Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery

摘要

A lipid coated calcium phosphate (LCP) nanoparticle (NP) formulation was developed for efficient delivery of small interfering RNA (siRNA) to a xenograft tumor model by intravenous administration. Based on the previous formulation, liposome-polycation-DNA (LPD), which was a DNA–protamine complex wrapped by cationic liposome followed by post-insertion of PEG, LCP was similar to LPD NP except that the core was replaced by a biodegradable nano-sized calcium phosphate precipitate prepared by using water-in-oil microemulsions in which siRNA was entrapped. We hypothesized that after entering the cells, LCP would deassemble at low pH in the endosome, which would cause endosome swelling and bursting to release the entrapped siRNA. Such a mechanism was demonstrated by the increase of intracellular Ca2+ concentration as shown by using a calcium specific dye Fura-2. The LCP NP was further modified by post-insertion of polyethylene glycol (PEG) with or without anisamide, a sigma-1 receptor ligand for systemic administration. Luciferase siRNA was used to evaluate the gene silencing effect in H-460 cells which were stably transduced with a luciferase gene. The anisamide modified LCP NP silenced about 70% and 50% of luciferase activity for the tumor cells in culture and those grown in a xenograft model, respectively. The untargeted NP showed a very low silencing effect. The new formulation improved the in vitro silencing effect 3–4 folds compared to the previous LPD formulation, but had a negligible immunotoxicity.