Glucose-Responsive Polymer Gel Bearing Phenylborate Derivative as

Biomacromolecules 2004, 5, 1038-1045

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Glucose-Responsive Polymer Gel Bearing Phenylborate Derivative as a Glucose-Sensing Moiety Operating at the Physiological pH Akira Matsumoto, Ryo Yoshida, and Kazunori Kataoka* Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan Received December 20, 2003; Revised Manuscript Received February 29, 2004

The work attempts to prepare a totally synthetic, glucose-responsive polymer gel bearing a phenylborate derivative as a sensor moiety to glucose, for future use as a self-regulated insulin delivery system. The molecular strategies to enable the system to be operated under physiological conditions (pH 7.4, 37 °C) are presented that involve the use of a novel phenylborate derivative {4-(1,6-dioxo-2,5-diaza-7-oxamyl) phenylboronic acid: DDOPBA} possessing an appreciably low pKa (∼7.8), the adoption of poly(Nisopropylmethacrylamide) (PNIPMAAm) for the main chain, which itself undergoes a sharp thermo-induced phase transition at its LCST around 40 °C, as well as the introduction of a carboxyl group of methacrylic acid as the third comonomer. Glucose-responsive behaviors of the obtained gels were evaluated based on the changes in the equilibrium swelling degree determined in the presence and the absence of glucose, for various pH and temperature conditions. As a consequence of the combined molecular effects, a sufficient sensitivity of the system was accomplished at physiological pH and in the temperature range close to the physiological condition such as 30 °C. Furthermore, the glucose-induced continuous volume changes of the gels were demonstrated under those conditions, which occurred in a remarkably concentration-dependent manner. In these experiments, the critical glucose concentrations to induce the gels’ responses in the range of normoglycemic sugar level were observed. These observations may provide us with an excellent prospect for the use of the gel as a self-regulated, insulin-delivery system discretely switching the release at the normoglycemia. Introduction The attempts to utilize stimuli-responsive polymer gels for constructing self-regulated, hence, “smart” systems have long been research subjects. A number of stimuli-types have thus far been demonstrated to induce abrupt changes in the physicochemical properties of a polymer gel. Above all, chemical-stimuli responsive systems mimicking the biofeedback systems, such as the recognition of the concentration change in a certain molecule, have become increasingly important with their potential utilities in many applied fields, including drug delivery systems.1 One challenging molecule is glucose, i.e., change in blood sugar level. The treatment for insulin dependent diabetes mellitus (IDDM) actually conducted today is limited to insulin injection that significantly burdens patients. A more problematic feature of the treatment is the difficulty in controlling the insulin dosage, where an overdose causes an excessively decreased blood sugar level, leading to serious hypoglycemia. The situation has inspired a great deal of research to establish glucose-responsive polymer (gel) systems for achieving self-regulated insulin delivery.1,2 The common methodologies involve two major types. One is the utilization of an enzymatic reaction between glucose oxidase * To whom correspondence should be addressed. Phone: +81-3-58417138. Fax: +81-3-5841-7139. E-mail: [email protected].

(GOD) and glucose,1-8 and the other is based on the competitive and complementary binding properties to concanavalin A (Con A) of synthetically glycosylated insulin and glucose.1,2,9-13 These protein-based components, however, yield a poor liability of the system due to the denaturalizing and antigenic nature of the proteins when used and stored for an extended period of time. Hence, to overcome this limitation, an alternative glucosesensor moiety should be prepared from a somewhat nonnatural or synthetic component. Phenylboronic acid may serve as a potential candidate for such an objective. Phenylboronic acid and its derivatives are known to form reversible covalent complexes with diol units, such as glucose.14-16 Based on this property, they have been primarily utilized as ligand moieties for affinity chromatography of polyol compounds.17-19 More recently, such applications as glyco-responsive polymer complex systems20,21 and synthetic mitogens for lymphocytes22,23 have been reported. As shown in Figure 1, phenylboronic acid compounds in water exist in equilibrium between the uncharged and the charged forms. Here only the latter (charged) form can make a relatively stable complex with glucose through a reversible covalent bonding, whereas the complex between the former (uncharged) form and glucose is unstable in water due to its high susceptibility to hydrolysis.24 Because the complex between the charged phenylborate and glucose itself is also

10.1021/bm0345413 CCC: $27.50 © 2004 American Chemical Society Published on Web 04/15/2004

