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We developed a lipid nanoparticle formulation (LNPK15) to deliver siRNA to a tumor for target gene knock down. LNPK15 is highly PEGylated with 3.3% 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-(polyethylene glycol-2000) (PEG-DSPE) and shows a long duration: the half-lives of siRNA in LNPK15 were 15.2 and 27.0h in mice and monkeys, respectively. Although LNPK15 encapsulating KRAS-targeting siRNA (LNPK15/KRAS) had very weak KRAS gene knock down activity in MIA PaCa-2 cells in vitro, LNPK15/KRAS showed a strong anti-tumor efficacy in MIA PaCa-2 tumor xenograft mice after intravenous administration at 5mg/kg twice weekly. KRAS mRNA and protein knock down was observed in tumor tissue, suggesting on-target anti-tumor efficacy. In order to elucidate the in vitro-in vivo discrepancy, we performed ex vivo knock down assay using serum samples obtained after intravenous administration of LNPK15/KRAS to mice and monkeys. The collected samples were added to MIA PaCa-2 cells, and KRAS gene knock down was evaluated after a 24-h incubation period. The knock down efficacy was weak (≈20%) with serum samples at initial sampling point (2h), and it became much stronger (∼90%) with serum samples at later time points. Lipid composition of LNPK15 in the serum samples was also investigated. Among the five lipids incorporated in LNPK15, PEG-DSPE was degraded more rapidly than siRNA and the other lipids in both mice and monkeys. In vitro lipase treatment of LNPK15/KRAS also hydrolyzed PEG-DSPE and enhanced knock down activity. From these results, it was concluded that LNPK15 acquires increased knock down activity after undergoing PEG-DSPE hydrolysis in vivo, and that is the key mechanism to achieve both long circulation and potent knock down efficiency. We also proposed an in vitro assay system using lipase for quality control of LNP to ensure biological activity.

To achieve delivery of doxorubicin (DXR), a very commonly used anticancer agent, to tumor tissues, it was intercalated to plasmid DNA to obtain a plasmid DNA/DXR complex. The cytotoxic effects of DXR, DNA and their complex were examined in colon26/Luc cells, a murine adenocarcinoma clone stably expressing firefly luciferase, co-cultured with RAW264.7 murine macrophage-like cells. Both CpG motif-containing plasmid DNA (CpG plasmid DNA) and DXR significantly inhibited the proliferation of colon26/Luc cells, but their complex was the most effective among those examined. Non-CpG plasmid DNA was less effective than the CpG plasmid DNA. When injected into mice bearing hepatic metastases of colon26/Luc cells, the CpG plasmid DNA/DXR complex produced a significant level of IL-12 in the serum and liver. The amount of DXR delivered to tumor tissues in the liver was greater when DXR was injected as a CpG plasmid DNA/DXR complex than as free DXR. The CpG plasmid DNA/DXR complex effectively inhibited the proliferation of colon26/Luc cells in the liver compared with free DXR, CpG plasmid DNA, or non-CpG plasmid DNA/DXR complex. These results indicate that CpG plasmid DNA is an effective polymer that inhibits tumor growth by delivering both a proinflammatory signal and anticancer agent to tumor tissues.