H6R6短肽修饰的壳聚糖纳米粒用于siRNA的递送及其在肿瘤治疗中的应用

发布时间：2018-01-14 12:22

本文关键词：H6R6短肽修饰的壳聚糖纳米粒用于siRNA的递送及其在肿瘤治疗中的应用　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：近年来,基因疗法已成为癌症治疗的重要手段之一,其中,小干扰RNA(siRNA)药物已受到医药领域广泛关注。但是,siRNA药物在体内稳定性差、易被核酸酶降解,半衰期短,这些缺陷限制了 siRNA的临床应用。因此,发展能够高效递送siRNA药物的载体已成为本领域急需解决的难题。本课题构建了能够高效递送siRNA的非病毒载体,并应用筛选出的载体加载siRNA制备纳米粒,进行体内外抗肿瘤的研究。首先,将6-聚精氨酸-6-聚组氨酸(H6R6)短肽偶联到壳聚糖高分子链游离氨基末端,制备H6R6-CS共聚物作为siRNA递送载体,并利用1H-NMR分析方法对其结构进行鉴定。然后选用不同类型的细胞,通过MTT方法对H6R6-CS共聚物进行细胞毒性评价,研究结果表明,合成的H6R6-CS共聚物安全无毒。采用酸碱滴定法考察H6R6-CS共聚物的缓冲能力,发现其在pH3.5-6.5之间存在明显缓冲能力。以复凝聚法制备载siRNA的纳米粒,并对其表面形态、粒径、Zeta电位、稳定性等理化性质进行考察,结果显示所制备的纳米粒的平均粒径在175nm左右,Zeta电位在+14.8mV左右、粒径分布范围较窄,纳米粒呈圆球状,且表面光滑。选择能够稳定表达Luciferase基因的4T1细胞,考察H6R6短肽修饰后的纳米粒对内源基因的表达的抑制情况,研究结果表明,其抑制率可达50%。同时利用流式细胞术和激光共聚焦显微镜法考察纳米粒的入胞情况,利用激光共聚焦法考察了纳米粒的溶酶体逃逸能力,研究结果表明,H6R6短肽修饰后的纳米粒被肿瘤细胞的摄取率显著增强,并在转染6h后,能迅速逃离溶酶体。选择靶向Survivin和BRAF基因的功能性siRNA制备纳米粒,研究纳米粒对4T1和A375两种肿瘤细胞的体外杀伤效果。研究结果表明:转染48h后,两种纳米粒抑制细胞增殖的效率达到最大,分别为49%和51%。细胞凋亡实验结果表明:修饰后的纳米粒能够显著诱导肿瘤细胞凋亡。选择靶向Survivin和BRAF基因的功能性siRNA制备纳米粒,研究纳米粒对乳腺癌和黑色素瘤的抑制效果。研究结果显示:H6R6-CS/siRNASur纳米粒能够显著抑制荷瘤小鼠肿瘤的增长和转移,并能有效延长荷瘤小鼠的生存周期。此外,选择靶向BRAF基因的功能性siRNA制备纳米粒,对荷瘤裸鼠进行瘤内注射观察其对黑色素瘤的抑制情况,研究结果显示:H6R6-CS/siRNABRAF纳米粒对黑色素瘤同样具有显著抑制作用。
[Abstract]:In recent years, gene therapy has become one of the important means of cancer treatment, among which, small interference RNA-siRNAs have been widely concerned in the field of medicine. SiRNA drugs have poor stability in vivo, are easily degraded by nuclease, and have a short half-life. These defects limit the clinical application of siRNA. The development of efficient delivery of siRNA drug vectors has become an urgent problem in this field. In this paper, a non-viral vector which can efficiently deliver siRNA has been constructed. The nanoparticles were prepared by loading siRNA with the selected carriers, and the anti-tumor effect was studied in vitro and in vivo. First of all. The short peptide of 6-arginine-6-polyhistidine (H6R6) was coupled to the free amino terminal of chitosan polymer chain to prepare H6R6-CS copolymers as siRNA delivery carrier. The structure of H6R6-CS copolymers was identified by 1H-NMR, and the cytotoxicity of H6R6-CS copolymers was evaluated by MTT method. The synthesized H6R6-CS copolymer was safe and non-toxic. The buffer capacity of H6R6-CS copolymer was investigated by acid-base titration. It was found that there was an obvious buffer between pH3.5-6.5 and siRNA nanoparticles were prepared by complex coacervation, and the surface morphology, particle size and Zeta potential of the nanoparticles were investigated. The results showed that the average particle size was about 175 nm and Zeta potential was about 14.8 MV, and the particle size distribution was narrow. The nanoparticles were spherical and smooth. 4T1 cells which could stably express Luciferase gene were selected to investigate the inhibition of endogenous gene expression by H6R6 short peptide modified nanoparticles. The results showed that the inhibition rate could reach 50%. At the same time, flow cytometry and laser confocal microscopy were used to investigate the entry of nanoparticles, and the ability of lysosome escape of nanoparticles was investigated by laser confocal method. The results showed that the uptake rate of H6R6 short peptide modified nanoparticles was significantly increased by tumor cells and was transfected for 6 h. Survivin and BRAF gene targeting functional siRNA were selected to prepare nanoparticles. To study the killing effect of nanoparticles on 4T1 and A375 tumor cells in vitro. The results showed that after 48 hours of transfection, the efficiency of inhibition of proliferation of 4T1 and A375 tumor cells was the highest. The apoptotic results showed that:. The modified nanoparticles could significantly induce apoptosis of tumor cells. The functional siRNA targeting Survivin and BRAF genes was selected to prepare the nanoparticles. To study the inhibitory effect of the nanoparticles on breast cancer and melanoma, the results showed that: H6R6-CSR / siRNASur nanoparticles could significantly inhibit tumor growth and metastasis in tumor-bearing mice. In addition, the functional siRNA targeting the BRAF gene was selected to prepare the nanoparticles, and the tumor bearing nude mice were injected intratumoral to observe the inhibition of melanoma. The results showed that BRAF nanoparticles had significant inhibitory effect on melanoma.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R943;R96

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