Introduction: RNA interference technology is underway in clinical trials for limited diseases. RNAi technology for solid tumors, especially in systemic administration, still faces hurdles and has not emerged on the clinical stage. Here we introduce an in vivo pH sensitive delivery system of siRNA and microRNA using super carbonate apatite (sCA) nanoparticles (mean size: 10 nm), which simply consist of inorganic ions. In order to show the therapeutic superiority of the sCA system over currently available in vivo siRNA delivery systems, we perform further experiments comparing the sCA system with Invivofectamine 2.0 (IF) and Atelocollagen (Atelo). Finally we examine the toxicity of sCA nanoparticle system in mice and monkeys.
Methods: To assess the bio-distribution of fluorescently labeled siRNA in normal organs and tumor tissues, we used the IVIS Spectrum CT system (PerkinElmer) and fluorescence microscopy. In addition, the multiphoton live imaging system (SP5; Leica) and light sheet fluorescence microscopy (Carl Zeiss) were employed for detailed analysis and clear visualization of extravasation and intracellular distribution of fluorescent siRNA in tumor cells. To examine therapeutic potential of the sCA system, we administered survivin siRNA in pre-established HCT116 tumor mice models.
Results & Discussions: Ex vivo imaging analysis using IVIS Spectrum CT showed that the fluorescence of sCA-siRNA was highly accumulated into the tumors as early as 90 min. Light sheet fluorescence microscopy revealed that sCA-siRNA accumulated in the cytoplasm of HCT116 cells with considerable green fluorescence signals at 4 h after intravenous injection. On the other hand, IF-siRNA and Atelo-siRNA did not accumulate in tumor cells. sCA-survivin-siRNA exhibited anti-tumor effects on day 11 and the tumor volume of mice treated with sCA-survivin-siRNA was significantly smaller than in mice treated with sCA-control-siRNA (P = 0.0008), IF-survivin-siRNA (P = 0.0028), or Atelo-survivin-siRNA (P = 0.0046) on day 18. For in vivo safety, we carried out a toxicity study by intravenous injection of sCA to mice. Blood chemistry tests and HE stained sections of the liver, kidney and spleen showed no physiologically significant difference between the saline and sCA treated groups. To aim the final goal of therapy and diagnosis of human tumors, we made up approximately 70-fold sCA of that administered in mouse per injection and administered it to monkeys on 4 consecutive days (n = 3). Consequently intravenous administer of sCA did not cause any serious adverse events in monkeys.
Conclusion: We have demonstrated a highly efficient in vivo siRNA delivery system targeting solid tumor models, which is characterized as 10 nm minimal particle size and quick accumulation in tumors, thus it can realize to deliver abundant intact siRNA into tumor cells.
Citation Format: Hirofumi Yamamoto, Xin Wu, Susumu Miyazaki, Mamoru Uemura, Taishi Hata, Junichi Nishimura, Ichiro Takemasa, Tsunekazu Mizushima, Yuichiro Doki, Masaki Mori. Super carbonate apatite as simplified systemic nanoparticle carrier for therapy of solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4460. doi:10.1158/1538-7445.AM2014-4460