阳离子聚合物基因转染载体的研究进展

安全有效的基因载体是实现基因治疗的必要条件,由于阳离子聚合物易于合成和改性,无免疫原性,可以方便地与DNA形成紧密的超分子复合物,保护DNA免受核酸酶的降解,并促进其进入细胞,从而成为非病毒基因载体中的一个重要类型;但阳离子聚合物基因载体,对细胞具有电荷相关的毒性,转染效率低于病毒载体,这成为限制其进入临床使用的瓶颈.本文从提高阳离子聚合物作为基因载体时的转染效率及降低其毒性方面综述了阳离子聚合物基因载体的研究进展,归纳了改善阳离子聚合物基因载体转染特性的八种方法,预测了阳离子聚合物基因载体的发展前景.     

响应性阳离子聚合物及聚合物纳米胶束基因载体

人类多种疾病都与基因的结构或功能改变密切相关,基因治疗已经成为改善人类健康的新兴医学治疗手段,基因治疗的关键在于构筑高效的基因载体.发展生物可降解、具有高转染效率和低毒性的阳离子聚合物基因载体已经成为当今该领域的主要任务.本文主要介绍了对细胞内外的环境差异和外界刺激具有响应性的阳离子聚合物基因载体的合成方法,实现DNA在细胞内的有效释放;构筑不同拓扑结构的聚合物,运用聚合物的拓扑结构来调节DNA与聚合物组装复合体形貌,实现DNA的可控压缩和提高载体的转染效率;通过两亲性阳离子聚合物的自组装形成稳定的阳离子纳米胶束,增加聚合物表面电荷密度,从而有效增强聚合物和DNA结合能力,实现低正负电荷比条件下的DNA压缩和基因的高效表达.     

纳米阳离子多聚物在基因载体系统的应用

阳离子多聚物能与DNA通过静电吸附作用而自组装成纳米微粒,防止DNA被核酸酶降解.阳离子多聚物由于具备合成简便、储存稳定、基因荷载率高、靶向性强、免疫原性低等优点而被用作基因载体.阳离子多聚物按特性可分为两类：合成型和天然型.经典的人工合成型阳离子多聚物基因载体主要有：多聚乙烯亚胺、多聚左旋赖氨酸和树状大分子等;天然生物型阳离子多聚物基因载体主要有壳聚糖及其衍生物和明胶等.本文详细论述了各种阳离子聚合物用作基因载体的性能特点、自身缺陷、介导基因进入细胞的机理和靶向性策略,并对非病毒基因载体的发展作出展望.     

基于门舒特金反应的酶触发自降解高分子载体及其在基因输送中的应用

用邻位苄基溴与双胺进行门舒特金反应,合成了2种线性的季铵盐阳离子聚合物.其中,含有酚基酯键的阳离子聚合物,一旦进入细胞后,可以在细胞内的酯酶催化下快速水解,使得聚合物自降解断裂为不带电的非季铵盐小分子,从而快速释放DNA,最终达到提高转染效率的目的.通过对复合物纳米颗粒的粒径和电势测定,证明了这2种阳离子聚合物都能够有效地结合DNA形成表面带正电的复合物纳米颗粒.凝胶阻滞电泳实验表明,所合成的阳离子聚合物都能稳定地包裹DNA.而在酯酶条件下,含有酚基酯键的阳离子聚合物可以发生降解,使得纳米复合物释放出DNA.同时,含有酚基酯键的阳离子聚合物由于其独特的可降解性,相比于PEI,降低了细胞毒性.在体外细胞转染实验中,2种阳离子聚合物都有较好的转染效果.其中酯酶响应的载体在高N/P下依然表现出较高的转染效率,说明该阳离子载体能够在细胞内有效降解并释放出DNA.     

