In this study, we examined the usefulness of a tissue-clearing technique for the evaluation of the lung distribution of aerosolized drugs. An aerosol formulation of TexasRed dextran (70 kDa), a model compound of drug carrier for aerosolized drugs, was administered intrapulmonarily to mice using a MicroSprayer, and then DyLight 488-conjugated tomato lectin was administered intravenously to visualize general lung structure via the fluorescent labeling of alveolar and bronchial epithelial cells. Tissue clearing followed by laser scanning confocal microscopy enabled the three-dimensional visualization of intrapulmonary TexasRed dextran and the evaluation of its distribution at the alveolar scale without the preparation of thin tissue sections. These findings suggest that tissue-clearing techniques are useful for the evaluation of intrapulmonary distribution and development of pulmonary drug delivery systems.Key words pulmonary drug delivery system; intrapulmonary pharmacokinetics; Clear
T2; laser scanning confocal microscopy; alveolus; alveolar epithelial cell Respiratory diseases such as lung cancer, pulmonary fibrosis, pulmonary emphysema, and respiratory infections cause considerable morbidity and mortality worldwide.1-3) Effective therapy of these diseases requires the selective, efficient, and safe delivery of drugs to the focus site via drug delivery systems such as the intrapulmonary administration of aerosolized drugs. Various drug carriers for the pulmonary delivery to the focus site have been investigated. [4][5][6][7] As part of the development processes of these systems, drug delivery efficiency in the lungs must be evaluated via visualization at the alveolar scale. Although X-ray computed tomography (CT), positron emission tomography (PET), and single-photon emission computed tomography (SPECT) enable the three-dimensional (3D) visualization of lung structure and the distribution of contrast agents administrated intrapulmonarily on a macroscopic tissue scale, 8) visualization at the alveolar scale for the evaluation of drug distribution at focus sites is impossible with present technologies owing to their low resolution. On the other hand, evaluating drug distribution with micrographs of thin tissue sections of the lungs is neither as technically completely nor as accurate owing to the loss or distortion of sections that become torn or folded.Optical tissue-clearing techniques have been developed for the depth-independent visualization and 3D imaging of fluorescent proteins and dyes in large tissues without the preparation of thin tissue sections.9,10) These techniques enable whole-organ imaging at a single-cell resolution using by laser scanning confocal microscopy. However, no studies have assessed the applicability of these techniques for the evaluation of drug distribution in tissues. In the present study, we examined the usefulness of a tissue-clearing technique (Clear T2 ) 11) for evaluating the intrapulmonary distribution of aerosolized drugs. Mouse Lung Sample Preparation The mice were anestheti...