Modulatory Factors on Temperature-Synchronized Degradation of

important factor for modulating enzymatic degradation of dextran in synchronization with temperature. Introduction. Dextran, composed of R-1,6-linked ...
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Biomacromolecules 2001, 2, 874-879

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Modulatory Factors on Temperature-Synchronized Degradation of Dextran Grafted with Thermoresponsive Polymers and Their Hydrogels Yoshikazu Kumashiro, Kang Moo Huh, Tooru Ooya, and Nobuhiko Yui* School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1, Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan Received February 28, 2001; Revised Manuscript Received June 8, 2001

Several types of dextran grafted with poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide [dextran-gpoly(NIPAAm-co-DMAAm)] with different graft lengths and numbers were synthesized in a preciously controlled fashion, and their enzymatic degradation was examined by viscosity measurement and gel permeation chromatography as a function of temperature. Degradation of dextran-g-poly(NIPAAm-coDMAAm)s decreased with increasing the graft length below their lower critical solution temperatures (LCSTs). Above the LCST, enzymatic degradation was independent of the graft length. A larger amount of the graft chain with increasing the graft length rather than the graft number was effective to modulate the temperaturesynchronized degradation. Hydrogels were prepared by cross-linking the graft copolymers using 1,6hexamethylenediamine. While all the hydrogels have water content of about 93-96% in a wide range of temperatures, their degradation behaviors show a significant dependence on a temperature change. Such a unique property is closely related to the structure of graft copolymers such as graft lengths. Consequently, introducing thermoresponsive grafts with longer length to dextran and its hydrogels is suggested to be an important factor for modulating enzymatic degradation of dextran in synchronization with temperature. Introduction Dextran, composed of R-1,6-linked D-glucopyranose residues, has been extensively investigated for biomedical applications such as polymeric carriers in the field of drug delivery systems due to its good biocompatibility.1-3 For example, Hennink et al.4-8 developed nondegradable and degradable hydrogels using methacrylated dextrans for the controlled release of proteins. Dextranase (EC 3.2.1.11) hydrolyzes 1,6-R-glucosidic linkage of dextran and has often been used as one of biochemical stimuli inducing degradation of such hydrogels. Stimuli-responsive polymers are defined as polymers which can respond to environmental conditions such as pH, temperature, electric fields, and so on.9 Over the past years, stimuli-responsive polymers, especially thermoresponsive polymers, have become very attractive as polymeric materials for modulated drug delivery. Poly(N-isopropylacrylamide) [poly(NIPAAm)] and its copolymers exhibit a lower critical solution temperature (LCST) in aqueous media in the vicinity of 32 °C. While they hydrate and form an expanded structure below the LCST, they dehydrate and form a compact structure above the LCST. Such a conformational change in response to temperature has been extensively used to modulate physicochemical properties of polymeric materials such as swelling/deswelling of hydrogels.10,11 Okano et al. demonstrated temperature-modulated drug release from polymeric micelles12-14 and cell manipulation15,16 using * To whom correspondence should be addressed: tel, +81-761-51-1640; fax, +81-761-51-1645; e-mail, [email protected].

hydration-dehydration properties of poly(NIPAAm) chains. They also investigated the swelling behavior of poly(NIPAAm) hydrogels with comb-typed grafts.17,18 The fast deswelling mechanism was explained in terms of rapid hydrophobic aggregation of freely mobile poly(NIPAAm) grafts and an operation of intrinsic elastic forces of the polymer network. We have studied dual-stimuli-responsive polymeric systems consisting of dextran and NIPAAm copolymers for biomedical application such as drug delivery.19 We have demonstrated modulated degradation of dextran hydrogels grafted with poly(NIPAAm-co-N,N-dimethylcrylamide) [poly(NIPAAm-co-DMAAm)]. Degradation of the hydrogels by dextranase was found to be synchronized with a temperature change, which induces the conformational change of freely mobile poly(NIPAAm-co-DMAAm) graft chain. The hydrogels were prepared by the addition of methacrylated poly(NIPAAm-co-DMAAm)s during cross-linking reaction with methacrylated dextran. However, adequate control of structural variables such as graft number and length is not easy due to low reactivity of the methacrylated poly(NIPAAmco-DMAAm). To overcome the lower graft number, a semitelechelic poly(NIPAAm-co-DMAAm) with an amino end group was directly coupled to carboxymethyl dextran.20 Further, the grafting method was developed in a more tailormade manner by coupling between a semitelechelic poly(NIPAAm-co-DMAAm) with a hydrazide end group and p-nitrophenyl chloroformate-activated dextran.21 In this method, precise control of molecular weight of poly(NIPAAm-co-DMAAm) was achieved by using methyl

