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Novel Preparation Method for Obtaining pH-Responsive Core-Shell Microcapsule Reactors Kazuki Akamatsu and Takeo Yamaguchi* Department of Chemical System Engineering, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
We have developed a new procedure using the plasma-graft polymerization method to investigate the preparation of core-shell enzyme-loaded microcapsule reactors with environment-responsive membranes. The conventional methods used to prepare this type of microcapsule lead to the deactivation of enzymes; therefore, we employed a novel preparation method to prevent any loss of enzymatic activity. First, coreshell microcapsules were prepared by combining a Shirasu porous glass membrane emulsification step and an interfacial polymerization step. The “bottle-in” method was used to load the microcapsules with glucose oxidase, and the “plasma-graft polymerization” method was used to fill in the pores of the shell membranes with a copolymer of N-isopropylacrylamide and acrylic acid to achieve a pH-responsive gating function. The plasma operation in this step was conducted on the enzyme-loaded microcapsules, and this formed environmentresponsive gates while maintaining the enzymatic activity. These microcapsule reactors were composed of a porous shell membrane that had a pH-responsive gating function that controlled the rate of reaction occurring in the core, and this pH-responsive reactivity was achieved by the gating function’s ability to recognize the pH of the medium from the change in diffusivity of the pH-responsive polymer gates at different pHs. 1. Introduction Immobilized biocatalysts have many advantages compared to untreated natural biocatalysts: (1) they can be used in a continuous process; (2) they can be recovered and reused after the reaction has taken place; (3) their activity or durability can be enhanced using appropriate preparation conditions; and (4) the most appropriate immobilization form can be chosen according to the desired process from the many available immobilization forms available, such as membranes, gels, or beads. Since Chang first prepared enzyme-loaded microcapsules,1 many attempts to form biocatalyst-loaded microcapsules2-9 for drug delivery systems (DDSs), bioreactors, or artificial cells have been reported. In regard to microcapsule reactors with immobilized enzymes, a variety of microcapsule reactors with different structures have been proposed and employed by many researchers.10-14 Some of these have been environment-responsive-type reactors, responding to changes in temperature,15,16 pH,17,18 a specific ion,19 or glucose concentration.20,21 These abilities were acquired by some form of immobilization of an environment-responsive polymer. However, a severe problem occurs in the preparation of environment-responsive microcapsule reactors using conventional methods: the deactivation of enzymes, cells, or microorganisms. This problem is usually attributed to the preparation method used. However, microcapsule systems have been developed by trial and error. In general, enzyme-loaded microcapsules are prepared by (1) an interfacial polymerization method, (2) an in-liquid drying method, or (3) a phase separation method. Each of these preparation methods involves many factors that can lead to the deactivation of enzymes. The organic solvent and surfactants used to stabilize emulsions have a deleterious effect on enzymes, and the amino groups of an enzyme can react with the monomers used to form the capsule * To whom correspondence should be addressed. E-mail: yamag@ chemsys.t.u-tokyo.ac.jp. Tel.: +81-3-5841-7345. Fax: +81-3-58417227.