Glucose-Responsive Polymer Gel

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group carrier, was introduced into the system. All of the chemical methodologies intended to allow the system to be operated under the physiological conditions are presented here with a systematic correlation between the modulated structures and the glucose-responsive behaviors. Experimental Section

Figure 1. Equilibria of (alkylamido)phenylbronic acid in an aqueous solution in the presence of glucose.

anionically charged, the further addition of glucose induces a shift in the equilibrium to the direction of increasing the fraction of the charged forms, and vice versa. Therefore, the introduction of the phenylborate group into an amphiphilic polymer gel structure, such as that of poly(N-isopropylacrylamide) (PNIPAAm) gel, brings about a reversible volume transition of the gel, which is mainly due to the change in the counterions’ osmotic pressure synchronized with the change in the glucose concentration.25-27 Our earlier work demonstrated such a copolymer gel system composed of PNIPAAm and 3-acrylamidophenylboronic acid (AAPBA) (9:1 in molar ratio), referred to as an NB10 gel.25 A remarkable on-off regulation of insulin release was achieved by the use of the NB10 gel in a synchronizing manner, with the change in the external glucose concentration under the proper pH and temperature conditions.25 The NB system, however, was not able to adequately function under the physiological pH and temperature conditions (pH 7.4, 37 °C) for some molecular limitations as will be described in the latter part of this paper. In our previous report, in an attempt to enable the system to be operated under these conditions, some modifications were made to the chemical-structural design of the system.28 The strategy involved the utilization of a newly synthesized phenylborate derivative as to possess an appreciably low pKa and also the adoption of a different type of main chain structure.28 The effects of the thus modified structures on the glucose-responsive behavior were studied for the copolymer systems. As a result, a drastic improvement in the operational condition was achieved, leading to significant glucose sensitivities of the system under the physiological pH or temperature conditions.28 Thus, the validity of the modulated structural design was confirmed. Accordingly, in the present study, we prepared a series of chemical-structured copolymer gels based on the same molecular strategies. To elucidate the operational conditions of these gels as the glucose-responsive system, the equilibrium swelling degrees were evaluated for various pH and temperature conditions in the presence and the absence of glucose. Continuous volume changes of the gel upon the addition of the varied glucose concentrations were demonstrated at a physiological pH of 7.4. Additionally, for the further optimization of the operational conditions of the system, the third comonomer, methacrylic acid as a carboxyl

Materials. N-Isopropylacrylamide (NIPAAm) was purchased from Kojin (Japan) and recrystallized from n-hexane. N-Isopropylmethacrylamide (NIPMAAm) was prepared by the reaction of methacryloyl chloride (Wako, Japan) with isopropylamine (Wako, Japan) in the same manner as has been reported elsewhere29-31 and then recrystallized in a 20/ 80 mixture of toluene and hexane. 2,2′-Azobisisobutyronitrile (AIBN; Wako, Japan) was recrystallized from methanol. Methacrylic acid (Wako, Japan) was distilled under reduced pressure. 3-Acrylamidophenylboronic acid (AAPBA), N,N′methylene-bis-acrylamide (MBAAm), and dimethyl sulfoxide were all supplied from Wako (Japan) and were used without further purification. 4-(1,6-Dioxo-2,5-diaza-7-oxamyl) phenylboronic acid (DDOPBA) was synthesized by a two-step reaction as has been previously described; 4-(Chloroformyl) phenylboronic acid was first reacted with an excess amount of ethylenediamine to obtain 4-[(2-aminoethyl)carbamoyl] phenylboronic acid (AECPBA), followed by the reaction with acryloyl chloride to yield DDOPBA.28 Polymer Synthesis. Each copolymer was prepared by radical polymerization in methanol in a sealed glass ampule in vacuo at 60 °C for 24 h using AIBN as an initiator. The detailed reaction conditions are summarized in the footnote of Table 1. After the polymerization, the reacted mixture was poured into a large excess of diethyl ether. The precipitated polymer was filtered off and dried in vacuo. The copolymer compositions were determined by 1H NMR (EX400, JEOL, Tokyo, Japan, DMSO-d6) from the peak intensity ratios between the methine proton on the isopropyl group either of NIPAAm or NIPMAAm (-CH(CH3)2: δ ) 3.8 ppm) and the aromatic protons either of AAPBA or DDOPBA (COC6H4: δ ) 7.8-7.9 ppm). Preparation of Capillary Gels. Capillary gels were prepared by radical copolymerization in 1-mm-diameter glass capillaries using ′AIBN as an initiator, and in the presence of ¢MBAAm as a cross-linking agent. The feed monomer compositions for each gel are all summarized in Table 2 together with the reaction conditions. The monomer structures are displayed in Figure 2. The obtained gels were washed with an excess amount of distilled water to thoroughly exchange the solvent from DMSO to water and to remove the unreacted monomers. Potentiometric Titration. A potentiometric titration of each copolymer was performed at various temperatures using an automatic titrator system (DL25, Mettler Toledo, Germany) equipped with a pH electrode (Glass electrode DG111SC, Mettler Toledo, Germany). The calibration of the electrode was carried out using carbonate and phthalate buffers (Beckman, USA) at each experimental temperature. The measurement error was less than 0.01 pH units. Samples were prepared by dissolving 100 mg of each copolymer in

