结缔组织生长因子多肽（P1c）修饰的脂质体制备及整合素ανβ3靶向性研究

发布时间：2018-06-03 08:02

本文选题：盐酸阿霉素 + 整合素ανβ3　；参考：《东南大学》2016年博士论文

【摘要】：研究目的：整合素ανβ3在血管生成相关疾病中发挥着重要作用,可被视为血管生成的一个特征性标记物。本研究制备了整合素ανβ3亲和肽P1c(本实验室发现并制备的结缔组织生长因子多肽,命名为P1c)修饰、PEG化的盐酸阿霉素长循环脂质体,以期通过靶向肿瘤细胞和肿瘤新生血管中过度表达的整合素ανβ3受体,增加药物向肿瘤部位的转运,达到特异性地治疗肿瘤的目的,从而为血管生成相关疾病的诊疗提供新方案。研究方法：1、采用薄膜分散法和硫酸铵梯度法制备P1c修饰的盐酸阿霉素长循环脂质体,并对其粒径、外观形态、包封率、偶联率及体外释放度等理化表征进行考察。2、选用高表达整合素ανβ3的人脑胶质瘤U87MG细胞及低表达整合素ανβ3的人乳腺癌MCF-7细胞作为体外考察的细胞模型,研究该载体的体外靶向性及抗肿瘤活性。通过流式细胞实验和激光共聚焦实验研究U87MG细胞及MCF-7细胞细胞对P1c靶向及非靶向盐酸阿霉素脂质体的摄取,通过CCK-8法检测脂质体的细胞毒性。3、建立裸鼠U87MG脑胶质瘤模型,考察P1c靶向盐酸阿霉素长循环脂质体的体内抗肿瘤效果,盐酸阿霉素、非靶向盐酸阿霉素脂质体和生理盐水作为实验对照,以肿瘤体积作为主要考察指标,并对肿瘤组织进行免疫组化及免疫荧光等病理学检查,比较各组制剂的抗肿瘤效果。研究结果：1、P1c修饰的盐酸阿霉素脂质体和非靶向脂质体平均粒径分别为131.2 m和128.4nm,Zeta电位分别为-19.7±2.8 mV和-33.1±1.6 mV。透射电镜观察脂质体形态圆整且分散均匀,包封率均在95%以上,P1c偶联率为66.8±1.6%,体外释放结果表明所制备的脂质体在37℃磷酸盐缓冲液(pH7.4)中有明显的缓释效果。2、流式细胞实验发现,将脂质体与细胞共同孵育1小时后,在U87MG细胞中,P1c修饰的盐酸阿霉素长循环脂质体组的平均荧光强度高于非靶向脂质体组及ανβ3单克隆抗体阻断组(P0.05),在MCF-7细胞各脂质体组的荧光强度无明显差异(P0.05),激光共聚焦实验与流式细胞分析结果一致。体外细胞毒性实验结果显示,在U87MG细胞,Plc修饰的盐酸阿霉素长循环脂质体组的细胞毒性高于非靶向脂质体组,在MCF-7细胞各脂质体组细胞毒性无明显差异。在两种肿瘤细胞,游离盐酸阿霉素的细胞结合能力和细胞毒性均明显高于各脂质体组。3、U87MG肿瘤模型鼠体内药效学结果显示,Plc修饰的盐酸阿霉素脂质体组、非靶向盐酸阿霉素脂质体组和盐酸阿霉素溶液组的抑瘤率分别为48.08%、31.03%和22.9%,肿瘤组织的免疫组化及免疫荧光结果显示Plc修饰的盐酸阿霉素脂质体组整合素ανβ3和CD31表达量最低,表明Plc修饰的盐酸阿霉素脂质体具有较好的抗肿瘤活性。研究结论：以P1c多肽修饰的长循环脂质体作为药物载体,能够通过靶向肿瘤细胞和肿瘤新生血管中过度表达的整合素ανβ3受体,提高药物的体内抗肿瘤作用,是一种具有应用前景的主动靶向药物传递系统。
[Abstract]:Objective: integrin V beta 3 plays an important role in angiogenesis related diseases and can be considered as a characteristic marker of angiogenesis. This study prepared the integrin alpha v beta 3 affinity peptide P1c (the connective tissue growth factor polypeptide, named P1c), which was discovered and prepared in our laboratory, and PEG hydrochloride adriamycin long circulation fat Plastids, in order to increase the transport of integrin alpha v beta 3 by targeting tumor cells and tumor neovascularization, increase the transport of drugs to the tumor site, and achieve the purpose of specific treatment of tumors, thus providing a new scheme for the diagnosis and treatment of vascular related diseases. Research methods: 1, preparation of membrane dispersion and ammonium sulfate gradient method. P1c modified adriamycin long circulating liposome was investigated, and its particle size, appearance, encapsulation rate, coupling rate, and in vitro release were investigated for.2. The human glioma U87MG cells with high expression of integrin V beta 3 and human breast cancer MCF-7 cells with low expression of integrin V beta 3 were selected as the cell models for the study. The targeting and antitumor activity of the carrier in vitro was studied by flow cytometry and laser confocal experiment to study the uptake of P1c targeting and non targeting adriamycin liposomes by U87MG cells and MCF-7 cells. The cytotoxic.3 of liposomes was detected by CCK-8, and U87MG glioma model was established in nude mice, and P1c target doxorubicin was examined by P1c. The antitumor effect of long circulating liposomes in vivo, adriamycin hydrochloride, non targeting doxorubicin liposomes and physiological saline as experimental control, tumor volume as the main investigation index, and immunohistochemical and immunofluorescence examination of tumor tissue, compare the antitumor effect of each group. The results of the study: 1, P1c modification The average particle size of doxorubicin and non targeted liposomes was 131.2 m and 128.4nm respectively. The Zeta potential was -19.7 + 2.8 mV and -33.1 + 1.6 mV. respectively. The liposomes were round and distributed evenly, the encapsulation efficiency was above 95% and the P1c coupling rate was 66.8 + 1.6%. The release results showed that the prepared liposomes were at 37 C. In the acid salt buffer solution (pH7.4), there was a significant release effect of.2. Flow cytometry found that after incubating the liposomes with the cells for 1 hours, the average fluorescence intensity of the P1c modified adriamycin long circulating liposome group was higher than that of the non targeted liposome group and the alpha Nu beta 3 monoclonal antibody block group (P0.05) in the U87MG cells, and in the MCF-7 cell lipids. The fluorescence intensity of the plastid group was not significantly different (P0.05), and the laser confocal experiment was consistent with the flow cytometry. In vitro cytotoxicity test showed that in U87MG cells, the cytotoxicity of Plc modified adriamycin long circulating liposome group was higher than that of non targeted liposome group, and there was no significant difference in cytotoxicity between the liposomes of MCF-7 cells. The cell binding capacity and cytotoxicity of doxorubicin in the two tumor cells were significantly higher than those of the liposome group.3. The pharmacodynamic results of the U87MG tumor model mice showed that the Plc modified adriamycin liposome group, the non targeting doxorubicin liposome group and the salt acid adriamycin group were 48.08%, 31 respectively. 3% and 22.9%, the immunohistochemical and immunofluorescence results of the tumor tissue showed that the expression of integrin alpha v beta 3 and CD31 in the adriamycin liposome group of Plc modified hydrochloric acid was the lowest, indicating that the adriamycin liposome with Plc modified adriamycin had better anti-tumor activity. Overexpression of the integrin - V beta 3 receptor in tumor cells and neovascularization is an active targeting drug delivery system, which improves the antitumor effect of the drug in vivo.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R943

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