walls. Furthermore, it is very difficult to deposit an environmentresponsive gating function on enzyme-loaded microreactor systems without deactivating the enzyme. Thus, a process for preparing enzyme-loaded microcapsules that does not deactivate enzymes is desirable, because there are many applications for this type of microcapsule reactor. Therefore, we have proposed a new procedure to prevent enzymatic deactivation. The first stage is the preparation of the microcapsule. Then, the enzymes are loaded into the microcapsules, and an environment-responsive gate polymer is grafted onto the microcapsule using plasma-graft polymerization. As far as we are aware, the use of this type of plasma operation on enzymes in the solid state has not previously been employed. Indeed, there have been many reports of surface modification using charged groups employing base-activated chemistry,22 using polymers to obtain an environment-responsive ability employing the UV-graft method,23-25 or using the radiationinduced graft polymerization method.26 In previous studies, we determined that both grafting a stimulus-responsive polymer and filling the pore using plasma-graft polymerization were important for achieving an on-off permeability behavior of solutes across an environment-responsive membrane.15,16,19 Therefore, this method is a suitable candidate for the preparation of an environment-responsive microcapsule reactor without loss of any enzymatic activity, on the condition that the enzyme can retain its original activity. To elucidate the efficiency of our proposed method, we have used it to prepare a pH-responsive microcapsule reactor system that can recognize changes in pH in a medium and, accordingly, can convert chemical signals. For this system, we selected acrylic acid (AA), N-isopropylacrylamide (NIPAM), and glucose oxidase (GOD) to form the pH-responsive device, gating device, and reaction device, respectively. Figure 1 shows the concept of our pH-responsive microcapsule reactor. After preparation of the microcapsule structure, the materials were integrated in the core-shell microcapsule space, and GOD was loaded inside the core space. The pores in the shell layer were then filled with poly(N-isopropylacrylamide)-co-AA linear grafted chains
10.1021/ie060857q CCC: $37.00 © 2007 American Chemical Society Published on Web 12/06/2006
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Figure 1. Schematic representation of our pH-responsive core-shell microcapsule reactor system. The microcapsule reactor is composed of a core-shell microcapsule with a porous shell membrane and a grafted pH-responsive linear polymer in the pores, with the enzymes being loaded in the core. The pH-responsive polymer grafted in the pores acts as a pH gate: the polymer is hydrated at pH 5.0 and dehydrated at pH 4.0. Thus, this microcapsule reactor has a high rate of reaction at high pH because of the high diffusivity of the solute across the hydrophilic gate polymer. Conversely, the reactor has a low rate of reaction at pH 4.0 because of the low diffusivity of the solute across the hydrophobic gate polymer, i.e., this system is the microcapsule reactor that can sense the pH of its medium and change its rate of reaction using the pH-sensitive gate polymer controlling the diffusion of the solute.
acting as the pH-responsive gates using the plasma-graft polymerization method.27 Poly(N-isopropylacrylamide) (PNIPAM) is a popular temperature-responsive polymer, because it shows a very sharp phase transition at 32 °C. In addition to this property, a significant feature of P-NIPAM is the ability to tailor its lower critical solution temperature (LCST) by incorporating suitable comonomers. AA is a well-known vinyl monomer that has ionizable carboxyl groups and is sensitive to its environment, as the degree of ionization of carboxylic acid is determined by the pH of the environment. Consequently, the copolymer poly(NIPAM-co-AA) (P-NIPAM-co-AA) is a pHresponsive phase transition polymer.28-30 Judging from the properties discussed above, this type of microcapsule reactor is expected to function as shown in Figure 1. When dispersed in a medium at pH 5.0, the P-NIPAM-coAA gate polymer is hydrophilic, so the microcapsule reactor allows glucose to penetrate into the core space and a reaction with GOD takes place. On the other hand, when dispersed in a medium at pH 4.0, the P-NIPAM-co-AA gate polymer becomes hydrophobic, and the microcapsule reactor prevents glucose penetration into the core space, so that the reaction with GOD does not proceed. Thus, it can be said that this system is a microcapsule reactor that can sense slight changes in pH and change its rate of reaction accordingly by using its pHresponsive gates to control the diffusion of the solute. This function is realized only when a microcapsule reactor with this type of structure is prepared. In this article, we discuss the efficiency of our proposed method and the performance of the microcapsule reactors prepared using our method. 2. Experimental Section 2.1. Materials. Terephthaloyl dichloride (TDC) was purchased from Tokyo Kasei Kogyo Co. Ltd., Tokyo, Japan. β-D(+)-Glucose used was purchased from Sigma Chemicals Corp., St. Louis, MO. Glucose oxidase was from Aspergillus sp. and was purchased from Toyobo Co. Ltd., Osaka, Japan. Ethylenediamine (EDA), sodium dodecyl sulfate (SDS), poly(vinyl alcohol) (PVA, average molecular weight of 123000, 8690% hydrolysis), benzene, xylene, sodium carbonate, and acrylic acid (AA) were purchased from Wako Pure Chemical Industries Ltd., Osaka, Japan. The AA was used after distillation, and all other chemicals were used as received without further purifica-
Figure 2. Schematic illustration of the preparation of the monodisperse core-shell microcapsules. In the first step, an O/W emulsion containing TDC monomer with a narrow size distribution was prepared using the SPG membrane emulsification technique. In the second step, EDA monomer was added to the emulsion to form a polyamide shell membrane using the interfacial polymerization technique.
