| Abstract [Objective]To prepare fotemustine-PLGA sustained-release microsphere, and to explore the effect of fotemustine on LN229glioma cells including the cells proliferation, apoptosis, and drug resistance. Relevant results will provide evidence for further study of fotemustine-PLGA sustained-release chemotherapy on gliomas.[Method]Fotemustine-PLGA sustained-release microsphere was prepared first by solvent volatilization extraction method and its morphology was observed with scanning electron microscope, and the fotemustine content in fotemustine-PLGA sustained-release microsphere and coating rate was determined with high-performance liquid chromatography (HPLC). The toxic effects of fotemustine-PLGA sustained-release microsphere to LN229cell lines and the cells apoptosis and necrosis by double staining of PI and Hochest were detected with fluorescence microscope. MGMT expression after the treament of fotemustine-PLGA sustained-release microsphere liquid for14days,21days, PLGA vector liquid for14days,21days and teniposide were also detected.[Results](1) The characters of fotemustine-PLGA sustained-release microsphere conformed to the requirements of the experiments.(2) The linear range of fotemustine-PLGA sustained-release microsphere is0.1-100ug/ml, r=0.9995. The recovery rate of low, medium and high concentration were morer than98%, and The precision in daytime and days are all less than3.5%. The drug-loading rate and coating rate of fotemustine-PLGA is respectively (4.81+0.36)%and (87.15+5.12)%. The1day release in vitro was11.3%,28d acculted released was88.47%。(3) The inhibition rate of LN229glioma cell line for24h of teniposide (including0.1g/ml,0.5g/ml,2.5g/ml,12.5gg/ml,62.5g/ml)was0,5.15%,19.77%,52.17%,90.15%; 14day fotemustine-PLGA sustained-release microsphere (including0.27g/ml,0.27g/ml,27g/ml,270g/ml,2700g/ml) was0,8.74%,44.09%,47.06%and8.74%respectively;21day fotemustine-PLGA sustained-release microsphere (including0.27g/ml,0.27g/ml,27g/ml,270g/ml,2700g/ml was0,10%,10.648%,2.315%,10.648%; the PLGA vector was0.(4)14day fotemustine-PLGA sustained-release microsphere induced LN229glioma cell apoptosis, inhibited cells proliferation and reduced MGMT expression in dose-dependent manner,21day fotemustine-PLGA sustained-release microsphere (2700g/ml) significantly induced LN229glioma cell apoptosis, inhibited its growth and decreased MGMT expression. Teniposide with different dose induced LN229glioma cell apoptosis, inhibited its growth but no effect on the MGMT expression. PLGA vector had no effects on all the inditators.[Conclusion]1.The morphology, diameter and so on indicators of fotemustine-PLGA sustained-release microsphere is ready for use;2. Fotemustine-PLGA sustained-release microsphere peak, drug-loading rate, recovery rate, microspheres coating rate, release in vitro and so on are all in good range which was determined by HPLC;3.Fotemustine-PLGA sustained-release microsphere induced LN229glioma cell apoptosis, inhibited its proliferation, reduced MGMT expression which provided a new way for fotemustine chemotherapy Part I The studies of fotemustine-PLGA sustained-release micro sphere preparation and release in vitro[Abstract] Objective:To prepare fotemustine-PLGA sustained-release microsphere, determine its characterization and study its release in vitro. Methods:HPLC method was established and its specificity, precision, recovery and stability was observed. Fotemustine-PLGA sustained-release microsphere was prepared first by solvent volatilization extraction method and its morphology was observed with scanning electron microscopy, and the fotemustine content in fotemustine-PLGA sustained-release microsphere and coating rate was determined.. Fotemustine-PLGA sustained-release microsphere was put in PBS solution and the curve of release in vitro was drew. Result:The HPLC method had good specificity, high sensitivity, good reproducibility. Fotemustine-PLGA sustained-release microsphere peak shape is good, no impurity peak interference, whose linear range was0.1-100g/ml, r=0.9995. The recovery of low, medium and high concentrations was all more than98%, and The precision in daytime and days are all less than3.5%. The drug-loading rate and coating rate of fotemustine-PLGA is respectively (4.81+0.36)%and (87.15+5.12)%. The lday release in vitro was11.3%,28d acculted released was88.47%.Conclusion:The established HPLC method is simple, accurate and reliable; Fotemustine-PLGA sustained-release microsphere preparation technology is feasible, particle size is small, encapsulation efficiency and drug-loading rate is high and has good release properties in vitro. Part II The toxic effects of fotemustine-PLGA sustained-release microsphere on LN229glioma cell lines and expression of MGMT[Abstract] Objective:To explore the toxic effects of fotemustine-PLGA sustained-release microsphere on LN229glioma cell lines and the changes of MGMT expression. Methods:The toxic effects of fotemustine-PLGA sustained-release microsphere to LN229cell lines were detected by MTT and IC50of it in LN229cell lines was calculated. Then the cells apoptosis and necrosis was observed by double staining of PI and Hochest with fluorescence microscope. Finally expression of MGMT was detected after the treament of fotemustine-PLGA sustained-release microsphere liquid for14days,21days, PLGA vector liquid for14days,21days and teniposide by Western Blot. Results:14days fotemustine-PLGA sustained-release microsphere induced LN229glioma cell apoptosis, inhibited cells proliferation and reduced MGMT expression in dose-dependent manner,21days fotemustine-PLGA sustained-release microsphere (2700g/ml) significantly induced LN229glioma cell apoptosis, inhibited its growth and decreased MGMT expression. Teniposide with different dose induced LN229glioma cell apoptosis, inhibited its growth but no effect on the MGMT expression. PLGA vector had no effects on all the inditators. Conclusion:Fotemustine-PLGA sustained-release microsphere induced apoptosis of LN229glioma cells, inhibited its proliferation and reduced MGMT expression which provided a new way for fotemustine-PLGA chemotherapy... |
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