Efficient non-viral gene delivery mediated by nanostructured calcium carbonate in solution-based transfection and solid-phase transfection

Abstract

Among different non-viral gene delivery methods, the technique of co-precipitation of Ca²⁺ with DNA in the presence of inorganic anions is an attractive option because of the biocompatibility and biodegradability. In this study, nano-sized CaCO₃/DNA co-precipitates for gene delivery were prepared. The effect of Ca²⁺/CO₃²⁻ molar ratio on the gene delivery was investigated. The mechanism of the transfection mediated by CaCO₃/DNA co-precipitates was studied by treatment of the cells with chloroquine, wortmannin and cytochalasin D, respectively. The in vitrogene transfections in different cells were carried out for both solution-based transfection and solid-phase transfection. The gene expression of the calcium carbonate based approach is strongly affected by the Ca²⁺/CO₃²⁻ ratio because the size of CaCO₃/DNA co-precipitates is mainly determined by the Ca²⁺/CO₃²⁻ ratio. In addition, the encapsulation efficiency of DNA increases with decreasing Ca²⁺/CO₃²⁻ ratio. With a suitable Ca²⁺/CO₃²⁻ ratio, CaCO₃/DNA co-precipitates could effectively mediate gene transfection with the expression levels higher than that of Lipofectamine 2000 in the presence of serum. The mechanism study shows that CaCO₃/DNA co-precipitates are internalized viaendocytosis of the cells and macropinocytosis is the main route of internalization. Compared with the solution-based transfection, CaCO₃/DNA co-precipitates in the solid-phase transfection exhibit a lower gene expression level. The calcium carbonate based approach has great potential in gene delivery.