tion. N-isopropylacrylamide (NIPAM) was kindly provided by the Kohjin Co. Ltd., Fuji, Japan, and was used after being purified by recrystallization from hexane and acetone and then dried under vacuum at room temperature. 2.2. Preparation of the Core-Shell Microcapsules. Monodisperse oil-in-water (O/W) emulsions containing TDC monomer were first prepared using the Shirasu porous glass (SPG) membrane emulsification method. In this step, the SPG membrane (SPG Technology Co. Ltd., Miyazaki, Japan) and the SPG membrane emulsification kit (Kiyomoto Iron Works Co. Ltd., Miyazaki, Japan) shown in Figure 2 were used. The SPG membrane was a hollow-fiber type of membrane, with an outer diameter of 10 mm and a pore size of 11.9 µm. A volume of 10 mL of the organic mixture [benzene/xylene ) 2:1 (v/v)] that was 1.5 M in the TDC monomer was pressurized using nitrogen gas to a continuous phase. This continuous phase consisted of 150 mL of water with 0.50 wt % SDS and 0.50 wt % PVA. The pressure at which the emulsion particles formed on the surface of the SPG membrane was 0.01-0.02 MPa, and membrane emulsification began after stirring. Next, the monodisperse core-shell microcapsules were prepared using the interfacial polymerization method. A volume of 20 mL of water that was 1.18 M in sodium carbonate and 32 mL of EDA monomer were added to the emulsion. The polymerization time was 5 min. Then, the microcapsules were separated by centrifugation and washed three times to remove any emulsifier, stabilizer, and unreacted monomer.
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of glucose in the medium was measured using a glucose concentration analyzer (GLU-12, TOA Electronics Ltd., Tokyo, Japan), and the permeability coefficient, P, was calculated using the following equation derived from Fick’s first law of diffusion15,16
P)
Figure 3. Schematic illustration of the apparatus for adding a pH-responsive gating function to the microcapsules by the plasma-graft polymerization method. Enzyme-loaded and freeze-dried microcapsules were treated with radio-frequency plasma (30 W, 60 s) under argon atmosphere, and then the microcapsules were immersed in the monomer solution for 1 day.
The microcapsules were observed using a field-emission scanning electron microscope (FE-SEM, model S-900, Hitachi, Tokyo, Japan) and analyzed using a Micromeritics adsorption apparatus (ASAP-2010, Shimadzu Corporation, Kyoto, Japan) to determine their morphology. 2.3. Loading GOD into the Microcapsules: The “Bottlein” Method. In our work, the bottle-in method was used to load GOD into the microcapsules. The bottle-in method refers to the infusion of GOD into the microcapsules by diffusion. A GOD solution was prepared using an acetate buffer at pH 4.6, and the microcapsules were immersed in the GOD solution after the solution had been degassed. After GOD had been infused into the microcapsules, the microcapsules were washed repeatedly with acetate buffer and dispersed in an alkaline solution to remove any GOD activity left on the outer surface of the microcapsules. 2.4. Plasma-Graft Polymerization for Adding a pH-Gating Function to the Microcapsules. To confirm the possibility of grafting NIPAM-AA onto the organic material, plasma-graft polymerization was employed according to a previously published method.27 The apparatus used for plasma-graft polymerization is shown in Figure 3. A 5 wt % NIPAM-AA solution was prepared using deionized water and acetate buffer at pH 4.6. A porous polyethylene film (supplied by Asahi Chemical Co. Ltd., Tokyo, Japan) was used as the test organic material to enable easy examination and confirmation of the grafting behavior using Fourier transform infrared (FTIR) spectroscopy (Magna IR 560 with a Nic-Plan microscope, Thermo Electron Corporation, Waltham, MA). After confirmation of the graft polymerization of NIPAM-AA, a pH-gating function was added to the GOD-loaded microcapsules by this method. Enzymeloaded and freeze-dried microcapsules were placed into the glass tube, and about one-seventh of the glass tube (by volume) was occupied with the microcapsules. They were treated with plasma under argon atmosphere to generate activated points, after which the monomer solution was added to the microcapsules, and then the mixture was kept at 30 °C for 1 day to allow the graft reaction to proceed. The plasma power used was 30 W, and the treatment time was 60 s. 2.5. Performance of the pH-Gating Function. To check the performance of the pH-responsive P-NIPAM-co-AA gate polymer, P-NIPAM-co-AA-grafted microcapsules formed using plasma-graft polymerization were dialyzed against an aqueous glucose solution with a known concentration. The concentration
