Medical Genetics Laboratory, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
Appl Biochem Biotechnol. 2013 Jun;170(4):841-53. doi: 10.1007/s12010-013-0224-0. Epub 2013 Apr 25.
Leukemic cells are hard-to-transfect cell lines. Many transfection reagents which can provide high gene transfer efficiency in common adherent cell lines are not effective to transfect established blood cell lines or primary leukemic cells. This study aims to examine a new class of cationic polymer non-viral vector, PEGylated-dextran-spermine (PEG-D-SPM), to determine its ability to transfect the leukemic cells. Here, the optimal conditions of the complex preparation (PEG-D-SPM/plasmid DNA (pDNA)) were examined. Different weight-mixing (w/w) ratios of PEG-D-SPM/pDNA complex were prepared to obtain an ideal mixing ratio to protect encapsulated pDNA from DNase degradation and to determine the optimal transfection efficiency of the complex. Strong complexation between polymer and pDNA in agarose gel electrophoresis and protection of pDNA from DNase were detected at ratios from 25 to 15. Highest gene expression was detected at w/w ratio of 18 in HL60 and K562 cells. However, gene expression from both leukemic cell lines was lower than the control MCF-7 cells. The cytotoxicity of PEG-D-SPM/pDNA complex at the most optimal mixing ratios was tested in HL60 and K562 cells using MTS assay and the results showed that the PEG-D-SPM/pDNA complex had no cytotoxic effect on these cell lines. Spherical shape and nano-nature of PEG-D-SPM/pDNA complex at ratio 18 was observed using transmission electron microscopy. As PEG-D-SPM showed modest transfection efficiency in the leukemic cell lines, we conclude that further work is needed to improve the delivery efficiency of the PEG-D-SPM.
白血病细胞是难转染的细胞系。许多在常见贴壁细胞系中提供高基因转移效率的转染试剂对已建立的血细胞系或原代白血病细胞无效。本研究旨在研究一类新型阳离子聚合物非病毒载体，聚乙二醇化葡聚糖-精胺（PEG-D-SPM），以确定其转染白血病细胞的能力。在此，研究了复合物制备（PEG-D-SPM/质粒 DNA（pDNA））的最佳条件。制备了不同重量混合（w/w）比的 PEG-D-SPM/pDNA 复合物，以获得理想的混合比，以保护包封的 pDNA 免受 DNase 降解，并确定复合物的最佳转染效率。在琼脂糖凝胶电泳中检测到聚合物和 pDNA 之间的强复合物形成以及聚合物对 pDNA 的保护免受 DNase 的降解，在 25 至 15 的比例下检测到。在 HL60 和 K562 细胞中，w/w 比为 18 时检测到最高的基因表达。然而，来自两种白血病细胞系的基因表达均低于对照 MCF-7 细胞。使用 MTS 测定法在 HL60 和 K562 细胞中测试最适混合比下的 PEG-D-SPM/pDNA 复合物的细胞毒性，结果表明 PEG-D-SPM/pDNA 复合物对这些细胞系没有细胞毒性。在比例为 18 时，使用透射电子显微镜观察到 PEG-D-SPM/pDNA 复合物的球形形状和纳米性质。由于 PEG-D-SPM 在白血病细胞系中显示出中等的转染效率，因此我们得出结论，需要进一步的工作来提高 PEG-D-SPM 的递送效率。
