Synthesis of calcium phosphate nanoparticles

Calcium phosphate nanoparticles were precipitated from aqueous solutions of calcium nitrate (6.25 mM) and diammonium hydrogen phosphate (3.74 mM) and then functionalized with DNA or siRNA (1 g l⁻¹) and PEI (25 kDa, 2 g l⁻¹) as described earlier⁷⁵ with the following modifications: CaP/DNA/CaP/PEI nanoparticles were separated from nonadsorbed molecules (including excess nucleic acid or PEI) by centrifugation (5000 g, 4 °C, 15 min) and then dispersed in the half of the original volume of water by ultrasonication (UP50H, Hielscher, Teltow, Germany; Ultrasound Technology; sonotrode 2, cycle 0.8, amplitude 80%, 10 s). CaP/siRNA/CaP/PEI nanoparticles were isolated by centrifugation (20 000 g, 4 °C, 15 min) and then dispersed in the original volume of water by ultrasonication with the same conditions.

Model DNA leading to the expression of eGFP (pcDNA3-eGFP) was used. For the gene-silencing experiments the eGFP-siRNA: sense, 5′-GCAAGCUGACCCUGAAGUUCAU-3′ antisense, 5′-AUGAACUUCAGGGUCAGCUUGC-3′ (Invitrogen, Paisley, UK) was used. Lipofectamine 2000 was obtained from Life Technologies (Invitrogen, Carlsbad, CA, USA) and used according to the manufacturer's specifications.