Preparation Of10-hydroxycamptothecin Loaded Poly (D, L-lactide-co-glycolide) Microspheres For The Inhibition Of Angiogenesis In The Liver Tumor Treated With Embolic Therapy

Part I Preparation and Characterization of the HCPT Loaded PLGA microspheresObjective:To prepare the HCPT loaded PLGA microspheres and blank PLGA microspheres with the emulsion solvent evaporation method. The microspheres were characterized after the preparations.Materials and methods:The microspheres were prepared with O/W emulsion solvent evaporation method. The factors affecting the formation of microspheres including organic solvent ratio of dichloromethane and dimethylformamide, polymer concentration, emulsification time, stirring rate were screened to get suitable programs for the best parameter to control the microsphere morphology, drug loading and particle size distribution. The blank microspheres and drug loaded microspheres were prepared with the screened method. The microspheres were characterized with the fluorescence microscopy to detect the loaded HCPT, electron microscopy to observe the morphology, laser particle size analyzer determination of particle size distribution. In vitro drug release was performed within the dialysis bag in a constant temperature shaker with PBS at37℃. The concentration of samples at different time points were calculated the cumulative amount of drug release to create a release curve.Results: The blank PLGA microspheres and HCPT loaded microspheres were successfully prepared with the emulsion solvent evaporation method. The average diameter of the PLGA loading HCPT microspheres was85±37μm (mean±standard deviation), and average diameter the blank PLGA microspheres was90±41μm (mean±standard deviation). The drug loading rate was0.4%and the encapsulation rate was65%. HCPT distributed uniformly in the microspheres with green fluorescence in the fluorescent microscope. The microspheres showed smart surface in the scanning electron microscope. HCPT were sustained released form the microsphere and29.2%drugs were released from the microspheres in the25th days in the vitro release system.Conclusion:The emulsion solvent evaporation method can be used to prepared PLGA drug-loading microspheres that were suitable for the transcatheter arterial embolization. Part II Experimental Evaluation of the PLGA microspheres for Embolization in the Rabbit VX2Liver Tumor ModelObjective:To investigate the feasibility of blank PLGA microspheres in the rabbit VX2implanted liver tumor model embolism. Compare the embolic effect of the PLGA microspheres with PVA particles which is commonly used in clinic as embolic agent by evaluating the tumor growth rate.Materials and mthods:Thirty tumor-bearing rabbits which were conformed with MRI were divided into threes groups randomly (10in each group). The rabbits in the group A were treated with false embolization with saline. The transcatheter arterial embolizaton was performed in the group B with blank PLGA microspheres. The rabbits in the group C received transcatheter arterial embolizaion with PVA particles (150-250μm). The rabbits were humanly sacrificed in seventh days after the embolization and the liver scan was performed with MRI before the sacrifice. The tumor growth rate was calculated by comparing the tumor volume before and after the embolic therapy.Results:PLGA microspheres can be subjected to2.7F micro-catheter to perform embolization. Three groups of tumor growth rates were372.5±80.1%,175.3±80.9%and164.1±57.2%in group A, group B and group C, respectively. There was statistically significant difference among groups (P<0.05). Although tumor growth rate in group B was greater than that in group C, there was no significant difference (P>0.05). Ther was sigfificant difference among groups for the tumor necrosis rate, but there was no significant difference between group B and group C.Conclusion:PLGA microspheres can be used as the embolic agent in liver cancer animal models for experimental research. Part III Experimental evaluation the inhibition of angiogenesis in residual tumor after embolization with HCPT loaded PLGA microspheresObjective:To investigate the inhibition of angiogenesis in the residual tumor after embolization with HCPT loaded PLGA microspheres.Materials and methods:A total of40tumor bearing rabbits of VX2carcinoma was divided into4groups randomly (10in each group). The rabbits in group A received saline false embolism. The rabbits in group B were embolized with PLGA microspheres. The rabbits in group C were treated with PLGA microspheres containing HCPT. For the animals in group D, emulsion containing HCPT and Lipiodol was infused to the hepatic artery and then the150-250μm PVA particles were used to embolize the tumor feeding arteries. Five animals were sacrificed in1st or5th days after embolization with sodium pentobarbital (100mg/kg). The tumor samples were harvested and fixed in4%paraformaldehyde. Then the samples were embedded in paraffin and sliced for immunohistochemical staining to detect the expression of HIF-1α and VEGF and MVD.Results:The interventional therapy was successfully performed in all animals. And the animals survived to the end of the experimental observation. Large area necrosis was found in the animals slice that receiving emblolic therapy and residual tumor cells scattered in the tumor. Immunohistochemical staining showed that HIF-1expression in both the nucleus and cytoplasm in the residual tumor cells but not in the liver tissue and tumor cells near the normal liver tissues. VEGF staining was mainly found in the residual tumor cells and vascular endothelial cells of the residual tumor tissues, and a few in normal liver tissue and adjacent tumor tissue. The neovascularization localized in the tumor edge predominantly. There was significant difference for the expression of HIF-la and VEGF and MVD respectively (P<0.05). The expression of HIF-1α and VEGF and MVD in the animals treated with PLGA microspheres was significantly higher than the experimental group in the residual tumor tissues. There was no significant difference for the expression of HIF-1α and VEGF and MVD in group1,3and4(P>0.05).Conclusion:HCPT loaded PLGA microspheres showed the inhibition of angiogenesis in the residual tumor by inhibiting the HIF-1α with the HCPT released from the microspheres, therefore enhancing the effect of embolization.