维甲酸/聚乙二醇-聚乳酸二嵌段共聚物胶束的制备及其体外性质

Preparation and in vitro characterization of ATRA loaded mPEG-PLA diblock copolymeric micelles

目的：制备甲氧基聚乙二醇-聚乳酸[methoxyl poly（ethylene glycol）-poly（lactic acid）,mPEG-PLA]二嵌段共聚物及其载全反式维甲酸（all-trans-retinoic acid,ATRA）的胶束,验证共聚物的结构,并考察胶束的形态、粒径、载药量、包封率、体外释放特性及其溶血性质。方法：采用辛酸亚锡催化的开环聚合法制备不同嵌段比例的mPEG-PLA二嵌段共聚物,用核磁共振氢谱（1HNMR）和傅利叶红外光谱（FTIR）验证其结构,凝胶渗透色谱（GPC）考察其相对分子质量及分布。用透析法、丙酮溶剂蒸发法及丙酮挥发透析法制备空白及载ATRA胶束。透射电子显微镜（TEM）观察胶束的形态,动态光散射仪（DLS）测定其粒径分布。采用紫外分光光度法测定胶束的载药量和包封率,并研究其体外释放性质及溶血性质。结果：制备了5种不同嵌段比例的mPEG-PLA共聚物,TEM结果显示ATRA胶束呈类球形。胶束的粒径及载药量受制备方法、药物/嵌段共聚物比例以及嵌段共聚物性质的影响,通过筛选得到丙酮挥发透析法及嵌段共聚物/药物的比例为5∶2为最适制备条件,得到胶束的平均粒径在267.5-431.8 nm内,并随着嵌段共聚物疏水部分的增大而增加;胶束的载药量在11.23%-20.01%内,包封率为31.79%-56.57%,也具有随疏水嵌段增大而增加的趋势。载药胶束在PBS-乙醇（9∶1）的释放液中48 h体外累积释药率为61.6%-94.1%,载药胶束与50%血浆混合后药物释放趋于零级,药物的释放随聚合物相对分子质量的减小和载药量的减小而增加。100-150mg·L^-1的载药胶束无溶血现象,而5 mg·L^-1的ATRA溶液即出现溶血。结论：通过研究嵌段比例对mPEG-PLA嵌段共聚物载ATRA胶束一系列体外性质的影响,为选择最适的嵌段共聚物比例提供了依据。载药量,体外释放及溶血性质结果显示mPEG-PLA嵌段共聚物载ATRA胶束具有明显的增溶,缓释作用,并能够减少药物的毒副作用。

Objective：To prepare all-trans-retinoic acid （ATRA） loaded methoxyl poly （ethylene glycol）- poly （lactic acid） （mPEG-PLA） diblock copolymeric micelles and characterize its structure, morphology, size, drug contents, loading efficiency, drug release characteristics in vitro and hemolytic potential. Methods：The series mPEG-PLA diblock copolymers with different block ratio were synthesized by ring-opening polymerization catalyzed by stannous 2-ethyl-hexanmoate. The identification of the copolymers was investigated by ^1H nuclear magnetic resonance （ ^1H NMR） and fourier transform infrared microspectroscopy （FTIR）. The molecular weight （Mw） and its distribution of the copolymers were determined by gel permeation chromatography （ GPC）. Empty and ATRA loaded micelles were prepared by dialysis method, acetone rotary evaporation method and acetone volatilization dialysis method. Transmission electron microscope （TEM） was used to assess the shape of the nanoparticles. Dynamic light scattering （DLS） Malvern Zetasizer Nano-ZS was performed to measure the particle size distribution. The drug contents and loading efficiency were investigated with UV spectrophotometry. The drug release characteristic and hemolytic potential in vitro were also evaluated. Results：Five mPEG-PLA diblock copolymers with different segment ratio were synthesized and identified by ^1HNMR and FTIR. The molecular weight and its distribution were determined by ^2HNMR and GPC. According to ^1H NMR using CDCl3 and D2O hydrophobic core and hydrophilic shell formed in water with ATRA in the core. The micelles were spherical observed by SEM. Size and drug contents of micelles were effected by preparation method, drug/copolymer ratio and polymer used. The appropriate preparation condition was using acetone volatilization dialysis method with the ratio of copolymer/drug = 5 ： 2. The size of micelles was from 267.5 to 431.8 nm, the drug contents and loading efficiency was 11.23% - 20.01% and 31.79% - 56.57% , respectively. The size and drug contents were all increased as the increase of the Mw of PLA block of mPEG-PLA. 61.6% - 94.1 % of ATRA was released from micelles over a period of 48 h from PBS-ethanol = 9： 1. With 50% plasma as the release media, ATRA followed the zero-order release model. ATRA release was faster at smaller Mw of copolymer and lower drug contents. The hemolysis of RBCs was observed within 3 h of incubation with 5 mg·L^-1 ATRA and was not observed with 100 - 150 mg·L^-1 ATRA loaded micelles. Conclusion：A series of in vitro characterization of ATRA loaded mPEG-PLA copolymeric micelles were affected by the block ratio of PEG to PLA. It provides some evidence to choose the appropriate block ratio of PEG to PLA using the above results. The drug loading, in vitro drug release characteristics and hemolytic toxicity suggested that ATRA loaded mPEG-PLA micelles showed obviously solubility enhancement, sustained release and reduction of drug toxicity.