曲妥珠单抗修饰的紫杉醇脂质纳米粒的制备及其靶向性的初步研究

发布时间：2018-02-25 06:16

本文关键词： 紫杉醇曲妥珠单抗聚乙烯亚胺脂质纳米粒靶向传递系统　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：乳腺癌发病率呈逐年上升趋势,已成为威胁女性健康的最大杀手。紫杉醇是乳腺癌化疗治疗的一线用药,单用和联合应用均显示很好的疗效,但其非选择性会引起严重的全身性毒副作用。肿瘤生物靶向治疗是以肿瘤标志性分子作为靶点,将药物特异性地运送到肿瘤部位,而不影响正常细胞。HER2是公认的重要乳腺癌分子标志物,HER2靶向的单克隆抗体药物曲妥珠单抗已获得FDA批准为一线治疗药物。曲妥珠单抗联合紫杉醇化疗显著提高了乳腺癌的临床疗效,可紫杉醇的靶向性仍旧不足。虽然抗体药物偶联物(ADC)靶向性强,但连接物的构建技术壁垒高,偶联物的位置和数量不易控制。脂质纳米粒作为新型给药系统,具有良好的生物相容性、低毒性及缓控释作用。曲妥珠单抗作为靶向配体与脂质纳米粒结合,不仅实现了化疗药物的缓释性和靶向性,而且制备工艺相对简单。现有配体偶联方法是将曲妥珠单抗通过化学键结合在纳米粒表面,化学反应易导致抗体变性失活,非特异性连接部位也可能出现在抗原识别区域。如何最大程度的保留抗体的生物活性,成为抗体作为化疗药物载体靶向配基的应用关键。本文先后采用聚乙烯亚胺(PEI)和阳离子磷脂(DODMA)作为基质,与其它材料混用制备阳离子纳米粒,通过静电作用与带负电的曲妥珠单抗相结合。通过对曲妥珠单抗的结构及生物活性进行表征,证明了温和的静电力能够很好的保护抗体的生物活性。通过对纳米粒的基质处方、抗体连接策略及连接配比进行优化,制备高效低毒的靶向纳米粒,并对其体外抗肿瘤效应和肿瘤靶向进行初步研究。具体内容主要包括以下几个部分:1.紫杉醇PEI阳离子纳米粒的制备和评价本章选用四种类型PEI(800-b PEI,2000-b PEI,25k-b PEI和25k-l PEI)与PLGA、Egg PC混用制备PEI阳离子纳米粒,以粒径电位为指标,对PLGA/Egg PC含量比、PEI类型及含量进行优化,最佳的阳离子纳米粒处方为PLGA/Egg PC(60:20),PEI类型为25k-b PEI,PEI含量为5%。阳离子纳米粒的粒径大小为249.9±4.76 nm,电位为35.3±2.3 m V。与游离PEI相比,空白阳离子纳米粒对A549细胞和MCF7细胞无明显毒性,相对细胞活力均在90%以上。稳定性结果表明,静置2周后,阳离子纳米粒在PBS(10 m M,p H 7.4)中粒径大小和PDI均未发生明显变化。细胞毒性及细胞摄取实验中,由25k-b PEI制备的紫杉醇PEI阳离子纳米粒在A549和MCF7细胞中均具有最高的细胞毒性(P0.001),其细胞摄取效率明显高于其它三种PEI制备的纳米粒(P0.01)。2.曲妥珠单抗修饰的紫杉醇PEI阳离子纳米粒的制备和评价本章分别通过静电吸附作用和化学交联键将曲妥珠单抗与紫杉醇PEI阳离子纳米粒相结合。粒径增加、电位下降以及TEM观察到的双层核壳结构均证明了抗体的成功连接。静电吸附法制备的靶向纳米粒粒径大小为280.7±7.8nm,表面电位1.00±0.73 m V。阳离子纳米粒与曲妥珠单抗的最佳连接配比为1:1,BCA法检测此时抗体结合效率为92.7±0.6%。24 h时,曲妥珠单抗在生理条件下的解离百分率为23%。靶向纳米粒体外释放呈双相缓释模式,6 h内突释率为28.6%,120 h内累计释放率为71.2%。细胞毒性试验中,选用HER2过表达的BT474细胞及低表达的MCF7细胞为实验对象。靶向纳米粒的细胞增殖抑制作用呈受体依赖性和时间依赖性。72 h后,静电吸附法和化学交联法制备的靶向纳米粒对BT474细胞的抑制率分别为53.3%和34.9%(P0.001)。采用双荧光模式标记靶向纳米粒:FITC标记曲妥珠单抗,Rhod·B标记阳离子纳米粒。在BT474细胞中,静电吸附法制备的靶向纳米粒的FITC和Rhod·B平均荧光强度均明显高于化学交联法(P0.001)。MCF7细胞中,二者显示了同等水平的荧光强度。在细胞摄取过程中,靶向纳米粒表面的曲妥珠单抗没有发生解离现象。3.曲妥珠单抗修饰的紫杉醇阳离子脂质纳米粒的制备和评价本章采用毒性更低、生物相容性更好的阳离子磷脂DODMA替代阳离子聚合物PEI与PLGA混用制备阳离子脂质纳米粒,随后结合曲妥珠单抗。为了准确控制纳米粒表面抗体数目,采用三种不同抗体连接策略制备靶向脂质纳米粒:(1)将曲妥珠单抗与DSPE-PEG2000-Mal分子连接,再通过静电吸附作用结合到DODMA/PLGA纳米粒表面,即Improved pre-conjugation strategy(Imp);(2)将曲妥珠单抗与DSPE-PEG2000-Mal分子连接,然后与其它基质(DODMA/PLGA)通过物理混合制备纳米粒,即Pre-conjugation strategy(Pre);(3)曲妥珠单抗通过化学交联键结合到DSPE-PEG2000-Mal/DODMA/PLGA纳米粒表面,即Pos-conjugation strategy(Pos)。抗体密度分析结果显示,Imp、Pre和Pos总抗体装载量分别为17.8%,14.5%和4.1%,其中表面抗体含量分别为17.8%,6.12%和4.1%。Pre中,8.38%抗体在制备过程中浪费在纳米粒内部。Pos显示了较低的抗体连接活性。Imp中,对抗体装载量与投药量进行线性拟合,曲线R2为0.995。结果显示通过Imp定量控制纳米粒表面抗体的数目是可行的。细胞毒性试验中,靶向纳米粒的细胞增殖抑制作用呈受体依赖性与剂量依赖性。Imp中,当曲妥珠单抗装载量增加17.8%时,48 h后BT474细胞抑制率提高了7.3%(P0.001)。Imp灵活的给药方式,有利于实现曲妥珠单抗和紫杉醇之间的协同效应。三种策略构建的纳米粒的细胞毒性由高到低顺序依次为ImpPrePos,均高于无抗体包被的纳米粒及游离紫杉醇。在BT474细胞中,Imp内化荧光水平最高,其次为Pre和Pos,分别为30.3,23.6和20.7。靶向纳米粒在MCF7细胞中整体内化荧光水平比BT474细胞中要低。最后考察了细胞内吞抑制剂对靶向纳米粒的内吞途径的影响,结果表明靶向纳米粒主要是通过HER2受体及网格蛋白介导的内吞作用进入肿瘤细胞。目前,国内外关于配体偶联策略的研究报道主要集中在化学修饰法连接靶向配体。然而在化学反应中,抗体的活性极易遭到破坏,从而影响其靶向性。高强度的化学键也会阻碍受体-配体识别过程及后续化疗药物的释放。本文采用静电力将抗体结合到阳离子纳米粒表面,极大的提高了抗体的结构稳定性及生物活性。此外,Imp能够准确控制纳米粒表面抗体数目,在一定范围内具备调控纳米粒的靶向能力。曲妥珠单抗修饰的紫杉醇阳离子脂质纳米粒为靶向载体的研发提供理论依据、方法参考和技术借鉴。
