本文關(guān)鍵詞：基于硅質(zhì)體的siRNA遞送載體的研究　出處：《哈爾濱工業(yè)大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 陽(yáng)離子脂質(zhì) 硅質(zhì)體 陽(yáng)離子復(fù)合脂質(zhì)納米盤(pán) 基因載體 si RNA遞送

【摘要】：RNA干擾(RNA interference,RNAi)是一種重要的基因表達(dá)調(diào)控機(jī)制,自1998年被發(fā)現(xiàn)后,極大地促進(jìn)了生命科學(xué)的發(fā)展。然而,能夠?qū)崿F(xiàn)RNAi的si RNA容易被核酸酶降解、被血清蛋白吸附和被腎臟排出體外,極大地制約了RNAi技術(shù)在各方面的應(yīng)用,因此,si RNA遞送系統(tǒng)的研究將大大促進(jìn)RNAi技術(shù)的發(fā)展。以脂質(zhì)材料為基礎(chǔ)的si RNA遞送系統(tǒng)具有生物相容性好、毒性低、轉(zhuǎn)染效率高等優(yōu)點(diǎn)。然而,脂質(zhì)體容易受外界條件和環(huán)境干擾而穩(wěn)定性較差,其與si RNA結(jié)合后容易發(fā)生融合形成較大顆粒,會(huì)影響血管流動(dòng)性和細(xì)胞攝取敏感性。近年來(lái),有學(xué)者合成了一種新型的有機(jī)-無(wú)機(jī)復(fù)合脂質(zhì),其制備的硅質(zhì)體具有儲(chǔ)存周期長(zhǎng)、穩(wěn)定性好等優(yōu)勢(shì),克服了很多傳統(tǒng)脂質(zhì)體的缺陷,但利用這種復(fù)合脂質(zhì)在si RNA遞送方面的應(yīng)用研究還處于空白狀態(tài)。此外,si RNA能否從溶酶體成功逃逸是siRNA進(jìn)入細(xì)胞后發(fā)揮作用前最大的障礙。有學(xué)者發(fā)現(xiàn),一種含有羥基頭部的陽(yáng)離子膽固醇衍生物在用于DNA遞送時(shí)表現(xiàn)出了較好的溶酶體逃逸效果,但其對(duì)si RNA的遞送是否也存在這種作用尚不知曉。為驗(yàn)證復(fù)合脂質(zhì)和含羥基頭部陽(yáng)離子脂質(zhì)在si RNA遞送方面的作用,并利用二者的各自優(yōu)勢(shì),研究有效的si RNA遞送系統(tǒng)成為了本論文的主要目標(biāo)。因此,本論文設(shè)計(jì)合成了一種新型的含羥基頭部基團(tuán)的陽(yáng)離子脂質(zhì),這種陽(yáng)離子脂質(zhì)具有與復(fù)合脂質(zhì)相似的結(jié)構(gòu),其能夠與復(fù)合脂質(zhì)摻雜形成陽(yáng)離子硅質(zhì)體(cationic cerasomes,CCs),利用這兩種脂質(zhì)制備的載體,既可以發(fā)揮陽(yáng)離子脂質(zhì)的作用,又可以充分利用復(fù)合脂質(zhì)的各種優(yōu)勢(shì)。本論文主要通過(guò)薄膜水化法制備了復(fù)合脂質(zhì)和陽(yáng)離子脂質(zhì)不同摻雜比例構(gòu)成的陽(yáng)離子硅質(zhì)體。為克服單一屬性脂質(zhì)載體在體內(nèi)循環(huán)時(shí)間短的缺陷,同時(shí)制備了PEG化的陽(yáng)離子硅質(zhì)體(PEGylated cationic cerasomes,PCCs)。通過(guò)表征發(fā)現(xiàn),CCs和PCCs在與si RNA結(jié)合前后都呈單分散狀態(tài),說(shuō)明載體表面被覆的Si-O-Si網(wǎng)狀結(jié)構(gòu)能夠有效防止該載體的自聚集和融合。轉(zhuǎn)染效果評(píng)價(jià)結(jié)果表明:CCs在體外轉(zhuǎn)染實(shí)驗(yàn)中對(duì)熒光素酶表達(dá)的抑制效率高達(dá)72.80%;在小鼠體內(nèi)分布的熒光圖像顯示PCCs表現(xiàn)出了優(yōu)異的肝臟富集效果,且肝靶向功能研究結(jié)果表明PCCs能夠有效遞送si RNA至肝臟并發(fā)揮抑制靶蛋白表達(dá)的作用。