Dual-stimuli-responsive drug release from interpenetrating polymer network-structured hydrogels of gelatin and dextran

Journal of Controlled Release 54 (1998) 191–200

Dual-stimuli-responsive drug release from interpenetrating polymer network-structured hydrogels of gelatin and dextran Motoichi Kurisawa, Nobuhiko Yui* School of Materials Science, Japan Advanced Institute of Science and Technology, 1 -1 Asahidai, Tatsunokuchi, Ishikawa 923 -12, Japan Received 25 September 1997; received in revised form 11 November 1997; accepted 21 November 1997

Abstract Interpenetrating polymer network (IPN)-structured hydrogels of gelatin (Gtn) and dextran (Dex) were prepared with lipid microspheres (LMs) as a drug microreservoir, and LM release from these hydrogels was examined in relation to their dual-stimuli-responsive degradation. A phase morphology in the IPN-structured hydrogels was varied with the preparation temperature, i.e. above or below the sol-gel transition temperature (T trans ) of Gtn. The IPN-structured hydrogel prepared below T trans exhibited a specific degradation-controlled LM release behavior: LM release from the hydrogel in the presence of either a-chymotrypsin or dextranase alone was completely hindered, whereas LM release was observed in the presence of both enzymes. It is concluded that dual-stimuli-responsive drug release can be achieved by specific degradation of a particular IPN-structured hydrogel.  1998 Elsevier Science B.V. Keywords: Dual-stimuli-responsive degradation; Biodegradable hydrogels; Interpenetrating polymer network; Gelatin; Dextran

1. Introduction Biodegradable polymers have been studied in the development of drug delivery systems. The use of biodegradable polymers in controlled drug delivery systems is desirable, since the devices will be degraded in a living body after their use [1–3]. The majority of studies concerning biodegradable polymers have focused on water-insoluble polymers such as poly(lactic acid) and poly(glycolic acid) [4]. Recently, hydrogels have received much attention for the development of new applications concerned with the dosage form and drug release in response to *Corresponding author. Tel.: 181 761 51 1621; fax: 181 761 51 1625; e-mail: [email protected]

biological and external stimuli [5–7]. Hydrogels are conventionally prepared by cross-linking hydrophilic polymers, and are believed to exhibit good biocompatibility as well as high responsibility for external stimuli. More effective drug therapies for complicated diseases may require polymeric materials, the functions of which are variable or switchable in response to many kinds of stimuli. Indeed, the diagnosis of patients suffering from some diseases is generally achieved by monitoring several physiological changes [8]. However, previously reported stimuliresponsive polymers are designed to release a drug in response to a single stimulus. Thus, the integration of some information in the living body is thought to be important in the design of intelligent drug delivery

0168-3659 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 97 )00247-2

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systems. Such multi-stimuli responsive functions will act as a fail-safe mechanism for guaranteed drug delivery for a given disease. It can be indispensable to prevent the disorder of drug delivery when complicated diseases are being suffered, because much more physiological changes will occur spontaneously at the same time. From these perspectives, our recent studies have focused on dual-stimuli-responsive degradation of biodegradable hydrogels with an interpenetrating polymer network (IPN) [9–12]. The concept of dualstimuli-responsive drug release is illustrated in Fig. 1. In our first study, the in vitro degradation of IPN-structured hydrogels consisting of Nmethacryloyl-glycylglycylglycyl-terminated poly(ethylene glycol) and dextran (Dex) was examined using papain and dextranase [9,10]. Specific degradation in the presence of papain and dextranase was observed in the IPN-structured hydrogel with a particular composition of oligopeptide-PEG and Dex. This hydrogel was not degraded by one of the two enzymes. It is considered that the dual-stimuli-responsive degradation observed in the IPN-structured hydrogel is achieved by controlling the chain entanglements between the two biodegradable polymers. Our second approach for dual-stimuli-responsive degradable hydrogels was performed by combining gelatin (Gtn) and Dex as the constituents of IPNstructured hydrogels [11,12]. Gtn–Dex systems have important applications in the food and photographic industries [13]. Their binary behavior in aqueous solutions is well known to exhibit a specific phase separation that is closely related to the sol-gel transition temperature (T trans ) of Gtn [13]. The IPNstructured hydrogels were prepared below or above the T trans of Gtn, and their enzymatic degradability was examined. Dual-stimuli-responsive degradation was achieved in the IPN-structured hydrogels with