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Table 1. Composition of Phenylborate-Containing Polymersa feed composition [molar ratio]

a

abbreviation

NIPAAm

PNIP-A-10.5 PNIP-D-9.5 PNIPM-D-10.0

90 90

NIPMAAm

AAPBA

DDOPBA

yield [wt%]

borate contents in copolymera [mol%]

10

10.5 9.5 10.0

10 10

90

Initiator (AIBN) concentration of 10 mM; monomer concentration of 1.0 M; reaction temperature of 60 °C; reaction time of 24 h.

Table 2. Composition of Phenylborate-Containing Polymer Gelsa feed composition [molar ratio] abbreviation gPNIP-A-10 gPNIP-D-10 gPNIPM-D-10 gPNIPM-D-20 gPNIPM-D-M-20-2 gPNIPM-D-M-20-5

NIPAAm NIPMAAm AAPBA DDOPBA MAAc 90 90

10 10 10 20 20 20

2 5

changes in the gel, the swelling degree was defined as (d/ d0),3 where d0 is the diameter as obtained in a glass capillary (1 mm for all in DMSO), and d is the observed diameter. Evaluations of the glucose-induced volume changes were conducted by exchanging the suspending solution of the gel with the different glucose concentration-conditioned buffer solutions, which had been, prior to the addition, thermocontrolled at each experimental temperature.

a Initiator (AIBN) concentration of 7.5 mM; (acrylamide) concentration of 30 mM; monomer concentration of 1.5 M; reaction temperature of 60 °C; reaction time of 24 h.

Results and Discussion

40 mL of freshly prepared 0.01N NaOH solution (150 mM NaCl) adjusted to a series of glucose concentrations. The water was purified by a Milli-Q instrument (Millipore Corporation, USA). The titrant (0.01N HCl, 150 mM NaCl) was added in 0.05 mL portions at 12-120 s intervals when the drift equilibrium reached the rate of 1 mV/s. Observation of the Volume Changes in the Capillary Gel. Diameter changes in the capillary gels were observed under a microscope (Nikon, Diaphot) equipped with a blackwhite CCD camera (Hamamatsu photonics, C2400). The obtained images were processed through analytical software (Hamamatsu photonics, Image Processor Argus C5510) and then recorded by a video recorder. For the temperature control, a water-flow chamber with four cavities on the top plate made of acrylic resin was placed on the microscope stage. Gel capillaries were cut into 5 mm long (on a hydrated swollen state) pieces and put in the cavities filled with each experimental buffer solution. Prior to each experiment, the gels were kept in the solution under the controlled temperature for a sufficient period of time to reach their equilibrated state. Equilibrium swelling degrees of the gels under the various pH and glucose concentrations were recorded while decreasing the temperature. Assuming symmetric shape

The limited operational pH condition under which the NB system can reveal a considerable glucose-sensitivity is derived from a relatively high intrinsic pKa of the phenylborate group.25 The pKa value of the utilized phenylborate in the system (AAPBA) was formerly determined to be 8.2. In our previous report, with an attempt to achieve an appreciable sensitivity of the system at lower pH conditions, a novel phenylborate-containing monomer, [4-(1,6-dioxo2,5-diaza-7-oxamyl) phenylboronic acid (DDOPBA)], was synthesized.28 The chemical-structural design of the DDOPBA for decreasing the pKa was based on the introduction of a stronger electron-withdrawing substituent group (parapositioned carbamoyl group) into the phenyl ring, instead of the meta-positioned amide group as in the case for the AAPBA. The monomer-state pKa value of the DDOPBA was determined to be 7.8.28 The use of the DDOPBA as a 10 mol % pendent group to the PNIPAAm main chain copolymer has revealed a more appreciable glucose-sensitivity (a greater glucose-induced changes in the LCST) under the lower pH conditions close to the physiological condition, in comparison to that of the same amount of the AAPBApendent (NB-10) system.28 Figures 3 and 4 show apparent pKa’s (pKaapp) of the PNIPAAm-based copolymers with each

Figure 2. Monomer structures of the main chain components (NIPAAm and NIPMAAm) and of the phenylborate-containing monomers (AAPBA and DDOPBA).


