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 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 calciumphosphate 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 un-targeted 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.

We have developed a self-assembled nanoparticle (NP) that efficiently delivers small interfering RNA (siRNA) to the tumor by intravenous (IV) administration. The NP was obtained by mixing carrier DNA, siRNA, protamine, and lipids, followed by post-modification with polyethylene glycol and a ligand, anisamide. Four hours after IV injection of the formulation into a xenograft model, 70-80% of injected siRNA/g accumulated in the tumor, approximately 10% was detected in the liver and approximately 20% recovered in the lung. Confocal microscopy showed that fluorescent-labeled siRNA was efficiently delivered into the cytoplasm of the sigma receptor expressing NCI-H460 xenograft tumor by the targeted NPs, whereas free siRNA and non-targeted NPs showed little uptake. Three daily injections (1.2 mg/kg) of siRNA formulated in the targeted NPs silenced the epidermal growth factor receptor (EGFR) in the tumor and induced approximately 15% tumor cell apoptosis. Forty percent tumor growth inhibition was achieved by treatment with targeted NPs, while complete inhibition lasted for 1 week when combined with cisplatin. The serum level of liver enzymes and body weight monitoring during the treatment indicated a low level of toxicity of the formulation. The carrier itself also showed little immunotoxicity (IMT).

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