非病毒载体在siRNA体内递送中的应用

RNAi是一种由siRNA触发的特异基因沉默机制。自其发现以来,RNAi技术迅速发展为药物靶标确认和功能基因组学研究的新策略,并有望成为一种疾病基因治疗学方法。然而,由于siRNA体内不稳定的理化性质,递送siRNA成为RNAi发展的最大障碍。因此研发能够保护siRNA、促进细胞吸收、正确细胞定位和包内体逃逸,结合具有高生物相容性与减少非特异沉默和毒性反应的体内递送系统是RNAi成功用于临床治疗的关键。本文就体内非病毒载体方式递送siRNA的研究进展进行综述。     

纳米基因载体的研究现状

基因治疗正成为当今医学研究的热门课题,载体问题是制约基因治疗成功的关键.目前,常用的载体包括病毒载体和非病毒载体,后者以其无病毒毒性和免疫原性等优越性而倍受瞩目.随着纳米生物技术的发展和多种纳米材料的涌现,以纳米颗粒为基础的非病毒基因载体倍受研究者推崇.综述了纳米基因载体的研究现状,重点总结阐述了阳离子聚合物、无机纳米颗粒及磁性纳米颗粒载体系统,并探讨了其在应用研究方面的现状和前景.     

纳米载体SWCNT投递Bcl-2 siRNA促进U251细胞凋亡

目的设计和构建特异性siRNA纳米载体SWCNT-siRNA,通过体外实验,研究其对脑胶质瘤的作用。方法设计合成特异性Bcl-2 siRNA,利用超声技术合成SWCNT-siRNA复合物,动态散射粒度分析仪进行粒径分析;体外培养U251胶质瘤细胞,SWCNT-siRNA复合物处理细胞后,激光共聚焦显微镜追踪siRNA进入细胞的分布。PCR检测靶基因Bcl-2沉默情况,Annexin-V FITC与PI联合标记细胞后,流式细胞仪检测细胞凋亡。结果通过粒径分析证明siRNA成功缠绕到SWCNT上形成稳定的复合物siRNA-SWCNT。利用激光共聚焦显微镜对标记红色荧光的siRNA进行追踪,发现siRNA成功投递到细胞内。PCR验证了被投递到胞浆的siRNA很好的沉默目标基因Bcl-2。通过流式细胞仪分析发现,与对照组比较发现其可以抑制肿瘤细胞生长,促进肿瘤细胞早期凋亡。结论 SWCNT作为纳米载体投递Bcl-2 siRNA进入U251胶质瘤细胞,通过降解Bcl-2 mRNA抑制Bcl-2的表达并促进胶质瘤细胞的凋亡。     

病毒纳米颗粒在医学领域的潜在应用

过去,对于病毒的研究主要集中在它的传染性和将其作为工具来加深对细胞生物学的理解,然而随着近年来对于病毒结构的研究深入,病毒在纳米科技领域的应用日益受到人们的关注。通过化学或基因方法修饰过的病毒纳米颗粒能够作为潜在的优秀载体应用到多个方面,例如:药物/基因输送载体、高级疫苗载体以及各种具有特殊功能的无机、有机纳米材料等。本文综述了近来病毒纳米颗粒在医学领域的应用情况。     

纳米基因载体的研究进展

由于使用病毒载体难以避免机体对病毒微粒的免疫反应和由病毒介导的随机整合或野生型病毒重整等潜在危害,因此应用纳米技术进行新型非病毒基因载体的研究发展迅速。本文就纳米微粒应用于基因载体的研究作一综述。     

免疫纳米微粒靶向胰腺癌细胞输送siRNA的方法研究

 目的：构建一种向胰腺癌细胞安全、高效输送siRNA的免疫纳米载体。方法：检测纳米载体IONP-PEI（非靶向组）及其与siRNA复合物的表征;通过琼脂糖凝胶电泳检测siRNA结合力、MTS法检测细胞活力和流式细胞术检测转染率以确定其复合siRNA的最佳N/P比值;细胞免疫荧光和普鲁士蓝染色观察scFvCD44v6偶联IONP-PEI的靶向纳米载体（靶向组）在细胞内分布;流式细胞术、荧光显微镜观察、real-time PCR和Western blotting检测靶向组和非靶向组的转染率和转染siKRAS后的干扰效果。结果：IONP和PEI的最适质量比为0.75;纳米载体复合siRNA的最佳N/P比值为20;IONP-PEI/siRNA复合物的电位为（21.73±8.07）mV,粒径为（51.3±2.2）nm。荧光显微镜显示,非靶向组和靶向组转染后均在细胞内,靶向组的转染率为（89.75±1.81）%,高于非靶向组的（59.87±4.52）%,且靶向组的荧光强度高于非靶向组。靶向组的KRAS mRNA的相对表达量为（34.02±615）%,低于非靶向组的（51.09±6.70）%;Western blotting显示靶向组的KRAS蛋白表达量低于非靶向组。结论：非靶向组和靶向组均能够将siRNA转染进细胞内,且靶向组具有更高的转染效率和更好的干扰基因表达效果。本课题构建的scFvCD44v6-IONP-PEI是一种高效、安全和靶向识别胰腺癌细胞的免疫纳米载体。     

