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.     

ATRP法制备聚(甲基丙烯酸氨乙酯-co-甲基丙烯酸N,N-二乙基氨乙基酯)基因载体

为得到低毒、高效的聚阳离子基因载体,以甲基丙烯酸氨乙酯(AMA)和甲基丙烯酸N,N-二乙基氨乙基酯(DEAEMA)为单体,以2-溴代异丁酸乙酯(EBIB)为引发剂,通过原子转移自由基聚合(ATRP)制备了两种聚(甲基丙烯酸氨乙酯-co-甲基丙烯酸N,N-二乙基氨乙基酯)阳离子无规共聚物(P(AMA-co-DEAEMA),简称P).琼脂糖凝胶电泳实验结果表明聚合物P作为阳离子载体可以有效地络合DNA,通过粒径仪测定的复合物粒子的尺寸在400 ～ 600 nm之间.扫描电镜观察的P/DNA复合物形貌是分散均匀的球形颗粒.以25kDa PEI为阳性参照,利用MTT比色法考察了聚合物P对HEK293T细胞的毒性.结果表明,聚合物P的细胞毒性低于25 kDa PEI的细胞毒性.以25 kDa PEI和裸质粒DNA作为参照,我们进一步考察了聚合物P与DNA形成的复合物在HEK293T细胞中的转染效率.结果表明P/DNA复合物在HEK293T细胞中的转染效率远远高于裸质粒DNA的转染效率,并且接近于25 kDa PEI/DNA复合物的转染效率.     

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.     

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.     

季铵盐阴离子交换膜研究进展

近年来,阴离子交换膜燃料电池的发展受到了广泛关注。开发具有碱稳定性能优异,电导率高的阴离子交换膜(AEMs)材料成为了研究的热点。AEMs主要由聚合物骨架和阳离子基团组成,这两者是影响膜碱稳定性和电导率的重要因素。本文综述了季铵盐类阴离子交换膜聚合物骨架结构中含有醚氧键和不含醚氧键的烷基季铵盐AEMs、N-螺环季铵盐AEMs和环季铵盐AEMs的碱稳定性、电导率等性能;总结了不同骨架结构季铵盐AEMs碱稳定性的差异;分析了季铵盐的降解机理。同时对于含有季铵盐阳离子交换基团的AEMs的结构设计进行了分析和展望。     

阳离子基因载体的pH敏感遮蔽体系的制备及表征

合成了一种pH敏感的遮蔽体系-谷氨酸苄酯/谷氨酸共聚物(PBLG-co-PGA), 用于对DNA/阳离子基因载体复合物颗粒表面正电荷的遮蔽, 以提高其在体内的稳定性. 研究表明, PBLG-co-PGA (PGA(x), x为PGA占共聚物中摩尔百分数)具有pH敏感性. 并以pH敏感点接近生理pH值的PGA(60)为遮蔽体系进行研究. PGA(60)能够对DNA/PEI(1:1)复合物颗粒表面正电荷进行有效遮蔽. 凝胶阻滞电泳显示, 用PGA(60)对DNA/PEI复合物进行不同比例遮蔽, 没有发生与DNA的链交换作用. MTT细胞毒性测试表明, PGA(60)和三元复合物DNA/PEI/PGA(60) 在测试范围内几乎没有细胞毒性. 荧光素酶转染实验表明, 部分遮蔽后转染效率有所提高; 用PGA(60)对DNA/PEI复合物完全遮蔽为负电后, 由于同细胞表面的电荷排斥作用, 三元复合物不易被细胞内吞, 导致不发生细胞转染. 因其合适的pH响应性, PGA(60)将可能成为一种能随pH值的变化, 实现对聚阳离子基因载体进行电荷遮蔽/智能释放的遮蔽材料.     

刺激响应性非病毒转基因载体

常见的阳离子聚合物能够通过静电作用有效缩合DNA,形成聚电解质复合物（PECs）。这些复合物易于细胞内在化,从内涵体中逃逸,并能保护DNA免受DNA酶的降解。但是,强烈的静电作用也限制了基因进入细胞核之后从复合物中的释放,限制了基因的表达。鉴于此,科学家们设计了一类“智能”高分子载体,这些智能高分子载体能够响应外界微环境温度、pH值和氧化还原环境变化的刺激,其自身大分子构象发生改变,进而促进DNA从复合物中逃逸,提高了转染率。本文介绍了近年来有关聚异丙基丙烯酰胺基温度响应性载体以及光、pH和响应胞内谷胱甘肽（GSH）等氧化还原反应的非病毒转基因载体的研究进展。     

