Efficient oncogene silencing and metastasis inhibition via systemic delivery of siRNA

Abstract

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.

Figure 1. Immunohistochemical analysis of the lung metastasis

Immunohistochemical staining of MDM2, c-myc, and vascular endothelial growth factor (VEGF) in the B16F10 lung metastatic nodules after treatment with small interfering RNA (siRNA) in different formulations. Original magnification = ×100. NP, nanoparticle.

Figure 2. Western blot analysis of the tumor-loaded lung

Western blot analysis of MDM2, c-myc, and vascular endothelial growth factor (VEGF) in the B16F10 tumor-loaded lung after treatment with small interfering RNA (siRNA) in different formulations. NP, nanoparticle.

Figure 3. Antimetastasis efficacy of different small interfering RNA (siRNA) formulations

(a) Images of lungs excised from the tumor-bearing mice on day 17 after two consecutive treatments. (b) Photographs of the hematoxylin and eosin–stained tissue sections processed from the excised lungs. Original magnification = ×40. (c) Luciferase activity in the tumor-loaded lungs on day 17 after two consecutive intravenous (IV) injections of siRNA in different formulations on days 10 and 11. n = 4–9. ***P < 0.0001 as compared to the siRNA in phosphate-buffered saline (PBS) formulation. Formulations from left to right: untreated control (black), siRNA in PBS (red), siRNA in nontargeted nanoparticle (NP) (green), siRNA in targeted NP (purple), and control siRNA in targeted NP (blue). (d) Survival analysis of B16F10 lung metastases–bearing mice. Tumor-bearing mice were IV injected with different siRNA formulations on days 10, 11, 17, and 18. n = 10. *P < 0.05, ***P < 0.0001 as compared to the siRNA in PBS formulation. Formulations: untreated control (black), siRNA in PBS (red), siRNA in nontargeted NP (green), siRNA in targeted NP (purple), and control siRNA in targeted NP (blue).

Figure 4. Serum cytokine analysis

Serum cytokine levels of C57BL/6 mice 2 hours after receiving intravenous injections of small interfering RNA in targeted nanoparticle at different doses. The data are given as mean values ± SD, n = 4. *P < 0.05; **P < 0.01 as compared to the untreated control. IFN-α, interferon-α; IL, interleukin; TNF, tumor necrosis factor.