Figure 3. Temperature dependencies of the apparent pKa values (pKaapp) of PNIP-A-10.5 as determined from ptentiometric titration in the absence (opened circles) and the presence (5 g/L) of glucose (fixed circles).

Figure 4. Temperature dependencies of the apparent pKa values (pKaapp) of PNIP-D-9.5 as determined from ptentiometric titration in the absence (opened circles) and the presence (5 g/L) of glucose (fixed circles).

phenylborate moiety that are PNIP-A-10.5 and PNIP-D-9.5 bearing 10.5 and 9.5 mol % of AAPBA and DDOPBA, respectively, as determined from the potentiometric titration in the presence (5 g/L) and the absence of glucose for various temperatures. The pKaapp values significantly decrease upon the addition of 5 g/L glucose for both copolymers, due to the stabilization of the charged (dissociated) phenylborates through complex formation with glucose. Both copolymers show somewhat higher pKaapp values (in the absence of glucose) than those of the corresponding monomers (AAPBA ) 8.2, DDOPBA ) 7.8 in the absence of glucose). This should be attributed to the nature of the polyelectrolytes where the phenylborate moieties are spatially trapped, namely, as the dissociation of the phenylborate proceeds, the concentration of anionic charges in the immediate vicinity of the phenylborates increases and thus further dissociation of the phenylborates is impeded.32 Nevertheless, PNIP-D9.5 reveals lower pKaapp values than those for the PNIP-A10.5 over the entire range of evaluated temperatures in the absence and presence of glucose, consistent with the lower pKa of DDOPBA than that of AAPBA. More noteworthy is that the PNIP-A-10.5 displays a tendency of increasing pKaapp as the temperature increases closer to the precipitation point (lower critical solution temperature: LCST) as seen in Figure 3, whereas the PNIP-D-9.5 sustained an almost constant


value over the evaluated temperature range. This may be caused by a less expanded state of the polymer chain due to the relatively reduced degree of hydration as the temperature increases closer to the precipitation point (LCST).29 This may lead to a decreased local dielectric constant around the phenylborate moieties or an even more emphasized polyelectrolyte effect that is described above, resulting in an increased pKaapp value with a temperature increase for PNIPA-10.5. The negligible temperature-induced increase in the pKaapp for PNIP-D-9.5 may then be due to the increased distance from the main chain (see Figure 2) and, therefore, the greater mobility of the phenylborate moieties, reducing a microenvironment effect induced by the polymer strands. This temperature-independency of the DDOPBA-based polymer becomes a great advantage in order to achieve the glucose-induced phenylborate fraction changes at as low pH conditions as possible and in the higher temperature range closer to the physiological temperature. In the same way, the PNIPAAm-based copolymer gels bearing 10 mol % of each phenylboate with different pKa’s, namely, AAPBA (pKa ) 8.2) and DDOPBA (pKa ) 7.8), were prepared. Thus obtained copolymer gels were referred to as gPNIP-A-10 (or NB-10) and gPNIP-D-10, respectively. Figure 5 shows changes in the equilibrium swelling degree of these gels (phase diagrams) as a function of the temperature with (glucose concentrated 5 g/L) and without glucose at various pHs conditions [(a) pH 7.4, (b) pH 8, and (c) pH 8.5]. The overall tendency is that gels become more swollen as the temperature decreases, because of the thermo-sensitive nature of the PNIPAAm main chain. The completely shrunken-state volumes of the gels appear to converge on the same degree for all of the cases, consistent with that the total monomer concentrations and the cross-linker densities in the feed were all fixed at the same amounts. For both gels, temperature ranges where the gels undergo volume changes shift toward higher temperature upon the addition of glucose and with increased pH due to the increased fraction of the charged phenylborate that are schematically represented in Figure 1. The same reason accounts for increasingly remarkable glucose-dependent volume changes with increased pH. What should be noted is that a comparison of these two gels clearly reflects the effects of their different pKa’s of the phenylborates utilized in each structure. As can be seen in Figure 5, the gPNIP-D-10 gel bearing DDOPBA as a phenylborate moiety shows greater values of the swelling degree as compared to those of the gPNIP-A-10 gel at any fixed pH and temperature conditions. This can be explained by the increased fraction of the charged phenylborates in the gPNIP-D-10 gel thus causing a greater degree of counterions’ osmotic pressure in the gPNIP-D-10 gel than that in the gPNIP-A-10 gel, being consistent with the observed difference in the pKaapp values assessed for the two different-structured copolymer systems in Figures 3 and 4. Moreover, at the physiological pH of 7.4, sufficient glucosedependent changes in the swelling degrees are visible for the gPNIP-D-10 gel in the temperature range of 10-20 °C, whereas almost no glucose-dependency on the swelling degrees can be observed for the gPNIP-A-10 gel. This