含氟高分子基因载体

阳离子高分子被广泛应用为非病毒类基因载体,但这类高分子材料的转染效率与细胞毒性之间通常存在"恶性"关联,即获得高转染效率时往往会伴随严重的细胞毒性.如何制备兼具高效、低毒特点的高分子载体是成功实施基因治疗的关键.含氟高分子是一类具有独特理化性质的高分子,能够在低电荷密度条件下与核酸形成稳定的复合物,从而实现高效、低毒的基因转染.含氟功能基团可帮助阳离子高分子改善复合物稳定性、细胞内吞、内涵体逃逸、胞内核酸释放等多个环节,从而赋予了含氟高分子在基因递送过程中的氟效应.该专论系统地总结了含氟高分子基因载体的研究,介绍了含氟高分子的基因递送性能、作用机理以及在基因治疗、基因编辑中的应用,并对含氟高分子载体的未来发展进行了展望.     

金属配合物作为核酸释放的载体

基因治疗是指利用一种载体将健康的基因载入细胞替换致病的基因.由基因缺陷导致的人类疾病达1200多种,最合理的选择是采用基因替换的方法进行治疗.基因治疗的关键问题是解决"使用何种载体才能安全有效地将治疗基因载入靶细胞".非病毒基因载体主要是一些有机阳离子物种,一直受到极大重视;近年来,磷酸钙、纳米粒子和金属配合物释放核酸的功能也开始受到关注.本文总结了金属配合物作为非病毒基因载体使用的研究进展,希望由此理解配合物释放核酸的优势和不足之处.     

交联型聚乙烯亚胺智能基因载体的制备及PEG化影响

使用胱胺双丙烯酰胺(CBA)对低分子量聚乙烯亚胺(PEI)进行交联反应制备智能降解型聚阳离子基因载体.通过与聚乙二醇(PEG)反应得到不同程度PEG化的聚阳离子载体.利用核磁、黏度测试、粒度仪、zeta电位仪和凝胶电泳对聚阳离子载体及其与DNA的复合物进行了表征.研究表明随着PEG含量的增加,聚阳离子载体/DNA复合物颗粒粒径变小、表面正电荷降低,PEG具有明显的屏蔽作用,但过多的PEG也使载体与DNA复合能力下降.通过MTT细胞毒性测试和荧光素酶质粒转染实验得出,含二硫键的交联型阳离子聚合物在测试范围内显示了非常低的细胞毒性,最佳转染效率是PEI25k的4倍,PEG化后其细胞毒性得到进一步改善,转染效率却明显降低.     

含巯基/二硫键聚合物生物材料

含巯基/二硫键聚合物生物材料具有多种良好的性能,作为药物、基因等的释放载体在生物医学领域具有广泛的应用前景。随着基因工程和组织工程的发展,含巯基/二硫键聚合物生物材料的可生物降解性得到高度重视,而怎样改善其降解性能成为限制其应用的关键因素。由于二硫键在细胞外环境里保持稳定,在细胞溶质的还原环境中容易发生断裂,因此在制备新型基因、药物等释放载体上,二硫键充当了重要的角色,它的引入为聚合物生物材料的生物降解性能的设计与改善提供了一条重要的途径。本综述重点以聚合物水凝胶、聚合物微胶束、囊泡等为例,从巯基/烯的光聚合反应、Michael加成反应、氧化还原反应的角度,介绍了巯基/烯在聚合物中形成二硫键的不同途径的研究进展,并详细论述了基因载体、蛋白质载体、小分子药物载体三种还原敏感型材料的制备、表面修饰和改性的进展情况,进一步强调含巯基/二硫键聚合物生物材料的研究在生物医学领域应用的重要性。     

