Photoresponse of an Aqueous Two-Phase System Composed of

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Photoresponse of an Aqueous Two-Phase System Composed of Photochromic Dextran Jun-ichi Edahiro, Kimio Sumaru,* Toshiyuki Takagi, Toshio Shinbo, and Toshiyuki Kanamori Research Center of AdVanced Bionics, National Institute of AdVanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8565, Japan ReceiVed February 1, 2006. In Final Form: April 28, 2006 A novel aqueous two-phase system, which exhibits a reversible photoinduced phase separation, has been developed with photochromic dextran synthesized by substituting 0.3 mol % of the hydroxyl groups with the photochromic chromophore, 6-nitrospiropyran (NSp). For an aqueous solution containing this photochromic dextran and poly(ethylene glycol), it was observed that the solution, which had been uniform in the dark, quickly separated into two phases through blue light irradiation within 1 min and returned to the former uniform state spontaneously after heating at 50 °C for 1 h. Photoisomerization of NSp was confirmed to shift the phase separation temperature of this aqueous two-phase system by up to 30 °C.

Many types of water-soluble polymer mixed aqueous solutions separate into two or more phases based on the affinity among the polymers and solvent, and these systems are called aqueous two-phase systems.1,2 Dextran is a hydrophilic polymer that has been intensively investigated as a component of aqueous twophase systems.1,2 Altered affinity with other polymers through chemical modification of dextran has been reported, as have changes in phase separation behavior.3 Thereupon, through the introduction of a functional group into dextran to change its hydrophilicity through an external stimulus, it is possible to control the phase separation behavior exhibited with other polymers in an aqueous two-phase system. Since light irradiation can be applied instantaneously only for the time required, it is a suitable means for an accurate and immediate manipulation method, and Vesperinas et al. reported that phase separation could be induced in a mixture of nonpolymeric surfactants in aqueous solution through the photolytic reaction of the component.4 On the other hand, in the research field of polymers, many researchers have been intensively studying photoresponsive polymers, the structures of which can be controlled by light irradiation.5-11 In these studies, photoresponses were reported to be reversible since the polymers were composed of photochromic chromophores, which exhibit reversible photochromism, as the photoresponsive components.12,13 * Corresponding author: E-mail: [email protected]. Phone: +81298-61-6373. Fax: +81-298-61-6278. (1) Albertsson, P. A. Partition of Cells, Particles and Macromolecules, 3rd ed.; John Wiley & Sons: New York, 1986. (2) Zaslavsky, B. Y. Aqueous Two-Phase Partitioning; Marcel Dekker: New York, 1995. (3) Zhang, J.; Pelton, R.; Wagberg, L. Colloid Polym. Sci. 1998, 276 (6), 476-482. (4) Vesperinas, A.; Eastoe, J.; Wyatt, P.; Grillo, I.; Heenan, R. K.; Richards, J. M.; Bell, G. A. J. Am. Chem. Soc. 2006, 128 (5), 1468-1469. (5) Irie, M.; Menju, A.; Hayashi, K. Macromolecules 1979, 12, 1176-1180. (6) Menju, A.; Hayashi, K.; Irie, M. Macromolecules 1981, 14, 755-758. (7) Irie, M.; Hosoda, M. Makromol. Chem., Rapid Commun. 1985, 6 (8), 533-536. (8) Irie, M.; Kunwatchakun, D. Macromolecules 1986, 19, 2476-2480. (9) Kroger, R.; Menzel, H.; Hallensleben, M. L. Macromol. Chem. Phys. 1994, 195 (7), 2291-2298. (10) Fissi, A.; Pieroni, O.; Angelini, N.; Lenci, F. Macromolecules 1999, 32, 7116-7121. (11) Sumaru, K.; Kameda, M.; Kanamori, T.; Shinbo, T. Macromolecules 2004, 37 (13), 4949-4955. (12) Shipway, A. N.; Willner, I. Acc. Chem. Res. 2001, 34 (6), 421-32. (13) Rosario, R.; Gust, D.; Hayes, M.; Jahnke, F.; Springer, J.; Garcia, A. A. Langmuir 2002, 18 (21), 8062-8069.