VsVm A(Vs + Vm)t
log
(
)
C f - Ci C f - Ct
where Ci, Ct, and Cf are the initial concentration, an intermediate concentration at time t, and the final concentration, respectively, of glucose in the surrounding medium. The parameters Vm and A are the total volume and the total surface area of microcapsules, respectively, and Vs is the volume of the surrounding medium. Our experiments were carried out at pH 4.0 and pH 5.0 at 40 °C. The pH of the medium was adjusted using acetate buffer. 2.6. Effect of Plasma-Graft Polymerization on GOD. To examine the influence of the plasma-graft polymerization conditions on GOD, the activities of (1) solid GOD with no treatment, (2) solid GOD treated with an Ar plasma, and (3) solid GOD treated with an Ar plasma and then immersed in the NIPAM-AA monomer solution for a period of 1 day were measured as a function of time in terms of the concentration of dissolved oxygen (DO). This is because the reaction catalyzed by GOD is as follows
glucose + O2 f glucono-δ-lactone + H2O2 The concentration of DO was measured using a DO meter (DO24P; TOA Electronics Ltd., Tokyo, Japan). For activities 1 and 2, the activity was measured 1 h after the preparation of the GOD solution with acetate buffer, and for activity 3, the activity of the solution was measured after the treatment described above had been carried out. The plasma treatment of the solid enzymes was conducted using the same apparatus as in the case of grafting pH-responsive polymers shown in Figure 3. The plasma power used for activities 2 and 3 was 30 W, and the treatment time was 60 s. These conditions were the same as those used in the plasma-graft polymerization method. 2.7. Performance of the pH-Responsive Microcapsule Reactor. P-NIPAM-co-AA was grafted onto the pores of the shell layer of the GOD-loaded core-shell microcapsules using the procedure described. The microcapsule reactors were dispersed in a beaker, and glucose was then added. The apparent activity of the microcapsules was measured at pH 4.0 and pH 5.0 at 40 °C. The apparent activity was also calculated from the concentration of DO, using the method described above. The pH in the medium was adjusted using acetate buffer. 3. Results and Discussion 3.1. Preparation of Core-Shell Microcapsules. CCD camera observations showed that monodisperse O/W emulsions containing the TDC monomer were successfully prepared using the SPG membrane emulsification method. In addition, monodisperse microcapsules were successfully prepared using the interfacial polymerization method. Figure 4 shows FE-SEM micrographs of the general morphology and cross-sectional views of the outer and inner surfaces of the microcapsules. The SPG membrane emulsification technique provided emulsions with a narrow size distribution because of the uniform, controlled pores of the SPG membranes.31 Furthermore, the average emulsion particle size was determined by the pore size
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Figure 5. Relationship between the inclusion time and the inclusion amount of GOD using the bottle-in method. The prepared core-shell microcapsules were immersed in a GOD solution under vacuum, and the GOD diffused into the microcapsules. Figure 4. FE-SEM micrographs of core-shell microcapsules prepared using a two-step SPG membrane emulsification and interfacial polymerization method: (a) general morphology, (b) cross-sectional view, (c) outer surface, and (d) inner surface. Table 1. Structural Parameters of the Microcapsules Determined by FE-SEM Observations and BET Surface Area Measurements parameter
value
diameter (µm) shell thickness (µm) BET surface area (m2/g) average pore diameter (nm)
42 1 13 16
of the SPG membrane, as there is a linear relationship between the membrane pore size and the average emulsion size.31 In our two-step microcapsule preparation method, the microcapsules were prepared from monodisperse and size-controlled emulsions, which led to monodisperse and size-controlled microcapsules. This monodispersity was also verified experimentally in our previous study.16 We confirmed the monodispersity of the prepared emulsions statistically by Coulter counter analysis, and FE-SEM observations also revealed the monodispersity.16 As can be seen in Figure 4, the diameter of our microcapsules was approximately 42 µm. The microcapsules had a porous shell membrane and a core space. The shell membrane had larger pores at the outermost surface as can be seen in Figure 4; however, many smaller pores exist in the inner layer of the shell membrane as a result of the membrane formation mechanism. The formation of a complicated porous structure probably resulted from the mechanism of the polymerization-induced