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目的以一种新型两亲性大分子材料普鲁兰多糖接枝聚乳酸(pullulan-grafted-poly(D,L-lactide),PPLA)为载体材料,制备普鲁兰多糖接枝聚乳酸紫杉醇(paclitaxel,PTX)大分子胶束(PPLAPTX),以期降低紫杉醇的不良反应。方法采用薄膜水化法制备PPLA-PTX,以粒径、粒径分布(particle distribute index,PDI)、载药量(drug loading,DL)、包封产率(encapsulation efficiency,I.E)为指标,应用单因素联合星点设计效应面法,对胶束的处方及工艺进行优化,采用多元线性回归及二项式拟合,预测较优参数。对按照最优处方所制备的PPLA-PTX的结构特性和制剂学性质进行表征,并以紫杉醇注射液(PTX-solution,PTX-S)为对照,考察PPLA-PTX的体内毒性。结果二次多项式非线性回归模型是描述因素与指标关系的最佳模型,紫杉醇在PPLA-PTX中以分子或无定型形式存在,根据所得优化处方制得共聚物胶束的包封产率约为86.39%,载药量质量分数约为7.58%,透射电镜下粒子呈圆形均匀分布,采用马尔文激光粒度仪测定胶束粒径约为117.7 nm,PDI值0.158,Zeta电位为-3.43 mV,PPLA-PTX体外释放分为两阶段,第一阶段(0~4 h)符合Higuchi释放动力学,第二阶段(4~12 h)符合一级释放动力学。小鼠体内毒性实验表明,PPLAPTX的给药剂量为PTX-S的1.8倍时,前者的小鼠存活率为70%,后者全部死亡,连续给药7 d,前者的白细胞数约为后者的1.6倍,给药结束后7 d,PPLA-PTX组白细胞数回升至生理盐水组的83%。结论通过单因素与星点设计效应面法对处方和工艺优化,成功制备了普鲁兰多糖接枝聚乳酸紫杉醇大分子胶束,其粒径小而均匀,且具有较高的载药量和包封产率,与PTX-S相比,其具有更高的耐受剂量,更低的骨髓毒性,具有潜在的研究价值。
OBJECTIVE To prepare pullulan-grafted-poly (D, L-lactide) (PPLA), a novel amphipathic macromolecule material, to prepare pullulan-grafted polylactic acid paclitaxel paclitaxel, PTX) macromolecular micelles (PPLAPTX), with a view to reduce the side effects of paclitaxel. Methods PPLA-PTX was prepared by membrane hydration method. Particle diameter, particle distribution index (PDI), drug loading (DL) and encapsulation efficiency (IE) Single-factor combined with the design of the effect of surface area method, the micelles prescription and process optimization, using multiple linear regression and binomial fitting, prediction of optimal parameters. The structure and properties of PPLA-PTX prepared according to the optimal formulation were characterized and PTX-solution (PTX-S) was used as a control to investigate the in vivo toxicity of PPLA-PTX. Results The quadratic polynomial nonlinear regression model was the best model to describe the relationship between factors and indexes. Paclitaxel existed in molecular or amorphous form in PPLA-PTX. The encapsulation yield of copolymer micelles was about 86.39% and the mass fraction of drug loading was about 7.58%. The particles were round and evenly distributed under transmission electron microscope. The diameter of micelles was 117.7 nm, the PDI was 0.158 and the Zeta potential was -3.43 mV by Malvern laser particle sizer. The in vitro release of PPLA-PTX was divided into two phases. The first phase (0 ~ 4 h) was consistent with Higuchi release kinetics and the second phase (4 ~ 12 h) was consistent with the first-order release kinetics. In vivo toxicity test in mice showed that when the dose of PPLAPTX was 1.8 times that of PTX-S, the survival rate of the former mice was 70%, the latter all died, and the continuous administration for 7 days, the former was about the latter 1.6 times. After 7 days, the number of white blood cells in PPLA-PTX group rose to 83% of the saline group. Conclusion The single factor and the asterisks design effect surface method for prescription and process optimization, successful preparation of pullulan polysaccharide-grafted polylactic acid paclitaxel macromolecular micelles, the particle size is small and uniform, and has a high drug loading and Encapsulation yield, compared with PTX-S, which has a higher tolerance dose, lower bone marrow toxicity, has potential research value.