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Figure 6. Changes in the equilibrium swelling degrees (phase diagrams) of the PNIPMAAm-based copolymer gels bearing DDOPBA as a phenylborate moiety: gPNIPM-D-10 (squares) and gPNIPM-D20 (circles), as a function of the temperature in the presence (fixed: glucose concetration of 5 g/L) and the absence (opened) of glucose at the physiological pH of 7.4.

Figure 7. Temperature dependence of the apparent pKa values (pKaapp) of PNIPM-D-10.0 as determined from ptentiometric titration in the absence: opened circles and the presence (5 g/L) of glucose: fixed circles.

Figure 5. Changes in the equilibrium swelling degrees (phase diagrams) of the PNIPAAm-based copolymer gels bearing different phenylborate moieties: gPNIP-A-10 (squares) and gPNIP-D-10 (circles), as a function of the temperature in the presence (fixed: glucose concetration of 5 g/L) and the absence (opened) of glucose for various pH conditions: (a) pH 7.4, (b) pH 8 and (c) pH 8.5.

implies that the gPNIP-D-10 can be operated as a glucosesensitive system at the physiological pH condition, although the temperature range is still far below the physiological temperature of 37 °C. To improve the operational temperature conditions of the system, we took notice of a different type of main chain structure, PNIPMAAm, which undergoes a sharp thermosensitive phase transition at its LCST around 40 °C, approximately 10 °C higher than that of the PNIPAAm. The well-accepted molecular reason for the higher LCST of the PNIPMAAm is the restricted free rotation of the main chain induced by the R-methyl group, which prevents the hydrophobic groups from aggregating in the most favorable manner.29-31 Figure 6 shows phase diagrams obtained at the physiological pH for the PNIPMAAm-based copolymer gels

bearing different amounts of DDOPBA (10 and 20 mol % in feed) as a phenylborate moiety. As expected, for the gPNIPM-D-10 gel, glucose-dependent volume changes were observed in a considerably closer temperature range to the physiological condition as compared to the gPNIP-D-10 gel bearing the same amount of phenylborate (DDOPBA, 10 mol %) [Figure 5a], in accordance with the higher LCST of the PNIPMAAm than that of the PNIPAAm. Figure 7 indicates temperature dependencies of the pKaapp for the PNIPMAAmbased copolymer bearing 10 mol % DDOPBA, referred to as PNIPM-D-10.0, in the absence and the presence (5 g/L) of glucose. The pKaapp values of this copolymer were obtained in the similar ranges to those observed for PNIP-D-9.5 in Figure 4. Therefore, the effect of the modified main chain structure (from PNIPAAm to PNIPMAAm) on the disassociative behavior of the (approximately 10 mol %) pendent phenylborate (DDOPBA) moieties should not be significant. On the other hand, for the gPNIPM-D-20 gel with the higher phenylborate content, the temperature range where the volume changes occur has been significantly further decreased below the physiological temperature, due to the increased hydrophobicity of the network caused by a larger content of the uncharged DDOPBA. Indeed, no volume



Figure 8. Series of glucose concentrations (diamonds) 0.5, (squares) 1, (triangles) 3, and (circles) 5 g/L, induced changes in the volume of gPNIPM-D-20 gel, all with the initial glucose concentrations of 0 g/L, as a function of time at (a) 20 and (b) 25 °C at the physiological pH of 7.4. The values (dt) are standardized with those of the initially equilibrated state (di) in the absence of glucose at each temperature.

