大黄酸PEG-PCL-PEI纳米粒的制备及体外评价

Preparation and in vitro evaluation of rhein-loaded PEG-PCL-PEI nanoparticles

合成聚乙二醇-聚己内酯-聚乙烯亚胺(PEG-PCL-PEI)三嵌段聚合物载体材料,制备包载大黄酸(RH)的PEGPCL-PEI纳米粒(PPP-RH-NPS),并评价其理化性质和体外生物学特性。通过开环聚合和麦克尔加成反应合成得到PEG-PCL-PEI聚合物,相对分子质量为9.5×10~3,临界胶束浓度为0.723 nmol·L^(-1)。包载RH制备得到PPP-RH-NPS,外观浅黄、乳光明显,透射电镜观察纳米粒分散均匀圆整无团聚,粒径为(118.3±3.6)nm,PDI为(0.19±0.08),Zeta电位为(6.3±1.5)mV,包封率为(93.64±5.28)%,载药量为(8.57±0.53)%。透析法考察PPP-RH-NPS体外释药特征,48 h内累积释放率为75.92%,释药曲线符合Higuchi模型方程:Q=0.121 6t^(1/2)+0.069 5(R^2=0.887 4),呈缓释特性。选用兔红细胞考察其溶血率,MTT法评价其对HK-2细胞的生物安全性,在0~0.05 mmol·L^(-1)不会引起红细胞溶血和细胞毒性。流式细胞仪考察其摄取效率,PPP-RH-NPS可被细胞迅速内吞,摄取效率高,30 min内摄取完全,经激光共聚焦显微镜观察,PPP-RH-NPS能够从溶酶体进入细胞质,具有溶酶体逃逸特性。因此,该研究成功合成PEG-PCLPEI聚合物和制备得到PPP-RH-NPS,粒径分布均匀,包封率及载药量较高,摄取迅速,具有缓释特征、溶酶体逃逸特性、优良的生物安全性,是一种良好研究前景的新型纳米制剂。

This study was aimed to synthesize the polyethyleneglycol-polycaprolactone-polyethyleneimine（ PEG-PCL-PEI） three block polymer material,prepare Rhein（ RH）-loaded PEG-PCL-PEI nanoparticles（ PPP-RH-NPS）,and then evaluate their physical and chemical properties and biological characteristics in vitro. PEG-PCL-PEI polymer was obtained by adopting thering-opening polymerization and Michael addition reaction,and their physical and chemical properties were analyzed by using NMR and gel permeation chromatography. PEG-PCL-PEI was then used as the carriers to prepare PPP-RH-NPS by applying spontaneous emulsification solvent diffusion method. The results showed that molecular weight of PEG-PCL-PEI polymer was 9. 5 × 10~3,and critical micelle concentration was 0. 723 mmol· L^（-1）. PPP-RH-NPS had pale yellow,opalescence facade,round and smooth without aggregation,formed of（ 118. 3（ 3. 6） nm in particle size with PDI of（ 0. 19 ± 0. 08）,Zeta potential of（ 6. 3 ± 1. 5） mV,entrapment efficiency of（ 93. 64 ±5. 28） %,and drug loading of（ 8. 57 ± 0. 53） %. The accumulative release percentage of PPP-RH-NPS was 75. 92% in 48 h,and the release profiles in PBS conformed to the Higuchi equation： Q = 0. 121 6t^（1/2）＋ 0. 069 5（ R^2= 0. 887 4）,presenting slow release characteristics. Within the scope of the 0-0. 05 mmol·L^（-1）,the nanoparticles had no obvious hemolysis on rabbit red blood cells and toxicity on HK-2 cells. In the investigation of uptake efficiency by flow cytometry,nanoparticles can be absorbed into cells quickly and internalized within 30 minutes fully,with a high uptake efficiency. In confocal laser scanning microscope observation,the nanoparticles can escape from lysosome into cytoplasm. Herein,this study synthesized the PEG-PCL-PEI polymer and prepared PPP-RH-NPS successfully; the nanoparticles showed uniform particle size,higher encapsulation efficiency and drug-loading rate,slow release characteristics,quick uptake and internalization,lysosome escape property and good biocompatibility. PPP-RH-NPS will be a promising pharmaceutical formulation for further development.