A new Tool for Transfection of Corneal Endothelial Cells using Calcium Phosphate Nanoparticles

Abstract

Purpose: : Calcium phosphate nanoparticles (CaP-NPs) are an ideal tool for transfection thanks to their distinct biocompatibility. After transfection, the particles dissolve in calcium and phosphate, and it has been shown that intracellular Ca²⁺ - levels do not affect cell viability. The aim of this study was to compare the transfection efficiencies of different CaP-NPs in corneal endothelial cells.

Methods: : Different CaP-NPs with pcDNA3-EGFP (triple-shell CaP/DNA/CaP/DNA in different preparations and those coated by various dispersions of polyethylenimine (PEI) were prepared. Polyfect-pcDNA3-EGFP served as positive control. Human and murine corneal endothelial cells (suspensions and donor tissue) were transfected with varying concentrations of CaP-NPs, for different transfection periods. The transfection efficiency was measured by EGFP-expression measured by quantitative flow cytometry and fluorescence microscopy. To evaluate a possible cell toxicity, apoptosis was studied by propidium iodide or TUNEL staining.

Results: : Coating with PEI significantly increased the transfection efficiency of triple shell CaP-NPs. Thus, after transfection with triple shell CaP-NPs/PEI(0.5), up to 50% of corneal endothelial cells showed EGFP expression. However, the cell viability in cell suspensions decreased with increasing dispersion of PEI. As corneal endothelial cells are a cell type with minimal proliferative capacity, the EGFP expression in tissues remained considerably stable.

Conclusions: : CaP-NPs are suitable tools for the transfection of corneal endothelial cells and may offer an alternative to viral transfection which is safe for patient use. Further studies are necessary to carefully evaluate the aspects of functional application and of biosafety.

Keywords: cornea: endothelium • calcium