Novel Method Using a Temperature-Sensitive Polymer

Biomacromolecules 2004, 5, 1917-1925

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Novel Method Using a Temperature-Sensitive Polymer (Methylcellulose) to Thermally Gel Aqueous Alginate as a pH-Sensitive Hydrogel Hsiang-Fa Liang, Min-Hao Hong, Rong-Ming Ho, Ching-Kuang Chung, Yu-Hsin Lin, Chun-Hung Chen, and Hsing-Wen Sung* Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan 30013, Republic of China Received March 30, 2004; Revised Manuscript Received May 7, 2004

A novel method using a temperature-sensitive polymer (methylcellulose) to thermally gel aqueous alginate blended with distinct salts (CaCl2, Na2HPO4, or NaCl), as a pH-sensitive hydrogel was developed for protein drug delivery. It was noted that the salts blended in hydrogels may affect the structures of an entangled network of methylcellulose and alginate and have an effect on their swelling characteristics. The methylcellulose/alginate hydrogel blended with 0.7 M NaCl (with a gelation temperature of 32 °C) demonstrated excellent pH sensitivity and was selected for the study of release profiles of a model protein drug (bovine serum albumin, BSA). In the preparation of drug-loaded hydrogels, BSA was well-mixed to the dissolved aqueous methylcellulose/alginate blended with salts at 4 °C and then gelled by elevating the temperature to 37 °C. This drug-loading procedure in aqueous environment at low temperature may minimize degradation of the protein drug while achieving a high loading efficiency (95-98%). The amount of BSA released from test hydrogels was a function of the amount of alginate used in the hydrogels. The amount of BSA released at pH 1.2 from the test hydrogel with 2.5% alginate was relatively low (20%), while that released at pH 7.4 increased significantly (86%). In conclusion, the methylcellulose/alginate hydrogel blended with NaCl could be a suitable carrier for site-specific protein drug delivery in the intestine. 1. Introduction Along with the recent advance of recombinant DNA techniques, production of therapeutically active peptides and proteins in large quantities has become feasible. 1 It is known that the oral route is the most convenient and comfortable way of administering drugs. However, peptide and protein drugs must be protected from the harsh environment of gastric media in the stomach before they can be absorbed in the intestine, if given orally.2 In the design of oral delivery of peptide or protein drugs, pH-sensitive hydrogels have attracted increasing attention recently. A variety of synthetic or natural polymers with acidic or basic pendent groups have been employed to fabricate pH-sensitive hydrogels.3,4 Among them, alginate is one of those commonly used. Alginate, a polyanionic copolymer of mannuronic and guluronic sugar residues, has been widely used in biomedical applications.5 The most common methods used to load drugs involve the use of organic solvents6 or loading during cross-linking of the polymeric hydrogels.7 It is known that organic solvents may cause degradation of peptide or protein drugs that are unstable and sensitive to their environments. Additionally, the use of cross-linking agents to form polymeric hydrogels may lead to toxic side effects (due to residual cross-linking agents) or to unwanted reactions with drugs. Therefore, it is * To whom correspondence should be addressed. Telephone: 886-3574-2504. Fax: 886-3-572-6832. E-mail: [email protected].

desirable to search for appropriate measures to form hydrogel networks while loading the drug.8 In this study, a novel method using a temperature-sensitive polymer (methylcellulose) to thermally gel aqueous alginate as a pH-sensitive hydrogel was developed. This allows manipulation of temperature in aqueous solution to load a model protein drug (bovine serum albumin, BSA) in polymeric hydrogels. The system has the advantage that only physical interaction between polymers and proteins is used to entrap drugs in the hydrogels. No chemical modification of the drugs is necessary which usually leads to a decrease in bioactivity of the drugs. Methylcellulose forms aqueous solutions, which demonstrate a unique property of forming reversible physical gels due to hydrophobic interactions when heated above a particular temperature.9,10 Tate et al. have studied the use of methylcellulose as a temperature-sensitive scaffolding material.11 In their study, methylcellulose solutions were produced to reveal low viscosity at room temperature and form a soft gel at 37 °C, thus making methylcellulose attractive for minimally invasive procedures in vivo. Acellular 2% aqueous methylcellulose was microinjected into the brains of rats 1 week after cortical impact injury and examined at distinct durations postoperatively. Their data indicated that methylcellulose is well-suited as a biocompatible injectable scaffold for the repair of defects in the brain. The aim of the study was to develop a pH-sensitivehydrogel-based controlled release system for protein drug