PEI基因载体靶向遮蔽体系的制备和表征

将RGD短肽接枝到聚谷氨酸(PGA)上,制备了一种靶向性的基因载体遮蔽材料PGA-RGD.通过凝胶电泳实验及体外转染实验证明得出RGD的引入增加了载体材料与细胞表面受体的特异性作用,在载体表面正电荷得到遮蔽的同时,转染效率还得到了一定程度的增加.同时,对转染了48h的三元复合物进行MTT细胞毒性测试表明,PGA遮蔽的基因载体体系(PGA/PEI/DNA)和PGA-RGD遮蔽的基因载体体系(PGA-RGD/PEI/DNA)的细胞毒性均低于PEI/DNA复合物体系.本文开发的基因载体改性方法不仅可以对复合物颗粒表面的正电荷进行遮蔽,从而降低复合物体系对非目标组织的非特性异作用;同时引入的RGD靶向短肽还可以提高载体的靶向性,这一改性策略对推动阳离子聚合物基因载体在体内的应用具有重要意义.     

阳离子聚合物修饰纳米Pt颗粒的合成

纳米Pt;季铵阳离子;合成;阳离子聚合物修饰纳米Pt颗粒的合成     

六氯环三磷腈交联寡聚乙烯亚胺的制备及基因载体应用

将六氯环三磷腈与分子量为600的超支化寡聚乙烯亚胺在干燥氯仿中反应,合成了可降解的交联型聚合物.利用1HNMR表征了聚合物的结构,并用GPC测试了聚合物的分子量.研究了其作为非病毒基因载体的性能,聚合物载体与DNA形成的复合物颗粒粒径为150nm,Zeta电位为30～40mV,凝胶阻滞电泳显示聚合物/DNA在质量比为0.4时能够将DNA完全阻滞.体外转染实验结果表明,载体对HeLa细胞的最佳转染效率为PEI-25K的3倍;聚合物浓度为20μg/mL时,细胞存活率仍然大于80%,材料的细胞毒性低,生物相容性好,具有良好的生物医学应用前景.     

Hydrolyzable p(DMAPEMA) polymers for gene delivery

The efficiency of cationic polymers as transfectants is thought to be closely related to their DNA association/dissociation properties. An incomplete polymer-DNA dissociation could explain the relatively low gene expression obtained with p(DMAEMA) polymers. Our approach was to synthesize a p(DMAEMA) analogue, p(DMAPEMA), bearing an hydrolyzable cationic group incorporated into the pendant chain with a view to improving transfection. The complexation of DNA with both polymers was studied by agarose gel electrophoresis, size and zeta potential measurements, as well as the dissociation of the polyplexes, after treatment by an anionic polymer, sodium hydroxide or heat. The transfection efficiencies of the polyplexes were evaluated with 293T and BHK21 cells in comparison with Exgen 500. P(DMAPEMA) polymers were able to complex DNA and to release it in a free intact form after an alkaline treatment or storage at 37 degrees C. Poly(aspartic acid) was unable to dissociate p(DMAPEMA) based polyplexes, in contrast to p(DMAEMA) ones. No transfection was obtained with p(DMAPEMA) with both cell lines. A slow hydrolysis under physiological conditions resulting in the absence of DNA unpacking or endosomal entrapment could explain these results. Better transfection results were obtained with polyplexes which were able to be dissociated by electrostatic interactions rather than ones which required the hydrolysis mechanism to release free DNA into cells. Scheme of hydrolyzable p(DMAPEMA) polymer.     

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.     

Time-resolved fluorescence spectroscopy reveals functional differences of cationic polymer-DNA complexes

Cationic polymers bind DNA and form compacted nanoparticulates (i.e., polyplexes). Polyplexes augment DNA delivery into the cells as a nonviral method of gene therapy. DNA packing and release are the key factors in polyplex-mediated gene delivery, but they are poorly understood due to the lack of physical methods of investigation. We used time-resolved fluorescence spectroscopy to study poly(ethylenimine) (PEI) and poly(L-lysine) (PLL) polyplexes. Analysis of fluorescence lifetimes and time-resolved spectra revealed that DNA exists in several different states in PEI polyplexes and only in one tightly bound state in PLL polyplexes. The observed difference in the nature of the polyplexes may explain why PEI releases DNA more easily than PLL even though both polycations condense DNA effectively. The present method utilizing time-resolved fluorescence spectroscopy gives information on the specific interactions between DNA and the cationic polymers in the polyplexes. This kind of information is very important in the development of biologically effective nonviral systems for DNA delivery.     

