本文主要研究内容
作者杜坤宇（2019）在《具有示踪靶向效应的蛋白类药物多功能给药体系的构建及评价》一文中研究指出：重组人血管内皮抑制素(rhEndostatin,rh-ES)是一种我国自主研发的具有抗非小细胞肺癌的蛋白类药物,目前大量的研究表明其对其它类的癌细胞也有抑制作用。重组人血管内皮抑制素为蛋白类药物,与其它蛋白类药物一样,稳定性较差,在体内循环时间短,目前通常将其制备成缓释靶向微球以提高药物的疗效、安全性与稳定性,但其制备工艺与添加剂等会对蛋白的活性造成影响,药物利用率低并产生突释现象,安全性低,同时制备的缓释靶向微球的体内靶向性评价比较繁琐,干扰较大,所以限制了其广泛的临床应用。本文通过静电喷雾技术在无添加剂、无乳化剂、无交联剂与低温的条件下实现一步法制备复合物载药微球,并通过新型的荧光成像材料量子点(QDs)标记rh-ES,共载于聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)中,制备具有缓释靶向可示踪的复合载药微球,通过活体成像技术对微球的靶向性进行评价,并对其抗肿瘤效应进行考察。全文共分为以下五部分:一、综述本部分首先对蛋白多肽类药物微球的制备方法与制剂体内靶向性评价普遍存在的问题进行了简要的概述;其次对新型的微纳米粒子制备技术静电喷雾技术与荧光成像材料量子点的特点与应用进行介绍;最后总结了蛋白质模型药物rh-ES在抗肺癌方面的研究进展。二、水溶性量子点的制备与表征本部分通过两亲性聚合物实现油溶性量子点的包裹,实现量子点在水溶液中稳定的存在,并能成功标记蛋白类药物。首先合成两亲性聚合物,通过核磁共振光谱(~1H-NMR)与傅里叶红外变换光谱(FT-IR)确定聚合物的成功合成。再通过自组装实现QDs的包裹,得到的QDs产物具有较好的水溶性,粒径为35 nm左右。通过紫外和荧光光谱可以看出,改性的QDs具有较好的光学性能。稳定性研究发现其在不同的pH和离子强度条件下稳定性较好,未发生聚集。另外通过MTT法对聚合物和修饰后的QDs进行安全性评价,结果表明浓度在5~200μg/mL范围内,细胞的存活率均大于85%,表明修饰后的QDs安全性高,满足运用于体内实验的要求。三、量子点与重组人血管内皮抑制素复合物的制备及其性质考察本部分通过共价结合法实现rh-ES与QDs的结合。通过对1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、N-羟基硫代琥珀酰亚胺(sulfo-NHS)的用量、反应时间、反应pH值等因素进行考察,优化工艺处方。通过琼脂糖凝胶电泳确定rh-ES-QDs复合物成功制备,另外通过粒径测定可以看出标记前QDs的粒径为35±2.4 nm,电位为-19.27±2.1 mv,标记后rh-ES-QDs的粒径为94±3.5 nm,电位为-22.92±1.3 mv,间接表明QDs与蛋白成功结合。通过间接法计算rh-ES-QDs的平均偶联效率为80%。rh-ES-QDs的荧光稳定性、偶联稳定性以及光学性能均较好,并且蛋白的二级结构未受到影响。四、基于静电喷雾技术重组人血管内皮抑制素复合微球的制备与表征本部分首先建立了测定微球中rh-ES的含量与体外释放度的方法,并对方法进行验证。采用静电喷雾技术实现一步法制备PLGA-rh-ES-QDs微球。以PLGA-rh-ES-QDs-MS的形态粒径和包封率作为指标,通过单因素考察和中心复合设计,确定了微球制备的最佳工艺。通过扫描电镜(SEM)观察微球的表面形态良好,分散比较均匀。荧光显微镜表征微球具有良好的荧光性能。激光粒径仪(DLS)测定微球的粒径在4-8μm范围内,满足肺部被动靶向的要求。PLGA-rh-ES-QDs微球的包封率为85.46±1.57%,载药量为23.95±2.32μg/mg。载药微球在PBS(含0.02%F-68)中体外释放实验表明PLGA-rh-ES-QDs-MS具有较好的缓释效果,在15天时rh-ES累积释放达80%以上,并且未出现明显的突释现象。通过MTT实验对微球的生物相容性和生物活性进行考察,结果表明微球和载体材料的安全性较好,微球的释放液与原注射液具有相当的细胞增殖抑制率,表明制备成微球后,蛋白的活性保持较好。五、体内药动学及靶向性与药效学评价本部分首先建立酶联免疫法(ELISA)测定SD大鼠血清中重组人血管内皮抑制素的浓度。体内药动学结果显示将其制备成微球后,在72 h时仍能检测到药物,消除半衰期从3.46 h增加到65.35 h,药物在体内的平均滞留时间(MRT)也从4.03 h增加到257.45 h,表明通过静电喷雾技术制备的PLGA-rh-ES-QDs-MS具有明显的缓释效果。其次通过活体成像技术对微球的靶向性进行评价,结果显示微球组经尾静脉注射吸收后大部分被肺部截留,原料组在肺部也有一定的分布,但是荧光强度明显低于微球组,表明通过静电喷雾技术制备的微球具有一定的肺部靶向性。最后通过荷瘤小鼠评价制备的PLGA-rh-ES-QDs-MS的药效学,从肿瘤的重量、体积变化以及肿瘤组织切片均可以判断微球具有较好的抗肿瘤效应,与生理盐水组相比抑瘤效率为38.4%。由小鼠体重的变化曲线和体内组织的HE染色可以看出,PLGA-rh-ES-QDs-MS的安全性较好。
Abstract