10.1021/bm015527y CCC: $20.00 © 2001 American Chemical Society Published on Web 07/19/2001

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3-mercaptopropionate as a chain transfer agent instead of 2-mercaptoethanol or 2-aminoethanetiol. Thus, tailor-made synthesis of the graft copolymers with controlled graft chain length and number was established. The objective of this paper is to clarify the effect of graft lengths and numbers on the enzymatic degradation of the dextran main chain and to evaluate temperature-synchronized degradation of both graft copolymers and their hydrogels. The enzymatic degradation of several types of dextran grafted with poly(NIPAAm-co-DMAAm) [dextran-g-poly(NIPAAmco-DMAAm)] with different graft chain lengths and numbers was examined by viscosity measurements. The hydrogels based on dextran-g-poly(NIPAAm-co-DMAAm) were prepared using 1,6-hexamethylenediamine as a cross-linker, and their enzymatic degradation was examined. In this study, the effect of structural factors on the dual-stimuli-responsive system is discussed. Materials and Methods Materials. Dextran (Mn ) 40 000 from Leuconostoc mesenteroides, Tokyo Kasei Kogyo Co., LTD, Tokyo, Japan) was purified by reprecipitation twice into diethyl ether. Dextranase (EC 3.2.1.11, from Penicillium sp.) was purchased from Sigma Aldrich Japan Co. (Tokyo, Japan), and its activity (one unit: 1.0 µmol of isomaltose (measured as maltose) per 1 min at pH 6.0 at 37 °C) is 10-25 units/mg of solid. N-Isopropylacrylamide (Wako Pure Chemical Industries, Osaka, Japan) was recrystallized twice from n-hexane. N,N-Dimethylacrylamide (Wako) was purified by vacuum distillation at 82 °C /20 mmHg. 2,2′-Azobisisobutyronitrile (AIBN, Wako) was recrystallized twice from methanol. Dimethyl sulfoxide (DMSO, Wako) was distillued under vacuum at 78 °C/12 mmHg. Methanol (Nakarai Tesque Inc., Kyoto, Japan) and pyridine (Wako) were dried over CaH2 and distilled after refluxing for 3 h. Methyl 3-mercaptopropionate (Aldrich, USA), hydrazine monohydrate (Wako), p-nitrophenyl chloroformate (Aldrich), (dimethylamino)pyridine (DMAP, Wako), 1,6-hexamethylenediamine (Wako), and the other chemicals were used as received without further purification. Synthesis and Characterization of Dextran-g-poly(NIPAAm-co-DMAAm). Several types of dextran-g-poly(NIPAAm-co-DMAAm) with different graft lengths and numbers were synthesized according to our previous report.21 The synthetic results of dextran-g-poly(NIPAAm-co-DMAAm)s are summarized in Table 1. LCSTs of the graft copolymers in 0.1 M phosphate-buffered saline (PBS, pH 7.4) were determined by measuring transmittance at 500 nm using a UV-vis spectrophometer (V-550, Jasco, Tokyo, Japan). Enzymatic Degradation of Dextran-g-poly(NIPAAmco-DMAAm). Dextran-g-poly(NIPAAm-co-DMAAm) (code GCM15N4-GC-M91N8 in Table 1) (50 mg) was dissolved in 10 mL of 0.1 M PBS, pH 7.4, in the presence of dextranase (0.5 U/mL) at 25 and 45 °C. Dextranase activity at 25 and 45 °C was checked by the determination of the reducing sugar units from colormetric assay, and there was no difference in the activity between the two temperatures. The

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solution at each temperature was equilibrated for 30 min, followed by adding dextranase (0.36 mg). Reduced viscosity was calculated by measuring solution viscosity at appropriate time using a Cannon-Fenske type viscometer only at 25 °C. The solution was heated at 80 °C after 5 min and cooled to 25 °C. The resulting solution was passed through a microfilter (COSMO nice filter, Nakarai Tesque, Japan (diameter 13 mm, pore size 0.45 µm)) to precipitate dextranase, equilibrated at 25 °C for 30 min in the viscometer, and measured the time of the solution drop. The reduced viscosity was measured in the manner similar to the case below the LCST. The reduced viscosity was calculated by the equation ηsp/c )

