Synthesis of Polysaccharide Chemical Gels by Gamma-Ray Irradiation

Chapter 13

Synthesis of Polysaccharide Chemical Gels by Gamma-Ray Irradiation 1

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 11, 2015 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0620.ch013

Annamaria Paparella and Kinam Park

2,3

1

Fidia Advanced Biopolymers, Via Ponte della Fabbrica 3/a, 35031 Abano Terme (PD), Italy School of Pharmacy, Purdue University, West Lafayette, IN 47907

2

The aim of this study was to compare the ability of different polysaccharides to form chemical gels by gamma-irradiation. Dextran, alginic acid, hyaluronic acid, benzyl esters of hyaluronic acid, and gellan were functionalized to introduce double bonds through reaction with glycidyl acrylate. All the polysaccharides used in our study formed chemical gels by gamma-irradiation, although the extent of gel formation wasdifferent.The effects of the polymer concentration, the amount of glycidyl acrylate, and the gamma-irradiation dose on the hydrogel formation were studied by determining the degree of swelling of gels in water. The acidification of polyelectrolytes to pH 3 allowed them to form chemical gels more easily. Alginic acid, hyaluronic acid, and benzyl ester of hyaluronic acid at 25% of esterification degree formed chemical gels at lower polymer concentrations and at lower gamma-irradiation dose at pH 3 than at pH 6. The ability to form chemical gels using various natural polymers may be useful in the development of biodegradable hydrogels for various applications. Hydrogels have been widely used in various applications rangingfromcontrolled drug delivery to agriculture. Recently many biodegradable hydrogel systems have been developed in the area of controlled drug delivery. Hydrogels made from natural polymers present advantages in controlling the drug release profiles and avoiding the necessity of removing the delivery systems after their use (1). Generally, the preparation of polymeric networks is carried out by direct crosslinking of homopolymers or copolymers in solution using a small amount of a crosslinking agent or by simultaneous copolymerization and crosslinking reactions of monofunctional and multifunctional monomers (2). Alternatively, modified biopolymers can be crosslinked by gamma-irradiation (3). Since the modified biopolymers can be purified before exposure to gamma-irradiation, the formed gels do not contain undesirable components, such as unreacted monomers or crosslinking agents (3). We have previously prepared chemical gels from dextran and gelatin by gammairradiation (3-4). The polymers were functionalized with glycidyl acrylate to introduce double bonds and then crosslinked by gamma-irradiation. This is a simple and efficient method of hydrogel formation which can be applied to a variety of water 3

Corresponding author 0097-6156/96/0620-0180$12.00/0 © 1996 American Chemical Society In Irradiation of Polymers; Clough, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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soluble polymers. The resulting gels do not need any further purification, since the functionalized polymers are purified prior to gel formation. In addition, this approach allows the preparation of hydrogels without the use of any externally added crosslinker. Drug molecules can be mixed with the functionalized polymers prior to the gel formation (3). This allows incorporation of a large amount of high molecular weight drugs such as proteins which are difficult to load by other approaches, e.g., equilibrium swelling of hydrogels in a drug solution. This paper describes the synthesis of gels by gamma-irradiationfromvarious polysaccharides such as dextran, gellan, alginic acid, hyaluronic acid and ester derivatives of hyaluronic acid. All the polysaccharides, except for dextran, are polyelectrolytes characterized by peculiar solution properties and ion sensitivity (5-7). Although gamma-irradiation provides a clean and efficient method for gel formation, it can also lead to degradation of polymer chains and loss of viscosity (2,8). We explored the pH-dependent ionization of polyelectrolytes to make chemical gels by gamma-irradiation under mild conditions which do not degrade polysaccharides. The formation of chemical gels at lower gamma-irradiation dose (i.e., less than 0.5 Mrad) will also minimize the loss of bioactivity of the incorporated drugs such as bioactive proteins (3,9,10). Materials and Methods Preparation of Functionalized Polymer Solutions Functionalization of proteins (11-13) and polysaccharides (14-16) has been reported in the literature. Polysaccharides were functionalized following the procedure established in our laboratory (3,4). The ability to form gels was studied for samples of dextran (Sigma, mol. wt. 2,000,000), gellan (Gelrite, Kelco), alginic acid (Sigma, Medium viscosity), hyaluronic acid (HA, mol. wt. of 200,000 and 2,000,000), and benzyl esters of hyaluronic acid at 25% and 50% of esterification degree (HYAFF11p25 and HYAFFll-p50, respectively). The benzyl esters of hyaluronic acid were prepared from HA of mol. wt. 200,000. Both HA and HAYFF11 samples were supplied by Fidia Advanced Biopolymers (Abano Terme, Italy). The polymers were dissolved in distilled deionized water to obtain the final concentrations of 5% (w/v) for dextran, alginic acid, HA 200,000, and HYAFF11p25, 1.5% for HA 2,000,000, and 3.5% for HYAFFll-p50 and gellan. The initial concentrations of polysaccharide solutions were chosen based on the viscosity of the solution. Glycidyl acrylate (Aldrich) was added directly to polysaccharide solutions while stirring. The amount of glycidyl acrylate was variedfrom0.8 ml to 1.3 ml/g of polymer. After 1 day (except for 2 days for dextran samples) glycine solution (20%) was added to stop the functionalization process. The solutions were then dialyzed against distilled deionized water. The final volumes of functionalized solutions of alginic acid, HA 200,000, HYAFFll-p25, and HYAFll-p50 increased significantly after dialysis. The solutions were then centrifuged and the precipitates removed. Finally the supernatants were concentrated using a rotoevaporator to obtain a concentration range between 1 and 3% (w/v). ThefinalpH of all solutions was 6. Portions of the functionalized solutions of gellan, alginic acid, hyaluronic acid, and benzyl esters of hyaluronic acid at different concentrations were acidified to pH 3 with citric acid The acrylic group content in the functionalized dextran was determined spectrophotometrically after bromination of the double bonds as well as spectroscopically by FT-lH-NMR analysis. The former method consisted in measuring the decrease in absorbance at 480 nm in methanol-water (80/20) solution of bromine (0.2% v/V) (17). Acrylamide was used as a standard for this assay. The bromination of polysaccharides did not work very well and often resulted in a slow and incomplete reaction (18). The proton NMR analysis of dextran solutions at the