[Abstract]:The incidence of breast cancer increased year by year, has become the biggest threat to women's health killer. Paclitaxel is the first-line chemotherapy of breast cancer treatment, the single and combined application showed good curative effect, but its non selective will cause serious side effects of systemic cancer. Targeted therapy with tumor markers molecular targeting drugs specifically delivered to the tumor site, without affecting the normal cells is.HER2 markers of breast cancer molecular recognized by monoclonal antibody trastuzumab HER2 targeted has been approved by the FDA as first-line therapy. Trastuzumab combined with paclitaxel chemotherapy significantly improved the clinical curative effect of breast cancer, paclitaxel can be targeted is still insufficient. Although antibody drug conjugates (ADC) targeted strong, but the construction technology of connection of high barriers, the position and number of conjugates of lipid nano is not easy to control. Rice as a new drug delivery system, has good biocompatibility, low toxicity and controlled-release effect. Trastuzumab as target binding to the ligand and lipid nanoparticles, not only to achieve sustained release and chemotherapeutic drugs to target, and the preparation process is relatively simple. The existing ligand coupling method is trastuzumab based on the surface of the nanoparticles by chemical bond, the chemical reaction can lead to inactivation of nonspecific antibody, joints may also occur in the area. Keep the antibody antigen recognition to the greatest degree of biological activity, become antibody as chemotherapeutic agents targeting carrier application of key ligands. This paper has by polyethyleneimine (PEI) and cationic phospholipid (DODMA) as the matrix, and other materials used for the preparation of cationic nanoparticles through electrostatic interaction with the negatively charged trastuzumab combination. Through the structure and life of trastuzumab Physical activity was characterized, proved moderate static electricity can be a very good protective antibody bioactivity. Through matrix formulation of nanoparticles, antibody connection strategy and connection ratio is optimized, the preparation of high efficiency and low toxicity targeted nanoparticles and its antitumor effect in vitro and tumor targeting of specific content. It includes the following parts: 1. paclitaxel PEI cationic nanoparticles were prepared and evaluated in this chapter selected four types of PEI (800-b PEI, 2000-b PEI, 25k-b PEI and 25k-l PEI) and PLGA Egg PC, mixed preparation of PEI cationic nanoparticles, with diameter of PLGA/Egg potential as an index, the content of PC, PEI type and the content is optimized, the best prescription for cationic nanoparticles PLGA/Egg PC (60:20), PEI type 25k-b PEI, PEI content of cationic 5%. nanoparticle size was 249.9 + 4.76 nm + 2.3 m V. potential was 35.3 compared with the free PEI