Singh Bijay, Maharjan Sushila, Kim You-Kyoung, Jiang Tai, Islam Mohammad Ariful, Kang Sang-Kee, Cho Myung-Haing, Choi Yun-Jaie, Cho Chong-Su
J Nanosci Nanotechnol. 2014 Nov;14(11):8356-64. doi: 10.1166/jnn.2014.9919.
Receptor-mediated endocytosis is a promising approach of gene delivery into the target cells via receptor-ligand interaction. Vimentins at the cell surface are recently known to bind N-acetylglucosamine (GlcNAc) residue, therefore, the cell surfaces of vimentin-expressing cells could be targeted by using the GlcNAc residue as a specific ligand for receptor-mediated gene delivery. Here, we have developed polymeric gene delivery vectors, based on poly(ethylene oxide)(PEO) and poly(aspartamide), namely poly[(aspartamide)(diethylenetriamine)]-b-[PEO-(GlcNAc)] (PADPG) and poly[(aspartamide)(diethylenetriamine)]-b-[PEO] (PADP) to elucidate the efficiency of GlcNAc ligand for gene delivery through receptor mediated endocytosis. To determine the efficiency of these polymeric vectors for specific gene delivery, the DNA condensation ability of PADPG and PADP and the subsequent formation of polymeric nanoparticles were confirmed by gel retardation assay and transmission electron microscopy respectively. Both PADPG and PADP had lower cytotoxicity than polyethylenimine 25 K (PEI 25 K). However, their transfection efficiency was comparatively lower than PEI 25 K due to hydrophilic property of PEO in the vectors. To observe the stability of polymeric nanoparticles, the transfection of PADPG and PADP was carried out in the presence of serum. Favorably, the interfering effect of serum on the transfection efficiency of PADPG and PADP was also very low. Finally, when the cell specificity of these polymeric vectors was investigated, PADPG had high gene transfection in vimentin-expressing cells than vimentin-deficiency cells. The high transfection efficiency of PADPG was attributed to the GlcNAc in the polymeric vector which interact specifically with vimentin in the cells for the receptor-mediated endocytosis. The competitive inhibition assay further proved the receptor-mediated endocytosis of PADPG. Thus, this study demonstrates that conjugation of GlcNAc is an effective and rational way to prepare a suitable vector for targeted gene delivery to vimentin-expressing cells.
受体介导的内吞作用是一种通过受体-配体相互作用将基因导入靶细胞的很有前景的方法。最近发现细胞表面的波形蛋白能结合N-乙酰葡糖胺（GlcNAc）残基，因此，利用GlcNAc残基作为受体介导基因递送的特异性配体，可以靶向波形蛋白表达细胞的细胞表面。在此，我们基于聚环氧乙烷（PEO）和聚天冬酰胺开发了聚合物基因递送载体，即聚[（天冬酰胺）（二乙烯三胺）]-b-[PEO-（GlcNAc）]（PADPG）和聚[（天冬酰胺）（二乙烯三胺）]-b-[PEO]（PADP），以阐明GlcNAc配体通过受体介导的内吞作用进行基因递送的效率。为了确定这些聚合物载体进行特异性基因递送的效率，分别通过凝胶阻滞试验和透射电子显微镜确认了PADPG和PADP的DNA凝聚能力以及随后聚合物纳米颗粒的形成。PADPG和PADP的细胞毒性均低于25K聚乙二胺（PEI 25K）。然而，由于载体中PEO的亲水性，它们的转染效率相对低于PEI 25K。为了观察聚合物纳米颗粒的稳定性，在有血清存在的情况下进行了PADPG和PADP的转染。有利的是，血清对PADPG和PADP转染效率的干扰作用也非常低。最后，当研究这些聚合物载体的细胞特异性时，PADPG在波形蛋白表达细胞中的基因转染率高于波形蛋白缺陷细胞。PADPG的高转染效率归因于聚合物载体中的GlcNAc，它与细胞中的波形蛋白特异性相互作用以进行受体介导的内吞作用。竞争性抑制试验进一步证明了PADPG的受体介导的内吞作用。因此，本研究表明GlcNAc的缀合是制备用于靶向基因递送至波形蛋白表达细胞的合适载体的有效且合理的方法。
