| Hepatocellular carcinoma is one of health threats in the worldwide. At present, the methods of clinical treatment include surgery, chemotherapy, percutaneous intratumoral injection therapy, radiation therapy, immune gene therapy, hepatic arterial embolization therapy. It can reduce the dose of medication and the side effects of drugs on the body by the intratumoral injection.The retention time of common preparation in tumor is short because of tissue proliferation and metabolism. So the chemotherapy cannot give the full play to their potential. In order to improve the effect of chemical medicine in the treatment of hepatocellular carcinoma, we envisaged to change the form of a drug to make the drug release slowly. The drugs can stay in the tumor for a long time by intratumoral injection to achieve the best treatment.The PLGA microsphere (Microspheres, MS), based on polymer materials as the carrier, has sustained release effects. PLGA is a synthetic biodegradable polymer material which has been approved by the FDA as a pharmaceutically acceptable adjuvant. The final degradation products of PLGA are water and CO2, nontoxic to humans. The drug dispersed or dissolved in carrier after made to be microspheres. The drug can not only rely on their own properties to spread, also can rely on the biodegradation, dissolution and osmotic pressure of the skeleton. So the drugs release slowly to achieve long-term and improve the drug efficacy and reduce the side effects. Tamibarotene has antiangiogenic properties that impact on tumor cell growth. It significantly inhibited proliferation of HCC cells in vitro and also inhibited VEGF-induced angiogenesis, promoted apoptosis.In this study, Am80 was selected as the model medicine, the PLGA was selected as the carrier material which has biological compatibility and biodegradability, using the O/W emulsification-solvent evaporation to make delayed-release and prolonged-action Am80 microspheres (Tamibarotene loaded PLGA microspheres, Am80-PLGA-MS). The main methods and results were elaborated from the following four parts:The preparation and physicochemical properties of Am80-PLGA-MS; The in vitro evalution and Preliminary stability study of Am80-PLGA-MS; The in vivo evalution of Am80-PLGA-MS; The pharmacodynamics study of Am80-PLGA-MS.1. The preparation and physicochemical properties of Am80-PLGA-MSThe encapsulation efficiency of Am80 was determined by the dissolution-ultracentrifugation method and the methodology was inspected. According to the results of the effects of different variables, with encapsulation efficiency and particle size used as the evaluation index, the orthogonal experimental design was applied to optimize the formulation and parameters of the preparation process based on the single factor experiment results. DSC results authenticated the formation of Am80-PLGA-MS. Microspheres appearances were observed using light microscope. The powder properties of microspheres (such as surface angle of slide, angle of repose, bulk density, mean diameter and size distribution) were studied. The results showed that the free drug and Am80-PLGA-MS can be completely separated by the centrifugation-filtration method. The good linearity of the drug concentration was obtained within the range of 4.4～28.6μg/ml. The recovery and the RSDs (intra-day and inter-day) meet the requirement. The optimal formulation was obtained by orthogonal experimental design studies, taking the ratio of Am80 and PLGA, the volume ratio of dichloromethane and ethyl acetate, the concentration of PLGA and concentration of PVA as influencing factors. The optimization was as follows:the ratio of Am80 and PLGA was 1:7; the volume ratio of dichloromethane and ethyl acetate was 1:1; Concentration of PLGA was 80 mg/mL; concentration of PVA was 1%, internal water phase-to-oil phase volume ratio was 1:3, shearing rate was 12000 r/min, shearing time was 1 min. The results of DSC indicated that Am80 existed in microspheres in the form of non-crystalline morphology, not crystal morphology. This DSC result authenticated the formation of Am80-PLGA-MS. Three batches of microspheres were prepared using the optimized formulation. Examination using light microscope showed that microspheres were spherical particles without adhering. The mean diameter was (7.04±0.03)μm. The entrapment efficiency and drug loading were (82.23±0.74)% and (11.74±0.11)% respectively.2. The in vitro evalution and Preliminary stability study of Am80-PLGA-MSIn vitro release properties of Am80 from microspheres were performed by dialysis method and the preliminary stability of Am80-PLGA-MS was studied by influencing factor test. The in vitro release behavior of Am80-PLGA-MS investigated in pH 5.0 phosphate buffer saline containing 1% Tween-80 and 0.1% NaN3 could be described by Higuchi equation. The release of Am80 solution is fast. The cumulative release percentage of 8h reached to 95.65%. However, the release of Am80-PLGA-MS is slow. The cumulative release percentage of first day is (21.59± 0.28)% and cumulative release percentage of the seventh days were released to (64.34 ±0.36%). The cumulative release percentage of 14th days cumulative release percentage reached to (96.17±1.49)%. It has a good sustained release effect. The results of microspheres stability study showed that microspheres were unstable under heat, high humidity conditions and should be stored in the cold, dry environment.3. The in vitro evalution of Am80-PLGA-MSA mouse tumor model was established by subcutaneous injection with H22 liver cancer cells. H22 liver cells in logarithmic growth phase were injected in the right. Am80 solution was used as control, and the amount of drug leaked to the circular system was studied after intratumoral injection of Am80-PLGA-MS. The accumulation amount of drug in plasma and tumor was investigated respectively. After intratumoral injection, in the tumor, the retention time of Am80-PLGA-MS is 8.84 times than Am80 solution. The AUC of Am80-PLGA-MS is 10.37 times more than the Am80 solution group. However, in the plasma, The Cmax of the Am80 solution is 2.31 times more than Am80-PLGA-MS group. It indicated more drugs leaked to the systemic cycle of mice from the tumor in the solution group.The results showed that Am80-PLGA-MS could reduce drug leakage to systemic cycle from the tumor, extend retention time of drug in the tumor, enhance local Am80 concentration in the tumor and improve drug efficacy and decrease drug side effects.4. The pharmacodynamics study of Am80-PLGA-MSFirstly, a mouse tumor model was established by subcutaneous injection with H22 liver cancer cells. H22 liver cells in logarithmic growth phase were injected in the right. PBS of pH 5.0 was used as control. After intratumoral injection Am80 solution and Am80-PLGA-MS,record the volume of the tumors at different time points, calculate the TGI, DT, and SGR to assessment the tumor growth inhibition of each groups. By recording the animal weight at different time points, evaluation the drug toxicity. HE staining of the tumor in different time points were observed under the microscope. According to the inflammation, necrosis and the repair of the tumor cells to evaluate the effect of drugs. The results showed that the DT of Am80-PLGA-MS is 1.34 times more than the Am80 solution group, the TGI of Am80-PLGA-MS is 2.63 times more than the Am80 solution group, the SGR of Am80-PLGA-MS is 0.72 times more than the Am80 solution group. The change of mice weight is not obvious. The toxicity of drug is low in mice. Pathology results show that microspheres group has a better therapeutic effect. Although in the early treatment the effect of Am80 solution group is better than Am80-PLGA-MS group, the half-life of drug is short and small molecular weight of drug spread easily through the organization. So the effect of Am80 solution group is short.In summary, this study has explored a new idea and method for intratumoral administration to treatment the hepatocellular carcinoma and brings hope to the patients with hepatocellular carcinoma. It has high scientific value and clinical application prospect. |
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