phase separation, and further studies are required to clarify this complicated pore formation mechanism. From the above findings, we can conclude that monodisperse and size-controlled core-shell microcapsules were prepared using our two-step method. In addition, the structures obtained are suitable for microcapsule reactors with enzyme-loaded cores and a pH-responsive porous shell membrane, because the larger pores of untreated microcapsules allow easy enzyme incursion and, once the pores are filled with polymers to work the pHresponsive gating function, larger molecules such as enzymes can never escape from the microcapsules. The structural parameters of the microcapsules determined by the FE-SEM observations and BET surface area measurements are shown in Table 1. 3.2. Loading GOD into the Microcapsules: The Bottle-in Method. From dynamic light scattering (DLS) data, Kamyshny et al. determined that the size of GOD is 7 ( 1 nm.32 As shown in Table 1, the average pore diameter of our microcapsules was 16 nm, and the thickness of the shell of the microcapsules was 1 µm. Therefore, it is reasonable to suppose that GOD could pass through the shell membrane. This prediction is supported
by the pore model proposed by Nakao et al.,33 which shows that the diffusion coefficient of GOD in a shell membrane is 0.32 times larger than its diffusion coefficient in a dilute solution and that a period of a few minutes is sufficient to load GOD into microcapsules using dialysis. Figure 5 shows the relationship between the inclusion time and inclusion amount. The bottle-in method required a period of only 30 min to load GOD. In our work, one unit led to the formation of 1 µmol of hydrogen peroxide per minute at pH 4.6 and 40 °C, and in this case, the pores of the microcapsules had not been modified by plasma-graft polymerization, so the pores were completely open and it was believed that substrate transport was not the time-limiting step. In general, when enzyme-loaded microcapsules are prepared from W/O emulsions and contain enzymes in their cores, the interfacial polymerization method is employed to form core-shell microcapsules, and in almost all cases, the reported experimental conditions result in a deactivation of the enzymes.34 Using the interfacial polymerization method, enzyme-loaded microcapsules are prepared as follows: First, a W/O emulsion containing the monomer and the enzymes is prepared, and to this emulsion is added a solution of the second monomer to form the membrane shell by interfacial polymerization. However, this preparation method has several disadvantages for maintaining the activity of the enzyme. For example, the surfactants used to stabilize the emulsion can cause an inactivation of the enzyme, and during the interfacial polymerization stage, the amino groups of the enzymes can react with the monomers that have to form the shell of the microcapsules. That is, conventional methods involve many means by which inactivation of enzymes can occur, and to avoid this problem, the most suitable method for maintaining the activity of an enzyme has been reached through a trial-and-error process. On the other hand, our bottle-in method is an effective strategy for preparing enzyme-loaded microcapsules with no deactivation problems. 3.3. Performance of the pH-Gating Polymers Prepared Using Plasma-Graft Polymerization. The FTIR spectra of the samples showed peaks occurring at 1650 and 1550 cm-1, which are characteristic of NIPAM, and a peak occurring at 1720 cm-1, which is characteristic of AA. This indicates that the P-NIPAMco-AA had successfully grafted, as has been reported in earlier studies using other substrates or monomers.15,16,20,27,35-40 Indeed, the peroxide method36,40 or Ce4+-ion-initiated grafting methods41,42 can be employed to prepare pH-responsive gates; however, both of these methods result in the deactivation of the enzyme. In the peroxide method, the grafting reaction is carried out at 80 °C. As enzymes are proteins, such high temperatures denature the enzyme. In addition, some heavy metals are known to be enzyme inhibitors. In contrast, the
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Figure 6. Plot of log[(Cf - Ci)/(Cf - Ct)] versus time at different pH levels to measure the release of glucose from the microcapsules. The microcapsules had grafted pH-responsive P-NIPAM-co-AA gate polymers in the pores of the shell membranes. The pH-responsive microcapsules had a known concentration of glucose in their cores and were dispersed in media at different pHs. The concentration of glucose in each medium was measured with time. The temperature was kept at 40 °C, and the pH was adjusted using an acetate buffer.