10.1021/bm049813w CCC: $27.50 © 2004 American Chemical Society Published on Web 06/25/2004

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delivery. This hydrogel was composed of methylcellulose and alginate blended with distinct salts to form a polymeric network. Preparation of the methylcellulose-/alginate-based hydrogels is reported. Swelling characteristics of these hydrogels as a function of pH values were investigated. Additionally, release profiles of a model protein drug (BSA) from test hydrogels were studied in simulated gastric and intestinal media. 2. Materials and Methods 2.1. Methylcellulose Solutions. Methylcellulose (with a viscosity of 4000 cP for a 2% (w/v) aqueous solution at 25 °C) was obtained from Sigma (St. Louis, MO). To prepare methylcellulose solutions blended with distinct salts, a stock aqueous methylcellulose (4% (w/v)) was first prepared. Methylcellulose solutions in different concentrations were prepared by adding the stock solution into beakers with proper amounts of cold deionized water. Subsequently, distinct salts (NaCl, CaCl2, or Na2HPO4) in varying concentrations were added, and the mixtures were gently stirred overnight at 4 °C. 2.2. Gelation Temperatures of Methylcellulose Solutions. The physical gelation phenomena of methylcellulose solutions were observed visually as per a method described in the literature.11 Methylcellulose solutions blended with distinct salts (5 mL samples) were exposed to elevating temperatures via a standard hot-water bath. Behavior was recorded at intervals of approximately 2 °C over the range of 25-70 °C. The heating rate between measurements was approximately 0.5 °C/min. At each temperature interval, the solutions/gels were allowed to equilibrate for 1 h. A “gel” criterion was defined as the temperature at which the clear solution did not flow upon inversion of the container. 11 2.3. Preparation of Test Hydrogels. Methylcellulose/ alginate hydrogels with distinct weight ratios were prepared as follows. A stock solution of aqueous methylcellulose (4% (w/v)) was prepared with the procedure described above. Subsequently, appropriate amounts of sodium alginate (0.65.0% (w/v)) were added to this stock solution and wellmixed. The mixed methylcellulose/alginate was examined by means of the Fourier transformed infrared spectroscopy (FT-IR) and the scanning electron microscopy (SEM, JEOL JSM-6587, Tokyo, Japan) equipped with an energydispersive X-ray spectrometry (EDS, Oxford6587), with methylcellulose and alginate as controls. Test samples used for the FT-IR analysis first were dried and ground into a powder form. The powder then was mixed with KBr (1: 100) and pressed into a disk. Analysis was performed on an FT-IR spectrometer (Perkin-Elmer Spectrum RX1 FT-IR System, Buckinghamshire, England). The samples were scanned from 400 to 4000 cm-1. In the SEM-EDS study,12 element compositions (C and O) of the mixed methylcellulose/alginate (2.0%:2.5% (w/v)) samples were analyzed. Each element produces characteristic X-rays that are displayed as peaks at one or more specific energy levels in the EDS spectrum. Additionally, the energy-dispersive X-ray mapping technique was used to image the spatial distribution of the sodium element