η - η 0 t - t0 ) cη0 ct0

(1)

where η is the solution viscosity, η0 the solvent viscosity, ηsp the specific viscosity, c the density of the solution (the density of dextran-g-poly(NIPAAm-co-DMAAm) solution is 5.0 mg/mL), t the measuring time of the solution, and t0 the measuring time of the solvent. Determination of the Degradation by Gel Permeation Chromatography. To analyze the enzymatic degradation quantitatively, the degradation product was traced by gel permeation chromatography (GPC). Dextran-g-poly(NIPAAmco-DMAAm) (50 mg) was dissolved in PBS (10 mL), and the solution was stirred in the presence of dextranase (0.5 U/mL) for 12 h at 25 and 45 °C. The resulting solution was heated at 80 °C for 5 min, cooled to 25 °C, and passed through the microfilter. Four microliters of samples from each solution was injected in the high-performance liquid chromatography (HPLC) system (intelligent pump, PC-980; degasser, DG-980-50; chiral detector, OR-990; Japan Spectroscopic Co., LTD, Tokyo, Japan) with a column ((25 mm × 200 mm) Sephadex G-50 (exclusion limit, 2.0 × 104; Amersham Pharmacia Biotech AB, Sweden)) at a flow rate of 0.4 mL/min. The degradation of dextran-gpoly(NIPAAm-co-DMAAm) was calculated by the peak integration of the degradation products (oligosaccharides; AGC) in comparison with the integration at time to complete degradation of dextran (ADex) using the following equation degradation (%) ) (AGC/ADex) × 100

(2)

In this condition, the elution times of the original peak of dextran and its degradation products were found to be 1924 and 28-41 min, respectively. Preparation of Dextran-g-poly(NIPAAm-co-DMAAm) Hydrogels. A p-nitrophenyl chloroformate-activated dextrang-poly(NIPAAm-co-DMAAm) (0.025 µmol) was dissolved in 7.0 mL of DMSO and added to 1.1 mL of DMSO containing 1,6-hexamethylenediamine (15 mg) (Scheme 1). The solution was injected into a spacer (15 mm diameter, 2 mm height) and kept for 24 h at 60 °C in a dry oven. The obtained hydrogels were removed from the spacer and immersed in a phosphate-buffered saline (PBS, 0.1 mol/L, pH ) 7.4) for 1 week to remove unreacted compounds. For swelling measurements, the hydrogels were tapped with filter paper to remove excess water on the surface and were weighted at a fixed temperature. The swelling ratio and water

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Scheme 1. Preparation of Dextran Hydrogels Containing Thermoresponsive Grafts

Table 1. Synthetic Results of Dextran Grafted with Poly(NIPAAm-co-DMAAm)

code GC-M15N4 GC-M15N8 GC-M38N4 GC-M38N8 GC-M38N14 GC-M91N4 GC-M91N8

no. of ST-polymera Mn of grafts/mol graftb of dextranc (mmol) 0.2 0.2 0.2 0.2 0.2 0.2 0.2

1510 1510 3800 3800 3800 9140 9140

4.0 8.2 4.4 8.0 13.8 3.7 7.6

Mnd

LCSTe (deg)

46 000 52 400 54 800 70 200 92 400 73 800 109 500

40.8 40.5 40.5 40.6 40.3 42.2 41.8

a Molar amount of semitelechelic polymers, poly(NIPAAm-co-DMAAm)NHNH2, in feed. b Number average molecular weight of graft or poly(NIPAAm-co-DMAAm). c Average number of grafts calculated from the peak integration of 1H NMR spectra. d Number average molecular weight of graft copolymers calculated from peak integration of 1H NMR spectra. e Determined by measuring UV-vis light transmittance.

content were calculated using the equations Ws - Wd Wd

(3)