Gtn and Dex networks that were prepared below T trans having increased miscibility [11,12]. It is suggested that the achievement in dual-stimuli-responsive degradation of the Gtn–Dex hydrogels was closely related to the miscibility between Gtn and Dex networks. Drug release from biodegradable polymers can usually be controlled by diffusion through a matrix or by degradation of the matrix [14]. In particular, surface-degradable polymers have received much attention as substrates for regulated drug release. However, hydrogels are not suitable for regulated release of low molecular weight- and water-soluble drugs. In order to avoid drug leakage, hyaluronic acid (HA) hydrogels with lipid microspheres (LMs) as a drug microreservoir were prepared and their degradation properties were examined [7]. The HA hydrogels were found to result in degradation-controlled LM release. In order to achieve drug release from our designed IPN-structured hydrogels in response to their dualstimuli-responsive degradation, IPN-structured hydrogels of Gtn and Dex were prepared with LMs as a drug microreservoir in this study. The IPN-structured hydrogels were prepared with LMs at different temperatures, and LM release from these IPN-structured hydrogels was examined in relation to dualstimuli-responsive degradation.

2. Materials and methods

2.1. Materials All chemicals were analytical-grade commercial materials and were used without further purification. ¯ Dextran (Dex) (Mn5400 000) was purchased from Tokyo Chemical Industry, Japan. a-Chymotrypsin (52 U / mg) and dextranase from penicillium sp.

Fig. 1. Concept of dual-stimuli-responsive drug release by IPN-structured hydrogel.

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(12.9 U / mg) were purchased from Sigma (St. Louis, MO, USA). Gelatin (Gtn) was purchased from Wako (Osaka, Japan). The molecular weight of Gtn was determined to be 1.0310 5 using Eq. (1) [15]. ¯ n ] 0.885 [h ] 5 1.66 3 10 25 [M

(1)

where [h ] is an intrinsic viscosity. A suspension of lipid microspheres (LMs) with an average diameter of 0.2–0.4 mm was purchased from Otsuka Pharm. (Tokyo, Japan), as a 20 wt% aqueous suspension (Intralipid, Kavi vitrum, Stockholm, Sweden). Methacryloyl Dex (Ma-Dex) was prepared according to a previous study [16]. The number of methacryloyl groups in 100 glucopyranosyl units in Ma–Dex was determined to be five from 1 H NMR measurement in D 2 O.

2.2. Preparation of IPN-structured hydrogels with LMs IPN-structured hydrogels with LMs were prepared by a two-step reaction. The preparation was performed with a total polymer concentration of 35 wt% in distilled water. Gtn (0.7 g), Ma–Dex (0.7 g) and 20 wt% LM suspension (0.5 ml) were dissolved in 2.6 ml of distilled water containing ammonium peroxodisulfate (200 mg) at 378C. The homogeneous solution was injected to a spacer covered on both sides with glass plates to cross-link Ma–Dex by photoirradiation: Irradiation was carried out using an ultra high-voltage Hg lamp (500 kW) at a distance of 20 cm above or below T trans (|208C) for 30 min. After completion of the first step, the hydrogel was carefully dislodged and immersed in a formaldehyde solution (37 wt%) for 24 h at 48C. After the reaction was completed, the resulting hydrogel was placed in an excess amount of distilled water to remove the unreacted substances and was finally swollen in distilled water.

