【作者】 刘薇；

【导师】 陈建明；

【作者基本信息】 第二军医大学 ， 药剂学， 2011， 硕士

【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。更多还原

【Abstract】 Gene therapy is recently considered as an effective way in treating both inherited and acquired diseases, especially malignant tumors. RNA interfering (RNAi), an efficient sequence specific gene silencing technology, has sparked an explosion of research in tumor gene therapy field. siRNA (small interfering RNA) is the effective molecule in the RNAi pathways, which is 21~23 bp nucleotide, double-stranded. It can specifically degrade complementary mRNA, thus inhibiting tumor associated gene expression, tumor progression, invasion and metastasis.However, siRNA cannot enter the tumor cells without the help of carriers and carrier is the main problem that hampers the development of gene therapy. So the investigation of the siRNA carrier is the hottest focus of tumor gene therapy. Cationic liposomes have been widely used as the non-viral carriers for siRNA delivery. They are non-toxic, naturally degradable, low-immunogenic and easy-to-produce generously. Recently, they attracted the serious attention of researcher.Previously our group is always going in for the research of preparation and characteristic of cationic liposome for siRNA delivery. We developed Lyophilized PEGylated Immunoliposomes (LPIL), according to PEGlyation, HER2 antibody modification and lyophilization. LPIL containing 2.5% PEG (2.5% PEG LPIL) can specifically delivering siRNA to HER2-overexpressing cancers and slience specific gene expression. As is reported, PEG can significantly enhance the stability of liposomes in plasma and prolong the in vivo half-life period. PEG is arranged in the brush mode with >8 mol% PEGylation for a 100 nm liposomal particle. The brush mode is the ideal configurations that ensures complete coverage of the surfaces of the nanoparticles providing full protection and have an excellent performance in vivo. However, the PEG content of LPIL was low, thus its in vivo application was hindered. Besides, LPIL cannot bear hight content of PEG owing to protect the physical stability of liposomes, and high content of PEG can not only destroy the stability of LPIL and also decrease the siRNA encapsulation efficiency. As a result, we should develop the novel cationic liposomes which possess hight content of PEG, siRNA encapsulation efficiency and good targeting. LPD (liposome-polycation-DNA) is a novel cationic liposomes for siRNA delivery. They composed of nucleic acids, a polycationic peptide and cationic liposome, and were prepared in a self-assembling process. This structure is different from the common liquid phase structure of liposomes, and is a negative core consisting of polycation, siRNA and DNA. More importantly, a post-insertion PEGlyation method was adopted, which provides a high siRNA encapsulation efficiency, liposome stability and was demonstrated to efficiently delivery siRNA to tumor cells. However, nobody investigate the characteristic and in vitro and in vivo activity of antibody-modified LPD. In this study, we finally obtained a final product TLPD-FCC which possesses high potent of PEG and antibody targeting, and investigated the siRNA encapsulation and in vitro and in vivo activity.Firstly, we prepared DOTAP/Chol (1:1 M ratio) cationic liposome, then mixed with protamine, calf thymus DNA, and siRNA in a self-assembling process to form naked LPD. NTLPD or TLPD was finally obtained from naked LPD by PEGlation, antibody modification, respectively. We investigated the effect of antibody type, conjugation strategy and amount on the essential physicochemical properties (such as size, charge, et al.) of liposomes. As a result, TLPD-FC (TLPD conjugated anti-EGFR Fab’with convetional strategy) which had a particle size between 150 and 160 nm, and the average zeta potential was near 10 mV, was used in the subsequent experiments.Secondly, we evaluated the presentation and integrity of Anti-EGFR Fab’on the surface of TLPD-FC by SDS-PAGE. Meanwhile, the siRNA binding af?nity of liposomes, siRNA encapsulation efficiency (EE) and the in vitro gene silencing were examined. Gel retardation assay result showed that naked LPD, NTLPD and TLPD-FC all had powerful binding af?nity to siRNA. The siRNA EE of liposomes was precisely calculated by subtracting unencapsulated siRNA from total siRNA. Consistent with results obtained in the gel retardation assay, the siRNA EE of all liposomes was very high(> 90%), indicating that naked LPD, NTLPD and TLPD-FC have potent encapsulation capacity to siRNA, and PEGylation and antibody conjugation have little adverse impact on siRNA EE for NTLPD and TLPD-FC. Accompanying with increased amount of conjugated antibody, the luciferase gene silencing activity of TLPD-FC in MDA-MB-231 cells gradually increased, TLPD-FCC possessed the best gene silencing activity among TLPD-FC (TLPD-FCA, TLPD-FCB, TLPD-FCC and TLPD-FCD) and was used in the subsequent experiments. Finally, we investigated the characteristic and in vitro and in vivo activity of TLPD-FCC. The size and morphology of liposomes were observed by TEM, which showed that there is no difference between NTLPD and TLPD-FCC in shape and size distribution, suggesting that antibody conjugation have little impact on the structure of liposomes. siRNA serum stability was detected by agarose gel electrophoresis, the result showed that siRNA in TLPD-FC and NTLPD was protected well in aqueous solution of 50% serum. The dynamic light scattering (DLS) showed the strong interaction exsited between naked LPD and bovine serum albumin (BSA), while NTLPD or TLPD-FC had little interaction with BSA and thus had a good stability. In vitro experiment, transfection efficiency and gene silencing were studied. TLPD-FCC possessed signi?cantly increased transfection ef?ciency and gene silencing activity compared with NTLPD. Subsequently, we successfully established EGFR-overexpressing tumor xenograft model, Immunofluorescence staining had been taken to show the high EGFR expression in MDA-MB-231 tumor tissues. In the IVIS imaging system, TLPD-FCC showed a much better accumulation and distribution in the tumor compared with NTLPD. Furthermore, in vivo uptake study showed that TLPD-FCC accumulated profusely throughout the tumors tissues in a pattern consistent with receptor-mediated endocytosis. However, NTLPD showed only minimal binding or uptake. Consistent with the in vitro gene silencing assay, TLPD-FCC showed significantly higher gene silencing activity in vivo than NTLPD.In this study, we obtained TLPD-FCC which could effectively deliver siRNA to EGFR-overexpressing cancer cells. Furthermore, TLPD-FCC showed a significantly enhanced EGFR targeting efficiency and gene silencing activity both in vitro and in vivo, which has the potential possibility to cure breast cancer in clinic.更多还原