纳米基因载体的研究进展

由于使用病毒载体难以避免机体对病毒微粒的免疫反应和由病毒介导的随机整合或野生型病毒重整等潜在危害，因此应用纳米技术进行新型非病毒基因载体的研究发展迅速。本就纳米微粒应用于基因载体的研究作一综述。     

基于非病毒载体的mRNA疫苗递送系统研究进展

mRNA疫苗是一类新型核酸疫苗。它具有通用性、开发迅速以及制造成本低等优点,目前已被广泛用于传染性疾病防治和癌症治疗的研究。但mRNA在体内的不稳定性和低表达限制其在临床的应用,因此开发出能高效转染mRNA的递送载体十分关键。目前的基因递送载体分为病毒载体和非病毒载体。本文对现有递送mRNA的非病毒载体进行综述,并对mRNA疫苗及其非病毒载体的研究前景进行展望。     

叶酸修饰的聚乙烯亚胺聚合物载PD-L1 siRNA体外靶向胃癌细胞的研究

目的:构建高效的siRNA纳米载体靶向SGC-7901胃癌细胞,并下调胃癌表达的程序性死亡配体1(PD-L1)。方法:检测叶酸(FA)-PEG-SS-PEI-SPION纳米载体与siRNA复合后的粒径、电位等表征;体外实验检验siRNA的结合能力、复合物细胞毒性、细胞摄入能力及转染效率;磁共振(MR)成像检测示踪能力;检验胃癌细胞PD-L1下调效应及共培养T细胞的细胞因子水平。结果:N/P比值为10时,FA-PEG-SS-PEI-SPION完全复合siRNA,形成电位为(9.14±0.80)m V、粒径为(116.7±2.5)nm的多聚复合物。靶向组的转染率为(95.06±0.44)%,与非靶向组的(93.87±1.05)%相当;平均荧光强度为1 892.67±81.51,高于非靶向组的1 324.33±186.58(P0.05)。普鲁士蓝染色和激光共聚焦显微镜成像证实了复合物的细胞摄入。体外MR成像验证了聚合物的MR造影成像能力。靶向组PD-L1的mRNA最低相对表达量为9.07%±0.79%,Western blot显示PD-L1的表达显著降低。共培养实验显示IFN-γ和TNF-α的分泌水平增加,IL-10的分泌水平降低(P0.05)。结论:本研究构建了FA-PEG-SS-PEI-SPION纳米载体,并证明了其体外靶向细胞及载siRNA下调PD-L1表达的能力和MR示踪的能力,是一种高效和安全的靶向治疗纳米载体。     

聚乙烯亚胺纳米粒递送siRNA的抗肿瘤生长作用

聚乙烯亚胺(polyethylenimine,PEI)是一种阳离子聚合物,能够结合并压缩siRNA,形成稳定的PEI/siRNA纳米复合物。PEI纳米复合物通过静电作用与细胞膜结合,以胞吞方式进入细胞内。在质子化作用下纳米复合物逃离溶酶体,使携带的siRNA免受溶酶体酶降解,从而靶向作用于目的mRNA。对PEI纳米粒进行表面修饰后,可显著降低其细胞毒性和提高siRNA递送效率。因此,这种PEI纳米粒递送siRNA先进技术的有效利用对于肿瘤治疗有着广阔的应用前景。     

非病毒型载体介导基因转染

基因载体是制约基因转移技术发展的关键。近年来,非病毒载体由于其安全、低毒、低免疫原性等特点而备受青睐。文章以脂质体和聚乙烯亚胺为代表,介绍了非病毒载体的性质、介导转染的机制。随着人们对细胞转染机制了解的深入以及生物材料科学的迅速发展,非病毒型载体将有望实现高效、低毒、靶向特异等特点,从而成为基因治疗中的理想载体。     

Supramolecular assemblies in functional siRNA delivery: where do we stand?

