Zanin H, Hollanda L M, Ceragioli H J, Ferreira M S, Machado D, Lancellotti M, Catharino R R, Baranauskas V, Lobo A O
Departamento de Semicondutores, Instrumentos e Fotônica, Faculdade de Engenharia Elétrica e de Computação, Universidade Estadual de Campinas, 13083-852 Campinas, SP, Brazil.
Laboratory of Biotechnology, Department of Biochemistry, Institute of Biology at UNICAMP, Rua Monteiro Lobato 255, Campinas, SP CEP 13083-862, Brazil.
Mater Sci Eng C Mater Biol Appl. 2014 Jun 1;39:359-70. doi: 10.1016/j.msec.2014.03.016. Epub 2014 Mar 12.
For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.
首次合成了氧端基纤维素碳纳米颗粒（CCN），并将其应用于pIRES质粒的基因转染。CCN通过化学气相沉积技术在聚苯胺催化下制备。该质粒包含一个在真核细胞中编码绿色荧光蛋白（GFP）的基因，使细胞发出荧光。这种新型纳米材料和pIRES质粒在磁力搅拌下分散于水中时形成π堆积。zeta电位的频率变化证实了质粒与纳米材料紧密相连。体外试验发现，这种结合物被NG97、NIH-3T3和A549细胞系吞噬，使其发出荧光，通过荧光显微镜可观察到。在转染试验之前，我们研究了CCN对细胞活力的影响。使用NG97、NIH-3T3和A549细胞系进行了MTT和中性红摄取试验。此外，我们利用代谢组学来验证少量纳米材料是否足以对NG97细胞造成一些细胞损伤。我们展示了CCN-DNA复合物的两种作用机制，即转染细胞产生外源性蛋白质以及代谢组学变化，这有助于更好地理解胶质母细胞瘤，这是本研究的主要发现。我们的结果表明，这种纳米材料作为非病毒基因载体具有巨大潜力，在代谢组学试验中具有低细胞毒性、良好的转染效率以及少量纳米材料对细胞损伤小的特点。
