Quatsomes as versatile fluorescent nanocarriers: stable Eosin Y loading and FRET with a membrane dye.

Fluorescent nanoprobes are key components in advanced bioimaging and optical sensing, enabling enhanced brightness, photostability, and multifunctionality beyond what is achievable with molecular dyes. Using multiple dyes in one nanocarrier boosts signal and enables multi-color imaging in a single system. However, the development of nanoplatforms capable of stably incorporating multiple fluorescent probes with different solubility and physicochemical properties remains a significant challenge. In this work, we demonstrate the efficient co-encapsulation of a hydrophilic dye (Eosin Y) and a hydrophobic dye (DiD) in quatsomes (QSs), a class of stable, non-liposomal nanovesicles composed of ionic surfactants and sterols. QSs are multifunctional nanocarriers of interest for drug delivery and bioimaging, since they can encapsulate molecules with different functionalities. Two different strategies are proposed for the loading of Eosin Y: (a) pre-assembly loading during the preparation of nanovescicles, and (b) post-assembly loading (incubation) of nanovescicles with a solution of the dye. While the hydrophobic probe DiD easily inserts into QS membranes thanks to its long alkyl chains, our results show that a hydrophilic dye like Eosin Y can also be efficiently and stably incorporated, even after vesicle formation. This opens the possibility of tuning the emission properties of QSs on demand, provided that suitable hydrophilic dyes with appropriate structural affinity for the QS system are employed. Moreover, Eosin Y and DiD are dyes compatible for Förster resonance energy transfer (FRET). FRET between two fluorophores on the same carrier provides a sensitive readout of nanoscale interactions, converting molecular proximity into a measurable optical signal. Together, these results position QS as a versatile and modular nanoplatform for multicolor bioimaging and optical sensing, combining stability with on-demand tunability of their emission properties.