Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery

Abstract

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 micro-emulsions in which siRNA was entrapped. We hypothesized that after entering the cells, LCP would de-assemble 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 Ca(2+) 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.

Figure 1

The formation process of liposome/calcium/phosphate (LCP) nanoparticles.

Figure 2

The hypothesized release process of siRNA entrapped in LCP after endocytosis to the endosome. There are four steps for siRNA released from LCP. (1) The LCP enters the cell through endocytosis and stays in the endosome. (2) The CaP core is dissolved at low pH, causing NP de-assembly. (3) The dissolved calcium and phosphate ions increase the osmotic pressure and cause endosome swelling. (4) The endosome bursts and releases the siRNA, calcium and phosphate ions into the cytoplasm.

Figure 3

The TEM imaging of (A) siRNA entrapped in calcium phosphate. (B) The LCP nanoparticle with negative staining. (C) Effect of lipid/siRNA molar ratio on particle size and zeta potential of LCP.

Figure 4

The release profile of LCP. Light scattering of CaP/siRNA (A) and siRNA in LCP (B) at different pH. The fluorescence images of Fura-2 labeled H-460 cells before (C) and after (D) the addition of LCP. The arrows indicate cells that changed color from red (low Ca²⁺) to green (high Ca²⁺).

Figure 5

(A) In vitro luciferase gene silencing effect of different formulations in H-460 cells by incubation at 37°C for 24 h (n=5). The amount of siRNA in LCP formulation was 99.8±6.9 nM by evaluating the entrapped fluorescently labeled siRNA. For the LPD formulations, the concentration of FAM labeled siRNA was 100 nM. (B) The measurement of IC50, half maximal inhibitory concentration, of LCP-PEG-AA and LPD-PEG-AA. * indicates p<0.05, ** indicates p<0.01.

Figure 6

(A) In vivo luciferase gene silencing effects of different formulations at the dose of 1.2 mg/kg. The luciferase activity in H-460 cells was measured after 24 hours of the i.v. injection with different siRNA formulations. (B) The serum IL-6 (solid line) and IL-12 (dash line) levels of the mice after 4 hours of the i.v. injection with targeted LCP (squares) and LPD (circles) formulations at different dose of siRNA. ** indicates p<0.01.