递送siRNA的EGFR靶向免疫脂质体的制备及其体内外活性研究

发布时间：2018-07-14 21:44

【摘要】：基因治疗是目前治疗遗传性疾病或后天获得性疾病比较理想的治疗手段,尤其是恶性肿瘤的治疗。RNAi(RNA interfering, RNA干扰)作为一种高效的序列特异性基因沉默技术在恶性肿瘤基因治疗领域发展掀起了一股研究热潮,其中,siRNA(small interfering RNA,小干扰RNA)是RNAi路径中的效应分子,它是一种21~23 bp的短片段双链RNA,能够特异性降解同源序列的mRNA,抑制特异肿瘤相关基因的表达,从而达到抑制肿瘤生长、侵袭和转移的目的。然而,如果没有载体的帮助,siRNA无法进入肿瘤细胞内,载体是制约siRNA基因治疗的首要问题,所以,siRNA的递送载体研究是目前肿瘤基因治疗研究的热点问题。阳离子脂质体是目前应用较为广泛的递送siRNA的一种非病毒载体,它具有无毒、可自然降解、无免疫原性、可以大量合成并放大生产等优点,近年来备受研究者的重视。本课题组前期一直致力于递送siRNA的阳离子脂质体的制备和活性研究,前期研究发现,通过对阳离子脂质体进行PEG(聚乙二醇)化、HER2抗体修饰以及冷冻干燥等处理后制备得到一种PEG化免疫冻干脂质体(Lyophilized PEGylated Immunoliposomes, LPIL),这种脂质体在PEG含量为2.5 mol%时,能有效的将siRNA特异性递送至高表达HER2乳腺癌细胞内并沉默相关基因的表达。PEG能显著提高脂质体在血浆中的稳定性,延长药物体内半衰期,当PEG含量大于8 mol%时,粒径为100 nm左右的脂质体表面呈刷状,这种刷状构象能完全覆盖脂质体表面,为脂质体免于网状内皮系统的吞噬提供更全面的保护。然而LPIL的PEG含量较低,体内应用受到限制。并且,LPIL无法引入较高含量的PEG,因为较高含量的PEG会破坏LPIL的物理稳定性,而且会大大降低siRNA的包封率。所以我们亟待发展一种高PEG含量、高siRNA包封率以及靶向性较好的阳离子脂质体。 LPD(liposome-polycation-DNA,脂质体-多聚阳离子-DNA复合物)是一种新型递送siRNA的阳离子脂质体,它的结构是PEG(聚乙二醇)化的包裹鱼精蛋白、siRNA/DNA复合物的阳离子脂质体,与传统阳离子脂质体不同的是,DNA在鱼精蛋白的作用下将siRNA压缩,三者形成一个紧密的带负电的核,与阳离子脂质体混合后通过自组装过程形成稳定的LPD,尤为重要的是,LPD采用后插入PEG的方法在其表面修饰了高含量的PEG,不但保证了siRNA的高包封率,而且有效地增加了脂质体的稳定性,在体内被证明能有效地递送siRNA至肿瘤细胞中。然而,抗体修饰的LPD尚无人系统探讨过其各种纳米表征和体内外活性。本研究正是在前期PEG化免疫阳离子脂质体和LPD的基础之上,通过一系列的处方筛选,首次优化出一种高PEG含量的EGFR靶向免疫脂质体TLPD-FCC,并对其各种纳米表征及其体内外活性进行了较为深入的研究和探讨。首先,我们将DOTAP/Chol阳离子脂质体与鱼精蛋白、小牛胸腺DNA、siRNA混合得到Naked LPD(非PEG化脂质体),然后通过PEG化和引入抗体(Anti-EGFR mAb或Fab’),制备得到EGFR靶向免疫脂质体TLPD,针对抗体对脂质体粒径大小以及zeta电位的影响,对抗体类型、连接方式和投入量进行优化,结果发现抗体类型为Anti-EGFR Fab’,且采用传统连接方式连接的抗体时制备得到的脂质体TLPD-FC,平均粒径在150 nm~160 nm之间,zeta电位在10 mV左右,为后续实验研究奠定了基础。然后,通过SDS-PAGE实验证实抗体确实已经连接到脂质体,同时考察了Naked-LPD、NTLPD(PEG化非靶向脂质体)、TLPD-FC对siRNA结合能力、siRNA的包封率以及体外基因沉默效率。凝胶阻滞实验结果表明Naked-LPD、NTLPD、TLPD-FC对siRNA均具有较强的结合能力,通过超滤离心的方法证实了siRNA包封率高达90%,以上实验证实上述样品对siRNA很强的包裹能力,并且PEG化或者抗体修饰对siRNA包封率影响较小。体外基因沉默效率考查了抗体投入量不同时TLPD-FC(包括TLPD-FCA、TLPD-FCB、TLPD-FCC、TLPD-FCD)在MDA-MB-231细胞中的基因沉默效率,结果表明TLPD-FCC具有最高的基因沉默效率。最后,对TLPD-FCC的相关性质和体内外活性进行了深入的研究和探讨。通过透射电镜观察TLPD-FCC和NTLPD的形态大小发现,两者在形态和大小分布上没有明显区别,表明了抗体连接对脂质体的结构几乎没有影响。琼脂糖凝胶电泳实验证实了siRNA在TLPD-FCC或NTLPD的保护下血清稳定性良好。脂质体的血清稳定性通过动态光散射实验得到结论:与Naked LPD相比,NTLPD或TLPD-FCC在PEG的保护下不易与BSA相互作用,稳定性好。体外基因转染效率和基因沉默效率结果表明与NTLPD相比,TLPD-FCC具有较高的特异性的转染效率和基因沉默活性。随后,通过MDA-MB-231乳腺癌肿瘤模型的建立,免疫荧光标记实验证实了体内肿瘤细胞的EGFR表达水平,体内分布实验也验证了TLPD-FCC通过受体介导内吞机制,随着时间的变化在肿瘤部位高度聚集,达到峰值,且激光共聚焦结果显示,TLPD-FCC显示出比NTLPD更高的肿瘤细胞靶向特异性和结合能力及内吞效率。最终,体内基因沉默效率的考察结果表明TLPD-FCC具有比NTLPD更高的基因沉默效率,具有特异性的基因沉默活性。本研究制备得到的TLPD-FCC能有效地递送siRNA至高表达EGFR的乳腺癌细胞,并具有良好的体内外基因沉默效率,有可能作为一种治疗高表达EGFR乳腺癌的基因载体用于临床。
