Preparation And In Vitro Evaluation Of Silk Fibroin Nanoparticles-L-Asparaginase Bioconjugates

L-Asparaginase is an effective drug in the treatment of Acute Lymphoblastic Leukemia (ALL). But its resistance to albumen enzyme hydrolysis is feeble, its half-life time is too short, and the side-effect is serious. In order to overcome these common shortcomings mentioned above, in this study, we prepared silk fibroin nanoparticles (SFNs) using silk fibroin of a kind of important economic insect Bombyx mori. to discuss the feasibility of SFNs being drug controlled-release carrier of L-asparaginase. With SFNs as carrier, glutaraldehyde and EDC as cross-linking reagent respectively, silk fibroin nanoparticles-L-Asparaginase (ASNase-SFNs) bioconjugates was synthesized. Furthermore, the systemic in vitro evaluation of ASNase-SFNs bioconjugates was studied in comparison to free L-Asparaginase.The experimental results showed that the optimal modifying condition of ASNase-SFNs bioconjugates synthesized with glutaraldehyde as cross-linking reagent was set up as follows: glutaraldehyde concentration was 0.75%, duration was 5h, temperature was 4℃, the U/mg ratio of L-Asparaginase and SFNs was 7~8:1, concentration of protection reagent L-Asparagine (L-Asp) was 2.0mg·mL-1; and that of EDC as cross-linking reagent was set up as follows: EDC concentration was 0.75%, duration was 9h, temperature was 25℃, the U/mg ratio of L-Asparaginase and SFNs was 8:1, concentration of protection reagent L-Asparagine (L-Asp) was 3.0mg·mL-1. The recovery of ASNase-SFNs bioconjugates are 43.7% and 32.9% respectively. The apparent Michaelis Constant of free L-asparaginase (Km) was 22.346×10-3 Mol·L-1, and the apparent Michaelis Constants of ASNase-SFNs bioconjugates were 14.665×10-3 Mol·L-1 and 9.117×10-3 Mol·L-1 respectively. Both the two Km of ASNase-SFNs bioconjugates were lower than that of free L-asparaginase, which indicated that the affinity to substrate L-asparagine elevated. The thermal stability of ASNase-SFNs bioconjugates enhanced obviously. The optimal pH scale widened to 6.0~8.0. The optimal reaction temperature of ASNase-SFNs bioconjugates was 10℃higher than that of free L-asparaginase. The resistance to trypsin hydrolysis increased obviously. Their half-life time were near 50h and 65h, much longer than that of free L-asparaginase of 3h. These results proved that silk fibroin nanoparticles-L-Asparaginase bioconjugates could overcome the common shortcomings of free L-asparaginase, which suggests that these modified enzymes are worth further studying and exploring. This technique is likely to provide a new approach for new type of anticancer drug.