Preliminary Study On The Preparation,Biological Activity Of PEGylated Cucurmosin

Objectives: To investigate the optimal reaction conditions of modifying cucurmosin(CUS) by monomethoxy polyethylene glycol butyraldehyde (mPEG-BAD)with relative molecular weights of20kD and the method for the purification of the modified products．And to study the antitumor activity and immunogenicity of the mono PEGylated CUS(the modified desired products) Providing a theoretical basis for designing a new dosage form of the (CUS).Methods:1. CUS was modified with mPEG-BAD under various conditions．To analyze the influence of various conditions on the components of modified products by SDS-PAGE.The reaction mixture was purified by ion-exchange column．2．The MTT assay was carried out to test the cell viability of HL-60、PANC-1in order to study the antitumor activity of the modified desired products in vitro.3. establishing mice tumor model with H22hepatoma carcinoma cells to study antitumor activity of the desired products in vivo.4. ELISA technology was applied to observe the immunogenicity of the desired products.Results:1.5:1for the mass ratio of mPEG-BAD and CUS,48hours for reaction time,2mg/ml for the concentration of CUS,phosphate buffer with pH6.5was elected as the optimal reaction conditions for modification of CUS with mPEG-BAD. the purity of modified desired products is higher than97%after two steps purification by ion-exchange column．The modification rate of the mono PEGylated CUS reaches about40%,its apparent relative molecular weights is about70KD was identified by SDS-PAGE.2. The antitumor activity of the mono PEGylated CUS in vitro reduce approximately30times comparing with the native CUS.3. The antitumor activity of the mono PEGylated CUS in vivo is more effective comparing with the native CUS.4. The analysis on the antigenecity denoted that when compared to CUS，the IgE and IgG level of the serum of the immunized mono PEGylated CUS of small rats was reduced to some degree. But reduction of the IgE level after the mono PEGylated CUS immunization was more distinct.Conclusion：5:1for the mass ratio of mPEG-BAD and CUS,48hours for reaction time,2mg/ml for the concentration of CUS,phosphate buffer with pH6.5was elected as the optimal reaction conditions for modification of CUS with mPEG-BAD. Compared the antitumor activity and immunogenicity with the native CUS,found that the antitumor activity of the mono PEGylated CUS in vitro decreased,while its antitumor activity in vivo increased；Immunogenicity was reduced to some extent.