The selective delivery of small interfering RNA (siRNA) to metastatic tumors remains a challenging task. We have developed a nanoparticle (NP) formulation composed of siRNA, a carrier DNA, a polycationic peptide, and cationic liposomes. The NP was obtained by a self-assembling process, followed by surface modification with a polyethylene glycol (PEG)-conjugated ligand, anisamide. The NP was PEGylated and a ligand was presented to target sigma receptor-expressing murine melanoma cells, B16F10. The lung metastasis model was established by intravenous (i.v.) injection of the B16F10 cells into C57BL/6 mice. A mixture of siRNA against MDM2, c-myc, and vascular endothelial growth factor (VEGF) co-formulated in the targeted NP caused simultaneous silencing of each of the oncogenes in the metastatic nodules. Two consecutive i.v. injections of siRNA in the targeted NP significantly reduced the lung metastasis (approximately 70-80%) at a relatively low dose (0.45 mg/kg), whereas free siRNA and the nontargeted NP showed little effect. This targeted NP formulation significantly prolonged the mean survival time of the animals by 30% as compared to the untreated controls. At the therapeutic dose, the targeted NP showed little local and systemic immunotoxicity and did not decrease the body weight or damage the major organs.

We have developed a nanoparticle formulation [liposomes-protamine-hyaluronic acid nanoparticle (LPH-NP)] for systemically delivering siRNA into the tumor. The LPH-NP was prepared in a selfassembling process. Briefly, protamine and a mixture of siRNA and hyaluronic acid were mixed to prepare a negatively charged complex. Then, cationic liposomes were added to coat the complex with lipids via charge-charge interaction to prepare the LPH-NP. The LPH-NP was further modified by DSPE-PEG or DSPE-PEG-anisamide by the post-insertion method. Anisamide is a targeting ligand for the sigma receptor over-expressed in the B16F10 melanoma cells. The particle size, zeta potential and siRNA encapsulation efficiency of the formulation were approximately 115 nm, +25 mV and 90%, respectively. Luciferase siRNA was used to evaluate the gene silencing activity in the B16F10 cells, which were stably transduced with a luciferase gene. The targeted LPH-NP (PEGylated with ligand) silenced 80% of luciferase activity in the metastatic B16F10 tumor in the lung after a single i.v. injection (0.15 mg siRNA/kg). The targeted LPH-NP also showed very little immunotoxicity in a wide dose range (0.15 -1.2 mg siRNA/kg), while the previously published formulation, LPD-NP (liposome-protamine-DNA nanoparticle), had a much narrow therapeutic window (0.15-0.45 mg/kg).

We have developed a nanoparticle (NP) formulation for systemically delivering siRNA into metastatic tumors. The NP, composed of nucleic acids, a polycationic peptide and cationic liposome, was prepared in a self-assembling process. The NP was then modified by PEG-lipid containing a targeting ligand, anisamide, and thus was decorated for targeting sigma receptor expressing B16F10 tumor. The activity of the targeted NP was compared with the naked NP (no PEGylation) and nontargeted NP (no ligand). The delivery efficiency of the targeted NP was 4-fold higher than the nontargeted NP and could be competed by excess free ligand. Luciferase siRNA was used to evaluate the gene silencing activity in the B16F10 cells, which were stably transduced with a luciferase gene, in a lung metastasis model. The gene silencing activity of the targeted NP was significantly higher than the other formulations and lasted for 4 days. While confocal microscopy showed the naked NP provided no tissue selectivity and non-targeted NP was ineffective for tumor uptake, the targeted NP effectively penetrated the lung metastasis, but not the liver. It resulted in 70-80% gene silencing in the metastasis model after a single i.v. injection (150 μg siRNA/kg). This effective formulation also showed very little immunotoxicity.

The influence of particle size and surface mannose modification on the uptake of liposomes by alveolar macrophages (AMs) was investigated in-vitro and in-vivo. Non-modified liposomes of five different particle sizes (100, 200, 400, 1000 and 2000 nm) and mannosylated liposomes with 4-aminophenyl-alpha-D-mannopyranoside (particle size 1000 nm) were prepared, and the uptake characteristics by rat AMs in-vitro and in-vivo were examined. The uptake of non-modified liposomes by rat AMs in-vitro increased with an increase in particle size over the range of 100-1000 nm, and became constant at over 1000 nm. The uptake of non-modified liposomes by AMs after pulmonary administration to rats in-vivo increased with an increase in particle size in the range 100-2000 nm. The uptake of mannosylated liposomes (particle size 1000 nm) by rat AMs both in-vitro and in-vivo was significantly greater than that of non-modified liposomes (particle size 1000 nm). The results indicate that the uptake of liposomes by rat AMs is dependent on particle size and is increased by surface mannose modification.

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