【作者】 刘颖；

【导师】 曹傲能；

【作者基本信息】 上海大学 ， 应用化学， 2014， 硕士

【摘要】 siRNA参与RNA干扰途径，干扰特定基因的表达，是如今基因治疗研究的热点。然而siRNA药物分子由于本身的亲水性、阴离子特性、尺寸原因等导致其难以进入细胞，且siRNA在生理环境中很容易被酶解而失活，因此设计合理的siRNA药物传递系统是一个非常重要的研究课题。虽然这方面的研究已经取得了不错的进展，但还是有其各自的应用局限：如病毒载体基因转染率高，但是具有潜在的致癌性和免疫反应；阳离子脂质体也较为常用但是具有较大的细胞毒性和不稳定性；聚乙烯亚胺（PEI）纳米材料载带效率高，但是其毒性作用也被普遍认同；碳纳米管材料在药物传递方面受到广泛关注，但是其安全性问题也饱受争议。所以我们需要找到一种更加安全、稳定、有效的核酸药物载体。近年来，使用阳离子聚合物壳聚糖作为核酸药物载体，无论在细胞水平还是活体水平实验都取得了不错的效果。壳聚糖是自然界中一种带正电荷的天然碱性多糖，它存在范围广、廉价易得、无毒副作用、生物相容性极好、易于表面修饰、并且可生物降解，具有十分优异的生物活性，在生物医学领域应用十分广泛。但是，目前的技术仅仅是利用共沉淀或者离子凝聚等传统方法制备壳聚糖纳米载体，共沉淀法的最大缺陷就是制备的壳聚糖纳米颗粒在转染介质中稳定性很差，最终沉默效率很低；复凝聚法虽然会大大提高纳米颗粒的稳定性，但是制备的颗粒粒径不均，尺寸较大，有些甚至达到微米级别，分散性和稳定性都不好，容易发生严重的团聚，不能长期存放。本论文设计了一种全新的利用蛋白质来控制壳聚糖纳米颗粒合成的方法，我们使用不同尺寸和带电量的蛋白质作为“核”，经质子化处理的壳聚糖会吸附于表面，交联以后形成“壳”，该方法制备的核壳结构的纳米颗粒可以调控尺寸在最优范围内（约20nm左右），分散性和稳定性良好，而且“非球体”的结构使得纳米颗粒对siRNA有更高的吸附量。激光共聚焦显微镜和流式细胞实验都表明该壳聚糖纳米颗粒可以有效地传递siRNA进细胞。实时荧光定量和蛋白免疫印迹实验结果也都展示了这种纳米颗粒传递siRNA沉默GAPDH基因具有较高的沉默效率，其效果优于目前最为常用的商业化的转染试剂Lipofectamine2000。同时我们还能观察到该载体能将siRNA传递进入细胞核，这样可以实现从细胞核转运至细胞质之前就干扰目标mRNA，因此得到更高的沉默效率。由于蛋白质和壳聚糖都具有良好的生物相容性和生物可降解性，即使浓度高达400μg/mL的纳米颗粒暴露在Hela与Caco-2细胞中也没有表现出任何毒性。这一切都展示其在核酸药物载带及基因治疗领域的良好应用前景。当“核”为荧光蛋白质时，就可以将其开发成生物荧光探针。我们发现壳聚糖的包裹不会影响RFP的蛋白质结构和荧光特性，而且还提高了蛋白质对变性剂、蛋白酶、荧光淬灭剂的稳定性，同时光稳定性、热稳定性以及pH稳定性都有很大程度的提高，所有的这些非常优异的性能都显示出壳聚糖纳米材料包裹荧光蛋白可以作为一种安全稳定的荧光探针。当然，我们还可以将上述应用结合起来：用壳聚糖包裹荧光蛋白质并在其表面吸附siRNA。这样我们就制备得到了一种兼具成像和载药作用的多功能纳米载体，可以实现在药物传递的同时监控其过程，这对于核酸药物传递和荧光探针的研发都具有非常重要的意义。更多还原

【Abstract】 RNA interference (RNAi) is a biological process in which RNA moleculesinhibit gene expression, typically by causing the destruction of specific mRNAmolecules. siRNA involved in RNAi process, inhibits the expression of specific genes,and is a hot issue for gene therapy. However, siRNA delivery remains highlychallenging. This is in part attributable to the nature of siRNA, which is sensitive tonuclease degradation. Moreover, siRNA is hydrophilic and anionic and is unable tocross the cellular membranes. Many previous studies in siRNA delivery achievedmuch progress, but there are limitations in applications. For example, viral vectorshave a high transfection efficiency, but the safety of virus, the high expense and thecomplicated precedure in viral vector preparation hamper its applicability. Thecommonly used cationic liposome carrier has the cell toxicity and instability. PEI andCNTs are also widely used for siRNA delivery, but the safety of them have beenhighly controversial. So it is important for us to explore the carriers with safety, highefficiency, and stability.Chitosan-based nanoparticles (NPs) have been widely described investigated inthe delivery of nucleic acids such as siRNA or DNA because of its cationic nature,low toxicity, biocompatibility, and biodegradability. One of the common methods ofnanoparticles formulation was coprecipitation, but the biggest weakness of NPssynthesed by this method is the poor stability in the transfection medium, and the lowfinal silencing efficiency. By adding a cross-linker agent in the formulation, such assodium tripolyphosphate (TPP), the interactions between siRNA and the polymer arestrengthened. This approach, called ionic gelation, permits higher NPs stability.However, the NPs generally have a large size and uniform size distribution, and causethe poor dispersion and stability.An efficient chitosan-based siRNA delivery nano vector, P@CS, has beensynthesized. Using proteins with different sizes and surface charges as the nucleationcores, the size of P@CS NPs have been tuned into around20nm, which is regardedas the optimal size for siRNA delivery. Originated from the size and charge- distribution of the protein cores, P@CS has a non-spherical shape, which provideshigh binding capacity for siRNA.Confocal fluorescence microscopy and flow cytometry experiments show thatP@CS NPs deliver siRNA into cells efficiently. Real-time PCR and Western-blotexperiments exhibit that P@CS NPs delivered siRNA silences GAPDH gene with anefficacy much higher than the commercial lipofectamine2000. Notably, some of thesiRNA are even delivered into the cell nuclei, where they may intercept the targetmRNA before it translocates from the nuclei to the cytoplasm, thus accounts partly forthe high gene silencing effect. Due to the good biocompatibility and biodegradabilityof both protein templates and chitosan, P@CS NPs are non-toxic to both HeLa andCaco-2cells at a dose up to400μg/mL. The good transfection efficacy, the ability todeliver nucleic acids into cell nuclei and excellent safety demonstrate the promisingpotentials of P@CS NPs for the gene therapy.We choose the RFP as the core to synthesis the PR@CS NPs to explore the newbiological fluorescence probe for imaging. It is found that the chitosan encapsulationdid not affect the fluorescence of RFP, and could significantly improve the stabilitiesagaint denaturant, proteinase, and photobleaching. All of these properties showing itspotential application for bio-imaging.RFP was chosen as the core for tracking the siRNA delivery by fluorescence. Theimproved stability and fluorescence performance demonstrate that PR@CS is a goodprobe to monitor the delivery of siRNA to cells. So we prepared the multi-functionalnanocarriers for siRNA delivery and imaging at the same time. It has the promisingpotentials for the gene therapy and fluorescence imaging.更多还原