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afterwards. The hydrogel was dried in vacuo at 608C for 24 h. The water content was calculated using Eq. (2). Ww 2 Wd water content(%) 5 ]]] 3 100 Ww

(2)

where Ww is the weight of swollen film and Wd is the weight of dry film. After freeze-drying, the morphology of the IPN-structured hydrogel was observed with a scanning electron microscope (S-4100, Hitachi, Tokyo, Japan). The cross-link density of the hydrogel was determined by the method of Cluff et al. [17] using a thermal mechanical analysis (TMA) apparatus (SSC / 5200, Seiko Instruments, Tokyo, Japan). Swollen sample with a disk-shape (5 mm diameter32 mm thickness) was allowed to equilibrate for 24 h in distilled water at 25628C. The sample was placed beneath the probe of the TMA apparatus, a force of 5.0 g was applied using a calibrated weight and the displacement was determined directly from the gauge. Measurements were performed in duplicate at 25628C. The initial linear portion of the resulting force–deformation curve was used to calibrate the compression modulus and cross-link density using Eqs. (3) and (4), respectively. The dry dimension of the sample was measured after drying in vacuo at 608C for 24 h. Ec 5 Sh s /A s

(3)

ne /V 5 Sh 0 / 3A 0 RT

(4)

where Ec is the compression modulus, S is the initial slope of a force–deformation curve, ne /V is the effective network chain concentration (cross-link density), A s is the swollen area, A 0 is the unswollen area, h s is the undeformed swollen height, h 0 is the undeformed unswollen height, R is the gas constant and T is the absolute temperature.

2.3. Characterization of IPN-structured hydrogels with LMs

2.4. In vitro degradation of IPN-structured hydrogels with LMs

To determine the water content, the resulting hydrogel was swollen in distilled water for 24 h at 25628C. The hydrogel was blotted in order to remove surface water and weighed immediately

An IPN-structured hydrogel with LMs (2032032 mm in size) was immersed in 40 ml of buffer solution (35 mmol / l KH 2 PO 4 , 65 mmol / l Na 2 HPO 4 , pH 7.4) to which the enzymes were added and the

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mixture was stirred at 378C. The degree of degradation of the IPN-structured hydrogel was estimated by measuring the residual weight. LM release from the IPN-structured hydrogel was estimated by measuring the transmittance of the sampling solution at 500 nm using a spectrophotometer (V-550, Japan Spectroscopy, Tokyo, Japan). The degradation and release experiments were performed in duplicate for all of the hydrogels.

3. Results and discussion

3.1. Preparation and characterization of IPNstructured hydrogels with LMs The preparation of IPN-structured hydrogels consisting of Gtn and Dex by sequential cross-linking reactions has been reported previously [11,12]. Phase separation of these hydrogels is dominated by the temperature-dependent gelation phenomenon of Gtn. In this study, the T trans of Gtn was determined to be 208C. Thus, IPN-structured hydrogels were prepared with LMs below or above the T trans of Gtn. The preparation of a Dex network was carried out by photoirradiation either below (48C) or above the T trans (25 and 378C), as shown in Table 1. Here, the IPN-structured hydrogels prepared at different tem-

peratures (4, 25 and 378C) were designated as IPNGtn / Dex-LM-T4, IPN-Gtn / Dex-LM-T25 and IPNGtn / Dex-LM-T37, respectively. The IPN-structured hydrogels were obtained as opaque sheets due to the existence of LMs. Fig. 2 shows SEM views of these hydrogel surfaces. From SEM observation, the average distance of LM dispersion was estimated to be 0.5–1.0 mm in all of the IPN-structured hydrogels. In our previous study, the IPN-structured hydrogel prepared at 48C without LMs (IPN-Gtn / Dex-T4) was shown to be homogeneous in terms of phasecontrast microscopic observation and small-angle light scattering (SALS) measurements. In contrast, the interdomain distances of the hydrogels prepared at 258C (IPN-Gtn / Dex-T25) and 378C (IPN-Gtn / Dex-T37) were found to be 3.5 and 5.7 mm, respectively, from SALS measurements [12]. Also, Dex domains with sizes of ca. 1 and 2 mm were observed to be dispersed into the Gtn matrix, from phasecontrast micrographs of IPN-Gtn / Dex-T25 and IPNGtn / Dex-T37. A difference between the interdomain distance and the Dex domain may reflect the size of the Gtn continuous matrix. From these results, the average distance of LMs dispersed in the IPN-structured hydrogels was estimated to be much smaller than the interdomain distance of Gtn and Dex in these IPN-structured hydrogels. The water content and cross-link density of the