Figure 7. Effect of the operating conditions during plasma-graft polymerization on the activity of GOD: (1) activity of natural GOD, (2) activity of GOD after Ar plasma exposure, and (3) activity of the GOD after the supply of the monomer solution.
plasma-graft polymerization reaction proceeds at 30 °C and does not require the presence of any heavy metals. In addition, environment-responsive membranes prepared using the plasmagraft polymerization method are stable,36 because this technique can chemically immobilize functional polymers on the porous membranes, rather than just physically immobilizing them. Thus, we conclude that the plasma-graft polymerization method is one of the best techniques for forming pH-responsive gates in porous microcapsules. Figure 6 shows the effects of pH on the permeability of glucose from the P-NIPAM-co-AA-grafted microcapsules. The grafting time was 1 day. The permeability was calculated from the gradient of each line. At pH 5.0, the permeability of glucose was 2.6 times larger than that at pH 4.0. This difference resulted from the change in hydrophobicity between pH 4.0 and pH 5.0; that is, at pH 4.0, the gate polymers were hydrophobic, so it was difficult for glucose to diffuse into the gate polymers across the shell. However, at pH 5.0, the gate polymers became hydrophilic, and so, it was easy for glucose to diffuse into the gate polymers across the shell. Thus, the P-NIPAM-co-AA polymers grafted using the plasma-graft polymerization worked as pH-responsive gates for substrate penetration, resulting in different diffusivities of glucose at the different pHs. This permeability ratio increases when a substrate with a higher molecular weight is used, as has been reported by Chu et al.15 3.4. Effect of Plasma-Graft Polymerization on the Activity of GOD. Figure 7 shows the effects of the operating conditions during plasma-graft polymerization on the activity of GOD. The activity shown is relative to that of untreated GOD. In this work, the activity was defined at pH 4.6 and 40 °C. Neither the Ar
Figure 8. Amount of oxygen consumed by the pH-responsive microcapsule reactor with time. The GOD-loaded and P-NIPAM-co-AA-grafted microcapsule reactors were dispersed in media at different pHs, and the apparent activity was measured. The temperature was kept at 40 °C, and the pH was adjusted using an acetate buffer.
plasma treatment in the solid state nor the supply of the monomer solution led to the deactivation of the GOD. To the best of our knowledge, there has been one report on plasma exposure to enzymes. The plasma-initiated polymerization method was employed by Osada et al.43 to invertase-trapped fibers and membranes without any loss of activity of invertase. However, Osada et al.43 conducted the plasma operation on an enzyme solution and not on enzymes in the solid state, as shown in this study for the first time. On the other hand, the effect of γ-ray irradiation on some enzyme solutions has been reported by Maeda et al.44 After irradiation with 1.0 × 106 rad of γ-rays, invertase and β-galactosidase maintained >80% of their original activities. However, after irradiation with 4.0 × 106 rad of γ-rays, the activities of invertase and β-galactosidase decreased to about 60% of their original values. Plasma treatment is known to be a useful process for surface modification, and active species, such as electrons, cations, anions, and radicals, in the plasma gas can attack only the surface of a sample. In addition, the important feature of plasma treatment is that the activated surface returns to its untreated status with time. In contrast, the activated layer of a substrate after γ-ray irradiation is generally deeper. It seems that this feature of γ-ray irradiation brings about the deactivation of the enzymes. Thus, our plasma treatment did not harm the enzymes, and the activity of the enzymes was maintained, even in an Ar plasma treatment. This means that the plasma-graft polymerization method is very useful for adding a gating function to GOD-loaded microcapsules without losing the activity of GOD. The structure of the enzyme after plasma treatment is not clear at this stage, and further studies are required to clarify this issue. 