However, all of these results indicated light-induced structural changes in the photoresponsive polymer alone, and, until now, there have been no known examples in the literature of being able to control the affinity of photoresponsive and nonphotoresponsive polymers in an aqueous solution through irradiation with light. With the objective of establishing a new aqueous two-phase system in which phase separation behavior could be controlled using light, we modified dextran with a spiropyran chromophore in the synthesis of a new photoresponsive polymer and investigated how the phase separation behavior of a mixed aqueous solution of this modified dextran and poly(ethylene glycol) (PEG) changed upon irradiation by light that induced isomerization of the spiropyran. The synthesis of dextran modified with the photochromic chromophore 6-nitrospiropyran (NSp) is detailed in the Supporting Information. Absorbance and turbidity spectra were measured by using a spectrophotometer (V570, JASCO, Tokyo). In this photochromic dextran (NSp-dex, Figure 1), the substitution ratio of NSp was only 0.3 mol % of the total number of hydroxyl groups in dextran, which was calculated from the absorbance spectrum in dimethyl sulfoxide solution. As shown in Figure 1, in acidic conditions, the NSp contained in NSp-dex can essentially adopt one of two stable states: the open-ring state, known as the protonated merocyanine (McH) form, and the closed-ring state, known as the spiro (Sp) form.10 In neutral conditions, NSp-dex coagulated as a result of the hydrophobic NSp functioning as a cross-linking point,14 and we did not conduct further investigations. McH chromophore is yellow and Sp chromophore is colorless. In the case of a 0.30% w/w NSp-dex aqueous solution, absorbance at 408 nm (A408), which is attributed to the McH chromophore, is essentially saturated at an HCl concentration of 7.5 mM if the HCl concentration is raised. Because open-ring NSp has a much higher proton affinity than closed-ring NSp,15,16 this result strongly suggests that all of the NSp chromophores exist in the McH form at this HCl concentration. On the basis of this assumption, the proportion of McH as a percentage of all the NSp chromophores was estimated to be 96% in 3.0 mM HCl (14) Hirakura, T.; Nomura, Y.; Aoyama, Y.; Akiyoshi, K. Biomacromolecules 2004, 5 (5), 1804-1809. (15) Menger, F. M.; Perinis, M. Tetrahedron Lett. 1978, 19 (47), 4653-4656. (16) Sumaru, K.; Kameda, M.; Kanamori, T.; Shinbo, T. Macromolecules 2004, 37 (21), 7854-7856.

10.1021/la060318q CCC: $33.50 © 2006 American Chemical Society Published on Web 05/12/2006

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Figure 2. Changes in the external appearance of a 0.30% w/w NSp-dex, 7.0% w/w PEG, 3.0 mM HCl mixed aqueous solution in response to irradiation with light and temperature change: (a) at 25° C, (b) after 15 s of irradiation with blue light, (c) after being heated to 50° C, (d) after being kept at 50° C for an extended period of time, and (e) after being returned to 25° C.

Figure 1. Chemical structure of NSp-dex and the photoisomerization scheme of an NSp chromophore, and the absorbance spectra of 0.30% w/w NSp-dex in 3.0 mM HCl aqueous solution at 25 °C: the solid line is the result of the measurements of the sample prior to irradiation; the dashed line is the result of the measurements conducted immediately after the sample irradiation with blue light (436 nm).