环糊精超分子聚合物在药物/基因递送载体领域的研究进展

超分子聚合物通常以非共价键作为构筑驱动力,其结构具有动态可逆的特点,在新型响应性聚合物材料中具有突出优势。环糊精可通过主客体识别作用与客体分子如二茂铁、偶氮苯、金刚烷、苯环等形成包合,以此构筑的超分子组装体展现出丰富的自组装-解组装特性、刺激响应性、较低的细胞毒性和较好的生物相容性,有望在药物/基因载体领域得到应用。本文从环糊精超分子聚合物的生物医用出发,着重对近年来环糊精超分子聚合物载体在药物控制释放、基因转染以及药物/基因共递送三方面的研究进展进行了总结和评述,并在此基础上展望了环糊精超分子聚合物的研究方向和发展趋势。     

基于环糊精的药物载体的研究进展

药物载体具有控制药物释放速度,靶向输送药物,减少药物降解及降低毒副作用等特点,对它的研究越来越受到重视。近年来,各种药物载体不断出现,其中由环糊精构筑的超分子体系被国内外研究者广泛关注,本文从基于环糊精的轮烷/聚轮烷、聚合物、纳米体系、脂质体以及水凝胶等方面对近几年来环糊精在药物载体中的应用研究进行综述,为丰富药物载体系统提供了新的依据,并对其应用前景作了展望。     

Amphiphilic Block Copolymer Micelles for Gene Delivery

Gene therapy is a promising method to treat acquired and inherited diseases by introducing exogenous genes into specific recipient cells. Polymeric micelles with different nanoscopic morphologies and properties hold great promise for gene delivery system. Conventional cationic polymers, poly(ethyleneimine)(PEI), poly(L-lysine)(PLL), poly(2-dimethyla-minoethyl methacrylate)(PDMAEMA) and novel cationic polymers poly(2-oxazoline)s(POxs), have been incorporated into block copolymers and decorated with targeting moieties to enhance transfection efficiency. In order to minimize cytotoxicity, nonionic block copolymer micelles are utilized to load gene through hydrophilic and hydrophobic interactions or covalent conjugations, recently. From our perspective, properties(shape, size, and mechanical stiffness, etc.) of block copolymer micelles may significantly affect cytotoxicity, transfection efficiency, circulation time, and load capacity of gene vectors in vivo and in vitro. This review briefly sums up recent efforts in cationic and nonionic amphiphilic polymeric micelles for gene delivery.     

Recent progress in cationic polymeric gene carriers for cancer therapy

In recent years,various carriers for gene delivery nave been developed for biomedical applications.Among all kinds of gene carriers,cationic polymeric carriers for delivery therapeutic gene as non-viral carriers have received growing interests due to their improved high transfection efficiency with the relative safety.In particular,the advancement of novel polymeric gene carriers has gained much progress in the development of effective anticancer therapy.Herein,this review focused on the development of cationic polymeric carriers for cancer therapy,including polyethylenimine(PEI),polyamidoamine(PAMAM) dendrimers,polylysine(PLL),chitosan and modified cationic polymers.And recent progresses in the development of novel polymeric carriers for gene delivery,such as targeted gene carriers,responsive gene carriers and multifunctional gene carriers,were summarized.Finally,the future perspectives in the development of novel polymeric carriers for delivery gene were presented.     

High DNA‐Binding Affinity and Gene‐Transfection Efficacy of Bioreducible Cationic Nanomicelles with a Fluorinated Core

During the last two decades, cationic polymers have become one of the most promising synthetic vectors for gene transfection. However, the weak interactions formed between DNA and cationic polymers result in low transfection efficacy. Furthermore, the polyplexes formed between cationic polymers and DNA generally exhibit poor stability and toxicity because of the large excess of cationic polymer typically required for complete DNA condensation. Herein, we report the preparation of a novel class of bioreducible cationic nanomicelles by the use of disulfide bonds to connect the cationic shell to the fluorocarbon core. These bioreducible nanomicelles form strong interactions with DNA and completely condense DNA at an N/P ratio of 1. The resulting nanomicelle/DNA polyplexes exhibited high biocompatibility and performed very effectively as a gene‐delivery system.     

