Research On Bioactivity Of Active Protein And Its Immobilization Onto The Surface Of The Plga Membrane

Poly-co-glycolic acid (PLGA) is considered as the ideal biomaterial because of its biodegradation, avirulence, biocompatibility, processability and excellent mechanical properties. Studies on its applications in biological materials and drug delivery materials have become hot research spots in recent years.Based on the excellent properties of PLGA and considered the problem that biological materials are sensitive to bacterial infections, PLGA particles which contain free carboxyl groups are chosen as the substrates to prepare PLGA membrane. And then with the use of EDC/NHS methods, the active protein bacitracin and Ricin which contain free amino groups will be separately bonded on the surface of PLGA membrane. The purpose of the experiment is to explore whether the properties of proteins are changed after they are co-immobilized onto the surface of PLGA film, and to explore the way of preparing antibacterial biodegradable materials and anticancer materials for drug release. The main work of this thesis has been fininshed is as follows:(1) Based on the PLGA particles which containe free carboxyl groups, investigated the way of preparing PLGA membrane and got the PLGA membrane, and then through total reflection Fourier transform infrared (ATR-FTIR) technology, the groups on the surface of membrane were characterized and the presence of free carboxyl groups were determined.(2) With the use of plate method ang the four kinds of Gram-positive bacteria in our laboratory, detected the antibacterial activity of Bacitracin, and then selected Staphylococcus aureus strain of significant antimicrobial effect as detection strains of the bacitracin co-immobilized to the surface of PLGA membrane.(3) Adopting affinity chromatography to extract and purify the ricin toxin isolated from the seeds of Ricinus communius, and then with the use of SDS-PAGE gel electrophoresis to identify its biochemical and biological activities. Employ human liver cancer SMMC7721 and esophageal cancer EC109 as the experimental cell lines to make a preliminary study of antitumor properties of Ricin, and make a comparison of the Ricin's ability to kill two kinds of tumor cells in vitro. SDS-PAGE gel electrophoresis showed that the material extracted is pure Ricin, the research of anti-tumor properties showed Ricin have strong killing effects to hepatoma cells and esophageal cancer cells, however, the killing effect of liver cancer is stronger than esophageal cancer cells, the median lethal concentration of liver SMMC7721 IC50 value is 5.9×10-10 mol/L while the IC50 value of esophageal cancer EC109 is 1.984×10-6 mol/L, indicating that Ricin may be have selective cell-killing effects on different cancers.(4) The possible anti-tumor mechanism of Ricin was studied. Giemsa staining and acridine orange (AO) fluorescence staining were used to observe the morphological changes of the liver cancer SMMC7721 and esophageal cancer EC109 treated with ricin, cell apoptosis and cell cycle distribution were detected by flow cytometry, whether DNA ladder is observed by Agarose Gel Electrophoresis if cells occur in apoptosis. Cell morphology and flow cytometry results of the test have indicated that at low concentrations Ricin inhibit the cell proliferation mainly by inducing cell apoptosis, but may lead to cell necrosis in higher concentrations or the effect time is too long.(5) In this work, the active protein Bacitracin and Ricin were separately co-immobilized to the surface of PLGA membrane by using the carbodiimide (EDC-NHS) two-step chemistry grafting method. Through total reflection Fourier transform infrared (ATR-FTIR) technology we have made surface groups analysis of the PLGA membrane after active proteins were immobilized onto its surface. And then Staphylococcus aureus were used to detest antibacterial properties and liver cancer cell SMMC7721 were used to detect anti-tumor properties respectively after active protein immobilized to PLGA film. We have made a contrast between control PLGA membrane and co-immobilized active protein ones by MTT assay. Then assisting in acridine orange fluorescence staining pictures and photos of MTT we made a visual description of bacteria and cell adhesion on the surface of PLGA membrane. ATR-FTIR group's characterization indicated that both of the active proteins were immobilized to the surface of PLGA film. MTT results showed that the active proteins have effective antibiotic or anti-tumor activity after co-immobilized to the surface of PLGA membrane.