Table 1 Preparation of IPN-structured hydrogels with and without LMs Code

Content in feed (g)

Preparation temperature (8C)

Gtn b

Ma-Dex c,d

1st step

2nd step

IPN-Gtn / Dex-LM-T4 IPN-Gtn / Dex-LM-T25 a IPN-Gtn / Dex-LM-T37 a Gtn hydrogel-LM Dex hydrogel-LM

0.7 0.7 0.7 0.7 0

0.7 0.7 0.7 0 0.7

4 25 37 – 4

IPN-Gtn / Dex-LM-T4 e IPN-Gtn / Dex-LM-T25 e IPN-Gtn / Dex-LM-T37 e Gtn hydrogel e Dex hydrogel e

0.7 0.7 0.7 0.7 0

0.7 0.7 0.7 0 0.7

4 25 37 – 4

a

a

Water content (%)

Cross-link density (mol / cm 3 )

4 4 4 4 –

92 91 91 89 90

3.2310 26 2.8310 26 3.4310 26 3.3310 26 3.0310 26

4 4 4 4 –

88 90 90 88 87

5.2310 26 5.4310 26 6.2310 26 5.6310 26 4.8310 26

Reaction mixture: 0.5 ml of 20 wt% LM suspension was added to 2.6 ml of distilled water containing 0.7 g of Gtn and 0.7 g of Ma-Dex. The molecular weight of Gtn was determined to be 1310 5 by intrinsic viscosity measurement. c The molecular weight of Dex was 4310 4 . d The number of methacryloyl groups was 5 per 100 glucopyranosyl units in Ma-Dex. e These hydrogels were previously reported [12]. b

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Fig. 2. SEM views of lipid microspheres in IPN-structured hydrogels prepared at (A) 48C, (B) 258C and (C) 378C.

IPN-structured hydrogels were invariable and was independent of the preparation temperature, as shown in Table 1. The cross-link density of IPNstructured hydrogels was slightly lower than that found in the absence of LMs. Such a decrease in the cross-link density is thought to be due to a decrease in the concentration of reaction mixture by the addition of the LM suspension. In our previous report, the water content and cross-link density of the IPN-structured hydrogels without LMs were also invariable and independent of the preparation temperature (below dashed line in Table 1), although their phase morphology was variable [12]. A feature of IPNs is that they comprise a mixture of polymers held together predominantly by permanent entanglements of cross-linked networks [18,19]. The crosslink density of IPN-structured hydrogels depends on the number of both chemical cross-links and physical chain entanglements. From the results of SEM

observation and cross-link density, it is considered that chain entanglements between two chemically different networks exist in IPN-Gtn / Dex-LM-T4, although chain entanglements occur only within each polymer (Gtn or Dex) network in IPN-Gtn / Dex-LMT25 and IPN-Gtn / Dex-LM-T37. The water content and cross-link density of Gtn and Dex hydrogels were similar to those of IPN-structured hydrogels, although the polymer concentrations for Gtn and Dex hydrogel were set up to be lower than those for IPN-structured hydrogels. On one occasion, the degradation of a semi-IPN-structured hydrogel of Gtn and Dex was examined before the cross-linking reaction of Gtn by formaldehyde was performed. This hydrogel was degraded by dextranase, and the degradation rate was found to be higher than that of Dex hydrogel (data not shown). This result suggests that the degree of cross-linking in these IPN-structured hydrogels could be lower than those of the

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corresponding Gtn and Dex hydrogels. The similarity in the water content and the cross-link density between the IPN-structured- and the corresponding Gtn- and Dex hydrogels supports this suggestion.

