Institute of Anatomy, Essen University Hospital, 45147 Essen, Germany.
Acta Biomater. 2012 Mar;8(3):1156-63. doi: 10.1016/j.actbio.2011.09.013. Epub 2011 Sep 17.
Calcium phosphate nanoparticles (CaP-NP) are ideal tools for transfection due to their high biocompatibility and easy biodegradability. After transfection these particles dissociate into calcium and phosphate ions, i.e. physiological components found in every cell, and it has been shown that the small increase in intracellular calcium level does not affect cell viability. CaP-NP functionalized with pcDNA3-EGFP (CaP/DNA/CaP/DNA) and stabilized using different amounts of poly(ethylenimine) (PEI) were prepared. Polyfect®-pcDNA3-EGFP polyplexes served as a positive control. The transfection of human and murine corneal endothelial cells (suspensions and donor tissue) was optimized by varying the concentration of CaP-NP and the duration of transfection. The transfection efficiency was determined as EGFP expression detected by flow cytometry and fluorescence microscopy. To evaluate the toxicity of the system the cell viability was detected by TUNEL staining. Coating with PEI significantly increased the transfection efficiency of CaP-NP but decreased cell viability, due to the cytotoxic nature of PEI. The aim of this study was to develop CaP-NP with the highest possible transfection efficiency accompanied by the least apoptosis in corneal endothelial cells. EGFP expression in the tissues remained stable as corneal endothelial cells exhibit minimal proliferative capacity and very low apoptosis after transfection with CaP-NP. In summary, CaP-NP are suitable tools for the transfection of corneal endothelial cells. As CaP-NP induce little apoptosis these nanoparticles offer a safe alternative to viral transfection agents.
磷酸钙纳米颗粒（CaP-NP）由于其高生物相容性和易生物降解性，是转染的理想工具。转染后，这些颗粒会解离为钙和磷酸离子，即每个细胞中都存在的生理成分，并且已经表明，细胞内钙水平的微小增加不会影响细胞活力。用不同量的聚乙烯亚胺（PEI）对 pcDNA3-EGFP 功能化的 CaP-NP（CaP/DNA/CaP/DNA）进行稳定化。聚阳离子- pcDNA3-EGFP 超螺旋体用作阳性对照。通过改变 CaP-NP 的浓度和转染时间来优化人角膜内皮细胞（悬浮液和供体组织）的转染。通过流式细胞术和荧光显微镜检测 EGFP 表达来确定转染效率。为了评估该系统的毒性，通过 TUNEL 染色检测细胞活力。由于 PEI 的细胞毒性，用 PEI 涂层显著提高了 CaP-NP 的转染效率，但降低了细胞活力。本研究的目的是开发具有最高转染效率和最低角膜内皮细胞凋亡的 CaP-NP。用 CaP-NP 转染后，组织中的 EGFP 表达保持稳定，因为角膜内皮细胞表现出最小的增殖能力和非常低的转染后凋亡。总之，CaP-NP 是转染角膜内皮细胞的合适工具。由于 CaP-NP 诱导的细胞凋亡很少，因此这些纳米颗粒为病毒转染剂提供了一种安全的替代方法。