change was observed for the gPNIPM-D-30 gel, preserving the completely collapsed state even at 5 °C (data not shown). Nevertheless, more drastic volume changes and thus larger glucose-sensitivities have been achieved for the gPNIPMD-20 gel, because of the more appreciable change in the amount of the phenylborate anions. Figure 8 demonstrates various glucose concentrations-induced volume changes of the gPNIPM-D-20 gel under the physiological pH at (a) 20 °C and (b) 25 °C. Upon the addition of a series of the glucose concentrations, the gPNIPM-D-20 gel starts swelling from the initially equilibrated state in the absence of glucose. These temperatures have been carefully chosen from the equilibrium state of volumes (with and without glucose) achieved in Figure 6, on the basis of revealing sufficient responses. Under both temperature conditions at (a) 20 °C and (b) 25 °C, the swelling rates remarkably increases with the increased glucose concentration due to the increased amount of the phenylborate anions, synchronized with the complex formation with glucose. After several hours, the gel reaches the equilibrium volumes, which can be observed to be in the corresponding order to the added glucose concentrations. In particular, when carried out at 25 °C [Figure 8b], while the gel reveals prompt increases in the volume upon the addition of and 5 g/L glucose, only a slight increase in the volume can be observed for the addition of 1 g/L glucose, which is an equivalent sugar level to normoglycemia. Moreover, no volume change was observed for the addition of 0.5 g/L glucose, thus retaining the completely collapsed state even after 24 h. We assume that a formation of a thin, dehydrated (collapsed) layer on the gel surface, or “skin layer”, plays a critical role in achieving a quickly and reversibly controlled insulin release in the present phenylborate-based system. Formation of the skin layer can be observed as a decreasing transparency of the gel, and it occurs due to that the glucoseinduced phase transition always commences at the gel surface when the glucose molecules diffuse out of the gel.27 Once the layer is formed on the gel surface, diffusion of the insulin

molecule is sustained, and thus, the release turns off, and vice versa. In such a way, the skin layer serves as a highly sensitive “releasing switch”. We have previously observed, in the NB system, such a skin-layer formation and, consequently, have achieved an effectively on-off regulated insulin-release in a synchronizing manner with the change in the external glucose concentration.25 Furthermore, it was revealed that the skin layer formed on the NB (micro-particle) gel could stably exist longer than 10 h. Indeed, the present gPNIPM-D-20 gel has also shown such a reversible and remarkable skin-layer formation, in particular, under the condition of 25 °C [Figure 8b]. Note that, as can be seen in Figure 6, the gPNIPM-D-20 gel is on the completely collapsed (shrunken) state, forming the skin layer on the surface, under the condition of 25 °C in the absence of glucose. It was actually observed that the skin layer promptly disappears within the time range of several minutes or so, upon the addition of higher glucose concentrations than the normoglycemic level, such as 3 and 5 g/L. This implies that the insulin release could be quickly initiated (in the time range of several minutes) upon increasing the glucose concentration. Consequently, these observations indicate a significant advantage for the use of the gel as an insulin delivery system achieving a quick on-off regulation at the critical sugar level (normoglycemia) with prolonged time intervals. Finally, for a further improvement of the operational temperature conditions of the system, we attempted to increase the overall hydrophilicity of the network by introducing carboxyl anions carried by methacrylic acid. The PNIPMAAm-based copolymer gels bearing 20 mol % DDOPBA were prepared with the introduction of 2 or 5 mol % methacrylic acid, referred to as gPNIPM-D-M-20-2 and gPNIPM-D-M-20-5, respectively. Phase diagrams of these gels investigated at the physiological pH are displayed in Figure 9 along with that of gPNIPM-D-20 (no methacrylic acid introduced) as the control. Consequently, over the entire temperature range evaluated, the swelling degrees of the gels

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Figure 9. Changes in the equilibrium swelling degrees (phase diagrams) of the PNIPMAAm-based copolymer gels bearing 20 mol % of DDOPBA with the varied content of methacrylic acid: (squares) no methacrylic acid introduced (gPNIPM-D-20 gel), (triangles) methacrylic acid content of 2 mol %, (gPNIPM-D-M-20-2) (circles) methacrylic acid content of 5 mol % (gPNIPM-D-M-20-5), as a function of the temperature in the presence (fixed: glucose concetration of 5 g/L) and the absence (opened) of glucose at the physiological pH of 7.4.