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on alginate on the superficial and cross-sectional surfaces of test samples. The mixed methylcellulose/alginate solutions were blended with distinct salts (0.7 M NaCl, 0.7 M CaCl2, or 0.2 M Na2HPO4), and their gelation temperatures were determined as aforementioned. To make the gelled methylcellulose/alginate films for the following studies, the methylcellulose/alginate solutions blended with distinct salts (all with a gelation temperature of about 32 °C) were sonicated to remove the trapped air bubbles. After thoroughly stirring, the air-bubblefree solutions were poured into shallow dishes (with a diameter of 8.5 cm) and allowed to equilibrate at 37 °C to become gelation. The gelled methylcellulose/alginate films were thoroughly rinsed with deionized water to remove residual salts. After drying at 37 °C, the dried methylcellulose/alginate films (about 0.35 mm in thickness) were cut into small disks (with a diameter of 8.5 mm). The prepared films can be stored at any temperature if they are in a dried form. In contrast, the undried films need to be kept above their gelation temperature (32 °C) to prevent them from disintegrating. 2.4. Swelling Characteristics of Test Hydrogels. The swelling characteristics of test methylcellulose/alginate hydrogels blended with distinct salts were determined by immersing dried test samples (∼20 mg) to swell in 5 mL of a solution (0.1 N HCl) at pH 1.2 for 2 h and subsequently in a phosphate-buffered saline (PBS, Sigma Chemical Co.) solution at pH 7.4 (I ) 0.165) at 37 °C, simulating gastrointestinal tract conditions.13,14 At specific time intervals, the samples were removed from the swelling medium and were blotted with a piece of paper towel to absorb excess water on the surfaces. The swelling ratios (Qs) of test samples were calculated from the following expression: Qs ) (Ws - Wd)/Wd × 100% where Ws is the weight of the swollen test sample and Wd is the weight of the dried test sample. Test samples, which had a better swelling characteristic for protein drug delivery among all studied groups, were subsequently selected for the BSA release profile study. 2.5. Release Profiles of BSA from Test Hydrogels. In the preparation of the drug-loaded methylcellulose/alginate hydrogel, BSA with a final concentration of 1% (w/v) was added to the dissolved methylcellulose/alginate solution blended with 0.7 M NaCl with continuous stirring at 4 °C. After dissolution of the drug, the blend (with a gelation temperature of 32 °C) was gelled by elevating the temperature to 37 °C as described before. The rest of the procedures used were similar to those in the preparation of test hydrogels without loading drug. For determination of drug loading efficiency, test hydrogels containing BSA were immersed in PBS. After stirring at 4 °C for 3 days, the solution was placed in a mortar and triturated thoroughly to take BSA out.15 Subsequently, the whole mixture was filtered and analyzed for the amount of BSA using the Bradford method. The calibration curve was made using distinct concentrations of BSA.16 To study the release profiles for test hydrogels, dried test samples (∼100 mg) were immersed in a 30 mL solution with

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Biomacromolecules, Vol. 5, No. 5, 2004 1919

Figure 1. Schematic illustrations of the physical structures of aqueous methylcellulose with and without addition of salts at lower temperatures (solution state) and at elevated temperatures (gel state).

a value of pH 1.2 for 2 h and subsequently in a solution of pH 7.4, as the procedures used in the study of their swelling characteristics.13,14 At predetermined time points, 100 µL of this solution was taken out and analyzed by the Bradford method for released BSA at 595 nm using a spectrophotometer.16 The percentage of the cumulative amount of released BSA was determined from standard calibration curves. The stability of the released BSA was determined by analyzing the conformation of the released BSA using an Aviv 202 spectropolarimeter and comparing the spectrum with that of standard BSA (0.2 mg/mL). The ellipticity (θ, mdeg) was measured from 260 to 195 nm.15 2.6. Statistical Analysis. Statistical analysis for the determination of differences in the measured properties between groups was accomplished using one-way analysis of variance and determination of confidence intervals, performed with a computer statistical program (Statistical Analysis System, Version 6.08, SAS Institute Inc., Cary,

NC). All data are presented as a mean value with its standard deviation indicated (mean ( SD). 3. Results and Discussion Investigations of hydrogels have been focused on functional hydrogels. These functional hydrogels may change their structures per the environments they are exposed to, such as temperature or pH.17-22 In this study, a novel method using a temperature-sensitive hydrogel (methylcellulose) to harden aqueous alginate as a pH-sensitive-based system for protein drug delivery was developed. 3.1. Gelation Temperatures of Methylcellulose Solutions. It was reported that the strength and toughness of methylcellulose films add strength to adhesives in which methylcellulose is compounded. Additionally, the physiological inertness and the storage stability of methylcellulose permit its use in cosmetics, pharmaceuticals, and food

