Synthesis and Biological Evaluation of a Bioresponsive and Endosomolytic siRNA-Polymer Conjugate
Synthesis and Biological Evaluation of a Bioresponsive and Endosomolytic siRNA-Polymer Conjugate
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DOI：
10.1021/mp9000124
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发表时间：
2009-05-01
影响因子：
4.9
通讯作者：
Wagner, Ernst
中科院分区：
文献类型：
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作者：
Meyer, Martin;Dohmen, Christian;Wagner, Ernst
在递送过程中，静电形成的siRNA多聚体的细胞外稳定性是一个重要的问题。为了克服细胞外环境中多链解离的风险，siRNA被共价掺入到pH和氧化还原响应型聚合物偶联物中。这种新型的siRNA偶联物由聚赖氨酸(PLL)作为RNA结合和保护聚阳离子，聚乙二醇(PEG)作为增溶和屏蔽聚合物，裂解肽蜂毒素(Melittin)被二甲基马来酸酐(DMMAn)掩盖，可在内体pH下移除，siRNA通过可生物还原的二硫键连接在正义链的5‘端。在聚阴离子肝素存在下，在类似的静电siRNA多聚体分解的条件下，纯化的siRNA结合物是稳定的。只有肝素和还原剂如谷胱甘肽的结合才能引发siRNA从结合物中释放出来。在-gt；=25 nM的siRNA时，发现了高的体外生物相容性(在中性pH下没有细胞毒性或溶血活性)和有效的序列特异性基因沉默，可与相应的静电多聚体相媲美。该制剂的体内毒性研究表明，结合物的治疗应用仍有待优化。
Extracellular stability of electrostatically formed siRNA polyplexes is a significant concern in the delivery process. To overcome the risk of polyplex dissociation in the extracellular environment, siRNA was covalently incorporated into a pH- and redox-responsive polymer conjugate. The novel siRNA conjugate consists of polylysine (PLL) as RNA binding and protecting polycation, polyethylene glycol (PEG) as solubilizing and shielding polymer, the lytic peptide melittin masked by dimethylmaleic anhydride (DMMAn) removable at endosomal pH, and the siRNA attached at the 5'-end of the sense strand via a bioreducible disulfide bond. The purified siRNA conjugate was stable in the presence of the polyanion heparin at conditions where the analogous electrostatic siRNA polyplexes disassemble. Only the combination of heparin plus a reducing agent such as glutathione triggered the release of siRNA from the conjugate. High in vitro biocompatibility (absence of cytotoxicity or hemolytic activity at neutral pH) and efficient and sequence-specific gene silencing was found at >= 25 nM siRNA, comparable to the corresponding electrostatic polyplexes. In vivo toxicity studies of this formulation demonstrated that conjugates remain to be optimized for therapeutic application.