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Biodistribution/Biostability Assessment of siRNA after Intravenous and Intratracheal Administration to Mice, Based on Comprehensive Analysis of In Vivo/Ex Vivo/Polyacrylamide Gel Electrophoresis Fluorescence Imaging.

We performed in vivo/ex vivo/polyacrylamide gel electrophoresis (PAGE) fluorescence imaging of near-infrared fluorescence (NIRF)-labeled siRNA (Cy5.5-siGL3) in mice to investigate the validity of each fluorescence imaging result as the biodistribution/biostability assessment of siRNA. Statistically significant correlations could be obtained between the in vivo and ex vivo fluorescence intensities of Cy5.5 in the relevant regions/tissues, except the lung region/tissue after intravenous administration. On PAGE fluorescence images with the naked formulation, there was no band corresponding to intact Cy5.5-siGL3 from all the tissues evaluated after intravenous administration, indicating that the fluorescence detected by in vivo and ex vivo fluorescence imaging was derived from degraded Cy5.5-siGL3 or free Cy5.5 cleaved from Cy5.5-siGL3. However, the band was detected from the lungs after intratracheal administration of the naked formulation, confirming higher stability of siRNA on the respiratory epithelium than in the blood. Regarding the polyethyleneimine formulation, the band was detected from all the tissues evaluated after intravenous administration and from the lungs after intratracheal administration, verifying the enhanced stability of siRNA in the body. These results clearly indicated the necessity of comprehensive analysis from in vivo/ex vivo/PAGE fluorescence imaging to precisely assess the distribution and stability of NIRF-labeled oligonucleotides including siRNA in the body.

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