Ws - Wd × 100 Ws

(4)

swelling ratio ) water content )

where Ws is the weight of swollen hydrogel and Wd the weight of dried hydrogel. Enzymatic Degradation of the Cross-Linked Hydrogels. Hydrogels (15 mm diameter, 2 mm height) in a nylon mesh bag were immersed to be equilibrated in 40 mL of PBS at different temperatures (25 and 45 °C). After dextranase was added (0.5 U/mL) to 40 mL of PBS, the solution was continuously stirred for degradation. The degradation of the hydrogels was estimated by measuring the residual weight of hydrogels. Results and Discussion Synthesis of Dextran-g-poly(NIPAAm-co-DMAAm).21 Semitelechelic poly(NIPAAm-co-DMAAm)s, STPs, were obtained by polymerization of NIPAAm and DMAAm in the presence of methyl 3-mercaptopropionate as a chain transfer agent. The hydrazinolysis reaction of STP-COOCH3 to STP-NHNH2 was performed by hydrazine monohydrate. Several types of dextran-g-poly(NIPAAm-co-DMAAm) with different graft lengths (Mn ) 1510, 3800, and 9140) and numbers (about 4 and 8) were synthesized through a coupling reaction between p-nitrophenyl chloroformate-activated dextran and STP-NHNH2. The synthetic results of dextran-gpoly(NIPAAm-co-DMAAm)s are summarized in Table 1. It was found that all dextran-g-poly(NIPAAm-co-DMAAm)s exhibited LCSTs at almost the same temperature region (around 40-42 °C) independent of graft number and chain

lengths as poly(NIPAAm-co-DMAAm)s, but their transition points were higher than those of poly(NIPAAm-co-DMAAm)s (data not shown). Effect of Graft Length on the Enzymatic Degradation. The enzymatic degradation of the graft copolymers (GCM15G8, GC-M38N8, and GC-M91N8 in Table 1) was carried out and the effect of the graft length on the enzymatic degradation was investigated by determination of the solution viscosity. Solution viscosity did not change without dextranase. In the presence of dextranase, a decrease in the viscosity was observed and that was proportional to the amount of the degradation products which were traced by GPC (data not shown). These results indicate that measurements of the change in solution viscosity of graft copolymers can be useful for the observation of degradation. Figure 1 shows a change in the reduced viscosity of each graft copolymer. During enzymatic degradation, temperatures were maintained below and above LCST (25 and 45 °C). In the degradation experiments below the LCST, the values of the viscosity decreased dramatically during the first 60 min of degradation for all the graft copolymers. However, after the initial degradation they showed quite different patterns depending on the graft length of each graft copolymer. As a result, GC-M91N8 with the longest graft length (Mn ) 9140) showed the smallest decrease in the viscosity (Figure 1a). It should be noted that such dependence of enzymatic degradation on the graft length was not observed above the LCST (Figure 1b). These results suggest that enzymatic degradation proceeds above the LCST independent of graft lengths but is reduced depending on the graft lengths below the LCST. In these degradation, dextranase may catalyze the hydrolysis of dextran to an oligosaccharide, especially isomaltose.23 The solution viscosity of isomaltose (5.0 mg/mL) and poly(NIPAAm-co-DMAAm) (Mw 1510, 3800, and 9140 (2.0 mg/ mL)) was found to be 7.3 and 0.5-1.0 cm3/g, respectively (data not shown). As shown in Figure 1b, the viscosity of dextran-g-poly(NIPAAm-co-DMAAm) that was degraded above the LCST reached 7-9 cm3/g. These results suggest that the contribution of the viscosity of poly(NIPAAm-coDMAAm) is low, and thus, the graft copolymers are hydrolyzed to mainly isomaltose and isomaltose-g-poly(NIPAAm-co-DMAAm) above the LCST.

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Figure 3. Determination of enzymatic degradation by GPC below and above the LCST. Asterisk indicates significantly different degradation at P < 0.05 from 25 and 45 °C, which were calculated using a t-test.

Figure 1. (a) Enzymatic degradation of dextran-g-poly(NIPAAm-coDMAAm) below the LCST. The graft chain lengths are Mn ) 1510 (open circle), 3800 (open triangle), and 9140 (close circle). (b) Enzymatic degradation of dextran-g-poly(NIPAAm-co-DMAAm) above the LCST. The viscosity measurements were carried out at 25 °C in both cases (see Materials and Methods).

Figure 2. GPC charts of dextran-g-poly(NIPAAm-co-DMAAm) and its degradation products.

