Highly efficient processing of silk fibroin nanoparticle-l-asparaginase bioconjugates and their characterization as a drug delivery system

Abstract

Silk fibroin (SF) when dissolved in highly concentrated CaCl₂ solution formed a series of degraded polypeptides with a molecular mass range of 10–70 kDa. After the liquid silk, mixed mildly with L-asparaginase (ASNase), was introduced rapidly into excess acetone, the enzyme not only was not inactivated but was also well immobilized in simultaneously formed silk fibroin nanoparticles (SFNs). The resulting SFN–ASNase bioconjugates were easily isolated from acetone solution by centrifugation. The bioconjugates were crystalline globular particles of 50–120 nm in diameter, with high enzyme activity and showed similar enzymatic kinetics such as pH, reactive temperature, thermostability in solution and Michaelis constant to the native form. The bioconjugates could resist a high temperature of 90 °C in dry conditions for 30 min, and had a high recovery (90%), a greatly increased resistance to trypsin digestion, and better stability in serum, and storage stability in solution. No leakage of the enzyme from the nanoparticles occurred. These results indicated that ASNase was uniquely and efficiently bioconjugated in the protein nanoparticles that possessed a fine crystallinity. Thus, the highly efficient processing technology and use of silk nanoparticles as a novel drug delivery system has potential in research and development.