靶向HER3适配体-药物偶联物递送系统的构建及抑瘤作用研究

发布时间：2018-03-06 06:03

本文选题：SELEX　切入点：适配体-药物偶联物　出处：《广西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：癌症是人类健康的最大威胁之一,伴随医学的发展,肿瘤治疗策略已经趋于多样化,有联合治疗、免疫疗法,甚至精准医疗,但在部分肿瘤患者中化疗依旧作为一线治疗方案被使用。如何尽量克服、避免化疗药物因非靶向性带来的毒副作用,一直是临床关注的热点之一。构建靶向递送系统,可提高靶组织局部药物浓度,减少全身毒副作用,实现药物的高效、安全递送,是一种很有前景的给药方法。目前已有两种抗体药物偶联物(Antibody-drug conjugates,ADC)经FDA批准上市且获得良好药效。但在抗体-药物制备过程中的DAR(Drug-anbibody ratio)均一性、裸抗体的免疫原性等问题还未得到良好控制。适配体(Aptamer,Apt)是一种独特的单链寡核苷酸,其功能类似抗体,有着靶分子范围广泛、高特异性及亲和性以及免疫原性低等优势。除可作为药物(例如FDA批准上市的Macugen)单独应用外,更多可作为药物递送系统中的靶向载体。本论文关注化疗药物的靶向递送系统的概念性验证研究,构建适配体-药物偶联物递送系统(Aptamer-drug conjugate,Ap DC),证明其显著提高药物治疗指数,降低药物毒副作用的应用前景。本文首先应用指数富集的配体系统进化技术(Systematic evolution of ligands by exponential enrichment,SELEX),以人的表皮生长因子受体3(human epidermal growth factor receptor 3,HER3)的胞外结构域(extracellular domain,ECD)为靶目标,筛选获得具有高亲和力和高特异性的候选Apt。选择阿霉素(Doxorubicin,DOX)为递送对象,薄膜分散法制备脂质体阿霉素(liposome-doxorubicin,Lip-DOX)。通过“后插入法”连接Apt,构建稳定的适配体-脂质体-阿霉素(Aptamer-liposome-doxorubicin,Apt-Lip-DOX)。通过包封率、粒径大小、zeta电势、释放度等理化性质指标,考察并优化Apt-Lip-DOX制备工艺。体外实验中,在MCF-7 HER3+、BT474 HER3+细胞及阴性对照293THER3-细胞中考察该Ap DC系统的入胞情况及半数抑制浓度(half maximal inhibitory concentration,IC50)并对内吞途径进行初步探索。体内实验中,选择MCF-7HER3+荷瘤小鼠模型,考察Ap DC针对其他对照组在靶向性、有效性及安全性方面的优势。第一部分HER3ECD Apt的筛选和鉴定。选择HER3ECD质粒瞬时转染293E细胞,表达回收HER3ECD蛋白,作为筛选的靶蛋白。采用经优化的“QPCR-磁珠分选”SELEX方法,经四轮十次筛选,获得靶向HER3ECD的候选Apt。通过酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)并拟合亲和力曲线,得出Apt#7及#13亲和力常数较佳,Kd值分别为116±23 n M、98±9.7 n M。通过细胞流式实验及激光共聚焦结果显示,该适配体针对高表达HER3的乳腺癌细胞系具有较高的亲和力及特异性。第二部分Ap DC递送系统的构建及优化。优化脂质体阿霉素的制备工艺,通过DSPE-PEG2000连接Apt,采用“后插入法”制备稳定的Ap DC,即Apt-Lip-DOX。考察Apt-Lip-DOX的理化性质,粒径为170±25 nm,zeta电势为-45.9±6.31 m V,结构稳定。超滤法结合高效液相色谱(High Performance Liquid Chromatography,HPLC)测定DOX药物包封率为55±5%。模拟体内环境药物释放度结果显示:同等条件下,游离的DOX在1h释放度接近100%,而Apt-Lip-DOX 24h释放度仅为20%,充分证明了该递送系统缓释递送的优势。第三部分Ap DC系统体外胞吞机制和药效实验。细胞水平上,选择MCF-7 HER3+细胞及阴性对照293THER3-,比较Apt的介导是否增加递送系统的入胞量。并通过不同内吞途径抑制剂结合活细胞观测站,初步探讨该Ap DC入胞途径机制。结果表明Ap DC的入胞依赖于HER3的表达及Apt的存在,突出HER3 Apt的优势,同时该Ap DC主要通过网格蛋白介导的内吞作用完成内吞。进一步选择MCF-7 HER3+、BT474HER3+两株细胞系经实时细胞检测(real time cell assay,RTCA)考察DOX不同递送形式的IC50值,同时加入293THER3-细胞作为对照。结果显示,在三株细胞系中,Lip-DOX针对游离DOX的IC50值降低,且均具有统计学差异(P0.05),体现脂质体递送系统的优势。Apt-Lip-DOX在HER3高表达的MCF-7、BT474中较游离DOX组出现显著性统计学差异(P0.01),在293T细胞中未表现出该优势。证明所筛选适配体在递送系统中发挥一定的靶向优势,增加体外靶向肿瘤细胞的杀伤力,为后续体内实验奠定基础。第四部分Ap DC系统荷瘤小鼠体内肿瘤靶向性和药效、毒性实验。动物水平上,选择MCF-7荷瘤小鼠,考察体内Ap DC的肿瘤靶向性、有效性及安全性。1)肿瘤靶向性:按照Ap DC制备工艺,选择包裹量子点(QD),制备Apt-Lip-QD,同时Lip-QD为对照组,尾静脉注射MCF-7荷瘤小鼠1h后进行活体成像,观察荧光分布判断药物的体内分布。结果显示Ap DC表现出良好的靶向性,解剖瘤体与Lip-QD相比,具有明显的统计学差异(P0.01)。2)体内抑制肿瘤药效实验:设置空白对照组(Blank,等体积生理盐水)以及实验组DOX、Lip-DOX、Apt-Lip-DOX共四个组别,实验组的DOX给药量为5mg/kg,每三天尾静脉注射给药。结果显示:给药18天后,Apt-Lip-DOX抑瘤效果针对Blank组具有非常显著的统计学差异(P0.01,P0.001),同时较DOX组也表现出较大的优势(P0.05)。3)体内毒性考察:分别考察小鼠给药期间体重状态,60天存活率,组织病理切片。结果显示:给药期间Apt-Lip-DOX组小鼠状态、体重维持在正常水平,DOX组则在20天出现体重急剧下降的现象。不同组别(DOX、Lip-DOX、Apt-Lip-DOX)小鼠60天以内的存活率分别为0、40%、100%。小鼠死亡后对各脏器做HE病理切片观察病变损伤情况,在DOX组中,心脏切片显示出现脂肪变性,散在出血点,血管扩张出血等病变;肝脏切片出现局灶肝细胞坏死。在Lip-DOX组中也出现轻微病变,Apt-Lip-DOX组所有组织均正常。综上体内实验结果,充分证明了该Ap DC的靶向性、高效性及安全性。综上,本研究建立并完善SELEX技术路线,为其他靶点筛选提供参考;靶向HER3的治疗策略将会为肿瘤靶向治疗或逆转EGFR抑制剂耐药带来新的思路;以化疗药(DOX)为例,通过构建Ap DC递送系统,对其靶向性、抑瘤作用等进行研究,为实现化疗药的高效低毒递送提供参考。
[Abstract]:Cancer is one of the biggest threat to human health, along with the development of medicine, therapeutic strategies have tended to be diverse, with combined therapy, immunotherapy, and accurate medical treatment, but still chemotherapy as first-line therapy is used in patients with tumor. How to overcome and avoid chemotherapy for non targeting of the side role is one of clinical focus. Construction of targeted delivery system, can improve the local drug concentration of target tissue, reduce systemic toxicity, drug efficiency, safe delivery, is a promising drug delivery method. There are two kinds of antibody drug conjugates (Antibody-drug, conjugates, ADC) by approved by the FDA and get a good effect. But the antibody drug in the preparation process of DAR (Drug-anbibody ratio) uniformity, immunogenicity and other issues have not been good naked antibody aptamer (Aptam control. Er, Apt) is a unique single stranded oligonucleotide, its function is similar with the target molecule antibody, wide range, high specificity and affinity and lower immunogenicity. In addition can be used as a drug (such as the FDA approved Macugen) alone, more can be used as a drug delivery system targeting carrier this paper focuses on. Chemotherapy drugs targeting research proof of concept delivery system, construct the aptamer conjugates drug delivery system (Aptamer-drug conjugate, Ap DC), that significantly improve the therapeutic efficacy, reduce the side effect of the drug application. This paper firstly applied evolution of ligands by exponential enrichment (Systematic evolution system of ligands by exponential enrichment, SELEX), human epidermal growth factor receptor 3 (human epidermal growth factor receptor 3, HER3) of the extracellular domain (extracellular, domain, ECD) as the target Target candidate Apt. with high affinity and high specificity for selection of adriamycin (Doxorubicin, DOX) for screening the delivery object by film dispersion liposomal doxorubicin (liposome-doxorubicin, Lip-DOX) method. Apt connection through the post insertion method, construct the aptamer liposome doxorubicin (Aptamer-liposome-doxorubicin, Apt-Lip-DOX) stable through. Encapsulation efficiency, particle size, zeta potential, release of physical and chemical properties, investigate and optimize the preparation process of Apt-Lip-DOX. In vitro experiments, in MCF-7 HER3+, BT474 HER3+ cell and negative control cell of the Ap into the DC system in 293THER3- cells and the half inhibitory concentration (half maximal inhibitory concentration, IC50) and the endocytic pathways were investigated. In vivo, MCF-7HER3+ tumor bearing mice model of Ap DC for the other control group to the target, effectiveness and safety The advantage of the first part. Screening and identification of HER3ECD Apt. HER3ECD plasmid was transfected to 293E cells, the expression of HER3ECD protein recovery, as the target protein screening. Using the optimized "QPCR- multisort" SELEX method, after four rounds of the ten screening, get targeted candidate Apt. HER3ECD by ELISA (enzyme linked immunosorbent assay, ELISA) and the Apt#7 curve fitting affinity, and the affinity constant #13 is better, Kd = 116 + 23 n M + 9.7 n, 98 M. by flow cytometry and confocal laser experiments showed that the affinity and specificity of the adapter body of high expression of breast cancer cell lines HER3 has a higher Ap DC. The second part delivery of construction and optimization of the system. Optimization of the preparation of liposomal doxorubicin, Apt connection through the DSPE-PEG2000, the "post insert method" the preparation of stable Ap DC, namely Apt-Lip-DO X. Apt-Lip-DOX on the physical and chemical properties, particle size of 170 + 25 nm, zeta potential is -45.9 + 6.31 m V, stable structure. Ultrafiltration combined with high performance liquid chromatography (HPLC High Performance Liquid Chromatography, DOX) determination of drug entrapment efficiency shows 55 + 5%. for simulating the in vivo environment drug release results: the same conditions next, the free DOX in the 1H release rate close to 100%, while the Apt-Lip-DOX 24h release rate is only 20%, it is proved that this delivery system sustained release delivery advantage. The third part of the Ap DC system in vitro endocytosis mechanism and experiment. The level of cell, MCF-7 HER3+ cell and negative control 293THER3-, Apt mediated whether increase the