Freeze-dried cationic calcium phosphate nanorods as versatile carriers of nucleic acids (DNA, siRNA)

Functionalized calcium phosphate (hydroxyapatite) nanorods were freeze-dried in the presence of the cryoprotectant trehalose, giving a storable and easily redispersible system which can adsorb nucleic acids for transfection and gene silencing. The nanorods were first surface-functionalized with a layer of polyethyleneimine (PEI), purified by ultracentrifugation and redispersion, and freeze-dried in the presence of trehalose. The nanorods can be easily redispersed in water. The concentration-dependent adsorption of nucleic acids (DNA and siRNA) onto the surface of the redispersed cationic nanorods was measured by dynamic light scattering (particle diameter and zeta potential). The transfection efficiency on epithelial cells (HeLa) and osteoblasts (MG-63) was systematically determined for increasing amounts of added nucleic acid, up to a charge reversal by the anionic nucleic acids. Both transfection and gene silencing efficiency increased with increasing amount of nucleic acid, but went through minimum around the point of zero charge where the particles agglomerated. The application of an additional outer layer of polyethyleneimine around the hydroxyapatite/PEI/nucleic acid-nanoparticles reversed the charge back to positive, resulting in even higher transfection rates with almost complete cell viability.