新型乳腺癌免疫脂质体的制备及体外性质研究

发布时间：2018-06-08 12:36

本文选题：阿霉素 + 免疫脂质体　；参考：《第四军医大学》2014年硕士论文

【摘要】：阿霉素(doxorubicine，DOX)是一种常用的抗肿瘤药物，属于周期非特异性药物，对RNA的抑制作用较强，抗瘤谱较广。但因其毒副作用较大，长期应用会使机体产生耐药性，导致其疗效和应用受到极大限制。脂质体作为新型的药物制剂，不仅能降低药物对机体的毒副作用，改善药代动力学分布还具有缓释控释作用，但是其缺乏肿瘤主动靶向性。免疫脂质体是近年来新兴的一个研究领域，将抗体或配体通过聚合物（如聚乙二醇衍生物）连接在脂质体上，利用抗体或配体的特异性靶向作用，与肿瘤细胞表面上的抗原或者受体结合，将药物送达病灶部位，具有靶向性强、毒副作用小、半衰期长、运载量大等优点。大约有20-30%的乳腺癌存在人类表皮生长因子-2(human epidermal growth factorreceptor-2，HER2)过度表达。HER2基因编码一种具有酪氨酸激酶活性的蛋白，能增强激酶活性，促进细胞分裂、增生和转化。该基因在成人正常组织中表达水平较低，在乳腺癌发生的早期阶段具有高表达现象，提高了乳腺癌细胞的转移潜能。近年来，根据HER2结构构建的具有靶向作用的单克隆抗体曲妥珠单抗(trastuzumab，TMAB)，曲妥珠单抗能与HER2受体结合，具有较强的亲合力和特异性，通过阻断人体表皮生长因子在HER2上的附着，产生抗肿瘤效应，为乳腺癌的治疗提供了一种新途径。曲妥珠单抗（即赫赛汀）已于1998年10月由美国FDA正式批准上市。本文通过薄膜超声分散法制备阿霉素脂质体， DSPE-PEG-NHS与TMAB通过酰胺键连接，将阿霉素脂质体与TMAB衍生物4℃恒温震荡6h，得到阿霉素免疫脂质体。阿霉素脂质体的包封率为89.85%、载药率为8.03%、平均电荷在-26mv。确定曲妥珠单抗活性氨基数目为85个，当n（DSPE-PEG-NHS）：n(TMAB)=10：1进行反应时，测得曲妥珠单抗上参与反应氨基数目为7个。制备三种不同抗体含量的阿霉素免疫脂质体，通过葡聚糖凝胶G150分离纯化，BCA法测定阿霉素免疫脂质体TMAB含量（抗体含量分别为37μg·mg-1、83μg·mg-1、108μg·mg-1），并进行体外性质研究。以AU565细胞为HER2阳性细胞，MDA-MB-231细胞为HER2阴性细胞，三种不同抗体含量的阿霉素免疫脂质体的激光共聚焦、流式细胞实验和细胞毒性实验表明：DOX-IML抗体含量83μg·mg-1时，药物入胞量为86.8%，其入胞能力和细胞毒性仅次于阿霉素。阿霉素、阿霉素脂质体、DOX IML进行体外性质实验表明：阿霉素免疫脂质体有较强的靶向性，且入胞能力和细胞毒性均比阿霉素脂质体强。本文还研究阿霉素（免疫）脂质体粒径和电位变化：包封阿霉素前后阿霉素脂质体的粒径和电位几乎无变化，键合曲妥珠单抗前后阿霉素免疫脂质体的粒径和电位变化较小。体外释药及稳定性研究得；阿霉素免疫脂质体具有一定的缓释作用，在12h内释药量累计达40%，，在72h时累积释药量几乎达到平衡，累积释药率达79%。阿霉素免疫脂质体在4℃下，稳定性较好。在25℃下，随着时间延长，粒径越来越大，颜色由红色变为暗红色，伴有大量沉淀产生。
[Abstract]:Doxorubicine (DOX) is a common antitumor drug, which is a periodic non specific drug. It has a strong inhibitory effect on RNA and a wide spectrum of antitumor. However, because of its toxic and side effects, long term application will cause resistance to the body, and the effect and application are very limited. As a new drug preparation, the liposomes can not only be reduced. The toxic side effects of drugs on the body and the improvement of the pharmacokinetic distribution also have controlled release effect, but they lack active targeting of tumor. Immunliposome is a new research field in recent years. Antibodies or ligands are connected to liposomes through polymers such as polyethylene glycol derivatives, and the specific targeting of antibodies or ligands is used. It combines with the antigen or receptor on the surface of the tumor cells to deliver the drug to the site of the lesion, which has the advantages of strong targeting, small side effects, long half-life and large carrying capacity.
About 20-30% of breast cancer, human epidermal growth factor -2 (human epidermal growth factorreceptor-2, HER2) overexpresses a protein that encodes a tyrosine kinase activity by the.HER2 gene, which can enhance the activity of the kinase and promote cell division, proliferation and transformation. The gene is expressed in normal tissues in adults with low levels and in breast cancer. The early stage of the occurrence has high expression, which improves the metastatic potential of breast cancer cells. In recent years, the monoclonal antibody to trastuzumab (TMAB), a monoclonal antibody against the target by the HER2 structure, can be combined with the HER2 receptor, with strong affinity and specificity, and by blocking the human epidermal growth factor. The attachment on HER2, which produces antitumor effects, provides a new way for the treatment of breast cancer. Trastuzumab (Herceptin) was officially approved by the US FDA in October 1998.
The liposome of doxorubicin was prepared by the thin film ultrasonic dispersion method. DSPE-PEG-NHS and TMAB were connected through the amide bond. The adriamycin liposome and the TMAB derivative oscillated at a constant temperature of 6h to obtain the adriamycin immunliposome. The encapsulation efficiency of adriamycin liposome was 89.85%, the drug loading rate was 8.03%, and the average charge was determined by -26mv. to determine the active amino group of the trastuzumab in -26mv.. The number was 85. When n (DSPE-PEG-NHS):n (TMAB) =10:1 was reacted, the number of amino groups involved in the reaction was 7. Three kinds of adriamycin immunliposomes with different antibody content were prepared and purified by dextran gel G150. The content of adriamycin free liposome TMAB was determined by BCA method (the antibody content was 37 g mg-1,8 respectively. " 3 mu g. Mg-1108 mu g. Mg-1) and study in vitro, with AU565 cells as HER2 positive cells, MDA-MB-231 cells as HER2 negative cells, and three kinds of adriamycin immunliposomes with different antibody content, laser confocal, flow cytometry and cytotoxicity test showed that when the content of DOX-IML antibody was 83 mu g. Mg-1, the drug intake was 86.8%, Its cellular and cytotoxicity were second only to adriamycin. Adriamycin, adriamycin liposome and DOX IML in vitro experiments showed that adriamycin immunliposome had stronger targeting and stronger cellular and cytotoxicity than doxorubicin liposomes. The particle size and potential of adriamycin liposomes were almost unchanged, and the size and potential of doxorubicin immune liposomes were small. The release and stability of adriamycin were studied in vitro. The release effect of adriamycin immunliposome was 40%, and the cumulative release amount was almost reached at 72h. Balance, the cumulative release rate reached 79%. adriamycin immunliposome at 4 C, and the stability was better. At 25 C, the particle size became larger and larger with time, and the color changed from red to dark red with a large amount of precipitation.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

【共引文献】