Abstract 5672: Superb in vitro transfection efficiency and in vivo target gene silencing effects on systemic siRNA delivery by 10-nm scale pH-sensitive ultra-nanoparticles.
Authors: Wu Xin , Yamamoto Hirofumi , Uemura Mamoru , Haraguchi Naotsugu , Hata Taishi , Nishimura Junichi , Takemasa Ichiro , Mizushima Tsunekazu , Doki Yuichiro , Mori Masaki 
Published: August 2014

Introduction: A common hurdle of recent siRNA carriers, including liposomes and micelles with mean diameters of 70-100 nm, whose size is thought to be optimal for in vivo delivery is that systemic administration often results in accumulation in reticuloendothelial system (RES) and kidney. We have introduced a delivery system using 10-nm scale super apatite (sApa) ultra-nanoparticles, which consist of inorganic ions and are highly pH-sensitive, to improve in vitro siRNA transfection efficiency in colon cancer cells. Moreover we have successfully conducted the first in vivo systemic administration of sApa ultra-nanoparticles to reveal the biodistribution at 4 h &12 h and the anti-tumor efficacy. As results, sApa was unlikely to be trapped by the RES and kidney. The continuously systemic administration of sApa incorporating anti-survivin siRNA could suppress solid tumor growth. However, the mechanism for the high in vitro transfection efficiency has to be further investigated, and whether the down-regulated protein expression of target gene could be achieved still remain to be clarified. Here, we uncover the mechanism of in vitro transfection efficiency and confirm the in vivo anti-tumor efficacy by clarifying the protein expression level of target gene.

Methods: Intracellular siRNA measurement assay was performed in KM12sm colon cancer cell line, which was resistant to transfection using Lipofectamine. The differential cellular uptake was further analyzed with high-resolution confocal microscopy. As for the in vivo assay of accumulation in tumor, live imaging was performed at 90 min and day 2, and the in vivo western blotting assay was performed at day 3 and day 19 after the systemic administration of sApa incorporating fluorescently labeled anti-survivin siRNA.

Results & Discussions: Unlike Lipofectamine 2000, the fluorescently labeled siRNA transfected by sApa appeared in the cytoplasm at 4 h, and subsequently accumulated in the entire cytoplasm at 12 h. The enhanced pH-sensitivity of sApa could contribute to the high transfection efficiency to the cells that are resistant to transfection using Lipofectamine 2000. As early as 90 min after administration, in vivo two-photon live imaging clearly demonstrated that green fluorescent siRNA spread out from vasculature in a sApa-siRNA–treated tumor, but not in a naked-siRNA–treated tumor. In vivo western blotting assay of sApa-survivin-treated tumor revealed the protein expression was down-regulated at day 4, and fluorescent immunostaining of survivin protein in the tumor tissues at day 19 exhibited the suppressed protein expression, which supported the anti-tumor efficacy.

Conclusion: The sApa delivery system is of vital use to experimental approaches both in vitro and in vivo, and 10-nm scale ultra-nanoparticles may constitute a new avenue in nanoparticle-based medicine.

Citation Format: Xin Wu, Hirofumi Yamamoto, Mamoru Uemura, Naotsugu Haraguchi, Taishi Hata, Junichi Nishimura, Ichiro Takemasa, Tsunekazu Mizushima, Yuichiro Doki, Masaki Mori. Superb in vitro transfection efficiency and in vivo target gene silencing effects on systemic siRNA delivery by 10-nm scale pH-sensitive ultra-nanoparticles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5672. doi:10.1158/1538-7445.AM2013-5672