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products.23 Commercial methylcellulose is a heterogeneous polymer consisting of highly substituted zones (hydrophobic zones) and less substituted ones (hydrophilic zones).24 Methylcellulose forms aqueous solutions exhibiting a unique property of forming reversible physical gels due to hydrophobic interactions when heated above a particular temperature.9,10 In the solution state at lower temperatures, methylcellulose molecules are hydrated and there is little polymer-polymer interaction other than simple entanglement (Figure 1). It was reported that when a methylcellulose solution is heated, the viscosity decreases to a minimum just below the gelation temperature and subsequently rises rapidly when the gel point is reached.23 As the temperature is increased, methylcellulose molecules absorb energy and gradually lose their water of hydration, resulting in lowering of the viscosity. Eventually, a polymer-polymer association takes place, due to hydrophobic interactions, causing cloudiness in the solution and an infinite network structure that results in a sharp rise in the viscosity and turbidity (gel state, Figure 1).25 It was reported that the addition of salts lowers the gelation temperature of methylcellulose due to its dehydration.23 On the addition of a salt, the water molecules will locate themselves around the salt, thus reducing the intermolecular hydrogen-bond formation between water molecules and the hydroxyl groups of methylcellulose. This can increase the hydrophobic interaction between methylcellulose molecules and lead to a lowering of their gelation temperature (Figure 1). Tate et al. reported that the gelation temperature values for a methylcellulose concentration series obtained by both physical observations (the earliest temperature at which the gel is formed) and rheological measurements (an abrupt increase in viscosity at a characteristic temperature) were found to be statistically equivalent, indicating a correlation between rheological and physical data.11 In the study, the physical behavior of aqueous methylcellulose solutions blended with distinct salts was investigated as a function of temperature as the polymer systems transitioned from the solution to gel states. All aqueous methylcellulose compositions changed from a clear solution at lower temperatures to an opaque gel at elevated temperatures. Both the methylcellulose concentration and the salt blended were found to play a role in the physical sol-gel behavior of these compounds. As shown in Figure 2a, the gelation temperature decreased significantly with increasing concentration of methylcellulose blended with 0.1 M NaCl, CaCl2, or Na2HPO4 (p < 0.05). In the preparation of aqueous methylcellulose, it was found that the solution was too viscous to be manipulated with when its concentration was greater than 2% (w/v). Therefore, a 2% (w/v) concentration of methylcellulose was used in the rest of the study. At the same concentration of methylcellulose, the gelation temperatures of aqueous methylcellulose solutions blended with distinct salts follow the order NaCl H CaCl2 > Na2HPO4 (p < 0.05). Normally, an electrolyte (the salt blended) has a greater affinity for water than polymers resulting from removing water of hydration from the polymer and thus dehydrating or “salting out” the polymer. The ability of an

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Figure 2. (a) Gelation temperatures of aqueous methylcellulose blended with 0.1 M NaCl, CaCl2, or Na2HPO4: effect of methylcellulose concentration. (b) Gelation temperatures of aqueous methylcellulose (2% (w/v)) blended with distinct salts: effect of salt concentration.

electrolyte to salt out a polymer from its solution generally follows the salts order in the lyotropic series.26 The cations follow the order Li+ > Na+ > K+ > Mg2+ > Ca2+ > Ba2+, and more common anions follow the order CNS- < I- < Br- < NO3- < Cl- < tartrate < SO42- < PO43-.26 Accordingly, more water molecules were removed from aqueous methylcellulose when Na2HPO4 was added in the polymeric hydrogel, resulting in a lower gelation temperature (Figure 1). Although NaCl is greater than CaCl2 in the salts order in the lyotropic series, the ionic strength of CaCl2 is estimated to be 3-fold of NaCl. Therefore, the gelation temperatures of aqueous methylcellulose solutions blended with NaCl or CaCl2 were approximately the same. As the molarity of the salt blended increased, the gelation temperature of aqueous methylcellulose (2% (w/v)) decreased significantly (p < 0.05, Figure 2b). As shown, the gelation temperature of aqueous methylcellulose was approximately 32 °C when blended with 0.7 M CaCl2, 0.2 M Na2HPO4, or 0.7 M NaCl. The aforementioned results indicated that the temperature at which gelation is initiated can be altered by the concentration of methylcellulose and the formulation of the aqueous solvent. 3.2. Preparation of Test Hydrogels. Results of analysis of FT-IR spectra for methylcellulose, alginate, and methylcellulose/alginate (2.0%:2.5% (w/v)) showed that the characteristic peak observed at 2826 cm-1 was the CH stretch in methyl ether (O-CH3) on methylcellulose (Figure 3).



