Polyplex Formation between PEGylated Linear Cationic Block Copolymers and DNA: Equilibrium and Kinetic Studies
Polyplex Formation between PEGylated Linear Cationic Block Copolymers and DNA: Equilibrium and Kinetic Studies
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DOI：
10.1021/jp501234p
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发表时间：
2014-06-26
影响因子：
3.3
通讯作者：
Dhara, Dibakar
中科院分区：
文献类型：
--
作者：
Dey, Debabrata;Kumar, Santosh;Dhara, Dibakar
了解非病毒基因传递途径的基本要求是对 DNA 复合物进行彻底的生物物理表征。在这项工作中，我们研究了小牛胸腺 DNA (ctDNA) 和一系列新的线性阳离子嵌段共聚物 (BCP) 之间的相互作用。使用[3-(甲基丙烯酰氨基)丙基]三甲基氯化铵(MAPTAC)和聚(乙二醇)甲醚(PEGMe)作为共聚单体，通过受控自由基聚合合成BCP。紫外可见光谱、溴化乙锭染料排除和凝胶电泳研究表明，这些阳离子 BCP 能够有效地与 DNA 结合。稳态荧光、紫外熔解、凝胶电泳和圆二色性结果表明，含有较高 PEG 的 BCP 的结合增加。 PEG 和 DNA 碱基对之间的疏水相互作用在两个大分子非常接近时变得显着，从而影响结合趋势。 DLS 研究表明，由于压缩，在较低电荷比（聚合物的“+”电荷与 DNA 的“-”电荷之比）下，DNA 分子的尺寸会减小，而在较高电荷比下，由于聚合物复合物之间的聚集，尺寸会增加。此外，我们还使用停流荧光方法对结合过程进行了动力学研究。 BCP-DNA 结合研究的所有结果表明了一个两步反应机制 - 阳离子块和 DNA 之间的快速静电结合，随后在后续步骤中聚合复合物发生构象变化，导致 DNA 缩合。第一步的相对速率常数（k(1)'）比第二步的相对速率常数（k(2)'）高得多，并且发现两者都随着BCP浓度的增加而增加。 BCP 中的电荷比以及 PEG 含量对 DNA-BCP 复合物形成的动力学具有显着影响。在合成的阳离子嵌段中引入所需的 PEG 链长度使它们有可能用作非病毒基因递送剂。
The basic requirement for understanding the nonviral gene delivery pathway is a thorough biophysical characterization of DNA polyplexes. In this work, we have studied the interactions between calf-thymus DNA (ctDNA) and a new series of linear cationic block copolymers (BCPs). The BCPs were synthesized via controlled radical polymerization using [3-(methacryloylamino)propyl]trimethylammonium chloride (MAPTAC) and poly(ethylene glycol) methyl ether (PEGMe) as comonomers. UV-visible spectroscopy, ethidium bromide dye exclusion, and gel electrophoresis study revealed that these cationic BCPs were capable of efficiently binding with DNA. Steady-state fluorescence, UV melting, gel electrophoresis, and circular dichroism results suggested increased binding for BCPs containing higher PEG. Hydrophobic interactions between the PEG and the DNA base pairs became significant at close proximity of the two macromolecules, thereby influencing the binding trend. DLS studies showed a decrease in the size of DNA molecules at lower charge ratio (the ratio of "+" charge of the polymer to "-" charge of DNA) due to compaction, whereas the size increased at higher charge ratio due to aggregation among the polyplexes. Additionally, we have conducted kinetic studies of the binding process using the stop-flow fluorescence method. All the results of BCP-DNA binding studies suggested a two-step reaction mechanism-a rapid electrostatic binding between the cationic blocks and DNA, followed by a conformational change of the polyplexes in the subsequent step that led to DNA condensation. The relative rate constant (k(1)') of the first step was much higher compared to that of the second step (k(2)'), and both were found to increase with an increase in BCP concentration. The charge ratios as well as the PEG content in the BCPs had a marked effect on the kinetics of the DNA-BCP polyplex formation. Introduction of a desired PEG chain length in the synthesized cationic blocks renders them potentially useful as nonviral gene delivery agents.