| Silk fibroin derived from Bombyx mori is a biomacromolecular protein with outstanding biocompatibility. When it was dissolved in highly concentrated CaCl2 solution and then the mixture of the protein and salt was subjected to desalting treatments for long time in flowing water, the resulting liquid silk was water-soluble polypeptides with different molecular masses, ranging from 10 to 200 kDa. When the liquid silk were introduced rapidly into acetone, silk protein nanoparticles with a range of 40～120 nm in diameter could be obtained. The crystalline silk nanoparticles could be conjugated covalently with insulin alone with cross-linking reagent glutaraldehyde. In vitro properties of the insulin-silk fibroin nanoparticles (Ins-SFN) bioconjugates were determined by Enzyme-Linked Immunosorbent Assay (ELISA). The optimal conditions for the biosynthesis of Ins-SFN bioconjugates were investigated. Ins-SFN constructs obtained by 8h of covalent crosslin- king time at the experimental conditions of 0.7% cross-linking reagent, 4℃and the proportion of insulin and SFN (30:15, IU/mg) showed much higher recoveries from 90~120%. When insulin was coupled covalently with silk nanoparticles, the thermostability and resistance of the modified insulin to trypsin digestion and in vitro stability in human serum were greatly enhanced as compared with insulin alone. The results in human serum indicated that the half-life in vitro of the biosynthesized Ins-SFN derivatives was about 2.5 times more than that of native insulin. Activity in vivo tests Shows that Induced by Alloxan diabetes model rats Ins-SFNs In vivo hypoglycemic Activity about 8 times more than that of free insulin.Therefore, the silk protein nanoparticles have the potential values of being studied and developed as a new bioconjugate for enzyme/polypeptide drug delivery system.
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