The discovery of RNA interference (RNAi) has excited the scientific field due to its potential for wide range of therapeutic applications. The pharmacological mediator of RNAi, short interfering RNA (siRNA), however, has faced significant obstacles in reaching its target site and effectively exerting its silencing activity. Effective pharmacological use of siRNA requires ‘carriers’ that can deliver the siRNA to its intended site of action. The carriers assemble the siRNA into supramolecular complexes that display functional properties during the delivery process. This review will summarize non-viral approaches to siRNA delivery, emphasizing the current obstacles to delivery and the mechanisms employed to overcome these obstacles. The carriers successfully pursued in pre-clinical (animal) models will be presented so as to provide a glimpse of possible candidates for clinical testing. Supramolecular assembly of nucleic acids with carriers will be probed from thermodynamics and computational perspectives to understand supramolecular structures and their dynamics. The delivery and trafficking requirements for siRNA are then dissected and engineering approaches to overcoming these barriers will be articulated. The latter has been attempted both at the cellular levels, focusing on intracellular barriers, as well as systemic level, emphasizing macroscopic challenges affecting siRNA delivery. Clinical experience with non-viral siRNA delivery is summarized, highlighting the nature delivery modes attempted in clinical settings. We conclude with a perspective on the future of siRNA therapeutics, specifically concentrating on the possible impact of non-viral carriers in the field.     

Cyclodextrins in non-viral gene delivery

Cyclodextrins (CDs) are naturally occurring cyclic oligosaccharides. They consist of (α-1,4)-linked glucose units, and possess a basket-shaped topology with an “inner–outer” amphiphilic character. Over the years, substantial efforts have been undertaken to investigate the possible use of CDs in drug delivery and controlled drug release, yet the potential of CDs in gene delivery has received comparatively less discussion in the literature. In this article, we will first discuss the properties of CDs for gene delivery, followed by a synopsis of the use of CDs in development and modification of non-viral gene carriers. Finally, areas that are noteworthy in CD-based gene delivery will be highlighted for future research. Due to the application prospects of CDs, it is anticipated that CDs will continue to emerge as an important tool for vector development, and will play significant roles in facilitating non-viral gene delivery in the forthcoming decades.     

非病毒型基因载体的生物安全性

近年来,基因载体的生物安全性越来越受到人们的关注,本文介绍了近年来非病毒型基因载体生物安全性的研究进展,综述了非病毒型基因载体的毒性、纳米效应、血液相容性以及免疫反应等方面的研究成果.     

Non-viral gene delivery to mesenchymal stem cells: methods, strategies and application in bone tissue engineering and regeneration

Mesenchymal stem cells (MSCs) can be isolated from several tissues in the body, have the ability to self-renewal, show immune suppressive properties and are multipotent, being able to generate various cell types. At present, due to their intrinsic characteristics, MSCs are considered very promising in the area of tissue engineering and regenerative medicine. In this context, genetic modification can be a powerful tool to control the behavior and fate of these cells and be used in the design of new cellular therapies. Viral systems are very effective in the introduction of exogenous genes inside MSCs. However, the risks associated with their use are leading to an increasing search for non-viral approaches to attain the same purpose, even if MSCs have been shown to be more difficult to transfect in this way. In the past few years, progress was made in the development of chemical and physical methods for non-viral gene delivery. Herein, an overview of the application of those methods specifically to MSCs is given and their use in tissue engineering and regenerative medicine therapeutic strategies highlighted using the example of bone tissue. Key issues and future directions in non-viral gene delivery to MSCs are also critically addressed.     

Polysaccharide gene transfection agents

Gene delivery is a promising technique that involves in vitro or in vivo introduction of exogenous genes into cells for experimental and therapeutic purposes. Successful gene delivery depends on the development of effective and safe delivery vectors. Two main delivery systems, viral and non-viral gene carriers, are currently deployed for gene therapy. While most current gene therapy clinical trials are based on viral approaches, non-viral gene medicines have also emerged as potentially safe and effective for the treatment of a wide variety of genetic and acquired diseases. Non-viral technologies consist of plasmid-based expression systems containing a gene associated with the synthetic gene delivery vector. Polysaccharides compile a large family of heterogenic sequences of monomers with various applications and several advantages as gene delivery agents. This chapter, compiles the recent progress in polysaccharide based gene delivery, it also provides an overview and recent developments of polysaccharide employed for in vitro and in vivo delivery of therapeutically important nucleotides, e.g. plasmid DNA and small interfering RNA.     

纳米生物技术基因治疗载体研究进展

基因治疗已在治疗多种人类重大疾病如遗传病、肿瘤等方面显示出良好的应用前景,但也面临着巨大的挑战,其中之一就是需要安全、高效、靶向的载体系统。纳米生物材料,如脂质体、聚丙交酯-乙交酯(PLGA),聚乳酸(PLA)等,由于具有良好的生物安全性、可方便有效地实现基因靶向性及高效表达和缓释,成为制备高效、靶向的基因治疗载体系统的良好介质,日益在基因治疗载体系统中受到广泛重视。本文综述了目前在基因治疗领域中常用的纳米载体的生物学特性,以及它们的最新研究进展。     

