携Gelsolin单抗载紫杉醇靶向超声造影剂的制备及体外显影实验研究

发布时间：2018-01-04 00:15

本文关键词：携Gelsolin单抗载紫杉醇靶向超声造影剂的制备及体外显影实验研究　出处：《大连医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:以高分子聚合物聚乳酸-羟基乙酸(Poly lactic-co-glycolic acid,PLGA)为成膜材料,制备一种以肿瘤淋巴道转移相关特异性膜抗原凝溶胶蛋白(Gelsolin,GSN)为靶点的高分子造影剂,并考察其体外寻靶及显影能力。方法:通过改良的双乳化法制备包裹紫杉醇(paclitaxel,PTX)的高分子PLGA-COOH造影剂,并以碳二亚胺法将载药造影剂与Gelsolin单抗连接,制备出靶向载药高分子超声造影剂(PTX-PLGA-GSN)。利用光镜及Malvern激光仪检测此造影剂的分散度、粒径、zeta电位等一般理化性质;取适量该靶向载药造影剂以甲醇溶解萃取包载的紫杉醇,以高效液相色谱仪分析其包封率、载药量及体外释药特性;采用免疫荧光法及流式细胞仪检测Gelsolin单抗与造影剂表面的连接情况;体外培养小鼠肝癌细胞系具备不同淋巴道转移的能力的Hca-F和Hca-P细胞,设立4组实验组,Hca-F细胞非靶向PLGA组、Hca-F细胞抗体封闭组、Hca-P细胞GSN-PLGA组、Hca-F细胞GSN-PLGA组,每组加入相应的香豆素-6标记的造影剂处理后,于激光共聚焦显微镜下观察每组细胞对造影剂的摄取情况,流式细胞仪检测各组细胞的荧光强度,评价靶向造影剂的体外寻靶能力;称取一定量造影剂重悬于双蒸水中,调整浓度分别为1mg/ml、0.5mg/ml、0.25mg/ml,并以脱气水为对照组,于超声诊断仪造影模式下观察体外显影情况。结果:PTX-PLGA-GSN纳米超声造影剂的平均粒径为(328.59±3.82)nm,zeta电位为(-11.46±1.19)mV,包封率及载药量分别为(83.1±2.12)%、(8.31±0.21)%,药物体外释放实验显示30d紫杉醇缓释率约为90.63%。流式细胞术测得PTX-PLGA-GSN表面GSN单抗连接率高达98.31%。体外摄取实验显示小鼠腹水型肝癌高淋巴道转移细胞株(Hca-F)可摄取较多的携Gelsolin单抗PLGA造影剂,流式细胞仪检测Hca-F细胞GSN-PLGA组细胞荧光强度平均为(189.09±1.14),明显高于Hca-P细胞GSN-PLGA组的(140.15±0.98),差异具有统计学意义(P0.05),Hca-F细胞非靶向PLGA组及Hca-F细胞抗体封闭组的细胞荧光强度分别为(108.39±1.29)、(123.31±1.25),亦显著低于Hca-F细胞GSN-PLGA组,差异具有统计学意义(P0.05)。体外显影实验显示造影剂回声均匀细腻,后方回声无衰减,成像效果较好,且随着造影剂浓度的增加,造影效果更优。结论:成功制备出携Gelsolin单抗载紫杉醇靶向超声造影剂且包封率及载药量均较高,且缓释性能较好,体外寻靶实验显示此造影剂的靶向性较强,体外显影效果亦较好。
[Abstract]:Objective: to polymer poly lactic acid glycolic acid (Poly lactic-co-glycolic, acid, PLGA) as membrane material, a preparation for lymphatic metastasis of tumor associated antigen specific membrane gelsolin (Gelsolin, GSN) as the polymer contrast agent targeting, and investigate its in vitro targeting and developing ability. By parcel of paclitaxel by double emulsification method improved (paclitaxel, PTX) PLGA-COOH polymer contrast agent, and the carbon two imide method drug loaded contrast agent with Gelsolin mAb connection, prepared targeting drug loaded polymer ultrasound contrast agent (PTX-PLGA-GSN). By using optical microscope and Malvern laser dispersion detection the agent particle size, zeta potential of the general physical and chemical properties; apply the drug targeting contrast agent dissolved in methanol extraction entrapped paclitaxel, with HPLC analysis of the encapsulation rate, drug loading and release characteristics in vitro by immunofluorescence; The optical method and flow cytometry Gelsolin monoclonal antibody and contrast agent surface connection; cultured mouse hepatocellular carcinoma cell lines with different lymph node metastasis in Hca-F and Hca-P cells, the establishment of the 4 experimental groups, Hca-F cell non targeting PLGA group, Hca-F cell antibody group, Hca-P GSN-PLGA cells group, Hca-F cells GSN-PLGA group, each group with the corresponding contrast agent of coumarin labeled -6, the laser scanning confocal microscope on the uptake of contrast agents in each cell, the fluorescence intensity of cells were detected by flow cytometry, evaluation of targeted contrast agent targeting; weigh a certain amount of resuspended in double contrast agent the water vapor, adjust the concentration were 1mg/ml, 0.5mg/ml, 0.25mg/ml, and the degassed water as control group, to observe the in vitro development in ultrasonic diagnosis instrument contrast mode. Results: the average PTX-PLGA-GSN nano ultrasound contrast agent Particle size (328.59 + 3.82) nm, zeta potential (-11.46 + 1.19) mV, the entrapment efficiency and drug loading were (83.1 + 2.12)% and (8.31 + 0.21)%, in vitro drug release experiments showed that 30d paclitaxel release rate of about 90.63%. were measured by flow cytometry PTX-PLGA-GSN surface GSN mAb connection rate is as high as 98.31%. uptake in vitro experiments showed that mouse hepatocarcinoma cell lines with high lymphatic metastasis (Hca-F) can absorb more with Gelsolin monoclonal antibody PLGA contrast agent, flow cytometry Hca-F GSN-PLGA cells fluorescence intensity average (189.09 + 1.14), was significantly higher in Hca-P cells group (GSN-PLGA 140.15 + 0.98), the difference was statistically significant (P0.05), Hca-F cell non targeting PLGA and Hca-F cell antibody group the cell fluorescence intensity were (108.39 + 1.29), (123.31 + 1.25), was significantly lower than that of Hca-F cells in GSN-PLGA group, the difference was statistically significant (P0.05) in vitro development. Experiments show that the contrast agent echo uniform fine, rear echo attenuation, good imaging results, and with the increasing concentration of contrast, contrast is better. Conclusion: we successfully synthesized with paclitaxel loaded Gelsolin monoclonal antibody targeted ultrasound contrast agent and the encapsulation efficiency and drug loading were higher, and the sustained release performance in vitro the target experiment showed that targeting the strong contrast agent, the in vitro development effect is also good.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R445.1

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