叶酸受体靶向载紫杉醇高分子造影剂体外寻靶及超声显影实验研究

目的探讨叶酸受体靶向载紫杉醇（PTX）高分子造影剂（FOL—PLGA—PTX）的体外寻靶能力与超声显影情况。方法通过单乳化法及碳二亚胺法制备叶酸受体靶向载PTX高分子纳米微球，利用Malvern激光仪检测造影剂平均粒径和表面电位，用HPLC检测包封率和载药量，并通过免疫荧光法和流式细胞术检测造影剂表面叶酸连接情况及和荧光抗体的结合率。体外培养人卵巢癌SKOV3细胞，观察靶向造影剂与细胞的结合情况，评价其体外寻靶能力。考察靶向造影剂经高强度聚焦超声（HIFU）辐照后增强超声显影特性，并以DFY型定量仪进行定量。采用两独立样本t检验及单因素方差分析分析数据。结果叶酸受体靶向载PTX高分子超声造影剂的平均粒径为（244．43±13．32）nm，包封率及载药量分别为（86．23±1．23）％和（8．62±0．12）％；流式细胞术测得FOL—PLGA-PTX表面叶酸平均连接率高达（98．49±1．28）％，体外细胞寻靶实验中FOL—PLGA-PTX与SKOV3细胞平均结合率为（84．32±4．25）％，高于非靶向造影剂组（16．45±2．89）％（F=289．45，t=10．654，P〈0．01）和游离叶酸干预组（36．33±3．23）％（t=8．923，P〈0．01）；FOL-PLGA—PTX经HIFU体外辐照前后平均灰度值分别为39．32±3．64和126．44±7．15，差异有统计学意义（t=4．829，P〈0．01）。结论成功制备了叶酸受体靶向载PTX高分子超声造影剂，其包封率与载药量均较高，具备良好的体外寻靶能力与HIFU辐照增强超声显影特性。

In vitro study of folate receptor-targeted and paclitaxel-loaded ultrasound contrast agent

Objective To prepare the folate receptor-targeted and paclitaxel-loaded ultrasound con- trast agent （folate-poly（lactide-co-glyeolide）-paclitaxel, FOL-PLGA-PTX） and to investigate its targeting and imaging performance in vitro. Methods Paclitaxel-loaded PLGA-COOH microcapsules with a core of liquid perfluorocarbon （PLGA-PTX） were prepared using single emulsion technique and then conjugated with folate by carbodiimide method. The size, surface potential, entrapment efficiency and drug loading effi- ciency were measured by Malvern laser detector and HPLC. The connectivity condition of PLGA-PTX with folate and the binding rate of fluorescent antibody were detected by immunofluorescence staining and flow cy- tometry. The targeting performance of FOL-PLGA-PTX was checked after co-incubated with human SKOV3 cell lines in vitro and compared with that of non-targeted group and free folic acid intervention group. In vitro experiments were performed to explore the effects of FOL-PLGA-PTX on the enhancement of ultrasound imaging after irradiation by high intensity focused ultrasound （HIFU）. Two-sample t test and one-way analysis of variance were used to analyze data. Results The average diameter of FOL-PLGA-PTX was （ 244.43 ±13.32） nm, with the drug entrapment efficiency of （86.23 ±1.23 ）% and loading amount of （8.62± 0. 12）%. The binding rate of folate was as high as （98.49± 1.28 ）%. The connection rate of FOL-PLGA- PTX on SKOV3 cells was higher than that of non-targeted group （ （84.32±4.25） % vs （16.45±2.89） % ; F= 289.45, t = 10.654, P〈0.01） and the free folic acid intervention group （ （36.33±3.23） % ; t = 8.923, P〈 0. 01 ）. During in vitro ultrasound imaging, the average grey scale of FOL-PLGA-PTX before HIFU irradia- tion was significantly lower than that after HIFU irradiation （39.32±3.64 vs 126.44±7.15; t = 4.829, P〈0. 01）. Conclusion FOL-PLGA-PTX has been prepared successfully, with high entrapment efficiency and much drug loading, which can targ