摘要/Abstract

摘要： 目的·构建一种细胞穿透肽Penetratin修饰的眼用脂质体（Penetratin-modified liposome，Pen-Lip），并初步评价其通过滴眼给药治疗眼底新生血管疾病的适用性。方法·通过二硬脂酰基磷脂酰乙醇胺-N-马来酰亚胺-聚乙二醇2000（DSPE-PEG₂₀₀₀-Mal）和Penetratin的加成反应合成共聚物DSPE-PEG₂₀₀₀-Pen，再通过薄膜分散法制备Pen-Lip。利用细胞摄取实验确定修饰脂质体的最佳Penetratin比例。用纳米粒度电位仪测定Pen-Lip的平均粒径、Zeta电位和多分散系数（polydispersity index，PDI），用透射电子显微镜（transmission electron microscope，TEM）观察Pen-Lip的形态。采用离心法测定Pen-Lip装载康柏西普的包封率和载药量。将Pen-Lip在PBS或含50%胎牛血清PBS中静置48 h，通过粒径变化和TEM下形态考察Pen-Lip的稳定性。采用CCK-8法检测Pen-Lip对人源角膜上皮细胞（human corneal epithelial cell，HCEC）和人视网膜色素上皮细胞（human retinal pigment epithelium，ARPE-19）的细胞毒性，利用激光共聚焦显微镜观察ARPE-19对Pen-Lip的摄取情况。使用扩散池装置评估Pen-Lip对离体兔角膜的渗透能力，计算表观渗透系数P_app，通过角膜水合值评价载有康柏西普的Pen-Lip对角膜组织的毒性。结果·修饰脂质体的最佳Penetratin比例为3%。Pen-Lip的平均粒径为（148.07±3.51）nm，Zeta电位为（5.66±0.91）mV，PDI为0.227±0.045。TEM结果显示其呈外表光滑的球形结构。Pen-Lip的包封率为（44.06±3.70）%，载药量为（2.84±0.24）%。Pen-Lip在PBS和含50%胎牛血清PBS中静置48 h后粒径无明显变化，TEM显示分散均匀，表明其稳定性较好。在15~500 μg/mL范围内时，Pen-Lip对HCEC、ARPE-19无明显细胞毒性。与未修饰脂质体相比，ARPE-19对Pen-Lip的摄取能力显著提高（P=0.000）。离体兔角膜渗透实验中，Pen-Lip的P_app显著大于未修饰脂质体（P=0.000）；载药Pen-Lip处理后的离体兔角膜的水合值与未处理组相比差异无统计学意义（P>0.05）。结论·Pen-Lip在体外具有良好的稳定性、生物相容性、角膜透过能力，且能够被视网膜细胞摄取，是一种潜在的能够通过滴眼给药治疗眼底新生血管疾病的药物递送系统。
关键词: 细胞穿透肽, 脂质体, 康柏西普, 眼底新生血管疾病, 滴眼给药
Abstract:
Objective·To prepare the cell-penetrating peptide Penetratin-modified liposome (Pen-Lip) for ophthalmic application and evaluate its applicability to treat fundus neovascularization diseases through topical instillation.
Methods·The copolymer DSPE-PEG₂₀₀₀-Pen was synthesized by the addition reaction of DSPE-PEG₂₀₀₀-Mal and Penetratin, and Pen-Lip was then prepared by the thin film dispersion method. The cellular uptake experiment was used to determine the optimal ratio of Penetratin to modify liposomes. The average particle size, Zeta potential and polydispersity index (PDI) of Pen-Lip were characterized by the nano particle size potentiometer, and the morphology of Pen-Lip was observed by using transmission electron microscope (TEM). The encapsulation efficiency and the drug loading of Pen-Lip with conbercept were determined by centrifugation. Pen-lip was incubated in the PBS or the PBS containing 50% fetal bovine serum for 48 h and the stability of Pen-Lip was investigated by the change of particle size and the morphology under TEM. The CCK-8 assay was conducted to detect the cytotoxicity of Pen-Lip to human corneal epithelial cell (HCEC) and human retinal pigment epithelium (ARPE-19), and the uptake of Pen-Lip by ARPE-19 was observed by using laser confocal microscope. The diffusion cell device was used to detect the permeability of Pen-Lip through the isolated rabbit cornea, and the apparent permeability coefficient (P_app) was calculated. The toxicity of Pen-Lip with conbercept to the corneal tissue was evaluated by the hydration value.
Results·The optimal ratio of Penetratin to modify liposomes was 3%. The average particle size of Pen-Lip was (148.07±3.51) nm, the Zeta potential was (5.66±0.91) mV, and the PDI was 0.227±0.045. TEM results showed that Pen-Lip had a smooth spherical structure. The encapsulation efficiency of Pen-Lip was (44.06±3.70) %, and the drug loading was (2.84±0.24) %. The particle size of Pen-Lip did not change significantly and TEM showed uniform dispersion after dispersing in the PBS or the PBS containing 50% fetal bovine serum for 48 h, exhibiting the good stability of Pen-Lip. When Pen-Lip was at the concentration of 15?500 μg/mL, Pen-Lip had no cytotoxicity to HCEC and ARPE-19. Compared with unmodified liposomes, ARPE-19 cells had significantly higher uptake of Pen-Lip (P=0.000). In the experiment of rabbit cornea penetration in vitro, the P_app of Pen-Lip was significantly higher than that of unmodified liposomes (P=0.000). There was no statistically significant difference in the hydration value of the rabbit cornea treated with drug-loaded Pen-Lip compared to the untreatd group (P>0.05).
Conclusion·Pen-Lip has good stability, biocompatibility and corneal permeability in vitro and can be taken up by retinal cells, which is a potential ocular drug delivery system to treat fundus neovascularization diseases through topical instillation.

Key words: cell-penetrating peptide (CPP), liposome, conbercept, fundus neovascularization disease, topical instillation


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