Preparation And Effect On Anti-hepatoma Of PLGA Microspheres Of Cobra Venom Cytotoxin

Objective1. To separate and purify cytotoxin from crude cobra venom, and identify its physicochemical properties and biologic activity;2. To prepare PLGA microspheres of cobra venom cytotoxin and study its characterization and characteristics of in vitro release;3. To evaluate the pharmacological effects and safety of intratumoral injection of PLGA microspheres of cobra venom cytotoxin to treat hepatoma;4. To approach the relevant mechanism of the anti-hepatoma effect of PLGA microspheres of cobra venom cytotoxin.Methods1. Separation, purification, identification and biologic activity determination of cobra venom cytotoxinCobra venom cytotoxin is separated and purified from the crude cobra venom with methods such as gel filtration and ion exchange; the purity and molecular weight of cytotoxin separated and purified are determined with SDS-PAGE method; and the toxicity of purified cobra venom cytotoxin on cells cultured in vitro are determined with MTT colorimetric method.2. The preparation, characterization and in vitro release of PLGA microspheres of cobra venom cytotoxinPLGA delayed-release microspheres of cobra venom cytotoxin are prepared with double emulsion - solvent volatilization method; the surface morphology of the microspheres is observed with a light microscope or a scanning electron microscope; the size and distribution of microspheres are determined with a laser particle size analyzer; and the encapsulation rate, drug loading and in vitro release of drug loaded microspheres are determined with BCA method.3.The effect of intratumoral injection of PLGA microspheres of cobra venom cytotoxin to treat hepatomaDelayed-release preparation of cobra venom cytotoxin is injected into the subcutaneously transplanted tumors of human hepatoma-bearing nude mice; the hepatoma volume of nude mice is determined with ultrasound regularly; 26 days later, the hepatoma tumor tissue of mice is dissected, its weight is determined, the inhibitory rate of tumor is calculated, the tumor tissue is dissected and the pathological examination is carried out ,and then the anti-tumor effect of cytotoxin is evaluated.4.Relevant mechanism of the anti-hepatoma effect of PLGA microspheres of cobra venom cytotoxin.The tumor tissue is dissected, and transmission electron microscope examination is carried out to observe the morphological changes of apoptotic cells; the apoptosis rate is determined with TUNEL method; the influence of cobra venom cytotoxin on the expression of apoptosis - related proteins cytochrome C, caspase-3 and caspase -9 is determined with Western blotting; the activity of caspase-3 and caspase-9 is determined with colorimetric method; the expression of PCNA antigen is determined with immunohistochemical method and the expression of tumor cell proliferation antigen is observed.Results1.It is confirmed with SDS-PAGE method that the cobra venom cytotoxin separated and purified is a uniform protein band with a molecular weight of about 7300u. It has intense cytotoxic effect on hepatoma carcinoma cells cultured in vitro. 2. The diameter of PLGA delayed-release microspheres of cobra venom cytotoxin is (34.45±9.85)μm, the encapsulation rate is up to (78.13±8.92) %, and the cumulative in vitro release amount of cobra venom cytotoxin during 30days is up to 84.3%.3. Intratumoral injection of PLGA delayed release microspheres of cobra venom cytotoxin can significantly inhibit the growth of the subcutaneously transplanted human hepatoma in nude mice, and the inhibition rate of tumor growth is up to 52.36%.4. PLGA delayed-release microspheres of cobra venom cytotoxin have the effect of inhibiting proliferation of hepatoma carcinoma cells, and it can also induce apoptosis and necrosis of human hepatoma carcinoma cells. It can be seen with immunohistochemical method that the expression of PCNA antigen decreases, and thus the proliferation of hepatoma carcinoma cells is inhibited. Under a light microscope or an electron microscope the typical morphological changes of cell apoptosis and necrosis can be seen; TUNEL examination shows that the apoptosis rate of the CTX-PLGA microspheres group significantly increases; it can be seen in Western blotting that CTX-PLGA microspheres can promote cytochrome C releases from mitochondrion to cytoplasm and the expression of caspase-3 and caspase-9 increases; it can be seen with colorimetric method that the activity of caspase-3 and caspase-9 increases.Conclusion1. The cytotoxin separated and purified from crude cobra venom has intense cytotoxic effect;2. PLGA microsphere is an ideal delayed-release carrier of cobra venom cytotoxin for local injection administration;3. Intratumoral injection of PLGA microspheres of cobra venom cytotoxin has significant anti-hepatoma effect;4. PLGA microspheres of cobra venom cytotoxin inhibit the growth of tumors through inhibiting proliferation of human hepatoma carcinoma cells and promoting apoptosis of human hepatoma carcinoma cells.