3.5. Performance of the pH-Responsive Microcapsule Reactor. Figure 8 shows the amount of oxygen consumed by our pH-responsive microcapsule reactor at different pHs with time. Oxygen is one of the substrates of this reaction. The temperature was fixed at 40 °C, and each gradient denotes the rate of reaction. The rate at pH 5.0 was 2.7 times higher than that at pH 4.0. On the other hand, the activity of untreated GOD at pH 5.0 was only about 1.3 times higher than that at pH 4.0. These results agree well with the test glucose permeation data. As shown in Figure 6, the permeability of glucose at pH 5.0 was 2.6 times higher than that at pH 4.0. These results can be explained as follows: When the pH in the medium was pH 5.0, the pH-responsive P-NIPAM-co-AA gate polymer was in the hydrophilic state, so glucose could penetrate through the shell layer and the GOD reaction proceeded. However, when the pH in the medium was pH 4.0, the pH-responsive P-NIPAMco-AA gate polymer became hydrophobic, so glucose could not penetrate through the shell layer and the GOD reaction did not
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proceed. That is, in this system, the diffusion of glucose is the rate-limiting step, and the pH-responsive reactivity of the microcapsule reactor is determined by the substrate diffusivity of the pH-responsive polymer gates, which depends on the pH of the medium, and not on the activity of GOD. Therefore, the difference in the reactivity would be as expected from the data shown in Figure 6. We can conclude that the bottle-in method and the plasma-graft polymerization method are proper novel methods for preparing environment-responsive enzyme-loaded microcapsule reactors with high activities. For performance improvement, the states of the enzymes loaded into the microcapsules are considered to be very important. We showed in this report that a novel preparation method using plasmagraft polymerization was efficient for environment-responsive and enzyme-loaded microcapsule reactors. Further research is required on the relationship between the conformation, stability, or geometry of the loaded enzymes determined by the preparation method or experimental conditions and reactor performance. 4. Conclusions We have successfully demonstrated a novel efficient preparation method for a pH-responsive microcapsule reactor using the plasma-graft polymerization method. First, core-shell microcapsules were prepared using SPG membrane emulsification and interfacial polymerization. We demonstrated the efficiency of the bottle-in method for loading the microcapsules with GOD and showed that, with this method, enzymes could be loaded in a short period. Second, the microcapsules, which were composed of a coreshell porous membrane and linear grafted P-NIPAM-co-AA chains in the pores acting as pH gates formed via a plasmagraft polymerization method, were used to control the permeability of glucose, depending on the pH of the medium. This was controlled by the different hydration states of the gates at different pHs. Third, we showed that the plasma-graft polymerization method is very useful for grafting functional polymers onto GOD-loaded microcapsules. This technique activates only the pores and the surfaces of the microcapsules to generate activation points for graft polymerization without any loss of GOD activity. Finally, we demonstrated our pH-responsive core-shell microcapsule reactor system. In developing this system, it is very important to design the diffusion-limiting step of the substrate. This type of microcapsule reactor can be used in various applications, e.g., in drug delivery systems, diagnostic products, or artificial cells, because the same preparation method as used in this study can be utilized, even if the enzymes or environmentresponsive polymers are changed to those of various specific targets. Acknowledgment The authors thank Professor Liang-Yin Chu of the School of Chemical Engineering, Sichuan University, Chengdu, China, for many helpful discussions and Dr. Tadao Nakashima and Dr. Masato Kukizaki of the SPG Research Laboratory at the Miyazaki Prefecture Industrial Technology Center, Miyazaki, Japan, for the SPG membrane emulsification technique. We also thank Kohjin Co. Ltd., Fuji, Japan, and Asahi Chemical Co. Ltd., Tokyo, Japan, for kindly supplying the N-isopropylacrylamide monomer and the porous polyethylene film, respectively.
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ReceiVed for reView July 3, 2006 ReVised manuscript receiVed September 27, 2006 Accepted October 10, 2006 IE060857Q