In Irradiation of Polymers; Clough, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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IRRADIATION OF POLYMERS

concentration of 10% (w/v) in deuterium oxide produced more precise results than the bromine analysis. The degree of substitution of dextran was obtained from the peak ratio between anomeric proton and the acrylic proton (19). The results indicated that one acrylic group was introduced per 20 glucose residues. For the other polysaccharides it was not possible to determine the degree of substitution by NMR spectroscopy because of high viscosity of the solutions. Preparation of Hydrogels The functionalized polymer solutions were gamma-irradiated from 1 h to 8 h using a dose rate of 0.0606 Mrad/h. The irradiation was performed with a Co source. The samples were not purged with nitrogen prior to irradiation because of the known damaging effect of nitrogen on hyaluronic acid solutions (20). The gels were air dried for 24 h and oven-dried at 60°C for 12 h. The effect of gamma-irradiation dose on the gel formation was studied by examining the equilibrium swelling of the dried hydrogels. The gels were allowed to swell in distilled deionized water at room temperature. The time required to reach the equilibrium was determined by monitoring the change in the weight of hydrogels. The swellingratio,Q, was calculatedfromthe following equation: Q=W*/W where W* and W are the weights of the swollen and dried gels, respectively (21).


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Results and Discussion The functionalized polysaccharides showed different abilities to form chemical gels. Of the polysaccharides tried in our study, dextran formed the gel most readily and did not degrade easily. Functionalized dextran solutions resulted in gel formation just after 0.12 Mrad of gamma-irradiation. As the gamma-irradiationtimeincreased from 0.24 Mrad to 0.48 Mrad, the swelling ratio decreased due to the higher crosslinking density by higher gamma-irradiation dose. The gel formation of functionalized alginic acid was dependent on the polymer concentration, gamma-irradiation dose, degree of functionalization, and the pH of the solution. Figure 1 shows the gel formation by alginate at two different pH values as a function of alginate concentration and gamma-irradiation dose. The lines indicate the minimum concentrations required to form a chemical gel at a given gamma-irradiation dose. At pH 6, at least 2% concentration of alginate was necessary to form gels. When the concentration of glycidyl acrylate used for alginate modification was 0.8 ml/g polymer instead of 1.3 ml/g polymer, the alginate concentrations required to form gels at pH 6 were at least 0.5% higher than those shown in Figure 1. When the functionalized solutions were acidified to pH 3, gels were formed more readily. At pH 3, the alginate concentrations required to form gels were lower than those at pH 6. For example, a solution of 1% alginate at pH 3 was able to form a gel even by 0.12 Mrad of gamma-irradiation, while a higher concentration (>2%) was necessary at pH 6. This behavior occurs probably because the acidification deionizes the carboxylic groups and thus decreases the repulsion between the molecules. The polymer chains can come closer to each other and this favors the crosslinking reaction (8). Furthermore, the acidification can cause the formation of hydrogen bonds between the polymer chains. The presence of physical interactions accelerates the chemical gel formation. As the gamma-irradiationtimeincreasedfrom0.12 Mrad to 0.48 Mrad at pH 3, a higher alginate concentration was required to form a gel. This is probably due to degradation of the polysaccharide at higher gamma-irradiation dose. In fact, the formed gels degraded and turned into very viscous solutions after long exposure to gamma-irradiation. The trend of the gel formation by HA (Mol. Wt. of 200,000) was similar to that by alginates as shown in Figure 2. Gels were formed at lower concentrations if pH


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Figure 1. Gel formation by alginate as a function of alginate concentration and gamma-irradiation dose at pH 3 (O) and pH 6 (•). Glycidyl acrylate used to fiinctionalize alginate was 1.3 ml/g polymer. The lines indicate the minimum concentration of alginate necessary to form a three-dimensional network.