delivery system into the cell. And through the different endocytic pathway inhibitors combined with living cell observation station, preliminary study of the Ap DC into the cell mechanism. The results showed that the expression of Apt and Ap in DC cell dependent on the presence of HER3 HER3 A, prominent The advantages of the Pt and the Ap DC endocytosis through clathrin mediated endocytosis. Further complete the selection of MCF-7 HER3+ and BT474HER3+ two cell lines by real-time cell detection (real time cell assay, RTCA DOX) on different delivery form of the IC50 value, while adding 293THER3- cells as control. The results showed that in three cell lines, Lip-DOX for free DOX IC50 value decreased, and there was significant difference (P0.05), liposome delivery system embodies the advantages of the high expression of.Apt-Lip-DOX in HER3 MCF-7, significant difference between DOX group compared with the free BT474 (P0.01), in 293T cells did not show the advantage. To prove that the screening aptamers in a delivery system play a targeted advantage, increase the in vitro targeting tumor cell destruction, which lays the foundation for further study in vivo. The fourth part of the Ap DC system in vivo tumor targeting and tumor bearing mice The efficacy, toxicity. Animal level, select the MCF-7 tumor bearing mice were investigated in vivo Ap DC tumor targeting, effectiveness and safety of.1) tumor targeting Ap DC: according to the preparation process, select the package of quantum dots (QD), the preparation of Apt-Lip-QD Lip-QD, at the same time as the control group, tail vein injection of MCF-7 mice after 1h in vivo imaging, observation of fluorescence distribution determine the distribution of drugs in the body. The results showed that Ap DC exhibited better targeting tumor anatomy, compared with Lip-QD, the difference is statistically significant (P0.01).2) in inhibiting tumor experiment: blank control group (Blank, volume etc. saline) and experimental group DOX, Lip-DOX, Apt-Lip-DOX a total of four groups, the experimental group of DOX dosage was 5mg/kg, every three days intravenous injection. Results: after 18 days of treatment, Apt-Lip-DOX inhibitory effect in Blank group was statistically significantly different (P0.0 1, P0.001), and compared with the DOX group also showed a greater advantage (P0.05).3) were investigated in vivo toxicity study: mice were administered during weight status, the 60 day survival rate, pathological sections. The results showed that during the administration of Apt-Lip-DOX mice, body weight was maintained at a normal level, DOX group showed weight a sharp decline in the phenomenon in 20 days. Different groups (DOX, Lip-DOX, Apt-Lip-DOX) the survival rate of mice within 60 days were 0,40%, 100%. after the death of mice HE pathological lesions of various organs in the DOX group, the heart slices showed steatosis, scattered hemorrhage, vascular dilatation and hemorrhage other diseases; liver biopsy and focal necrosis of liver cells. Slight lesions in the Lip-DOX group, Apt-Lip-DOX group all were normal. The results of in vivo experiments proved that targeting the Ap of DC, high efficiency and safety. In summary, this research The establishment and improvement of SELEX technology, provide a reference for other target screening; targeted treatment strategies will HER3 for tumor targeted therapy or reversal of EGFR inhibitor resistance brings new ideas; to chemotherapeutic drugs (DOX) as an example, by constructing Ap DC delivery system, the targeting and anti-tumor effect etc. study, provide a reference for the realization of high efficiency and low toxicity of chemotherapeutic drug delivery.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96

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