前面的結(jié)果顯示,5mol%的PEG添加雖然能有效增加載體靶器官富集,但也明顯降低了載體的體外轉(zhuǎn)染效果,陽(yáng)離子硅質(zhì)體為球形,添加的PEG鏈會(huì)分別朝向親水的核內(nèi)表面和外表面兩個(gè)方向,因此,不利于確定分布于外表面的PEG量對(duì)載體體外轉(zhuǎn)染效果的影響。研究表明,一種用于研究膜蛋白結(jié)構(gòu)的脂雙層圓盤(pán)狀結(jié)構(gòu)的納米粒子具有比球形結(jié)構(gòu)更快的細(xì)胞內(nèi)吞速率、更好的內(nèi)涵體逃逸效果和更長(zhǎng)的體內(nèi)循環(huán)時(shí)間,而且這種結(jié)構(gòu)決定所摻雜的PEG鏈能夠完全暴露在載體表面,因此,非常有利于精確評(píng)價(jià)PEG脂質(zhì)摻雜量對(duì)轉(zhuǎn)染效果的影響。為充分利用硅質(zhì)體和新合成的羥基化陽(yáng)離子脂質(zhì)的優(yōu)勢(shì),同時(shí),驗(yàn)證納米盤(pán)結(jié)構(gòu)能否用于si RNA遞送和改善載體的轉(zhuǎn)染效果,并測(cè)試不同PEG摻雜比例對(duì)載體轉(zhuǎn)染效果的影響,本論文利用復(fù)合脂質(zhì)和羥基化陽(yáng)離子脂質(zhì)經(jīng)反復(fù)凍融法制備了一種具有圓盤(pán)狀結(jié)構(gòu)的陽(yáng)離子復(fù)合脂質(zhì)納米盤(pán)(cationic bicellar nanodiscs,NDs)。所制備的具有Si-O-Si網(wǎng)狀表面結(jié)構(gòu)的NDs不僅克服了傳統(tǒng)脂質(zhì)制備的納米盤(pán)穩(wěn)定性差的缺陷,還表現(xiàn)出了較好的體外轉(zhuǎn)染效果、比陽(yáng)離子硅質(zhì)體更好的內(nèi)涵體逃逸效果。雖然這種復(fù)合脂質(zhì)納米盤(pán)并未表現(xiàn)出較陽(yáng)離子硅質(zhì)體明顯的體內(nèi)循環(huán)時(shí)間延長(zhǎng),但結(jié)合載體PEG完全暴露表面的結(jié)構(gòu)特點(diǎn),本論文對(duì)不同PEG摻雜比例的樣品對(duì)載體轉(zhuǎn)染效率、細(xì)胞攝取和體內(nèi)循環(huán)時(shí)間的綜合評(píng)估發(fā)現(xiàn),1mol%的PEG摻雜量足以提高載體在體內(nèi)的循環(huán)時(shí)間和在靶器官中的富集且對(duì)體外轉(zhuǎn)染效果影響小。綜上所述,新合成的羥基化陽(yáng)離子脂質(zhì)在制備成CCs和NDs后都表現(xiàn)出了較好的內(nèi)涵體逃逸效果,說(shuō)明陽(yáng)離子脂質(zhì)的羥基化基團(tuán)可能有助于內(nèi)涵體逃逸,對(duì)后續(xù)新型陽(yáng)離子脂質(zhì)的合成具有一定的指導(dǎo)意義。本論文成功的利用了硅質(zhì)體高穩(wěn)定性的優(yōu)勢(shì),通過(guò)制備陽(yáng)離子硅質(zhì)體和陽(yáng)離子復(fù)合脂質(zhì)納米盤(pán)驗(yàn)證了其用于si RNA遞送的可行性,為硅質(zhì)體用于si RNA遞送開(kāi)辟新的發(fā)展平臺(tái)提供了實(shí)驗(yàn)基礎(chǔ)。
[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.

【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R943;TB383.1

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