[Abstract]:Gene therapy is an ideal treatment for the treatment of hereditary diseases or acquired acquired diseases, especially the treatment of.RNAi (RNA interfering, RNA interference), as an efficient sequence specific gene silencing technique in the field of malignant tumor gene therapy, set off a hot research upsurge, of which, siRNA (small in). Terfering RNA, small interference RNA) is an effector in the RNAi path. It is a short segment double chain RNA of 21~23 BP, which can specifically degrade mRNA of the homologous sequence, inhibit the expression of specific tumor related genes, and thus inhibit the growth, invasion and metastasis of tumor. But, siRNA can not enter the tumor fine without the help of the carrier. In the cell, the carrier is the most important problem that restricts the siRNA gene therapy. Therefore, the research on the delivery carrier of siRNA is a hot issue in the research of tumor gene therapy. The cationic liposome is a non virus carrier which is widely used to deliver siRNA. It is non-toxic, can be degraded automatically and has no immunogenicity. It can be synthesized and magnified in large quantities. In recent years, the advantages of production have been paid much attention by researchers.
The research group has been working on the preparation and activity of cationic liposomes delivering siRNA in the early period. The previous study found that a PEG immunized freeze-dried liposome (Lyophilized PEGylated Immunoliposomes, LPIL) was prepared by the treatment of cationic liposomes, such as polyethylene glycol (PEG), HER2 antibody modification and freeze drying. When the content of PEG is 2.5 mol%, this liposome can effectively deliver siRNA specific delivery to HER2 breast cancer cells and silence the expression of related genes..PEG can significantly increase the stability of liposomes in plasma and prolong the half-life of the drug. When the PEG content is more than 8 mol%, the surface of liposomes with a particle size of 100 nm is brushed This brush conformation can completely cover the surface of the liposome and provide more comprehensive protection for the liposomes to avoid the phagocytosis of the reticuloendothelial system. However, the PEG content of LPIL is low and the application in the body is limited. And LPIL can not introduce a higher content of PEG, because the higher content of PEG will destroy the physical stability of LPIL and will greatly reduce the siRNA. Therefore, we urgently need to develop a cationic liposome with high PEG content, high siRNA entrapment efficiency and good targeting.