Blank, cationic nanoparticles has no obvious toxicity to A549 cells and MCF7 cells, the relative cell viability was above 90%. The stability results showed that after 2 weeks of incubation, cationic nanoparticles in PBS (10 m M, P H 7.4) in size and PDI were not significantly changed. The cytotoxicity and cellular uptake experiments by 25k-b PEI, the preparation of paclitaxel PEI cationic nanoparticles has the highest cytotoxicity in A549 and MCF7 cells (P0.001), the cellular uptake efficiency was higher than that of other three kinds of preparation of PEI nanoparticles (P0.01).2. trastuzumab paclitaxel modified PEI cationic nanoparticles were prepared and evaluated in this chapter respectively by electrostatic adsorption and chemical cross-linking to trastuzumab and paclitaxel combined with PEI cationic nanoparticles. The particle size increases, the potential drop and double core shell structure observed by TEM showed antibody successfully connected electrostatic adsorption. Preparation of targeted nano particle size is 280.7 + 7.8nm, 1 + 0.73 m surface potential V. cationic nanoparticles and trastuzumab optimal connection ratio is 1:1, the BCA method was used to detect the antibody binding efficiency was 92.7 + 0.6%.24 h, trastuzumab under physiological conditions the dissociation rate of 23%. per cent target the in vitro release showed a biphasic release pattern, 6 h burst release rate was 28.6%, 120 h cumulative release rate of 71.2%. cell toxicity test, selection of BT474 cells and low expression of HER2 overexpressing MCF7 cells as the experimental object. Nanoparticles targeting to cell proliferation inhibition in the receptor and time dependent..72 h, electrostatic adsorption and chemical crosslinking prepared by targeting nanoparticles on BT474 cells were 53.3% and 34.9% (P0.001). Using double fluorescence pattern marker targeting nanoparticles: FITC labeled trastuzumab, Rhod labeled B cation Sub nanoparticles. In BT474 cells, electrostatic adsorption of prepared nanoparticles targeting to FITC and Rhod. The mean fluorescence intensity of B was significantly higher than that of chemical crosslinking method (P0.001) in.MCF7 cells, two showed the fluorescence intensity of the same level. In the process of cell uptake, targeting nanoparticle paclitaxel lipid nanoparticles surface cationic surface trastuzumab no dissociation of trastuzumab modified.3. were prepared and evaluated in this chapter with less toxicity, better biocompatibility of the cationic phospholipid substitution of DODMA cationic polymer PEI and PLGA mixture preparation of cationic lipid nanoparticles, then combined with trastuzumab. In order to accurately control the number of nanoparticles surface antibody, using three different antibodies connection strategy for the preparation of targeting lipid nanoparticles: (1) the trastuzumab is connected with the DSPE-PEG2000-Mal molecule, then binds to DODMA/ PLGA nano by