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.

The simulator is simple to construct and can schematically reproduce human inspiratory flow patterns. Testing with a TSLI and the simulator is useful to evaluate dry powder formulations for clinical application.