The quantitative determination of the degradation by GPC corresponds well to the results of the change in viscosity. Figure 2 shows GPC curves for GC-M38N4 and its degradation products below the LCST. As degradation proceeds, a single peak at elution time of 18-22 min becomes smaller and a new broad peak appears at 28-41 min, which increases in time. Two peaks correspond to the graft copolymer and its degradation products (oligosaccharides or oligosaccharides grafted with poly(NIPAAm-co-DMAAm)s), respectively. The peak corresponding to dextran-g-poly(NIPAAm-coDMAAm) disappeared completely after 12 h, indicating that all the graft copolymer chains participated in degradation reaction. It was noted that poly(NIPAAm-co-DMAAm) itself was not detected by the chiral detector. The percentages of

degradation were calculated from the integration of the degradation products using dextran as a control (Figure 3). Below the LCST, the degradation of GC-M15N8, GCM38N8, and GC-M91N8 was reduced to 96, 83, and 40% in comparison with the control, respectively. Above the LCST, the determination was reduced only maximaly 10% in all the cases compared with control. The reduction of the degradation with longer graft chain is considered to be due to the flexible movement and/or chain entanglement of the graft chain with dextran main chain, which can lead to the steric hindrance of enzymatic accessibility. The shorter graft chain (Mn 3800 and 1510) may be not enough to prohibit the accessibility. The results above the LCST (Figures 1b and 3), i.e., complete degradation independent of graft lengths, suggest that the steric hindrance of the enzymatic accessibility is not so significant despite the heterogeneous conditions. In that state, while the graft chains may interact intra- or intermolecularly to induce a phase transition, the dextran main chains are still solubilized and exposed the enzyme. The graft length of Mn ) 9140 was the minimum requirement to modulate the enzymatic degradation when the graft number was 8 per dextran chain. Effect of the Number of Grafts on Enzymatic Degradation. Figure 4 shows the change in the viscosity of the graft copolymers with the different number of grafts below the LCST. As for the GC-M15 series, the number of grafts was independent of the viscosity change (Figure 4a). This result indicates that increasing the number of the grafts from 4 to 8 is not effective for the enzymatic accessibility. On the other hand, with increasing the graft chain length, a much larger graft number contributed to preventing the decrease in the viscosity, especially the GC-M91 series (parts b and c of Figure 4). Since the change in the number of graft chains from 4 to 8 per 1 mol of dextran (approximately 1.6-3.2 per 100 glucose units) (Figure 4a) or from 8 to 14 (approximately 3.2-5.6 per 100 glucose units) (Figure 4b) was likely to be not significant. Hennink et al. have studied quantitative analysis of the dextranase-catalyzed degradation of methacrylated dextran. They reported that the main degradation product of the methacrylated dextran, which is 6 per 100 glucose units, was isomaltose. This report suggests that dextranase can bind the chemically modified dextran without any steric hindrance below 6. Considering this report, it is suggested that an increase in the number of the graft chain by the covalent bonding with hydroxyl group of

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Kumashiro et al. Table 2. Swelling Ratio and Water Content of Dextran Hydrogel code control control control GC-M38N8 hydrogel GC-M38N8 hydrogel GC-M38N8 hydrogel GC-M91N8 hydrogel GC-M91N8 hydrogel GC-M91N8 hydrogel

Figure 4. Enzymatic degradation of dextran-g-poly(NIPAAm-coDMAAm) below the LCST: (a) the graft lengths are Mn ) 1510, the number of grafts are about 4 (closed circle) and 8 (open circle); (b) the graft lengths are Mn ) 3800, the number of grafts are about 4 (closed circle), 8 (open circle), and 14 (closed triangle); (c) the graft lengths are Mn ) 9140, the number of grafts are about 4 (closed circle) and 8 (open circle).

dextran does not cause the steric hindrance by the chemical modification. This consideration was supported by a report of Kopecek et al.24 They reported that the chemical modification of dextran with benzylamide did not cause steric hindrance until 18 per 1 dextran molecule. Therefore, it is suggested that preventing the decrease in the viscosity in parts b and c of Figure 4 is due to a larger amount of the graft chain with increasing the graft chain length rather than the graft number. Presumably, the enzymatic accessibility causes in increasing the physical steric hindrance due to the chain entanglements. Synthesis and Characterization of Dextran Hydrogels Grafted with Thermoresponsive Polymers. To evaluate the temperature-synchronized degradation of the graft copolymers in their cross-linked state, hydrogels of dextran-g-poly(NIPAAm-co-DMAAm) were prepared by cross-linking the activated dextran-g-poly(NIPAAm-co-DMAAm) (M38N8 and M91N8), of which the degree of substitution (the number of p-nitrophenyl groups per 100 dextran glucopyranosyl monomer units) is around 9, with hexamethylenediamine. All the obtained hydrogels were transparent below the LCST of the graft chains. But these hydrogels become opaque above