LPD (liposome-polycation-DNA, liposome polycationic -DNA complex) is a new type of cationic liposome that delivers siRNA. Its structure is PEG (polyethylene glycol) encapsulated protamine, siRNA/DNA complex and cationic liposome. Unlike the traditional cationic liposome, DNA is compressed by the protamine of protamine in the action of protamine. The three formed a tight negative nucleus and formed a stable LPD through the self assembly process after mixing with cationic liposomes. It is particularly important that LPD be inserted into PEG after the insertion of a high content PEG on its surface, which not only ensures the high encapsulation efficiency of siRNA, but also increases the stability of the liposomes, and is proved in the body. SiRNA can be effectively delivered to tumor cells. However, the antibody modified LPD has not yet systematically explored its various nanoscale characterization and in vivo and in vivo activity. This study is based on the early PEG immunization of cationic liposomes and LPD. Through a series of prescription screening, a EGFR targeted immunliposome with high PEG content was first optimized. TLPD-FCC, and its various nano characterization and in vivo and in vitro activities have been studied and discussed in depth.
First, we mixed the DOTAP/Chol cationic liposomes with protamine, calf thymus DNA, and siRNA to get Naked LPD (non PEG liposome), and then prepare the EGFR targeting immunliposome TLPD by PEG and introducing the antibody (Anti-EGFR mAb or Fab '). Against the effect of the antibody on the size of the liposomes and the potential of the liposome, the antagonism class The model, connection mode and input were optimized. The results showed that the antibody type was Anti-EGFR Fab ', and the liposome TLPD-FC was prepared when the antibody was connected by the traditional connection. The average particle size was between 150 nm~160 nm and the zeta potential was about 10 mV, which laid the foundation for the follow-up experimental research.
Then, the SDS-PAGE experiment confirmed that the antibody did connect to the liposomes, and also examined Naked-LPD, NTLPD (PEG non targeting liposome), TLPD-FC to siRNA binding capacity, the encapsulation efficiency of siRNA and the efficiency of gene silencing in vitro. The results of gel block experiment showed that Naked-LPD, NTLPD, TLPD-FC had strong binding ability to siRNA. The method of ultra filtration centrifugation confirmed that the encapsulation efficiency of siRNA was as high as 90%. The above experiments confirmed that the above samples had a strong encapsulation ability of siRNA, and PEG or antibody modification had little effect on the siRNA encapsulation efficiency. In vitro gene silencing efficiency examined the TLPD-FC (including TLPD-FCA, TLPD-FCB, TLPD-FCC, TLPD-FCD) in MDA-MB-231 (including TLPD-FCA, TLPD-FCB, TLPD-FCC, TLPD-FCD) in MDA-MB-231. The efficiency of gene silencing in cells shows that TLPD-FCC has the highest gene silencing efficiency.
Finally, the related properties and in vivo and in vivo activity of TLPD-FCC were deeply studied and discussed. The morphology and size distribution of TLPD-FCC and NTLPD were observed by transmission electron microscopy, and there was no obvious difference in the morphology and size distribution. It showed that the antibody connection had little effect on the structure of liposomes. The agarose gel electrophoresis test confirmed the siR The serum stability of NA was good under the protection of TLPD-FCC or NTLPD. The serum stability of liposomes was concluded by the dynamic light scattering experiment. Compared with Naked LPD, NTLPD or TLPD-FCC was not easy to interact with BSA under the protection of PEG. The results of gene transfection efficiency and gene silence efficiency in vitro showed that TLPD-FCC was compared with NTLPD. High specific transfection efficiency and gene silencing activity. Subsequently, the immunofluorescent labeling experiment confirmed the EGFR expression level of the tumor cells in the body through the establishment of the MDA-MB-231 breast cancer tumor model. In vivo distribution experiments also demonstrated that TLPD-FCC was highly aggregated with the time in the tumor site through the receptor mediated endocytosis. The peak value was reached, and the laser confocal results showed that TLPD-FCC showed the targeting specificity and binding ability and endocytosis efficiency of tumor cells higher than that of NTLPD. Finally, the results of gene silencing efficiency in the body showed that TLPD-FCC had higher gene silencing efficiency than NTLPD and had specific gene silencing activity.
The TLPD-FCC obtained in this study can effectively deliver siRNA to EGFR in breast cancer cells, and has good gene silencing efficiency in vitro and in vivo. It may be used as a gene carrier for the treatment of high expression of EGFR breast cancer.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

【共引文献】