Vernon Matthew M, Dean David A, Dobson Jon
Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL 32611, USA.
School of Medicine and Dentistry, University of Rochester Medical Center, Rochester, NY 14642, USA.
Int J Mol Sci. 2015 Aug 17;16(8):19369-86. doi: 10.3390/ijms160819369.
Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell's cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells.
目前,对大多数干细胞、祖细胞和原代细胞系进行高效的非病毒质粒DNA转染,在基因治疗研究的许多应用中都构成了障碍。从效率和细胞活力的角度来看,基于磁性纳米颗粒的DNA转染是一种很有前景的基因载体技术,因为与其他非病毒方法相比,它已显示出快速且改善的转染效果。最近,我们的研究小组引入了振荡磁体阵列,进一步改进了这种新型质粒DNA(pDNA)载体技术。纳米磁性转染技术的持续改进主要集中在磁性纳米颗粒(MNP)功能化和转染参数优化上:细胞汇合度、生长培养基、血清饥饿、磁体振荡参数等。注意到在细胞胞质中MNP-pDNA复合物解离后,这些参数都无法协助递送的pDNA进行核转位,因此在理论上有理由在pDNA中加入核转位的盒式结构。在本研究中,在转染质粒中加入DNA靶向序列(DTS)结构提高了模型神经元中的转染效率,推测这是由于核转位增加所致。当比较分裂的SH-SY5Y前体细胞与不分裂和分化的SH-SY5Y神经母细胞瘤细胞的反应时,这一观察结果最为明显。