A Compensated Förster Resonance Energy Transfer-Based Platform for Quantitative Assessment of Liposome Integrity in Inhalation Drug Delivery

The structural integrity of inhalable liposomal carriers is a key determinant of drug release behavior, pulmonary residence time, and therapeutic efficacy. This study aimed to establish a compensated Förster resonance energy transfer (FRET)-based platform for the quantitative assessment of liposome integrity throughout the inhalation delivery process. By compensation for donor fluorescence bleed-through and direct acceptor excitation, the method enables accurate quantification of FRET signals from liposomes co-loaded with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate (DiD, donor)/1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR, acceptor), using both spectrofluorometry and macroscopic imaging. Upon treatment with Triton X-100, decreases in the compensated FRET/donor ratio were detected at lower concentrations than those required to induce measurable changes in membrane anisotropy and within the same concentration range as the onset of encapsulated 6-carboxyfluorescein release. Using this platform, in vitro aerosol characterization with an Andersen cascade impactor enabled simultaneous analysis of aerodynamic particle size distribution and stage-specific liposome integrity. In vivo experiments in mice-including ex vivo lung imaging and bronchoalveolar lavage fluid analysis-revealed a time-dependent decline in liposome integrity during pulmonary residence. This ability to monitor carrier structural stability from initial deposition through residence-an aspect not readily achieved with conventional single-fluorophore labeling-offers significant advantages for formulation development, stabilization strategies, and dosing regimen optimization. The FRET-based platform could, in principle, be adapted for use with other standardized cascade impactors such as the Next Generation Impactor and is expected to be applicable to a wide range of lipid-based or polymeric nanocarriers, including inhalable vaccines and nucleic acid therapeutics.