基于硅质体的siRNA递送载体的研究

发布时间：2018-01-12 17:34

本文关键词：基于硅质体的siRNA递送载体的研究　出处：《哈尔滨工业大学》2017年博士论文　论文类型：学位论文

【摘要】：RNA干扰(RNA interference,RNAi)是一种重要的基因表达调控机制,自1998年被发现后,极大地促进了生命科学的发展。然而,能够实现RNAi的si RNA容易被核酸酶降解、被血清蛋白吸附和被肾脏排出体外,极大地制约了RNAi技术在各方面的应用,因此,si RNA递送系统的研究将大大促进RNAi技术的发展。以脂质材料为基础的si RNA递送系统具有生物相容性好、毒性低、转染效率高等优点。然而,脂质体容易受外界条件和环境干扰而稳定性较差,其与si RNA结合后容易发生融合形成较大颗粒,会影响血管流动性和细胞摄取敏感性。近年来,有学者合成了一种新型的有机-无机复合脂质,其制备的硅质体具有储存周期长、稳定性好等优势,克服了很多传统脂质体的缺陷,但利用这种复合脂质在si RNA递送方面的应用研究还处于空白状态。此外,si RNA能否从溶酶体成功逃逸是siRNA进入细胞后发挥作用前最大的障碍。有学者发现,一种含有羟基头部的阳离子胆固醇衍生物在用于DNA递送时表现出了较好的溶酶体逃逸效果,但其对si RNA的递送是否也存在这种作用尚不知晓。为验证复合脂质和含羟基头部阳离子脂质在si RNA递送方面的作用,并利用二者的各自优势,研究有效的si RNA递送系统成为了本论文的主要目标。因此,本论文设计合成了一种新型的含羟基头部基团的阳离子脂质,这种阳离子脂质具有与复合脂质相似的结构,其能够与复合脂质掺杂形成阳离子硅质体(cationic cerasomes,CCs),利用这两种脂质制备的载体,既可以发挥阳离子脂质的作用,又可以充分利用复合脂质的各种优势。本论文主要通过薄膜水化法制备了复合脂质和阳离子脂质不同掺杂比例构成的阳离子硅质体。为克服单一属性脂质载体在体内循环时间短的缺陷,同时制备了PEG化的阳离子硅质体(PEGylated cationic cerasomes,PCCs)。通过表征发现,CCs和PCCs在与si RNA结合前后都呈单分散状态,说明载体表面被覆的Si-O-Si网状结构能够有效防止该载体的自聚集和融合。转染效果评价结果表明:CCs在体外转染实验中对荧光素酶表达的抑制效率高达72.80%;在小鼠体内分布的荧光图像显示PCCs表现出了优异的肝脏富集效果,且肝靶向功能研究结果表明PCCs能够有效递送si RNA至肝脏并发挥抑制靶蛋白表达的作用。前面的结果显示,5mol%的PEG添加虽然能有效增加载体靶器官富集,但也明显降低了载体的体外转染效果,阳离子硅质体为球形,添加的PEG链会分别朝向亲水的核内表面和外表面两个方向,因此,不利于确定分布于外表面的PEG量对载体体外转染效果的影响。研究表明,一种用于研究膜蛋白结构的脂双层圆盘状结构的纳米粒子具有比球形结构更快的细胞内吞速率、更好的内涵体逃逸效果和更长的体内循环时间,而且这种结构决定所掺杂的PEG链能够完全暴露在载体表面,因此,非常有利于精确评价PEG脂质掺杂量对转染效果的影响。为充分利用硅质体和新合成的羟基化阳离子脂质的优势,同时,验证纳米盘结构能否用于si RNA递送和改善载体的转染效果,并测试不同PEG掺杂比例对载体转染效果的影响,本论文利用复合脂质和羟基化阳离子脂质经反复冻融法制备了一种具有圆盘状结构的阳离子复合脂质纳米盘(cationic bicellar nanodiscs,NDs)。所制备的具有Si-O-Si网状表面结构的NDs不仅克服了传统脂质制备的纳米盘稳定性差的缺陷,还表现出了较好的体外转染效果、比阳离子硅质体更好的内涵体逃逸效果。虽然这种复合脂质纳米盘并未表现出较阳离子硅质体明显的体内循环时间延长,但结合载体PEG完全暴露表面的结构特点,本论文对不同PEG掺杂比例的样品对载体转染效率、细胞摄取和体内循环时间的综合评估发现,1mol%的PEG掺杂量足以提高载体在体内的循环时间和在靶器官中的富集且对体外转染效果影响小。综上所述,新合成的羟基化阳离子脂质在制备成CCs和NDs后都表现出了较好的内涵体逃逸效果,说明阳离子脂质的羟基化基团可能有助于内涵体逃逸,对后续新型阳离子脂质的合成具有一定的指导意义。本论文成功的利用了硅质体高稳定性的优势,通过制备阳离子硅质体和阳离子复合脂质纳米盘验证了其用于si RNA递送的可行性,为硅质体用于si RNA递送开辟新的发展平台提供了实验基础。
[Abstract]:RNA interference (RNA interference RNAi) is an important mechanism for regulating gene expression, since 1998 after the discovery, which greatly promoted the development of life science. However, to achieve Si RNA RNAi to nuclease degradation by serum protein adsorption and renal excretion, greatly restricts the application of RNAi. Technology in all aspects of the Si RNA delivery system research will greatly promote the development of RNAi technology. Si RNA in lipid based delivery system has good biocompatibility, low toxicity, high transfection efficiency advantages. However, liposome easily affected by external conditions and environment disturbance and poor stability, which combined with Si RNA is prone to fuse into larger particles, affect vascular fluidity and cell uptake sensitivity. In recent years, there is a kind of organic inorganic composite lipid synthesize new scholars, the preparation of the silicon body is provided with a storage cycle Long period, good stability and other advantages, overcome the defects of many traditional liposomes, but the use of this compound in lipid Si RNA delivery application research is still in the blank state. In addition, Si RNA can successfully escape from lysosomes is siRNA into cells play a role before the biggest obstacle. Some scholars found that contains a the head of the hydroxyl cationic cholesterol derivative for DNA delivery showed a good effect but its lysosomal escape, delivery of Si RNA whether the existence of such effects is not known. In order to verify the composite lipid and hydroxyl containing cationic lipid head delivery role in Si RNA, and the use of their respective advantages of the two, Si RNA efficient delivery system has become the main target of this thesis. Therefore, a new type of hydroxyl containing cationic lipid head groups were designed and synthesized in this paper, the cationic lipid and has The structure of complex lipid similar, it is capable of forming a silicon body and composite doped cationic lipid (cationic, cerasomes, CCs) by using the two kinds of preparation