chong zu ren xie guan nei pi yi zhi su (rhEndostatin,rh-ES)shi yi chong wo guo zi zhu yan fa de ju you kang fei xiao xi bao fei ai de dan bai lei yao wu ,mu qian da liang de yan jiu biao ming ji dui ji ta lei de ai xi bao ye you yi zhi zuo yong 。chong zu ren xie guan nei pi yi zhi su wei dan bai lei yao wu ,yu ji ta dan bai lei yao wu yi yang ,wen ding xing jiao cha ,zai ti nei xun huan shi jian duan ,mu qian tong chang jiang ji zhi bei cheng huan shi ba xiang wei qiu yi di gao yao wu de liao xiao 、an quan xing yu wen ding xing ,dan ji zhi bei gong yi yu tian jia ji deng hui dui dan bai de huo xing zao cheng ying xiang ,yao wu li yong lv di bing chan sheng tu shi xian xiang ,an quan xing di ,tong shi zhi bei de huan shi ba xiang wei qiu de ti nei ba xiang xing ping jia bi jiao fan suo ,gan rao jiao da ,suo yi xian zhi le ji an fan de lin chuang ying yong 。ben wen tong guo jing dian pen wu ji shu zai mo tian jia ji 、mo ru hua ji 、mo jiao lian ji yu di wen de tiao jian xia shi xian yi bu fa zhi bei fu ge wu zai yao wei qiu ,bing tong guo xin xing de ying guang cheng xiang cai liao liang zi dian (QDs)biao ji rh-ES,gong zai yu ju ru suan -qiang ji yi suan gong ju wu (poly(lactic-co-glycolic acid),PLGA)zhong ,zhi bei ju you huan shi ba xiang ke shi zong de fu ge zai yao wei qiu ,tong guo huo ti cheng xiang ji shu dui wei qiu de ba xiang xing jin hang ping jia ,bing dui ji kang zhong liu xiao ying jin hang kao cha 。quan wen gong fen wei yi xia wu bu fen :yi 、zeng shu ben bu fen shou xian dui dan bai duo tai lei yao wu wei qiu de zhi bei fang fa yu zhi ji ti nei ba xiang xing ping jia pu bian cun zai de wen ti jin hang le jian yao de gai shu ;ji ci dui xin xing de wei na mi li zi zhi bei ji shu jing dian pen wu ji shu yu ying guang cheng xiang cai liao liang zi dian de te dian yu ying yong jin hang jie shao ;zui hou zong jie le dan bai zhi mo xing yao wu rh-ESzai kang fei ai fang mian de yan jiu jin zhan 。er 、shui rong xing liang zi dian de zhi bei yu biao zheng ben bu fen tong guo liang qin xing ju ge wu shi xian you rong xing liang zi dian de bao guo ,shi xian liang zi dian zai shui rong ye zhong wen ding de cun zai ,bing neng cheng gong biao ji dan bai lei yao wu 。shou xian ge cheng liang qin xing ju ge wu ,tong guo he ci gong zhen guang pu (~1H-NMR)yu fu li xie gong wai bian huan guang pu (FT-IR)que ding ju ge wu de cheng gong ge cheng 。zai tong guo zi zu zhuang shi xian QDsde bao guo ,de dao de QDschan wu ju you jiao hao de shui rong xing ,li jing wei 35 nmzuo you 。