A33抗体修饰的外泌体给药系统的构建及体外评价

Construction and in vitro evaluation of A33 antibody functionalized exosomes drug delivery system

目的:构建A33抗体修饰的外泌体(exosomes,EXO)包载多柔比星(doxorubicin,DOX)给药系统并进行体外评价。方法:通过化学交联法将A33抗体与羧基化的超顺磁性纳米粒(ultrasmall superparamagnetic iron oxide nanoparticles,USPIO)连接制备探针(A33-USPIO),并利用动态光散射和红外光谱法对连接前后的USPIO进行表征。采用超速离心法分离出人结肠癌细胞株LIM1215细胞来源的EXO包载DOX,并通过透射电镜观察形态。用ELISA法测定EXO和探针结合的最适比例。透析法测定A33抗体修饰的载DOX的EXO给药系统(A33-US-EXO/DOX)体外释放特性。将不同的DOX制剂分别与LIM1215共培养后,用流式细胞仪测定细胞摄取DOX的情况,利用荧光显微镜验证A33-US-EXO/DOX的体外靶向能力。结果:透射电镜下EXO直径约为100nm,2.5μg探针约能与32μg的EXO结合,结合后平均粒径为(187.83±6.76)nm,聚合物分散指数(polymer dispersity index,PDI)为0.21。在pH 7.4、6.0、5.0条件下,DOX组、EXO/DOX组和A33-US-EXO/DOX组的48h累积释药量分别为24.32%、34.07%、62.82%,细胞摄取效率分别为(13.10±1.08)%、(51.53±3.56)%和(85.11±3.91)%。荧光观察显示,A33-US-EXO/DOX与A33抗原阳性的LIM1215细胞大量结合,而与A33抗原阴性的RAW264.7细胞结合较少。结论:本研究成功构建了A33抗原靶向的EXO给药系统,体外实验表明其具有较高的摄取效率和良好的靶向性。

To prepare and evaluate in vitro A33 antibody functionalized exosomes(EXO)loading doxorubicin(DOX)for drug delivering.Methods:A33 antibodies were coupled to carboxyl-ultrasmall superparamagnetic iron oxide nanop-articles(USPIO)by chemical crosslinking method to prepare A33-USPIO.Size distribution and zeta potential of USPIO and A33-USPIO were determined by dynamical light scattering,and their structures were elucidated by infrared spectroscopy(IR).EXO from human colon carcinoma cell line LIM 1215 cells were isolated by ultracentrifugation and the morphology was observed by transmission electron microscope after DOX loading.Enzyme linked immunosorbent assay(ELISA)was used to detect the optimum proportion of A 33-USPIO and EXO.Drug release rate of A 33-US-EXO/DOX was examined by dialysis method.Cel-lular uptake was investigated by flow cytometry after different DOX formulations were co-cultured with LIM 1215 cells.Fluores-cence microscopy was used to observed the fluorescence in A 33 antigen positive and negative cells to verify the targeting ability of A33-US-EXO/DOX.Results:Under transmission electron microscope,the diameter of EXO was about 100 nm,and 32^g EXO could bind to 2.5 pg A33-USPIO.After binding,the particle size and polymer dispersity index(PDI)of A33-US-EXO/DOX were(187.83+6.76)nm and 0.21,respectively.At the conditions of pH 7.4,6.0 and 5.0,the cumulative releases of DOX from A33-US-EXO/DOX in 48 h were 24.32%,34.07%and 62.82%,respectively.The uptake efficiency of cells trea-tedwithDOX,EXO/DOX and A33-US-EXO/DOX were(13.10+1.08)%,(51.53+3.56)%and(85.11+3.91)^,respec-tively.The fluorescence images showed that a large amount of A33-US-EXO/DOX bound with A33 antigen positive LIM 1215 cells,while a very small amount bound with A33 antigen nagetive RAW264.7 cells.Conclusion:The A33 antigen targeting drug delivery system was successfully constructed and showed a high uptake efficiency and good targeting ability in vitro.