Abstract C137: Single-walled carbon nanotubes provide a safe and effective means for delivery of siRNA. (2011)

Abstract Carbon nanotubes have unique physical and chemical properties that are being widely explored for applications in biomedicine, especially as transporters of drugs, proteins, DNA and RNA. We have shown that siRNA/SWCNT complexes can be delivered safely to animals at high doses over an extended period with no weight loss or changes in blood hematology or chemistry, and produce excellent biological activity including target knockdown and antitumor activity. The current study aimed to prepare stable, optimized SWCNT solutions with consistent siRNA compositions and physical properties and explore cellular uptake. Atomic force microscopy revealed siRNA covers the SWCNT on a mass ratio basis and provided length distributions of the samples. Quantitation of siRNA payload on the SWCNT was achieved using electrophoretic separation of the siRNA from the SWCNT. The stability of siRNA/SWCNT was explored in ribonuclease at 37°C over time and found that siRNA in the complex was stable in 37°C with 80% remaining after 1 hr and 40% after 6 hr versus free siRNA where none remained in solution at 1 hr. Labeled Cy-3-siRNA whose fluorescence is quenched when complexed with SWCNT was used to determine whether the siRNA payload is released intracellularly from the SWCNT following delivery into cells. MiaPaCa pancreatic carcinoma and H2122 non-small cell lung cancer cells were exposed to Cy-3 siRNA/SWCNT for 30 min to 6 hrs. The cells were examined microscopically using near infrared fluorescence detection to observe intracellular SWCNT and visible fluorescence detection for intracellular Cy-3-siRNA. The siRNA/SWCNT complexes readily enter cells within 1 hr and the amount of intracellular SWCNT increase over time to 6 hr. Cy-3-siRNA was released within 1 hr and the siRNA is widely dispersed throughout the cytoplasm. Control cells showed no fluorescence under either condition and intact Cy-3-siRNA/SWCNT complex was not fluorescent due to quenching. We also used Cy-3 labeled siRNA to compare the transfection efficiency of siRNA/SWCNT to that of liposomal delivery. Using equivalent amounts of siRNA, SWCNT/siRNA complexes delivered and released siRNA intracellularly more quickly and distributed the siRNA more evenly within a 1 hr time frame as compared to liposomal delivery. Finally, in vivo delivery of Cy-3-siRNA/SWCNT appears to demonstrate a more prolonged and pronounced distribution of siRNA than that of free Cy-3 siRNA. The studies reported here provide data showing the stability of the SWCNT/siRNA complexes in vitro and in vivo and illustrate their cell penetrating ability with release of the payload intracellularly. We believe the low toxicity, the excellent membrane penetration ability, the protection afforded against blood breakdown of the siRNA payload and the good biological activity seen in vivo will allow SWCNTs to become universal transfection vehicles for siRNA and other RNAs for therapeutic applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C137.