
0
20
-20
-40 -60
-80 -100 200
10
[MGL]/mM
1.64
0
210
220
230
240
250
Wavelength/nm 40
60
Fig. 6. CD spectra of 0.11 mg L1 native RNase A (thick solid line) and released RNase A from AOT reverse micelles triggered by irradiation of 50-mM MGL (broken line). Both samples were in sodium phosphate buffer (pH 8.0, 0.1 M).
[MGL]/mM Fig. 5. Chemical shift of H2O protons as a function of MGL concentration in CDCl3 solution containing 1.0 mM AOT. The inset shows a semilogarithmic plot for MGL concentration <10 mM. Experimental errors in the chemical shift were within 0.01 ppm. Key: under dark conditions, closed squares; after UV irradiation, open squares.
concentration of ionized MGL is considered to be 0.2 mM for a system containing 50 mM of MGL. This means that the concentration of ionized MGL effective in disrupting the reverse micelles is less than the actual concentration. The reduced concentration is possibly due to the neutral MGL, which can act as a co-surfactant contributing toward the formation of the AOT reverse micelles. The AOT reverse micelles consequently persist under irradiated conditions, assisted by neutral MGL, which does not undergo photoionization. No chemical shift of water protons for MGL concentrations exceeding 40 mM was obtained under dark conditions because the magnetic resonance peaks of OCCH2C of the MGL alkyl chain, which was observed at 1.71–1.81 ppm, overlaps those of water, effectively hiding it in the spectrum. Although the intensity of the MGL peaks was strong for concentrations ranging from 40 to 60 mM, the water peak was distinguished from the MGL peaks under irradiated conditions because the water peak exhibited sufficient upfield shift, and thus separated itself from the MGL peaks.
Fig. 7 shows the percentage of released RNase A as a function of MGL concentration. Under dark conditions, the percentage released is low for all the measured MGL concentrations. However, UV irradiation results in an increase in the percentage released with increasing MGL concentration. We also examined the effect of CTAC on the release of encapsulated RNase A. By adding the equimolar cationic surfactant, the percentage released was 84%, which agrees with the reported value for RNase A released from AOT reverse micelles using counterionic surfactants [20]. In contrast to these systems, wherein AOT is completely neutralized by cationic surfactants, the MGL system exhibits a maximum release of 32% (Fig. 7). Therefore, the unreleased RNase A remains in the reverse micellar solution in the MGL system; otherwise, it is denatured and precipitates in between the organic and aqueous solutions so that it avoids detection by ellipticity at 210 nm. The percentage released at 60 mM has the maximum concentration of MGL, above which the percentage released decreases. Although the reason for the maximum percentage is not obvious, we anticipate that the denaturation of RNase A is the cause of this phenomenon. Ionized MGL plays a role in the disruption of AOT micelles, and it can affect RNase A to denature itself.
3.4. Photoinduced release of RNase A from AOT reverse micelles
%release ¼
RNase A released to aqueous phase RNase A encapsulated in reverse micelle
ð1Þ
RNase A release/%
40
CD spectra in the far-UV region of the released RNase A from AOT reverse micelles were measured and compared to the native RNase A. The CD spectra of all samples were the same as that of native RNase A: a representative spectrum is shown in Fig. 6. Because the CD spectrum in the far-UV region reflects the secondary structure of a protein, we conclude that the secondary structure of the released RNase A remains unchanged after the procedures of encapsulation and release. We also examined the reaction of the released RNase A with RNA, and found that the released RNase A exhibits enzyme activity (see Supplementary material). Therefore, the activity of RNase A suggests that the cyanide ion, which is released after irradiation of MGL (Scheme 1), does not inhibit the enzyme reaction of RNase A. The concentration of the released RNase A was determined by ellipticity at 210 nm by using the calibration curves prepared for native RNase A. The percentage released was calculated using the following equation.
30
20
UV
10
Dark 0
0
20
40
60
80
[MGL]/mM Fig. 7. Release of encapsulated RNase A plotted as a function of MGL concentration. Experimental errors in the release percentage were within 3%. Key: under dark conditions, closed squares; after UV irradiation, open squares.
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4. Summary
Appendix A. Supplementary material
We demonstrated that MGL undergoes photoionization and induces the disruption of the AOT reverse micelle, thereby triggering the release of encapsulated RNase A. This is a novel method to achieve the photoinduced release of an enzyme encapsulated in a reverse micelle. Photoinduced destabilization of an AOT water-in-oil microemulsion was performed by the photolysis of azosulfonate surfactant [13,14]. The photochemical control of the macroemulsion was also been investigated using an azobenzene derivative grafted to poly(sodium acrylate) [16] and a mixture of an azobenzene containing amphiphile and a block-copolymer micelle [17]. Although such studies have indicated the importance of photoresponsive molecules, few studies have focused on the targeted delivery using a photoresponsive reverse micelle. Seno and colleagues studied the photochemically controlled extraction of amino acids from reverse micelles [12], but, there are no such studies that use biopolymers. By comparing with other published studies, we conclude that the reverse micellar system presented herein offers the important understanding that a change in the net charge of the surfactant is effective in disrupting reverse micelle and results in a triggered release of the encapsulated enzyme. Moreover, if electrostatic interaction is preferred between the head groups of the reverse micelle and biopolymer, this system can be applied to a variety of biopolymers. For wider application, future studies will investigate the molecular design of MGL thereby avoiding the release of the cyanide ion after irradiation.
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jcis.2010.12.047.
Acknowledgments This work was supported by Grants-in-Aid for Scientific Research from the Japan Society of the Promotion of Science (No. 22750135). The authors thank Dr. K. Kimura for helpful discussions concerning the results.
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