have been increased with the increased methacrylic acid content. In particular, the gPNIPM-D-20-2 gel reveals a definite glucose-sensitivity in the temperature range closer to that of physiology as compared to the case without methacrylic acid (gPNIPM-D-20). The magnitude of the sensitivity (changes in the volume in the presence and the absence of glucose), however, is significantly decreased, especially for the gPNIPM-D-M-20-5 gel, as compared to the case with no introduced methacrylic acid. This should be caused by the apparently decreased fraction changes of the total anionic charges (phenylborate + carboxyl anion), as that the carboxyl groups should exist in the completely dissociated form at pH7.4 regardless of the change in the glucose concentration. The excessive introduction of methacrylic acid may also affect the ability of the main chain to produce a sharp transition. In the case of the gPNIPM-DM-20-5 gel with a 5 mol % methacrylic acid content, the volume change in the gel for various temperatures becomes

Matsumoto et al.

significantly dull as observed in Figure 9, broadening over a wide range of temperatures. This gel did not completely shrink even at 70 °C. Thus, in terms of optimizing the sensitivity, the critical content of methacrylic acid to be introduced into the present system should exist within the range of 2-5 mol %. Figure 10 shows the volume changing behavior of the gPNIPM-D-M-20-2 gel in response to the addition of various glucose concentrations studied at physiological pH either at (a) 25 or (b) 30 °C. In the same manner as observed in Figure 8, the volume of the gel increases in the corresponding rate to the added glucose concentrations until reaching each equilibrium state. Particularly, when conducted at 30 °C [Figure 10b], the volume change of the gel was hardly observed for glucose concentrations lower than 1 g/L, which is the normoglycemic sugar level, whereas the gel undergoes a marked swelling for the addition of higher glucose concetrations. Thus, it should follow from these observations that the gPNIPM-D-M-20-2 gel can be operated as a glucose-responsive system under the physiological pH, and in the temperature range around 30 °C. If the operational temperature condition of the system can somehow be raised to 37 °C, then it could be used in a selfregulated, insulin-delivery system discretely switching the insulin-release at the normoglycemic sugar level. Conclusion In an attempt to improve the glucose-sensitivity under the physiological (pH and temperature) conditions of the phenylborate-based glucose-responsive system, a series of structures-composed copolymer gels were prepared based on the same molecular strategy as has been previously applied and investigated for the copolymer systems. This strategy involved the use of a phenylborate derivative possessing an appreciably low pKa as well as the adoption of PNIPMAAm as the main chain component exhibiting a higher LCST than that of PNIPAAm. In addition, for the further optimization of the sensitivity, methacrylic acid was introduced into the system. As a consequence of the combined molecular effects,

Figure 10. Series of glucose concentrations (diamonds) 0.5, (squares) 1, (triangles) 3, and (circles) 5 g/L, induced changes in the volume of gPNIPM-D-M-20-2 gel, all with the initial glucose concentrations of 0 g/L, as a function of time at (a) 25 and (b) 30 °C at the physiological pH of 7.4. The values (dt) are standardized with those of the initially equilibrated state (di) in the absence of glucose at each temperature.


sufficient sensitivities of the system were accomplished at the physiological pH (7.4) and at the temperature range close to the physiological one (37 °C) such as 30 °C. In addition, the glucose-induced, at the same time, concentration-dependent volume changes of the gels were demonstrated under these conditions, with the exhibition of a critical glucose concentration to induce the response at the range of the normoglycemic sugar level. There seems to still be room left for further improvements in the chemical design of the system, such as the synthesis of a phenylborate monomer with a still lower pKa, or developing a different methodology to increase the hydrophilicity of the network while keeping the sharp volume-phase transition behavior. These ideas are now under investigation in our research group. Finally, we expect this totally synthetic system, which includes no natural components such as proteins or enzymes,3-12 to be highly stable in the human body with a reliable functionality. Hence, we may conclude that, upon further improvement, the system may serve as a landmark device overcoming many of the difficulties in the treatment of diabetes carried out today. Acknowledgment. The present work was financially supported in part by a Grant-in-Aid for Scientific Research on Priority Area (A) “Molecular Synchronization for Design of New Materials System” from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and also by a Grant for 21st Century COE Program “Human-Friendly Materials based on Chemistry” from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References and Notes (1) Miyata, T.; Uragami, T.; Nakamae, K. AdV. Drug DeliVery ReV. 2002, 54, 79-98. (2) For some examples of related approaches see: Qiu, Y.; Park, K. AdV. Drug DeliVery ReV. 2001, 53, 321-339. (3) Ishihara, K.; Muramoto, N.; Shinohara, I. J. Appl. Polym. Sci. 1984, 29, 211-217. (4) Kost, J.; Horbett, T. A.; Ratner, B. D.; Singh, M. J. Biomed. Mater. Res. 1985, 19, 1117-1133. (5) Albin, G.; Horbett, T. A.; Ratner, B. D. J. Controlled Release 1985, 2, 153-164.

