FRET imaging revealed that nanocrystals enhanced drug oral absorption by dissolution rather than endocytosis: A case study of coumarin 6.

Nanocrystals (NCs) exhibit potential in improving oral bioavailability for poorly water-soluble drugs. However, whether NCs improve oral absorption by quick dissolution or by endocytosis remains inconclusive because tracking of dissolved drugs and NCs particles cannot occur simultaneously. In this study, we aim to elucidate how NCs improve oral absorption by using coumarin 6 (C6), an aggregation-caused quenching fluorophore, and 2-((5-(4-(dip-tolylamino)phenyl)thiophen-2-yl)methylene)malononitrile (MeTTMN), an aggregation-induced emission fluorophore. C6 was used as a model drug to prepare NCs and MeTTMN was incorporated to construct fluorescence resonance energy transfer (FRET) pairs. Thus, the molecular absorption can be detected using the fluorescence signal of dissolved C6 and the NCs particles can be tracked simultaneously by monitoring FRET signals. The reliability of this tracking method was validated. Accordingly, in vitro dissolution, gastrointestinal traffic, and biodistribution studies were conducted. The results showed that dissolved C6 molecules were the main absorption mode of C6 NCs. Identification of such pathways bears considerable significance for the broad application of drug NCs in improving the druggability of insoluble drugs.