Malakooty Poor Elham, Baghaban Eslaminejad Mohamadreza, Gheibi Nematollah, Bagheri Fatemeh, Atyabi Fatemeh
Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR , Tehran , Iran.
Artif Cells Nanomed Biotechnol. 2014 Dec;42(6):376-84. doi: 10.3109/21691401.2013.832685. Epub 2013 Sep 3.
This study evaluated the potential for prepared chitosan-plasmid DNA (pDNA) nanoparticles to transfer an exogenous gene into human bone marrow-derived mesenchymal stem cells (MSCs).
Chitosan-pDNA nanoparticles were synthesized by the complex coacervation method. We used 18, 50 and 136 KD chitosan at concentrations of 0.05%, 0.1%, 0.5% and 1%, in addition to a pTracer-CMV2 plasmid that contained the green fluorescent protein (GFP) gene. To examine the complexation, samples were run through an agarose gel. The sizes and zeta potential of the nanoparticles were measured by a nanosizer. Scanning electron microscopy (SEM) imaging was used to observe the nanoparticle morphology. MSCs were prepared from human bone marrow and transfected with chitosan-pDNA nanoparticles. The cultures transfected by lipofectamine(2000) were taken as the control. Cell viability was determined by MTT assay and transfection efficiency by flow cytometry.
The smallest size of complexes was obtained with 18 KD chitosan (211 nm) and the highest zeta potential was observed with 136 KD chitosan (29.61 mV). The best transfection rate (18.43%) was achieved with the 0.1% concentration of 18 KD chitosan nanoparticles versus 40.57% for commercial lipofectamine (p < 0.01). The MTT assay indicated an average of 95.5% cell viability for 0.1% concentration of 18 KD chitosan compared with approximately 60% of Lipofectamine(2000).
Nanoparticles produced by 18 KD chitosan at the 0.1% concentration and pDNA may be a promising gene delivery system for human marrow-derived MSCs. Although transfection efficiency of such nanoparticles is lower than that of Lipofectamine(2000), however comparatively they possess less cytotoxic effects.
本研究评估了制备好的壳聚糖 - 质粒DNA（pDNA）纳米颗粒将外源基因导入人骨髓间充质干细胞（MSCs）的潜力。
通过复凝聚法合成壳聚糖 - pDNA纳米颗粒。除了含有绿色荧光蛋白（GFP）基因的pTracer - CMV2质粒外，我们使用了浓度为0.05%、0.1%、0.5%和1%的18、50和136 KD壳聚糖。为检测复合情况，样品进行琼脂糖凝胶电泳。通过纳米粒度仪测量纳米颗粒的大小和zeta电位。使用扫描电子显微镜（SEM）成像观察纳米颗粒形态。从人骨髓中制备MSCs并用壳聚糖 - pDNA纳米颗粒进行转染。以脂质体2000转染的培养物作为对照。通过MTT法测定细胞活力，通过流式细胞术测定转染效率。
18 KD壳聚糖形成的复合物尺寸最小（211 nm），136 KD壳聚糖的zeta电位最高（29.61 mV）。0.1%浓度的18 KD壳聚糖纳米颗粒的最佳转染率为18.43%，而商业化脂质体为40.57%（p < 0.01）。MTT法表明，0.1%浓度的18 KD壳聚糖的细胞活力平均为95.5%，而脂质体2000约为60%。
0.1%浓度的18 KD壳聚糖与pDNA制备的纳米颗粒可能是用于人骨髓来源MSCs的有前景的基因递送系统。虽然这种纳米颗粒的转染效率低于脂质体2000，但其细胞毒性作用相对较小。