of lipid carrier, can play the role of cationic lipid, but also can make full use of the advantages of various compound lipids. This thesis mainly through the thin film hydration preparation of cationic silica complex lipids and cationic lipids with different doping ratio of the body. In order to overcome the defects of single attribute lipid carrier in the short circulation time in vivo, and preparation of cationic PEG siliceous body (PEGylated cationic cerasomes, PCCs). The characterization of CCs and PCCs are found, monodispersed before and after combined with Si RNA, explained Si-O-Si mesh surface coated carrier can effectively prevent the carrier from aggregation and fusion. The transfection efficiency evaluation results showed that CCs of luciferase expression in vitro The inhibition efficiency is up to 72.80%; in vivo fluorescence images showed that PCCs showed a good liver targeting enrichment effect, and the liver function results show that PCCs can effectively deliver Si RNA to the liver and inhibit the expression of target protein function. Previous results showed that 5mol% PEG added although can effectively increase the carrier the target organ enriched, but also significantly reduce the carrier effect in vitro transfection, cationic silica body is spherical, add PEG chain respectively toward the hydrophilic core inner surface and the outer surface of the two directions, therefore, is not conducive to determine the distribution of PEG content on the surface of the carrier in vitro transfection effect. The results show that a with faster than spherical structure endocytosis rate of nanoparticles for lipid bilayer disc structure of membrane protein structure, better effect of endosomal escape and longer circulation time in vivo, and And this kind of structure decided by the doped PEG chain can be exposed on the surface of the carrier, therefore, is very conducive to the effect of PEG doping on the accurate evaluation of lipid transfection efficiency. In order to make full use of silica and the newly synthesized hydroxylation of cationic lipid advantages, at the same time, Si can be used to verify the disk structure and improve RNA delivery vector the transfection efficiency, and test the effect of PEG doping on the transfection effect, this paper uses complex lipids and hydroxylation of cationic lipid by repeated freezing with a disk like structure of cationic lipid nano composite plate by melt method (cationic bicellar Nanodiscs, NDs). The prepared with Si-O-Si mesh surface structure NDs not only overcomes the defects of traditional lipid preparation of nano disk stability is poor, also shows better effect than in vitro transfection, cationic CERASOME better endosomal escape. Fruit. Although this compound did not show lipid nano disk than cationic silica body circulation time was significantly prolonged, but the combination of vector PEG completely exposed surface structural characteristics, samples with different PEG doping ratio of the transfection efficiency, cell uptake and in vivo evaluation of cycle time, the doping amount of PEG 1mol% to improve the carrier circulation times in vivo and in the target organ and the enrichment of in vitro transfection of little effect. In summary, the new synthesis of hydroxyl cationic lipid in the preparation of CCs and NDs showed a good effect that endosomal escape, the hydroxyl groups of the cationic lipids may contribute to the endosomal escape. The following new cationic lipid synthesis has a certain guiding significance. This paper successfully used cerasomes high stability advantages, and the preparation of the cationic silica body The cationic composite lipid nanodisc has verified its feasibility for Si RNA delivery and provides an experimental basis for the development of a new development platform for the use of siliceous body for Si RNA delivery.

【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R943;TB383.1

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