Figure 3. FT-IR spectra of methylcellulose, alginate, and the well-mixed methylcellulose/alginate. Table 1. SEM-EDS Analysis of the Elemental Compositions (C and O) of Methylcellulose, Alginate, and the Well-Mixed Methylcellulose (MC)/Alginate (2.0%:2.5% (w/v); n ) 6)

C, at. % O, at. %

MC

alginate

MC/alginate (surface)

MC/alginate (cross-section)

56.9 ( 0.7 43.1 ( 0.7

38.6 ( 0.3 61.4 ( 0.3

47.2 ( 0.3 52.8 ( 0.3

48.5 ( 0.7 51.5 ( 0.7

Additionally, the spectrum of alginate showed a characteristic peak at 1615 cm-1 for the associated carboxylic acid salt (-COO- antisymmetric stretch, 1500-1650 cm-1). In contrast, the spectrum of the methylcellulose/alginate showed both the aforementioned characteristic peaks for methylcellulose and alginate. However, recognizable peak shifts were found in the spectrum: 2826-2834 cm-1 for the CH stretch in methyl ether on methylcellulose and 1615-1630 cm-1 for the associated carboxylic acid salt on alginate. This suggested that methylcellulose/alginate was well-mixed to lead to significant changes on molecular dynamics for the constituted components. The results of analysis of the surface (both superficial and cross-sectional surfaces) by semiquantitative SEM-EDS for methylcellulose, alginate, and methylcellulose/alginate are reported in Table 1. As shown, the elemental compositions of methylcellulose/alginate included carbon (∼48%) and oxygen (∼52%) that approximately matched their corresponding compositions of the constituted components (2% methylcellulose and 2.5% alginate (w/v)). Additionally, the results of EDS mapping (Figure 4a,b) showed that the sodium element of alginate was homogeneously distributed on both the superficial and cross-sectional surfaces of methylcellulose/alginate. These results indicated that the methylcellulose/ alginate was well-mixed in the preparation of test hydrogels. Thus, it is hypothesized that, in the preparation of the thermally gelled methylcellulose/alginate films, alginate entangled with methylcellulose, to form a networklike structure.

Figure 4. Results of the EDS mapping of the sodium element on alginate on (a) the superficial and (b) cross-sectional surfaces of methylcellulose/alginate.

It was found that the gelation temperatures of methylcellulose/alginate hydrogels blended with distinct salts were not significantly influenced by the amounts of alginate used. When blended with 0.7 M CaCl2, 0.2 M Na2HPO4, or 0.7 M NaCl, the gelation temperatures of aqueous methylcellulose (2% (w/v)) with different amounts of alginate (up to