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Figure 2. Gel formation by hyaluronic acid (HA, Mol. Wt. 200,000) as a function of HA concentration and gamma-irradiation dose at pH 3 (O) and pH 6 (•). Glycidyl acrylate used to functionalize alginate was 0.8 ml/g polymer. The lines indicate the minimum concentration of alginate necessary to form a three-dimensional network.


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was reduced to 3. It is interesting to notice that the trend of gel formation at pH 3 is the same as that of alginate. HA with higher molecular weight, however, showed different behavior. Solutions of functionalized HA (Mol. Wt. of 2,000,000) at pH 6 were very viscous and formed gels at 1.5% upon 0.06 Mrad of gamma-irradiation. As the gamma-irradiation dose increased more than 0.48 Mrad, the formed gels were found to degrade. The acidification of HA solutions before gamma-irradiation did not result in any improvement in the gel formation. This behavior also occurred at higher polymer concentrations. Comparison of the HA gels at two different molecular weights showed that HA with higher molecular weight degraded to a greater extent. Figure 3 shows the concentrations of HYAFFll-p25 necessary to form chemical gels at different gamma-irradiation doses. Solutions of HYAFFll-p50 at pH 3, however, formed microgels which are dispersed in solution. The gamma-irradiation did not result in a macroscopic three dimensional network formation. Functionalized gellan also did not form macroscopic three-dimensional networks at pH 6. The acidification of gellan resulted in formation of microgels. Further study is required to find out the reasons for the formation of microgels, instead of macroscopic hydrogels, by HYAFF1 l-p50 and gellan under acidic conditions. The characterization of polyelectrolytes gels was carried out by swelling studies. Figure 4 compares the equilibrium swelling ratios of alginate gels at two different concentrations (2.5% and 3% w/v) obtained by reactions with the same amounts of glycidyl acrylate (1.3 ml/g polymer). The equilibrium swelling ratio increased with decreasing polymer concentration. Moreover, as the gamma-irradiation dose increased from 0.12 Mrad to 0.48 Mrad, the equilibrium swelling ratio increased. This means that the gel crosslinking density is reduced by long exposure to gamma-irradiation. The only explanation for this is that crosslinking is increased by long exposure to gamma-irradiation but at the sametimealginate molecules are degraded. Thus, the overall effective crosslinking density is reduced. The results of the swelling study with HA (shown in Figure 5) shows that the degradation of HA by gamma-irradiation is more pronounced than that of alginate. Figure 6 shows the equilibrium swelling ratios of hyaluronic acid gels obtained at two different pH values. The gels prepared from solutions at pH 6 did not maintain their shape during the swelling and therefore it was difficult to recover them for the measurement. The higher mechanical strength and the higher equilibrium swelling ratios of gels prepared at pH 3 probably means that the degradation at pH 3 was reduced compared to that at pH 6. Our preliminary study on the formation of gels from various functionalized polysaccharides suggests that the gel formation is strongly dependent on the type of biopolymer. The strength of dextran gels did not change significantly as the gammairradiation dose increased up to 0.48 Mrad. On the other hand, gels formed from alginic acid, hyaluronic acid, and ester derivatives of hyaluronic acid degraded at high gamma-irradiation doses. These three polysaccharides formed gels more readily if the solutions were acidified to pH 3 before gamma-irradiation. The acidified solutions formed gels at lower polysaccharide concentrations and lower gamma-irradiation doses. The effect of gamma-irradiation on the degradation of polysaccharides varies depending on the type of polysaccharide. While more study is necessary to understand the mechanisms of degradation and formation of microgels instead of hydrogels, polysaccharide hydrogels with different properties can be prepared by adjusting the experimental conditions, such as the gamma-irradiation dose, the polymer concentration, the degree of functionalization, and the pH of the solutions.



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Figure 3. Gel formation by benzyl ester of hyaluronic acid at 25% of esterification degree (HYAFFll-p25) as a function of HYAFFll-p25 concentration and gamma-irradiation dose at pH 3 (O) and pH 6 (•). Glycidyl acrylate used to functionalize alginate was 0.8 ml/g polymer. The lines indicate the minimum concentration of alginate necessary to form a three-dimensional network.

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Figure 4. Equilibrium swelling ratio of alginate gels prepared at pH 3 as a function of the gamma-irradiation dose. The concentrations of alginate were 2.5% (O) and 3% (•). Glycidyl acrylate used to functionalize alginate was 1.3 ml/g polymer.


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Figure 5. Equilibrium swelling ratio of HA gels prepared at pH 3 as a function of the gamma-irradiation dose. The concentrations of HA were 1% (O), 1.5% (•) and 2% (A). Glycidyl acrylate used to functionalize alginate was 0.8 ml/g polymer.

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Figure 6. Equilibrium swelling ratio of HA gels prepared at pH 3 (O) and at pH 6 (•) as a function of the gamma-irradiation dose. The concentrations of HA was 2%. Glycidyl acrylate used to functionalize alginate was 0.8 ml/g polymer.


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Synthesis of Polysaccharide Chemical Gels by Gamma-Ray Irradiation

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