tong guo zi wai he ying guang guang pu ke yi kan chu ,gai xing de QDsju you jiao hao de guang xue xing neng 。wen ding xing yan jiu fa xian ji zai bu tong de pHhe li zi jiang du tiao jian xia wen ding xing jiao hao ,wei fa sheng ju ji 。ling wai tong guo MTTfa dui ju ge wu he xiu shi hou de QDsjin hang an quan xing ping jia ,jie guo biao ming nong du zai 5~200μg/mLfan wei nei ,xi bao de cun huo lv jun da yu 85%,biao ming xiu shi hou de QDsan quan xing gao ,man zu yun yong yu ti nei shi yan de yao qiu 。san 、liang zi dian yu chong zu ren xie guan nei pi yi zhi su fu ge wu de zhi bei ji ji xing zhi kao cha ben bu fen tong guo gong jia jie ge fa shi xian rh-ESyu QDsde jie ge 。tong guo dui 1-(3-er jia an ji bing ji )-3-yi ji tan er ya an yan suan yan (EDC)、N-qiang ji liu dai hu po xian ya an (sulfo-NHS)de yong liang 、fan ying shi jian 、fan ying pHzhi deng yin su jin hang kao cha ,you hua gong yi chu fang 。tong guo qiong zhi tang ning jiao dian yong que ding rh-ES-QDsfu ge wu cheng gong zhi bei ,ling wai tong guo li jing ce ding ke yi kan chu biao ji qian QDsde li jing wei 35±2.4 nm,dian wei wei -19.27±2.1 mv,biao ji hou rh-ES-QDsde li jing wei 94±3.5 nm,dian wei wei -22.92±1.3 mv,jian jie biao ming QDsyu dan bai cheng gong jie ge 。tong guo jian jie fa ji suan rh-ES-QDsde ping jun ou lian xiao lv wei 80%。rh-ES-QDsde ying guang wen ding xing 、ou lian wen ding xing yi ji guang xue xing neng jun jiao hao ,bing ju dan bai de er ji jie gou wei shou dao ying xiang 。si 、ji yu jing dian pen wu ji shu chong zu ren xie guan nei pi yi zhi su fu ge wei qiu de zhi bei yu biao zheng ben bu fen shou xian jian li le ce ding wei qiu zhong rh-ESde han liang yu ti wai shi fang du de fang fa ,bing dui fang fa jin hang yan zheng 。cai yong jing dian pen wu ji shu shi xian yi bu fa zhi bei PLGA-rh-ES-QDswei qiu 。yi PLGA-rh-ES-QDs-MSde xing tai li jing he bao feng lv zuo wei zhi biao ,tong guo chan yin su kao cha he zhong xin fu ge she ji ,que ding le wei qiu zhi bei de zui jia gong yi 。tong guo sao miao dian jing (SEM)guan cha wei qiu de biao mian xing tai liang hao ,fen san bi jiao jun yun 。ying guang xian wei jing biao zheng wei qiu ju you liang hao de ying guang xing neng 。ji guang li jing yi (DLS)ce ding wei qiu de li jing zai 4-8μmfan wei nei ,man zu fei bu bei dong ba xiang de yao qiu 。