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2.5% (w/v)) remained approximately 32 °C. This is because the salts blended in hydrogels have a much greater affinity for water than alginate. Therefore, the blended salts (rather than alginate) determined the gelation temperatures of methylcellulose/alginate hydrogels. For simulating gastrointestinal tract conditions, the swelling characteristics and the drug release profiles of methylcellulose/alginate hydrogels blended with distinct salts (with a gelation temperature of approximately 32 °C) were determined by immersing dried test samples to swell in a solution at pH 1.2 for 2 h and subsequently in another solution at pH 7.4 at 37 °C. 3.3. Swelling Characteristics of Test Hydrogels. Figure 5a-c gives the swelling characteristics of methylcellulose/ alginate hydrogels blended with distinct salts (0.7 M CaCl2, 0.2 M Na2HPO4, or 0.7 M NaCl). These test hydrogels all had a gelation temperature at about 32 °C. The salts blended in polymeric hydrogels may affect the structures of the entangled network of methylcellulose and alginate and have an effect on their swelling characteristics. It is known that contact between alginate (a polyanionic polymer) and Ca2+ in solution induces immediate ionic polymerization of alginate via binding of Ca2+ within the cavities of the guluronic residues of alginate.27 Thus, a semi-interpenetrating networklike structure is formed within the methylcellulose/ alginate hydrogel when blended with divalent cations such as CaCl2 (Figure 6). In this case, at 37 °C (beyond the gelation temperature of the test hydrogel), there are the hydrophobic interaction between methylcellulose molecules, the hydrogen-bond formation between -COOH and -OH groups in hydrogel, and the ionic cross-link between alginate molecules within the test hydrogel (Figure 6). In contrast, with univalent cations (Na2HPO4 or NaCl) blended in the methylcellulose/alginate hydrogels, no ionic cross-link between the carboxylate ions (-COO-) on alginate could be formed. Hence, there are only the hydrophobic interaction between methylcellulose molecules and the hydrogen bonds formed by the -OH and -COOH groups for the test hydrogels blended with Na2HPO4 or NaCl at 37 °C. As shown in Figure 5a, the test hydrogels blended with CaCl2 swelled only slightly at pH 1.2 (∼180%), due to the constraint of the hydrophobic interaction between methylcellulose molecules formed at 37 °C and the hydrogen bonds created between the -OH and -COOH groups in hydrogels. At pH 7.4, the increases in the swelling ratio for the test hydrogels were limited (∼280%). This is because the ionic cross-link between the carboxylate ions on alginate, in the presence of Ca2-, prevents the test hydrogels from swelling. No significant changes in swelling ratio were observed with an increasing amount of alginate used (p > 0.05). In contrast, the test hydrogels blended with Na2HPO4 or NaCl swelled significantly at pH 7.4 (Figure 5b,c, p < 0.05], since no ionic cross-link between alginate molecules could be formed in these cases (Figure 6). This is mainly resulting from the electrostatic repulsion between the negatively charged carboxylate ions on alginate (Figure 6). As demonstrated in Figure 7, at pH 1.2, the characteristic peak observed at 1734 cm-1 for the test hydrogel blended with

Liang et al.

Figure 5. Swelling characteristics of methylcellulose/alginate hydrogels blended with distinct salts: (a) 0.7 M CaCl2, (b) 0.2 M Na2HPO4, and (c) 0.7 M NaCl determined by immersing dried test samples to swell in a solution at pH 1.2 for 120 min and subsequently in another solution at pH 7.4. These test hydrogels all had a gelation temperature at about 32 °C.

NaCl was the carboxylic acid on alginate (CdO stretch of the carboxylic acid on alginate). At pH 7.4, the carboxylic acid groups on alginate in the test hydrogel became progressively ionized (-COO-, the peak observed at 1631 cm-1). Therefore, at pH 7.4, the hydrogels blended with Na2HPO4 or NaCl swelled significantly (Figure 5b,c). With an increasing amount of alginate used, which has a higher concentration of carboxylic groups, the swelling ratios of test hydrogels increased significantly (p < 0.05). At pH 1.2, the test hydrogels blended with Na2HPO4 swelled more promptly and significantly (swelling ratio,



Figure 6. Schematic illustrations of the physical structures of methylcellulose/alginate hydrogels blended with CaCl2, Na2HPO4, or NaCl at pH 1.2 and pH 7.4 at 37 °C.

600-700%) than their counterparts blended with NaCl (150-200%). Presumably this was due to the fact that Na2HPO4 entrapped within the test hydrogel has a greater affinity for water than NaCl as described above (Figure 6). A higher swelling of the hydrogel (e.g., the hydrogel blended with Na2HPO4) may fail to provide adequate retention of

encapsulated proteins at the lower end of the gastric pH range (pH 1.2). Therefore, the methylcellulose/alginate hydrogel blended with 0.7 M NaCl had a better pH-sensitive characteristic among all studied groups and was selected for the study of release profiles of a model protein drug, BSA.

