Evaluation of various tissue-clearing techniques for the three-dimensional visualization of liposome distribution in...

To develop a three-dimensional visualization method for evaluating the distribution of pulmonary drug delivery systems and compare four tissue-clearing techniques (Clear^T2, CUBIC, ScaleS, and SeeDB2) using intrapulmonary liposomes as drug carriers.

Rhodamine B-labeled liposomes were administered intrapulmonarily to mice using a MicroSprayer, and then fluorescent-labeled tomato lectin was administered intravenously to visualize the general lung structure. Tissue-clearing treatment of the mouse lungs was performed using the standard protocols of the Clear^T2, CUBIC, ScaleS, and SeeDB2 techniques. Lung clearing was clarified using laser-scanning confocal microscopy, and three-dimensional images were reconstructed.

Fluorescent-labeled tomato lectin was preserved using Clear^T2 and SeeDB2 but not using CUBIC and ScaleS. In addition, the liposomes were stable in Clear^T2 reagent, but they were mostly degraded in other reagents by surface-active agents. Clear^T2 treatment enabled the three-dimensional visualization of intrapulmonary rhodamine B-labeled liposomes at the alveolar scale.

These results suggest that the Clear^T2 tissue-clearing technique was appropriate for the three-dimensional visualization of intrapulmonary liposomes at the alveolar scale. This study provides important information for selecting and optimizing suitable optical tissue-clearing techniques in lungs for evaluating the distribution of pulmonary drug delivery systems.