Water soluble poly(histamine acrylamide) with superior buffer capacity mediates efficient and nontoxic in vitro gene transfection

Water‐soluble cationic polymers, poly(histamine acrylamide)s (PHAs), with superior buffer capacity at the endosomal pH range were designed, prepared, and investigated for non‐viral gene transfection. PHAs were obtained with molecular weights ranging from 9.2 to 28.7 kDa through controlled radical polymerization of histamine acrylamide (HA). Acid–base titration results displayed that all PHA polymers had a remarkably high buffer capacity of about 70% at pH 5.1–7.2. 12.7–28.7 kDa PHAs were able to effectively condense DNA into nano‐sized (<220 nm) polyplexes with moderate positive surface charges (+13–+19 mV) at N/P ratios ≥10/1. CCK assays indicated that polyplexes of 12.7 and 17.5 kDa PHAs were non‐toxic to COS‐7 cells up to a tested N/P ratio of 20/1. Interestingly, the in vitro transfection using pCMV‐Luc and pEGFP‐C1 plasmid DNA as reporter genes showed that polyplexes of 12.7 kDa PHA formed at an N/P ratio of 20/1 mediated efficient transfection in COS‐7 cells under 10% serum conditions, with transfection efficiencies comparable to that of 25 kDa polyethylenimine control. Their versatile design of structures, controlled synthesis, low cytotoxicity, and high transfection activity render PHA‐based cationic polymers particularly interesting for the development of safe and efficient non‐viral gene delivery systems. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011.     

Nonviral gene delivery using poly‐D/L aspartate‐diethylenetriamine cationic polymers and polyethylene glycol: A two‐step approach

Cationic polymers have received much attention as promising nonviral vectors for gene transfer. However, development of polymers with low cell toxicities and high transfection efficiencies continue to be a significant problem and a major hurdle to their success. Poly‐D /L aspartate‐diethylenetriamine poly(D /L Asp‐DET) polymers were synthesized and evaluated as nonviral gene delivery agents. Poly(D /L Asp‐DET) polymers display endosome buffering capacity. The polymers condense plasmid DNA above N:P ratios of 1 and form polyplex particles of ～50–100 nm, with zeta potentials between neutral and +40 mV. Transmission electron microscopy shows the polyplexes to be uniform in size and shape. Polyplexes maintain the structural integrity of DNA following incubation in nucleases and also show high transfection efficiencies with minimal toxicity in both HCT‐116 and PC‐3 cell culture. However, it is found that these poly(D /L Asp‐DET)/DNA polyplexes immediately aggregate in salt and serum conditions, making them unsuitable for use in vivo. Therefore, the polyplexes were further modified by covalent addition of polyethylene glycol (PEG). Introduction of this second step produces PEG‐polyplexes of uniform size (below 100 nm), with neutral zeta potentials that are also stable in both salt and serum conditions. These results suggest poly(D /L Asp‐DET) cationic polymers as potentially safe and efficient nonviral gene delivery agents. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Hydrogen bonds in polycation improve the gene delivery efficiency in the serum-containing environment

Cationic polymers with high charge density could effectively condense the DNA and achieve gene transfection; however, it often brings non-negligible cytotoxicity. Notably, the high charge density gene vector fails in the serum environment, limiting further application in vivo. In this paper, an efficient and reliable non-viral gene vector of poly (amidoamine) (PAA) was designed by introducing diacryolyl-2,6-diaminopyridine (DADAP) onto the PAA backbone through Michael-addition polymerization, which provides high transfection efficiency in a serum-containing environment. Diacryolyl-2,6-diaminopyridine and cationic parts provided multiple interactions between gene vectors and DNA, including hydrogen bond and electrostatic interactions. The introduction of hydrogen bonding can effectively reduce the charge density of polyplexes without reducing the DNA condensing ability, incorporating the diaminopyridine group and cationic part in PAA chains successfully consolidated cellular uptake, endosome destabilization, and transfection efficiency for the PAA/DNA complexes with low cytotoxicity. The constructed vector with multiple interactions presented 6 times higher transfection efficiency in serum-free and 9 times in serum-containing environment than that of branched polyethyleneimine (PEI 25K) in 293T cells in vitro. Therefore, introducing the hydrogen band to form low charge density polyplexes with high transfection efficiency and low cytotoxicity has a great potential in gene delivery.     