A biomimetic lipid library for gene delivery through thiol-yne click chemistry

The delivery of nucleic acids such as plasmid DNA and siRNA into cells is a cornerstone of biological research and is of fundamental importance for medical therapeutics. Although most gene delivery therapeutics in clinical trials are based on viral vectors, safety issues remain a major concern. Non-viral vectors, such as cationic lipids and polymers, offer safer alternatives but their gene delivery efficiencies are usually not high enough for clinical applications. Thus, there is a high demand for more efficient and safe non-viral vectors. Here, we present a facile two-step method based on thiol-yne click chemistry for parallel synthesis of libraries of new biomimetic cationic thioether lipids. A library of novel lipids was synthesized using the developed method and more than 10% of the lipids showed highly efficient transfection in different cell types, surpassing the efficiency of several popular commercial transfection reagents. One of the new lipids showed highly efficient siRNA delivery to multiple cell types and could successfully deliver DNA plasmid to difficult-to-transfect mouse embryonic stem cells (mESC). Analysis of structure-activity relationship revealed that the length of the hydrophobic alkyl groups was a key parameter for efficient cell transfection and was more important for transfection efficiency than the nature of cationic head groups. The correlation of the size and surface charge of liposomes with transfection efficiency is described.     

Structural contributions of blocked or grafted poly(2-dimethylaminoethyl methacrylate) on PEGylated polycaprolactone nanoparticles in siRNA delivery

The multiformity in polymer structure and conformation design provides a great potential in improving the gene silencing efficiency of siRNA by polymer vectors. In order to provide information on the polymer design for siRNA delivery, the structural contributions of blocked or grafted poly(2-dimethylaminoethyl methacrylate) on PEGylated polycaprolactone nanoparticles (NPs) in siRNA delivery were studied. Herein, two kinds of self-assembly nanoparticles (NPs) formed by amphiphilic cationic polymers, methoxy poly(ethylene glycol)-block-polycaprolactone-block-poly(2-dimethylaminoethyl methacrylate) (mPEG-PCL-b-PDMAEMA, PECbD) and methoxy poly(ethylene glycol)-block-(polycaprolactone-graft-poly(2-dimethylaminoethyl methacrylate)) (mPEG-PCL-g-PDMAEMA, PECgD), were used to deliver siRNA for in vitro and in vivo studies. The physiochemical properties including size and zeta potential of PECbD NPs/siRNA and PECgD NPs/siRNA complexes were characterized. In vitro cytotoxicity, cellular uptake and siRNA knockdown efficiency were evaluated in HeLa-Luc cells. The endosome escape and intracellular distribution of PECbD NPs/siRNA and PECgD NPs/siRNA in HeLa-Luc cells were also observed. In vivo polymer mediated siRNA delivery and the complexes distribution in isolated organs were studied using mice and tumor-bearing mice. At the same total degree of polymerization (DP) of DMAEMA, PECgD NPs/siRNA complexes possessed higher zeta potentials than PECbD NPs/siRNA complexes (at the same N/P ratio), which may be the reason that PECgD NPs/siRNA complexes can deliver more siRNA into the cytoplasm and lead to higher in vitro luciferase and lamin A/C silencing efficiency than PECbD NPs/siRNA complexes. The in vivo imaging measurement and histochemical analysis also confirmed that siRNA could be delivered to lungs, livers, pancreas and HeLa-Luc tumors more efficiently by PECgD NPs than PECbD NPs. Meanwhile, the PDMAEMA chains of PECgD could be shortened which provides benefits for clearing. Therefore, PECgD NPs have great potential to be used as efficient non-viral carriers for in vivo siRNA delivery.     

The effect of serum in culture on RNAi efficacy through modulation of polyplexes size

Serum in the culture medium is one crucial factor that compromises RNAi efficiency of non-viral vectors. However, mechanistic roles of serum in siRNA delivery remain unknown. In this work, we took one cationic polymer, pullulan chemically modified by spermine (termed as pullulan-spermine, Ps), as a siRNA carrier model to investigate the effects of serum on key steps in siRNA delivery including formation of Ps and siRNA polyplexes (Ps-siRNA), cellular uptake, lysosomal escape, and cytotoxicity. We demonstrate that low serum concentration (1.25% and 2.5%) in culture medium results in large particles of Ps-siRNA, while high serum concentration (10%–40%) leads to small particles of Ps-siRNA. The larger particles initiated the internalization of siRNA more effectively in comparison to the smaller ones. The engulfed Ps-siRNA particles mainly locate in lysosomes. The large particles exhibited stronger abilities of destabilizing lysosomes than that of the small particles as large Ps-siRNA particles contain more amines and subsequently elicit a stronger proton sponge effect which results in more effective lysosomal escape of siRNA. Despite the lower RNAi efficiency, the small particle of Ps-siRNA in the high serum medium generates much lower cytotoxicity. These findings explain why serum significantly affects RNAi and also propose a strategy for improving RNAi efficiency and safety by modulating serum concentration and enhancing lysosomal destabilization.