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Figure 7. FT-IR spectra of the methylcellulose/alginate hydrogel blended with NaCl at pH 1.2 and pH 7.4.

Figure 8. BSA release profiles from the methylcellulose/alginate hydrogels blended with 0.7 M NaCl with different amounts of alginate determined by immersing dried test samples to swell in a solution at pH 1.2 for 120 min and subsequently in another solution at pH 7.4.

At pH 1.2, with an increasing amount of alginate used (e.g., increasing the concentrations of -COOH and -OH groups), the swelling ratio of the test hydrogel blended with NaCl decreased due to a greater degree of hydrogen bonds formed between -COOH and -OH groups in the hydrogels. Thus the penetration of water into hydrogels is limited at low pH. It was found that there was no significant loss of material at pH 1.2, while disintegration of the polymeric hydrogel was observed at pH 7.4. 3.4. Release Profiles of BSA from Test Hydrogels. Figure 8 shows the BSA release profiles from the methylcellulose/alginate hydrogels blended with 0.7 M NaCl with different amounts of alginate used obtained at pH 1.2 and pH 7.4. A high drug-loading efficiency was achieved for all three test hydrogels: 98.2 ( 0.4, 96.3 ( 0.6, and 95.6 ( 0.8% (n ) 6) for the hydrogels with 0.5, 1.5, and 2.5% (w/ v) alginate used, respectively. As shown, at pH 1.2, the

Figure 9. CD spectra of the standard BSA, the loaded BSA when released at pH 1.2, and the loaded BSA released at pH 7.4 (at pH 1.2 for 2 h and then at pH 7.4).

amount of BSA released for the test hydrogel with a 0.5% alginate used was about 40%, which failed to provide adequate retention of encapsulated proteins. In contrast, those released at pH 1.2 for the test hydrogels with 1.5 and 2.5% alginate used were relatively low; only about 20% of encapsulated proteins released within 2 h. It was found that the cumulative release of BSA from these test samples was increased slightly to ∼25% when left at pH 1.2 for 24 h. The relatively low amounts of protein released were probably related to the comparatively low degrees of swelling of test hydrogels at pH 1.2 (Figure 5c). At pH 7.4, the amounts of BSA released increased significantly (p < 0.05): 66, 73, and 86% for the test hydrogels with 0.5, 1.5, and 2.5% alginate used, respectively. This is because the swelling of the test hydrogel network increased considerably due to ionization of carboxylic groups on alginate at neutral pH (Figure 5c). With an increasing amount of alginate used, this phenomenon was significantly more pronounced (p < 0.05).


The circular dichroism (CD) spectra (Figure 9) showed that there was a slight change in the conformation of the loaded BSA when released at pH 1.2 as compared to the standard BSA. It is known that the stability of proteins at pH 1.2 is poor.29 In contrast, no significant conformation change was noted for the loaded BSA released at pH 7.4 (at pH 1.2 for 2 h and then at pH 7.4). This result suggested that the secondary structure of BSA was preserved after the loading procedure despite prolonged contact of BSA with the polymer and release into a buffer solution at pH 7.4. 4. Conclusions In conclusion, the methylcellulose/alginate hydrogel blended with NaCl demonstrated excellent pH sensitivity and could be a suitable polymeric carrier for site-specific bioactive protein drug delivery in the intestine. The main advantage of this system is that it allows aqueous loading of drugs with a high loading efficiency while preserving the bioactivity of protein drugs. Acknowledgment. This work was supported partly by a grant from the National Science Council (NSC 92-2320-B007-004) and partly by another grant from the National Health Research Institute (NHRI-EX92-9221EI), Republic of China. References and Notes (1) Wang, W. J. Drug Targeting 1996, 4, 195. (2) Ramadas, M.; Paul, W.; Dileep, K. J.; Anitha, Y.; Sharma, C. P. J. Microencapsulation 2000, 17, 405. (3) Kimura, Y. In Biomedical Applications of Polymeric Materials; Tsuruta, T., Hayashi, T., Eds.; CRC Press: Boca Raton, FL, 1993. (4) Mi, F. L.; Tan, Y. C.; Liang, H. F.; Sung, H. W. Biomaterials 2002, 23, 181.