PLGA-rh-ES-QDswei qiu de bao feng lv wei 85.46±1.57%,zai yao liang wei 23.95±2.32μg/mg。zai yao wei qiu zai PBS(han 0.02%F-68)zhong ti wai shi fang shi yan biao ming PLGA-rh-ES-QDs-MSju you jiao hao de huan shi xiao guo ,zai 15tian shi rh-ESlei ji shi fang da 80%yi shang ,bing ju wei chu xian ming xian de tu shi xian xiang 。tong guo MTTshi yan dui wei qiu de sheng wu xiang rong xing he sheng wu huo xing jin hang kao cha ,jie guo biao ming wei qiu he zai ti cai liao de an quan xing jiao hao ,wei qiu de shi fang ye yu yuan zhu she ye ju you xiang dang de xi bao zeng shi yi zhi lv ,biao ming zhi bei cheng wei qiu hou ,dan bai de huo xing bao chi jiao hao 。wu 、ti nei yao dong xue ji ba xiang xing yu yao xiao xue ping jia ben bu fen shou xian jian li mei lian mian yi fa (ELISA)ce ding SDda shu xie qing zhong chong zu ren xie guan nei pi yi zhi su de nong du 。ti nei yao dong xue jie guo xian shi jiang ji zhi bei cheng wei qiu hou ,zai 72 hshi reng neng jian ce dao yao wu ,xiao chu ban cui ji cong 3.46 hzeng jia dao 65.35 h,yao wu zai ti nei de ping jun zhi liu shi jian (MRT)ye cong 4.03 hzeng jia dao 257.45 h,biao ming tong guo jing dian pen wu ji shu zhi bei de PLGA-rh-ES-QDs-MSju you ming xian de huan shi xiao guo 。ji ci tong guo huo ti cheng xiang ji shu dui wei qiu de ba xiang xing jin hang ping jia ,jie guo xian shi wei qiu zu jing wei jing mai zhu she xi shou hou da bu fen bei fei bu jie liu ,yuan liao zu zai fei bu ye you yi ding de fen bu ,dan shi ying guang jiang du ming xian di yu wei qiu zu ,biao ming tong guo jing dian pen wu ji shu zhi bei de wei qiu ju you yi ding de fei bu ba xiang xing 。zui hou tong guo he liu xiao shu ping jia zhi bei de PLGA-rh-ES-QDs-MSde yao xiao xue ,cong zhong liu de chong liang 、ti ji bian hua yi ji zhong liu zu zhi qie pian jun ke yi pan duan wei qiu ju you jiao hao de kang zhong liu xiao ying ,yu sheng li yan shui zu xiang bi yi liu xiao lv wei 38.4%。you xiao shu ti chong de bian hua qu xian he ti nei zu zhi de HEran se ke yi kan chu ,PLGA-rh-ES-QDs-MSde an quan xing jiao hao 。
论文参考文献
论文详细介绍
论文作者分别是来自江苏大学的杜坤宇，发表于刊物江苏大学2019-09-19论文，是一篇关于静电喷雾技术论文,重组人血管内皮抑制素论文,量子点论文,活体成像技术论文,肺靶向论文,江苏大学2019-09-19论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自江苏大学2019-09-19论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：静电喷雾技术论文; 重组人血管内皮抑制素论文; 量子点论文; 活体成像技术论文; 肺靶向论文; 江苏大学2019-09-19论文;