Polymers for DNA delivery

Nucleic acid delivery has many applications in basic science, biotechnology, agriculture, and medicine. One of the main applications is DNA or RNA delivery for gene therapy purposes. Gene therapy, an approach for treatment or prevention of diseases associated with defective gene expression, involves the insertion of a therapeutic gene into cells, followed by expression and production of the required proteins. This approach enables replacement of damaged genes or expression inhibition of undesired genes. Following two decades of research, there are two major methods for delivery of genes. The first method, considered the dominant approach, utilizes viral vectors and is generally an efficient tool of transfection. Attempts, however, to resolve drawbacks related with viral vectors (e.g., high risk of mutagenicity, immunogenicity, low production yield, limited gene size, etc.), led to the development of an alternative method, which makes use of non-viral vectors. This review describes non-viral gene delivery vectors, termed "self-assembled" systems, and are based on cationic molecules, which form spontaneous complexes with negatively charged nucleic acids. It introduces the most important cationic polymers used for gene delivery. A transition from in vitro to in vivo gene delivery is also presented, with an emphasis on the obstacles to achieve successful transfection in vivo.     

A reactive oxygen species-responsive dendrimer with low cytotoxicity for efficient and targeted gene delivery

Nonviral vectors have been attracting more attention for several advantages in gene delivery and the development of nonviral gene ca rriers with high delivery efficiency and low cytotoxicity has long been a key project.Starburst polyamidoamine dendrimers are a class of synthetic polymers with unique structural and physical characteristics.However,when they are used as gene carrier,the gene transfection efficiency is not satisfactory.Herein,a novel thioketal-core polyamidoamine dendrimer(i.e.,ROS-PAMAM)was synthesized and characterized.Compared to ethylenediamine-core dendrimers or widely used cationic polymers of polyetherimide,ROS-PAMAM showed lower cytotoxicity.Moreover,ROS-PAMAM demonstrated reactive oxygen species responsive characteristics,which can facilitate the release of siRNA in the tumor microenvironment.In vitro gene transfection experiments based on A549 cells confirmed that siRNA/ROS-PAMAM exhibits high gene transfection efficiency.It is concluded that ROS-PAMAM shows great potential as a generalizable vehicle for gene therapy applications.     

Self‐Assembled Fluorodendrimers Combine the Features of Lipid and Polymeric Vectors in Gene Delivery

An ideal vector in gene therapy should exhibit high serum stability, excellent biocompatibility, a desired transfection efficacy and permeability into targeted tissues. Here, we describe a class of low‐molecular‐weight fluorodendrimers for efficient gene delivery. These materials self‐assemble into uniform nanospheres and allow for efficient transfection at low charge ratios and very low DNA doses with minimal cytotoxicity. Our results demonstrate that these vectors combine the features of synthetic gene vectors such as liposomes and cationic polymers and present promising potential for clinical gene therapy.     

高聚物基因载体的功能化研究

寻找安全、高效的基因载体是基因治疗的关键问题之一，聚合物基因载体具有低毒、低成本、可设计性强等优良的性质，是未来基因载体发展的方向。传统的高聚物作为基因载体时，功能单一，不能完全满足基因治疗的需要，因此对载体进行功能化修饰是十分必要的。本文在分析基因治疗过程的基础上，详述了基因载体应具有的四种基本功能——在细胞外保持稳定的功能、进入细胞的功能、逃离溶酶体的功能、进入细胞核的功能，并详细介绍了有关基因载体功能化修饰的方法和目前国际上的进展情况，最后本文总结了高聚物载体目前存在的主要问题及未来的发展方向。     

Synergistic effects in gene delivery-a structure-activity approach to the optimisation of hybrid dendritic-lipidic transfection agents

Novel gene delivery agents based on combining cholesterol units with spermine-functionalised dendrons exhibit enhanced transfection ability-we report significant synergistic effects in mixed (hybrid) systems which combine aspects of both main classes of synthetic vectors, i.e., cationic polymers and lipids.