aqueous solution. Upon irradiating this solution with blue light (436 nm, 30 mW) for 15 s, A408 declined conspicuously, and the yellow solution quickly changed to a colorless one; clearly, irradiation with light isomerizes the NSp chromophore to the Sp form. Because the absorbance at 450-500 nm, which is attributed to McH, is almost 0, all of the NSp chromophores are estimated to adopt the Sp form in this light-irradiated condition. Next, when the temperature of the solution after irradiation with light was set at 50 °C, A408 slowly increased, and after 1 h, the absorbance spectrum returned to almost the same profile as that prior to irradiation. These results indicate that NSp, in which ring closing has been induced through irradiation with light, reverts back to the McH chromophore in the dark under elevated temperature spontaneously. From the above results, the NSp chromophore contained in NSp-dex in an acidic aqueous solution switches from the McH to the Sp form upon irradiation with light (436 nm), and from the Sp to the McH form upon heating, thereby indicating the ability to change structure reversibly. Because only the McH chromophore carries a net positive charge, the interaction (affinity) between NSp-dex, water molecules, and other polymers is expected to change as a result of this light irradiation and temperature variation. To investigate the effect of the NSp isomerization on aqueous two-phase separation, a mixed aqueous solution of NSp-dex and PEG 6000 (Mw ) 6000, NBS Biologicals, Cambs, UK) in dilute HCl was irradiated with blue light followed by temperature elevation. In the experiments described below, the turbidity at 800 nm (T800) of the solution stirred intensively, where NSp does not absorb any light (Figure 1), was used as a measure of phase separation. In addition, the net absorbance at 408 nm (A408cor), which is calculated from A408 by subtracting the turbidity originating from light scattering, was used as an indicator of structural change in the NSp chromophore. The definition of the A408cor is detailed in the Supporting Information. A mixed aqueous solution of 0.30% w/w NSp-dex, 7.0% w/w PEG, and 3.0 mM HCl is transparently yellow at 25 °C and takes on a stable single-phase composition (Figure 2a). When the solution was irradiated for 15 s with blue light, the solution became turbid within 1 min after the onset of irradiation (Figure 2b), indicating that ring closing occurs in the NSp, and, at the

Figure 3. Net absorbance (A408cor) after subtracting the turbidity at 408 nm, which is attributed to the open-ring NSp chromophore, in a 0.30% w/w NSp-dex, 7.0% w/w PEG, 3.0 mM HCl mixed aqueous solution; the turbidity at 800 nm (T800), which is used as the measure of phase separation; and their changes with respect to irradiation with light and temperature change. Closed circles indicate A408cor, open circles indicate T800, and the dotted line indicates temperature. The labels a-e in the figure correspond to a-e in Figure 2. Absorbance and turbidity spectra are also shown for the cases (a) before photo-irradiation and (b) immediately after irradiation.

same time, phase separation is induced (Figure 3a,b). Next, when the phase-separated solution was heated to 50 °C, it changed to a colorless transparent solution (single-phase composition) within several minutes of the onset of heating (Figures 2c and 3c). If the solution was kept at a temperature of 50 °C for an extended period of time, thermally induced ring-opening gradually occurred in the NSp chromophore within 1 h and the A408cor value returned to close to its original value (Figure 2d and Figure 3d). In this case, even if the temperature was returned to 25 °C, phase separation was not induced (Figures 2e and 3e). From the above results, it is indicated that the induction of phase separation is strongly correlated with an increase in Sp chromophores in NSpdex. In addition, we confirmed experimentally that this system could repeat the phase-separation and the reset process at least

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Figure 4. Relationship between Tp (the phase separation temperature at which two-phase separation is induced from a uniform single phase) and PEG concentration for a mixed aqueous solution containing 0.30% w/w NSp-dex, 3.0 mM HCl, and PEG for (1) the photoirradiated (Sp-rich) state and (2) in the dark (McH-rich state): The solid circles show the Tp in each PEG concentration. Each circle is connected with a solid line; this is defined as the boundary between the conditions of one phase and two phases in this phase diagram. The diamonds show the state of the solution. The labels a-e correspond to a-e in Figure 2.