Biomacromolecules, Vol. 5, No. 5, 2004 1925 (5) Mumper, R. J.; Hoffman, A. S.; Puolakkainen, A.; Bouchard, L. S.; Gombotz, W. R. J. Controlled Release 1994, 30, 241. (6) Peracchia, M. T.; Gref, R.; Minamitake, Y.; Domb, A.; Lotan, N.; Langer, R. J. Controlled Release 1997, 46, 223. (7) Vakkalanka, S. K.; Brazel, C. S.; Peppas, N. A. J. Biomater. Sci., Polym. Ed. 1996, 8, 119. (8) Esposito, E.; Cortesi, R.; Nastruzzi, C. Biomaterials 1996, 17, 2009. (9) Heymann, E. Trans. Faraday Soc. 1935, 31, 846. (10) Haque, A.; Morris, E. R. Carbohydr. Polym. 1993, 22, 161. (11) Tate, M. C.; Shear, D. A.; Hoffman, S. W.; Stein, D. G.; LaPlaca, M. C. Biomaterials 2001, 22, 1113. (12) Kennedy, S. K.; Walker, W.; Forslund, B. EnViron. Forensics 2002, 3, 131. (13) Yuk, S. H.; Cho, S. H.; Lee, H. B. J. Controlled Release 1995, 37, 69. (14) Kikuchi, A.; Kawabuchi, M.; Watanabe, A.; Sugihara, M.; Sakurai, Y.; Okano, Y. J. Controlled Release 1999, 58, 21. (15) Ramkissoon-Ganorkar, C.; Liu, F.; Baudysˇ, M.; Kim, S. W. J. Controlled Release 1999, 59, 287. (16) Bradford, M. M. Anal. Biochem. 1976, 72, 248. (17) Liu, S. Q.; Yang, Y. Y.; Liu, X. M.; Tong, Y. W. Biomacromolecules 2003, 4, 1784. (18) Gupta, K. C.; Khandekar, K. Biomacromolecules 2003, 4, 758. (19) Benns, J. M.; Choi, J. S.; Mahato, R. I.; Park, J. S.; Kim, S. W. Bioconjugate Chem. 2000, 11, 637. (20) Ramkissoon-Ganorkar, C.; Baudysˇ, M.; Kim, S. W. J. Biomater. Sci., Polym. Ed. 2000, 11, 45. (21) Brahim, S.; Narinesingh, D.; Guiseppi-Elie, A. Biomacromolecules 2003, 4, 1224. (22) Ju, H. K.; Kim, S. Y.; Lee, Y. M. Polymer 2001, 42, 6851. (23) High Polymers. Ott, E., Spurlin, H. M., Grafflin, M. W., Eds.; Cellulose and Cellulose DeriVatiVes; Interscience Publishers: New York, 1963; Vol. 5, pp 930-937. (24) Kundu, P. P.; Kundu, M. Polymer 2001, 42, 2015. (25) Sarkar, N.; Walker, L. C. Carbohydr. Polym. 1995, 27, 177. (26) Mitchell, K.; Ford, J. L.; Armstrong, D. J.; Elliott, P. N. C.; Rostron, C.; Hogan, J. E. Int. J. Pharm. 1990, 66, 233. (27) Hennink, W. E.; van Nostrum, C. F. AdV. Drug DeliVery ReV. 2002, 54, 13. (28) Kikuchi, A.; Okano, T. AdV. Drug DeliVery ReV. 2002, 54, 53. (29) Peppas, N. A.; Huang, Y.; Torres-Lugo, M.; Ward, J. H.; Zhang J. Annu. ReV. Biomed. Eng. 2000, 2, 9.

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Novel Method Using a Temperature-Sensitive Polymer

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