Gene Silencing in a Mouse Lung Metastasis Model by an Inhalable Dry Small Interfering RNA Powder Prepared Using the Supercritical Carbon Dioxide Technique

Abstract

In this study, a novel dry small interfering RNA (siRNA) powder for inhalation, containing chitosan and mannitol, was prepared using the supercritical carbon dioxide (CO 2 ) technique. Although the siRNA/chitosan powder was difficult to disperse because of a long needle-like structure, it could be reduced to fragments of 10-20 µm by manual grinding, which allowed for administration into mice. Electrophoresis revealed that the supercritical CO 2 technique and manual grinding didn't greatly affect the integrity of the siRNA. Furthermore, the siRNA was more stable in the lungs than in blood, suggesting the utility of pulmonary delivery. Biodistribution experiments using Cy5.5-labeled siRNA demonstrated that pulmonary administration of the powder achieved a prolonged exposure of the siRNA/chitosan complex on the lung epithelial surface at a higher concentration. For the evaluation of the in-vivo gene silencing effect of the siRNA/chitosan powder, mice bearing colon26/Luc cells were used. The powder significantly inhibited the increase in luminescence intensity in the lungs, but the siRNA/chitosan solution and a non-specific dry siRNA/chitosan powder didn't, indicating the effective and specific gene silencing against the tumor cells metastasized in the lungs of mice by the siRNA/chitosan powder. These results strongly indicate that inhalable dry siRNA powders have the possibility of effective pulmonary gene silencing and that the supercritical CO 2 technique can be applied to the production.Key words small interfering RNA; inhalable dry powder; supercritical fluid technique; pulmonary gene delivery; chitosan RNA interference (RNAi), first reported by Fire et al., is a unique cellular phenomenon in which double-stranded RNA (dsRNA) regulates gene expression, contributing to cellular defenses against viral infections and transposon expansion.

Histological Quantification of Gene Silencing by Intratracheal Administration of Dry Powdered Small-Interfering RNA/Chitosan Complexes in the Murine Lung

“…This powdering process was required to prepare siRNA/chitosan powders with good uniformity. In a previous study (10), we confirmed that the powdering process and dry grinding process had no effect on the particle size and Zeta potential of the siRNA/chitosan complex.…”

“…This effect was reported in a previous study (10); however, no studies have reported this effect using histological analysis. In the present study, we performed histological quantification of EGFP fluorescence in tumors of EGFP-LLC cells formed in the lung and found that intrapulmonary delivery of EGFPsiRNA/chitosan powder could reduce EGFP expression in tumors.…”

“…This approach has been reported in several other previous studies of multiple dosing regimens for pulmonary delivery of siRNA (10,11,13,14,23).…”

“…Preparation of siRNA/Chitosan Powder Using the Supercritical CO 2 Technique Dry powder of siRNA/chitosan was prepared as previously described (10). In brief, 50 mg of materials (composition ratio: siRNA 2%, chitosan 10%, mannitol 88%) was dissolved in 1 mL of water.…”

“…In a previous study, we used mannitol and low-molecular-weight chitosan as a nonviral carrier of nucleic acid and succeeded in preparing the dry powder of plasmid DNA (3,9) and siRNA (10) with this technique. Moreover, in another previous study (10), intratracheal administration of this siRNA/chitosan powder containing siRNA against luciferase was shown to effectively reduce the gross luminescence intensity in mouse lungs bearing metastatic tumors consisting of cells expressing firefly luciferase. This result indicated that intratracheal administration of the siRNA/chitosan powder is effective at silencing gene expression in the lung; however, the anatomical location in the lung where gene silencing occurs has not yet been determined.…”