three times. Although we observed a slight change in the absorbance spectra suggesting some degeneration of NSp during each process, the possible repetition of the process was estimated from the data to be more than 10 times. Next, we investigated the effect of the PEG concentration on the aqueous two-phase separation. The NSp-dex concentration was fixed at 0.30% w/w and the HCl concentration was fixed at 3.0 mM in polymer mixed aqueous solutions, and the correlation between the PEG concentration and the phase separation temperature (Tp) was sought. Phase separation from a uniform state was observed as a rise in T800 because the solution was stirred intensively. The temperature of the mixed aqueous solution was lowered gradually while measuring T800; the temperature where T800 increased suddenly was defined as Tp. Figure 4 shows that the induction of aqueous two-phase separation is dependent on the PEG concentration. Because the higher the PEG concentration, the higher the Tp, phase separation occurs more readily at high PEG concentrations. On the other hand, phase separation is not observed for PEG concentrations of 5.0% w/w or less, even in the case of irradiation with light, and this indicates that a relatively high concentration of PEG is required for the phase separation. These results indicate that this phase separation phenomenon of the polymer mixed aqueous solution via irradiation with light is not attributed to simple coagulation of the NSp-dex: it is an aqueous two-phase separation based mainly on the photoinduced change in the interaction between the NSpdex and PEG. In Figure 4, which is a phase diagram of this aqueous twophase system, the results shown in Figures 2 and 3 can be explained on the basis of NSp isomerization. Under the initial conditions (a), the solution appeared clear in the dark at 25 °C, indicating that the state is in one-phase. When the solution was irradiated with light and changed into the Sp-rich state, the boundary between the conditions of one phase and two phases shifted toward the higher temperature and the solution became turbid, indicating that the state was in two phases (b). Next, when the solution was heated to 50 °C, it reverted back to one phase, even in the Sp-rich state (c). Under these conditions, the proportion of Sp as a percentage of all the NSp chromophore was estimated to be 67%. In this case, it was confirmed that phase separation was once again induced through a return to 25 °C. When the solution was kept at a temperature of 50 °C for

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1 h, NSp chromophore changed to the McH-rich state. In this case, even if the temperature was returned to 25 °C, phase separation was not induced (d,e) because the boundary shifted toward the lower temperature. Furthermore, to investigate the mechanism of the photoinduced phase separation phenomenon, the concentrations of the polymer components in each separated phase were analyzed. The measuring method is detailed in the Supporting Information. It was found that 15.5% w/w of the overall NSp-dex introduced into the polymer mixed aqueous solution was in the lower phase, although this phase accounted for only 1% of the overall weight. This means that the NSp-dex was concentrated in the lower phase in a high ratio of 15 during the phase separation. In contrast, PEG was distributed almost equally in both the upper and lower phases after phase separation. These results suggest the phase separation mechanism under the aforementioned conditions in which NSpdex’s affinity to PEG decreases upon irradiation with light. Finally, we discuss the advantages of controlling the phase separation of an aqueous two-phase system by light irradiation as described above. Phase separation can be induced in this system at room temperature (25 °C) by irradiating with light without any need for a change in temperature; photoirradiation shifts the phase separation temperature (Tp) by up to 30 °C. The energy required for photoisomerization of NSp in 1 L of the aqueous solution containing 0.30% w/w NSp-dex was calculated roughly at 0.1 kJ when quantum efficiency is 50%. This is equivalent to 0.07% of the thermal energy that is required for changing the temperature of the same solution by 30 °C. In this system, aqueous two-phase separation can be induced by photoirradiation, the photon energy of which is less than a thousandth part of the thermal energy that is required for thermally induced phase separation. Further, after blue light irradiation, the phase-separated solution and the Sp-rich state of NSp in NSp-dex can be maintained in the dark at room temperature for a considerably long time (half a day). On the other hand, through heating at 50 °C for 1 h, the phase-separated solution changes to a uniform single phase, and it can thus be reset to the state it was in prior to irradiation. As discussed above, we have developed a novel aqueous two-phase system, whose phase separation can be controlled reversibly through two external stimuli, namely, irradiation with light and temperature variation. As a conclusion, a novel aqueous two-phase system that exhibits a reversible photoinduced phase separation has been developed with dextran modified with photochromic NSp and PEG. This system, which exists in a uniform, single phase at room temperature (25 °C) in the dark, separates into two aqueous phases through blue light irradiation and returns to the former uniform state spontaneously after heating at 50 °C in the dark. Photoisomerization of the NSp introduced to the dextran is confirmed to shift the phase separation temperature of this system by up to 30 °C. This research is strongly expected to provide important knowledge for the development of applications for photofunctional polymers. Acknowledgment. This work was supported by the 2005 Industrial Technology Research Grant Program of the New Energy Development Organization (NEDO) of Japan, and the 2005 Creation and Support Program for Startups from Universities of the Japan Science and Technology Agency. Supporting Information Available: Synthesis of Nsp-dex, complete definition of A408cor, and a description of the process used to determine the composition of each phase after photo-induced separation. This material is available free of charge via the Internet at http://pubs.acs.org. LA060318Q