temp (°C) swelling ratio water content (%) 25 35 45 25 35 45 25 35 45

21.8 21.9 21.7 18.1 17.9 16.2 16.5 16.4 14.3

95.6 95.6 95.6 94.8 94.7 94.2 94.3 94.3 93.5

the LCST. This phenomenon is considered to be due to hydration-dehydration behavior of the poly(NIPAAm-coDMAAm) graft in response to temperature. The water contents of the hydrogels in PBS are summarized in Table 2. The water content of the hydrogels was around 93-96% in a wide range of temperatures (25-45 °C). This result shows that a deswelling phenomenon, which is usually found in thermoresponsive hydrogels, is hardly observed in these hydrogels even though they exhibit a phase transition of transparent to opaque state. The contents of grafts in the hydrogels are ca. 43 (GC-M38N8) and 63 wt % (GCM91N8). Considering such high contents of thermoresponsive grafts, it is interesting that there was hardly a difference in water content between below and above the LCST. The slight decrease in swelling ratio may be due to aggregation the graft chain itself. At this time, the reason why the hydrogels kept higher water content above the LCST is unclear. Presumably, the coil-globule transition without intermolecular aggregation may contribute to remove little water from hydrogels and maintain the high water content. Enzymatic Degradation of Hydrogels of Dextran Grafted with Thermoresponsive Polymers. Degradation below or above the LCST was estimated by measuring the residual weights of the hydrogels (Figure 5). As shown in Figure 5a (below the LCST), the residual gel weight of GC-M38N8 hydrogel continuously decreased to 30% of the initial weight during 12 h, and that of GC-M91N8 kept higher level as 80%. However, the residual weights of two hydrogels were 0 and 20%, respectively, above the LCST (Figure 5b). These results suggest that longer grafts play an important role for steric hindrance on the enzymatic accessibility in the crosslinked state as well as solution state. Taking no difference in the water content between above and below the LCST into account, this temperature-synchronized degradation of the hydrogels was not caused by a change in the cross-linking density. It is suggested that a change in the chain entanglement between the longer graft chain and dextran matrix by temperature is a dominant factor to modulate the enzymatic degradation. Conclusion Several types of dextran-g-poly(NIPAAm-co-DMAAm) with variations in graft length and number were synthesized, and their enzymatic degradation was examined below and above LCST of the graft. The dextranase-catalyzed degradation below the LCST was prohibited under physiological conditions with enough graft lengths and numbers. The

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“Molecular Synchronization for Construction of New Materials System” (No. 404/11167238), from the ministry of Education, Science, Sports and Culture, Japan. References and Notes

Figure 5. (a) Enzymatic degradation of dextran-g-poly(NIPAAm-coDMAAm) hydrogels below the LCST. Closed circle is control and the graft lengths are Mn ) 3800 (open triangle) and 9140 (open circle). (b) Enzymatic degradation of dextran-g-poly(NIPAAm-co-DMAAm) hydrogels above the LCST.

modulatory factors of temperature-synchronized degradation of dextran-g-poly(NIPAAm-co-DMAAm) and its hydrogel are considered to include the following points: (1) longer graft chain with Mn over 9000, (2) flexible movement and/ or chain entanglement of the longer graft chain with dextran below the LCST, and (3) dehydration of the longer graft chain, followed by intra- or intermolecular compaction of graft chain itself. Thus, we could establish a temperature-synchronized degradation system using dextran-g-poly(NIPAAm-coDMAAm) with well-defined modulatory factors. In our hydrogels, LCST can be controllable by changing the comonomer of NIPAAm and the other stimuli-responsive cross linker such as Pluronic25 and degradable oligopeptide26,27 can be selective to modulate swelling. By the combination of the temperature-synchronized and/or swelling-deswelling materials with other stimuli responsiveness, development of a multi-stimuli-responsive material system is expected. Acknowledgment. This study was financially supported by a Grant-in-Aid for Scientific Research for Priority Areas

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