electrostatic adsorption The rice surface, (Imp) Improved pre-conjugation strategy; (2) the trastuzumab connected with DSPE-PEG2000-Mal molecules, and other matrix (DODMA/PLGA) nanoparticles prepared by physical mixing system, namely Pre-conjugation strategy (Pre); (3) trastuzumab combined with DSPE-PEG2000-Mal/DODMA/PLGA nanoparticles by chemical cross-linking, Pos-conjugation strategy (Pos). The results showed the antibody density, Imp, Pre and Pos antibodies loading were 17.8%, 14.5% and 4.1%, of which the surface antibody levels were 17.8%, 6.12% and 4.1%.Pre, 8.38% antibody in the preparation process of waste in the internal.Pos shows the nanoparticles showed low antibody activity in.Imp were connected. Linear fitting of the antibody load and the dosage of R2 curve 0.995. results showed that the number of control nanoparticles surface antibody by Imp quantitative is feasible. The cell toxicity test, target The nanoparticles cell proliferation inhibition was receptor dependent and dose-dependent.Imp, when trastuzumab loading increased 17.8%, 48 h after the BT474 cell inhibition rate was increased by 7.3% (P0.001).Imp flexible mode of administration, is conducive to the realization of the synergistic effect of trastuzumab and paclitaxel between cells. The toxicity of nanoparticles construction three strategies from high to low order of ImpPrePos, were higher than those of non antibody coated nanoparticles and free paclitaxel. In BT474 cells, Imp internalization fluorescence level is the highest, followed by Pre and Pos, respectively 30.3,23.6 and 20.7. targeted nanoparticles in MCF7 cells to internalize the overall level of fluorescence low than BT474 cells. Finally we investigated the endocytosis inhibitor on the endocytic pathway targeted nanoparticles. The results show that targeting nanoparticles mainly through HER2 receptor endocytosis and clathrin mediated into the tumor Cells. At present, the domestic and foreign research reports on the ligand coupling strategy mainly focuses on the chemical modification method to connect target ligands. However in a chemical reaction, the activity of antibody easily destroyed, thus affecting its targeting. Chemical bonds of high strength will hinder the process of receptor ligand recognition and subsequent chemotherapy drug release the static electricity will be. The antibodies bind to the cationic nanoparticles surface, which greatly improves the structure stability and biological activity of antibody. In addition, Imp can accurately control the number of nanoparticles surface antibody, in a certain range with the regulation of nanoparticle targeting ability. Paclitaxel cationic lipid nanoparticles modified with trastuzumab as target and provide a theoretical basis for the to support R & D, using the methods of reference and technology.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R943

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