A new tool for the transfection of corneal endothelial cells : calcium phosphate nanoparticles
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.
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