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# Synthesis and characterization of poly(gamma-glutamic acid) hydrogels and their application in slow-release of porcine somatotropin

Abstract (Summary)
Newly developed hydrogels, produced by cross linking purified poly ($\gamma$-glutamic acid) ($\gamma$PGA) with dihaloalkane compounds were utilized for slow-release of porcine somatotropin (pST). The polymer was produced by fermentation of Bacillus licheniformis. It was found that the rate of the crosslinking reaction and hydrogel yield were affected by several parameters, such as temperature, initial polymer and base concentrations as well as the type of crosslinkers. Reaction occurred rapidly at a polymer concentration of 40-80 mg/ml and temperature of 60-80$\sp\circ$C without hydrolysis of newly formed networks and polymer backbone. The optimal concentration for the base (NaHCO$\sb3$) was 1-2 mole per mole of carboxylic residues. Analysis of the hydrolysis of the hydrogels as a function of pH indicated that the hydrolysis occurred very slowly at neutral pH, but rapidly in both acidic and alkaline solution. The ester bonds were more sensitive to hydrolysis than the peptide bonds. The biodegradability of the hydrogels and polymer was further examined through enzymatic degradation by three enzymes (Cathepsin B, Pronase E and Trypsin), which were able to gradually cleave both ester and peptide bonds. Swelling of the hydrogel showed dramatic changes when experimental conditions such as the molecular weight of $\gamma$-PGA, nature of the crosslinkers and solutions used for swelling (ionic strength, organic solvent) were varied. The swelling degree was positively correlated with initial molecular weight of $\gamma$-PGA, but inversely correlated with the chain length of the crosslinkers and the crosslinking degree. Increasing the organic solvent or ionic concentration in solutions decreased the gel swelling, which was in agreement with general reports in literature. Porcine somatotropin (pST) is a very unstable hormone in aqueous environment. It was found in this research that the peptide underwent aggregation and decomposition at extreme pH, but was relatively stable in neutral pH. Aggregation was positively correlated with pST and ionic concentrations. The hormone monomer was stabilized to a certain degree in glucose solutions and at low concentration of urea. A quantitative ELISA method was established using anti-pST monoclonal and multiclonal antibodies. The method was simple and specific. The release profiles of $\gamma$-PGA based hydrogels for p-nitroaniline and BSA were studied in vitro. Hydrogels formed with longer chain crosslinkers released materials much slower than shorter chain dihaloalkanes, but unevenly release was observed for macromolecules. The hydrogels formed by dihalopentane was applied for slow-release of pST in vitro and in vivo. Although the aggregation and decomposition of pST were expected in the system, the hydrogel was able to release the hormone for a period of 20-30 days in vitro and in an in vivo rat experiment. In live piglets, the release system increased growth rate by 17% during 8-week experiment. This research demonstrated the potential application of $\gamma$PGA based hydrogels in slow-release systems for bioactive materials, especially macromolecules, such as peptides and proteins.
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