Influence of Alkyl Chains of Modified Polysuccinimide‐Based Polycationic Polymers on Polyplex Formation and Transfection

The development of polymers with low toxicity and efficient gene delivery remains a significant barrier of nonviral gene therapy. Modification and tuning of chemical structures of carriers is an attractive strategy for efficient nucleic acid delivery. Here, polyplexes consisting of plasmid DNA (pDNA) and dodecylated or non‐dodecylated polysuccinimide (PSI)‐based polycations are designed, and their transfection ability into HeLa cells is investigated by green fluorescent protein (GFP) expressing cells quantification. All cationic polymers show lower cytotoxicity than those of branched polyethyleneimine (bPEI). PSI and bPEI‐based polyplexes have comparable physicochemical properties such as size and charge. Interestingly, a strong interaction between dodecylated polycations and pDNA caused by the hydrophobic moiety is observed in dodecylated PSI derivatives. Moreover, the decrease of GFP expression is associated with lower dissociation of pDNA from polyplexes according to the heparin displacement assay. Besides, a hydrophobization of PSI cationic derivatives with dodecyl side chains can modulate the integrity of polyplexes by hydrophobic interactions, increasing the binding between the polymer and the DNA. These results provide useful information for designing polyplexes with lower toxicity and greater stability and transfection performance.     

Surface-mediated delivery of DNA: Cationic polymers take charge

The last several years have seen a significant increase in the number of reports describing the use of cationic polymers to design new materials and nanoscale assemblies that promote the surface-mediated delivery of DNA to cells and tissues. In general, these approaches fall into one of two broad categories: (i) methods based upon the physical adsorption of pre-formed, colloidal aggregates of cationic polymer and DNA (polyplexes) to surfaces, and (ii) methods for the layer-by-layer adsorption of DNA and cationic polymers on surfaces to fabricate multilayered thin films that provide control over the release of DNA into solution or to cells. In this Opinion, we discuss several recent examples of each of these approaches and provide commentary on ways in which the physical and chemical behaviours of cationic polymers have played key roles and present future opportunities to develop new methods for localized and surface-mediated cell transfection in vitro and in vivo.     

聚乙二醇嵌段树枝状聚赖氨酸共聚物的合成及其基因载体研究

采用液相多肽合成法制备得到窄分子量分布、结构可控的生物相容性聚乙二醇嵌段共聚树枝状聚赖氨酸阳离子功能大分子(PEG-b-Dendritic PLL). 运用¹H NMR核磁共振、凝胶电泳以及荧光淬灭滴定手段对所得阳离子两嵌段大分子的化学结构及其与质粒DNA (pDNA)结合作用与复合行为进行了研究. 结果表明聚乙二醇嵌段树枝状聚赖氨酸与pDNA分子可以在缓冲溶液中形成稳定的胶束, pDNA与阳离子树枝赖氨酸嵌段通过静电相互作用形成胶束核, 其水溶性聚乙二醇嵌段形成水溶性胶束壳, 提高了阳离子大分子/pDNA复合胶束的稳定性. 同时发现随着阳离子嵌段树枝状赖氨酸代数的增加, 阳离子两嵌段大分子与pDNA的结合作用增强, 有利于其作为基因转染生物功能载体的应用.     

DNA release dynamics from reducible polyplexes by atomic force microscopy

Controlled intracellular disassembly of polyelectrolyte complexes of polycations and DNA (polyplexes) is a crucial step for the success of nonviral gene delivery. Motivated by our previous observation of different gene delivery performances among multiblock reducible copolypeptide vectors ( Manickam, D. S. ; Oupicky, D. Bioconjugate Chem. 2006, 17, 1395- 1403 ), atomic force microscopy is used to visualize plasmid DNA in various decondensed states from reducible polypeptide polyplexes under simulated physiological reducing conditions. DNA decondensation is triggered by reductive degradation of disulfide-containing cationic polypeptides. Striking differences in DNA release dynamics between polyplexes based on polypeptides of histidine-rich peptide (HRP, CKHHHKHHHKC) and nuclear localization signal (NLS, CGAGPKKKRKVC) peptide are presented. The HRP and NLS polyplexes are similar to each other in their initial morphology with a majority of them containing only one DNA plasmid. Upon reductive degradation by dithiothreitol, DNA is released from NLS abruptly regardless of the initial polyplex morphology, while DNA release from HRP polyplexes displays a gradual decondensation that is dependent on the size of polyplexes. The release rate is higher for larger HRP polyplexes. The smaller HRP polyplexes become unstable when they are in contact with expanding chains nearby. The results reveal potentially rich DNA release dynamics that can be controlled by subtle variation in multivalent counterion binding to DNA as well as the cellular matrix.