Lavigne Matthieu D, Pennadam Sivanand S, Ellis James, Yates Laura L, Alexander Cameron, Górecki Dariusz C
School of Pharmacy and Biomedical Sciences, and Institute of Biomedical and Biomolecular Science, University of Portsmouth, St. Michael's Building, White Swan Road, Portsmouth PO1 2DT, UK.
J Gene Med. 2007 Jan;9(1):44-54. doi: 10.1002/jgm.992.
Successful non-viral gene targeting requires vectors to meet two conflicting needs-strong binding to protect the genetic material during transit and weak binding at the target site to enable release. Responsive polymers could fulfil such requirements through the switching of states, e.g. the chain-extended coil to chain-collapsed globule phase transition that occurs at a lower critical solution temperature (LCST), in order to transport nucleic acid in one polymer state and release it in another.
The ability of new synthetic polycations based on poly(ethyleneimine) (PEI) with grafted neutral responsive poly(N-isopropylacrylamide) (PNIPAm) chains to condense DNA into particles with architectures varying according to graft polymer LCST was assessed using a combination of fluorescence spectroscopy, dynamic light scattering (DLS), zeta sizing, gel retardation and atomic force microscopy studies. Transfection assays were conducted under experimental conditions wherein the polymer components were able to cycle across their LCST.
Two PEI-PNIPAm conjugate polymers with different LCSTs displayed coil-globule transitions when complexed to plasmid DNA, leading to variations in molecular architecture as shown by changes in emission maxima of an environment-sensitive fluorophore attached to the PNIPAm chains. Gel retardation assays demonstrated differences in electrophoretic mobilities of polymer-DNA complexes with temperatures below and above polymer LCSTs. Atomic force micrographs showed changes in the structures of polymer-DNA complexes for a polymer undergoing a phase transition around body temperature but not for the polymer with LCST outside this range. Transfection experiments in C2C12 and COS-7 cells demonstrated that the highest expression of transgene occurred in an assay that involved a 'cold-shock' below polymer LCST during transfection.
Designed changes in thermoresponsive polycation vector configuration via temperature-induced phase transitions enhanced transgene expression. The results indicate that changes in molecular architecture induced by a carefully chosen stimulus during intracellular trafficking can be used to enhance gene delivery.
成功的非病毒基因靶向需要载体满足两个相互矛盾的需求——在转运过程中具有强结合力以保护遗传物质,而在靶位点具有弱结合力以实现释放。响应性聚合物可以通过状态切换来满足这些要求,例如在较低临界溶液温度(LCST)下发生的链伸展线圈到链塌陷球体的相变,以便以一种聚合物状态运输核酸并以另一种状态释放它。
使用荧光光谱、动态光散射(DLS)、zeta 粒度分析、凝胶阻滞和原子力显微镜研究相结合的方法,评估了基于聚(乙烯亚胺)(PEI)并接枝有中性响应性聚(N-异丙基丙烯酰胺)(PNIPAm)链的新型合成聚阳离子将 DNA 浓缩成具有根据接枝聚合物 LCST 而变化的结构的颗粒的能力。在聚合物组分能够在其 LCST 上下循环的实验条件下进行转染试验。
两种具有不同 LCST 的 PEI-PNIPAm 共轭聚合物在与质粒 DNA 复合时显示出线团-球体转变,导致分子结构发生变化,这通过连接到 PNIPAm 链上的环境敏感荧光团发射最大值的变化得以体现。凝胶阻滞试验表明,聚合物-DNA 复合物在聚合物 LCST 以下和以上温度时的电泳迁移率存在差异。原子力显微镜图像显示,对于在体温附近发生相变的聚合物,聚合物-DNA 复合物的结构发生了变化,而对于 LCST 不在此范围内的聚合物则没有变化。在 C2C12 和 COS-7 细胞中进行的转染实验表明,转基因的最高表达发生在转染过程中涉及聚合物 LCST 以下“冷休克”的试验中。
通过温度诱导的相变对热响应性聚阳离子载体构型进行设计改变可增强转基因表达。结果表明,在细胞内运输过程中由精心选择的刺激引起的分子结构变化可用于增强基因递送。