Biomacromolecules, Vol. 5, No. 3, 2004 1045 (6) Hassan, C. M.; Doyle, F. J., III.; Peppas, N. A.; Macromolecules 1997, 30, 6166-6173. (7) Holtz, J. H.; Asher, S. A. Nature 1997, 389, 829-832. (8) Kokufuta, E.; Matsukawa, S.; Tanaka, T. Macromolecules 1995, 28, 3474-3475. (9) Brownlee, M.; Cerami, A. Science 1979, 206, 1190-1191. (10) Kim, S. W.; Pai, C. M.; Makino, K.; Sminoff, L. A.; Holmberg, D. L.; Gleeson, J. M.; Wilson, D. E.; Mack, E. J. J. Controlled Release 1990, 11, 193-201. (11) Makino, K.; Mack, E. J.; Okano, T.; Kim, S. W. J. Controlled Release 1990, 12, 235-239. (12) Nakamae, K.; Miyata, T.; Jikihara, A.; Hoffman, A. S. J. Biomater. Sci. Polym. Ed. 1994, 6, 79-90. (13) Miyata, T.; Jikihara, A.; Hoffman, A. S. Macromol. Chem. Phys. 1996, 197, 1135-1146. (14) Boeseken, J. AdV. Carbohyd. Chem. 1949, 47, 189-210. (15) Aronoff, S.; Chen, T.; Cheveldayoff, M. Carbohydr. Res. 1975, 40, 299-309. (16) Foster, A. B. AdV. Carbohyd. Chem. 1957, 12, 81-115. (17) Weith, H.; Wiebers, J.; Gilham, P.; Biochem. 1970, 9, 4396-4401. (18) Hageman, J. H.; Kuehn, G. D. Anal. Biochem. 1997, 80, 547-554. (19) Gelijkens, C.; Deleenheer, A. J. Chrom. 1980, 183, 78-82. (20) Kitano, S.; Kataoka, K.; Koyama, K.; Okano, T.; Sakurai, Y. Makromol. Chem. Rapid Commun. 1991, 12, 227-233. (21) Kitano, S.; Koyama, K.; Kataoka, K.; Okano, T.; Sakurai, Y. J. Controlled Release 1992, 19, 161-170. (22) Miyazaki, H.; Kikuchi, A.; Koyama, Y.; Okano, T.; Sakurai, Y.; Kataoka, K. Biochem. Biophys. Res. Commun. 1993, 195, 829-836. (23) Uchimura, E.; Otsuka, H.; Okano, T.; Sakurai, Y.; Kataoka, K. Biotechnol. Bioeng. 2000, 72, 307-314. (24) Lorand, J. P.; Edwards, J. D. J. Org. Chem. 1959, 24, 769-774. (25) Kataoka, K.; Miyazki, H.; Bunya, M.; Okano, T.; Sakurai, Y. J. Am. Chem. Soc. 1998, 120, 12694-12695. (26) Nakayama, D.; Takeoka, Y.; Watanabe, M.; Kataoka, K. Angew. Chem., Int. Ed. 2003, 42, 4197-4200. (27) Matsumoto, A.; Kurata, T.; Shiino, D.; Kataoka, K. Macromolecules 2004, 37, 1502-1510. (28) Matsumoto, A.; Ikeda, S.; Harada. A.; Kataoka. K. Biomacromolecules 2003, 4, 1410-1416. (29) Fujishige, S.; Kubota, K.; Ando, I. J. Phys. Chem. 1989, 93, 33113313. (30) Kubota, K.; Hamano, K.; Kuwahara, N.; Fujishige, S.; Ando, I. Polym. J. 1990, 22, 1051-1057. (31) Kubota, K.; Fujishige, S.; Ando, I. Polym. J. 1990, 2, 15-20. (32) Elias, H. G. Macromolecules. Synthesis and Materials; Plenum Press, New York, 1997; Vol. 2, pp 811-813.

BM0345413

Glucose-Responsive Polymer Gel Bearing Phenylborate Derivative as

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