3.2. Enzymatic degradation of IPN-structured hydrogels with LMs In our previous study [6], the degradation of crosslinked hydrogels was estimated by two independent procedures; one was the analysis of the degradation products by using a high-performance liquid chromatography method and the other was the measurement of changes in the residual weight of the hydrogel. Since results from these two procedures were in good agreement in terms of the degradation profiles, all of the hydrogels were monitored by measuring the residual weight in this study. Prior to the estimation of the enzymatic degradability of the IPN-structured hydrogels, the degradation of Gtn and Dex hydrogels with LMs was estimated by using a-chymotrypsin and dextranase as model enzymes of hydrolyzing Gtn and Dex, respectively (Fig. 3). The weight loss of these hydrogels (slab shape) increased linearly with time, indicating that degradation proceeded via a dissolving surface front. The degradation rate (B) of hydrogels with a slab-shape can be calculated from the following equation:

Fig. 3. Enzymatic degradation of hydrogels with LMs in phosphate buffer at 378C. s, Gtn hydrogel–LM by 5 U / ml achymotrypsin; n, Dex hydrogel–LM by 0.5 U / ml dextranase.

B 5 h / 2t `

(5)

where h is the initial thickness of the slab and t ` is the time it takes to complete degradation. This equation is based on the assumption that degradation takes place from both sides of the slab and not from the edges. The degradation rate of Gtn hydrogel by a-chymotrypsin (5 U / ml) was estimated to be 2.33 10 26 cm / s. Dex hydrogel prepared from Ma–Dex was observed to be degraded by dextranase, and the degradation rate by dextranase (0.5 U / ml) was estimated to be 1.9310 26 cm / s. Enzymatic degradation of the IPN-structured hydrogels with LMs was examined under three different conditions: In buffers containing a-chymotrypsin (case i), dextranase (case ii) and with both a-chymotrypsin and dextranase (case iii). The enzymatic activity of dextranase was confirmed to be almost invariant and independent of the a-chymotrypsin concentration (data not shown). Our previous study demonstrated dual-stimuli-responsive degradation of the IPN-structured hydrogels in relation to their phase morphology: an IPN-structured hydrogel with enhanced miscibility of Gtn and Dex (IPN-Gtn / DexT4) exhibited dual-stimuli-responsive degradation [12]. It was considered that the phase morphology in the IPN-structured hydrogels prepared at different temperatures is dominated by intermolecular hydrogen bonding of Gtn networks as the driving force for the miscibility of aqueous solutions of Gtn and Dex. Such a different phase morphology in the IPNstructured hydrogels leads to a variation in the physical chain entanglements between Gtn and Dex networks. In particular, the homogeneity observed in IPN-Gtn / Dex-T4 indicates the existence of effective chain entanglements between Gtn and Dex networks. Enzymatic degradability of the IPN-structured hydrogels with LMs was similar to that of the IPNstructured hydrogels without LMs. The degradation of the IPN-structured hydrogels was observed to proceed perfectly in the presence of both a-chymotrypsin and dextranase (case iii) and to be independent of the preparation temperature. In the case of the IPN-structured hydrogel prepared below the T trans (IPN-Gtn / Dex-LM-T4), no degradation was observed in the presence of a single enzyme (cases i and ii), as shown in Fig. 4. However, degradation was observed in the IPN-structured hydrogels pre-

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Fig. 4. Enzymatic degradation of IPN-Gtn / Dex-LM-T4 in phosphate buffer at 378C. s, 5 U / ml a-chymotrypsin10.5 U / ml dextranase; n, 5 U / ml a-chymotrypsin; h, 0.5 U / ml dextranase.

