Laboratory for Polymer Materials, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China.
Biomaterials. 2013 Oct;34(32):7923-38. doi: 10.1016/j.biomaterials.2013.06.064. Epub 2013 Jul 21.
In this work, we first synthesized a ω-amino group Boc-protected poly(ω-aminohexyl methacrylamide) PAHMAA-Boc precursor via controlled reversible addition-fragmentation chain transfer (RAFT) polymerization of the Boc-AHMAA monomer in THF solution, and poly(ω-aminohexyl methacrylamide) (PAHMAA) was then prepared via Boc deprotection, and was further conjugated with (L-)-arginines at the ω-amino group sites to give a series of new (L-)-arginine conjugated PAHMAA-R7, PAHMAA-R16 and PAHMAA-R22. Employing these PAHMAA and PAHMAA-Rs as functional gene vectors, their plasmid DNA binding affinities were examined by agarose gel retardant assay. By means of dynamic light scattering (DLS), mean particle sizes and zeta potentials of the polyplexes were analyzed. Moreover, cytotoxicities of the PAHMAA and PAHMAA-Rs were evaluated by MTT and lactate dehydrogenase (LDH) release assays with COS-7 cells, and luciferase and EGFP gene transfection efficacies by these vectors were assayed in COS-7 and HeLa cells. Furthermore, intracellular uptake of the vector/Cy3-labeled pDNA polyplexes was studied with a flow cytometer (FACS), and the most efficient PAHMAA-R16 vector was employed to investigate the endocytic gateways with various inhibitors. In addition, colocalization of the Cy3-labeled pDNA and Oregon Green labeled PAHMAA-R16 vector in the intracellular organelles of COS-7 cells was visualized on a fluorescence microscopy. As a result, it was revealed that the PAHMAA-R vectors showed lower cytotoxicities and transfection efficacies significantly higher than those of the PAHMAA, strongly depending on their percentage of arginine conjugation, and that the results of endocytic inhabitation and fluorescence colocalization in endoplasmic reticulum may suggest a caveolae-mediated efficient intracellular trafficking route for the synthesized PAHMAA-R vectors.
在这项工作中，我们首先通过 Boc-AHMAA 单体在 THF 溶液中的可控可逆加成-断裂链转移 (RAFT) 聚合合成了 ω-氨基 Boc 保护的聚（ω-氨基己基甲基丙烯酰胺）PAHMAA-Boc 前体，然后通过 Boc 脱保护制备了聚（ω-氨基己基甲基丙烯酰胺）（PAHMAA），并进一步在 ω-氨基位点与（L-）-精氨酸偶联，得到一系列新的（L-）-精氨酸偶联的 PAHMAA-R7、PAHMAA-R16 和 PAHMAA-R22。将这些 PAHMAA 和 PAHMAA-Rs 用作功能基因载体，通过琼脂糖凝胶阻滞实验研究其与质粒 DNA 的结合亲和力。通过动态光散射（DLS）分析了聚合物的平均粒径和 zeta 电位。此外，通过 MTT 和乳酸脱氢酶（LDH）释放实验评估了 PAHMAA 和 PAHMAA-Rs 在 COS-7 细胞中的细胞毒性，并用这些载体在 COS-7 和 HeLa 细胞中测定了荧光素酶和 EGFP 基因的转染效率。进一步通过流式细胞仪（FACS）研究了载体/Cy3 标记的 pDNA 聚合物的细胞内摄取，并用各种抑制剂研究了最有效的 PAHMAA-R16 载体的内吞途径。此外，在 COS-7 细胞的细胞内细胞器上用荧光显微镜可视化 Cy3 标记的 pDNA 和 Oregon Green 标记的 PAHMAA-R16 载体的共定位。结果表明，PAHMAA-R 载体的细胞毒性和转染效率明显低于 PAHMAA，这强烈依赖于它们的精氨酸偶联百分比，内吞抑制和荧光共定位在内质网中的结果可能表明合成的 PAHMAA-R 载体具有有效的细胞内运输途径。