Fig. 6. Enzymatic degradation of IPN-Gtn / Dex-LM-T37 in phosphate buffer at 378C. s, 5 U / ml a-chymotrypsin10.5 U / ml dextranase; n, 5 U / ml a-chymotrypsin; h, 0.5 U / ml dextranase.

pared above the T trans (IPN-Gtn / Dex-LM-T25 and IPN-Gtn / Dex-LM-T37) in the presence of a-chymotrypsin (case i) (Figs. 5 and 6). Thus, dual-stimuliresponsive degradation was achieved in the IPNstructured hydrogel prepared below the T trans (IPNGtn / Dex-LM-T4). In a previous study by us, the interdomain distance of IPN-structured hydrogels was observed above the T trans and it increased with the preparation temperature. Such a different phase morphology in the IPNstructured hydrogels has been thought to correlate directly with the degree of physical chain entangle-

ments between Gtn and Dex networks. The phase morphology in the IPN-structured hydrogels with LMs could not be estimated due to the turbidity of LMs in terms of phase-contrast microscopic observation and SALS measurements. The relationship between the degradation rates and the preparation temperature of the IPN-structured hydrogels with and without LMs is summarized in Fig. 7. Here, the degradation rate was calculated by dividing the time

Fig. 5. Enzymatic degradation of IPN-Gtn / Dex-LM-T25 in phosphate buffer at 378C. s, 5 U / ml a-chymotrypsin10.5 U / ml dextranase; n, 5 U / ml a-chymotrypsin; h, 0.5 U / ml dextranase.

Fig. 7. Relationship between the degradation rates and the preparation temperature of IPN-structured hydrogels. s, IPNstructured hydrogels with LMs by 5 U / ml a-chymotrypsin10.5 U / ml dextranase (case iii); n, IPN-structured hydrogels with LMs by 5 U / ml a-chymotrypsin (case i). d, IPN-structured hydrogels without LMs by 5 U / ml a-chymotrypsin10.5 U / ml dextranase (case iii); m, IPN-structured hydrogels without LMs by 5 U / ml a-chymotrypsin (case i).

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to complete degradation to the half of thickness of slab. In the case of (i), the degradation rates of the hydrogels with LMs were similar to those of the hydrogels without LMs. However, in the case of (iii), higher degradation rates were observed in the hydrogels with LMs prepared above the T trans . These results may be explained by the hydrogels with LMs having a slightly lower cross-link density.

3.3. Degradation-controlled release of lipid microspheres from IPN-structured hydrogels LM release from Gtn and Dex hydrogels varied depending on the degree of enzymatic degradation (Fig. 8). Fig. 9 shows LM release from the IPN-Gtn / Dex-LM-T4. LMs were released from the hydrogel in proportion to the degradation that occurred in the presence of both enzymes, whereas no LM release was observed in the presence of a single enzyme. LM release from IPN-Gtn / Dex-LM-T25 and IPNGtn / Dex-LM-T37 was observed in the presence of a-chymotrypsin (Figs. 10 and 11). Thus, such LM release from IPN-structured hydrogels was well correlated with the degree of enzymatic degradation. Fig. 12 shows the relationship between LM release and the degradation (weight loss) of IPN-structured hydrogels in the presence of both enzymes. Only the plot for IPN-Gtn / Dex-LM-T4 provided almost a linear relationship, suggesting that LM release was dominated by its dual-stimuli-responsive degrada-

Fig. 8. Lipid microsphere release from hydrogels with LMs in phosphate buffer at 378C. s, Gtn hydrogel–LM by 5 U / ml a-chymotrypsin; n, Dex hydrogel–LM by 0.5 U / ml dextranase.

Fig. 9. Lipid microsphere release from IPN-Gtn / Dex-LM-T4 in phosphate buffer at 378C. s, 5 U / ml a-chymotrypsin10.5 U / ml dextranase; n, 5 U / ml a-chymotrypsin; h, 0.5 U / ml dextranase.

tion. Non-linear relationships were observed in IPNGtn / Dex-LM-T25 and IPN-Gtn / Dex-LM-T37. This result indicates that there may be another factor, such as diffusion, involved in LM release from IPN-Gtn / Dex-LM-T25 and IPN-Gtn / Dex-LM-T37. The amount of LM release from IPN-Gtn / Dex-LM-T25 was larger than that from IPN-Gtn / Dex-LM-T37. The water content and the cross-link density of these hydrogels were almost constant, in spite of different preparation temperatures (4–378C). As mentioned above, the interdomain distance and domain sizes of IPN-Gtn / Dex-T25 were found to be smaller than those of IPN-Gtn / Dex-T37. Thus, it appears that

Fig. 10. Lipid microsphere release from IPN-Gtn / Dex-LM-T25 in phosphate buffer at 378C. s, 5 U / ml a-chymotrypsin10.5 U / ml dextranase; n, 5 U / ml a-chymotrypsin; h, 0.5 U / ml dextranase.

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Fig. 11. Lipid microsphere release from IPN-Gtn / Dex-LM-T37 in phosphate buffer at 378C. s, 5 U / ml a-chymotrypsin10.5 U / ml dextranase; n, 5 U / ml a-chymotrypsin; h, 0.5 U / ml dextranase.

smaller Dex domains cause enhanced LM diffusion during the degradation process. Fig. 13 demonstrates LM release from IPN-Gtn / Dex-LM-T4 in response to changes in buffers containing two different enzymes. LM release from the hydrogel was not observed in the presence of dextranase. LM release was found to occur at a constant rate when both enzymes were added to the buffer. However, in the presence of a-chymotrypsin alone, LM release immediately stopped, as did enzymatic degradation. This result demonstrates that

Fig. 12. Relationship between lipid microsphere release and the degradation of IPN-structured hydrogels in the presence of 5 U / ml a-chymotrypsin and 0.5 U / ml dextranase. s, IPN-Gtn / Dex-LM-T4; n, IPN-Gtn / Dex-LM-T25; h, IPN-Gtn / Dex-LMT37.

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Fig. 13. Dual-stimuli-responsive lipid microsphere release from IPN-Gtn / Dex-LM-T4 in phosphate buffer at 378C.

such dual-stimuli-responsive drug release is achieved only by specific degradation behavior of the IPNstructured hydrogel and may also be useful in preventing problems in drug release caused by a single enzyme.

4. Conclusion IPN-structured hydrogels of Gtn and Dex were prepared with LMs as a model of a drug substrate exhibiting dual-stimuli-responsive drug release. The IPN-structured hydrogel prepared below the T trans exhibited a specific degradation behavior, i.e., hydrogel degradation by either a-chymotrypsin or dextranase alone was completely hindered whereas the hydrogel was completely degraded in the presence of both enzymes. Regulated LM release in response to dual enzymes was achieved in the IPN-structured hydrogel prepared below the T trans , although LM release from the IPN-structured hydrogels prepared at temperatures above the T trans was observed, even in the presence of a single enzyme. The difference in the LM release behavior is thought to have been caused by the enzymatic degradability of the hydrogels being closely related to physical chain entanglements between chemically different polymer networks. Such a dual-stimuli-responsive drug release system can act in preventing drug release problems arising from the unexpected appearance of a single enzyme. It is believed that biodegradable hydrogels

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with a particular IPN structure may be promising as substrates for temporally controlled drug delivery with a fail-safe mechanism.

Acknowledgements The authors are grateful to Prof. Dr. Minoru Terano and Dr. Hideharu Mori, Japan Advanced Institute of Science and Technology, for their valuable suggestions and discussion throughout this study.

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