Optimizing the Fluorescent Properties of Nano-emulsions for Single Particle Tracking in Live Cells.

Nano-emulsions (NEs) are biocompatible lipid nanoparticles composed of an oily core stabilized by a surfactant shell. It is acknowledged that the surface decoration with poly(ethylene glycol) (PEG), through the use of non-ionic surfactants, confers a high stealth in biological medium with reduced non-specific interactions. Tracking individual NEs by fluorescence microscopy techniques would lead to a better understanding of their behavior in cells and thus require the development of bright single particles with enhanced photostability. However, the understanding of the relationship between the physicochemical properties and chemical composition of the NEs, on the one hand, and its fluorescence properties of encapsulated dyes, on the other hand, remains limited. Herein, we synthesized three new Dioxaborine Barbituryl Styryl dyes (DBS) that displayed high molar extinction coefficients (up to 120,000 M-1.cm-1) with relatively low quantum yields in solvents and impressive fluorescence enhancement when dissolved in viscous oils (up to 0.98). The reported screening of 9 different oils allowed disclosing a range of efficient "oil/dye" couples, and understanding the main parameters that lead to the brightest NEs. We determine vitamin E acetate / DBS-C8 as the representative most efficient couple combining high dye loading capabilities and low aggregation induced quenching leading to <50 nm ultrabright NEs (as high as 30×106 M-1.cm-1) with negligible dye leakage in biological media. Beyond a comprehensive optical and physicochemical characterization of fluorescent NEs, cellular two-photon excitation imaging was performed with poly-mer-coated cell penetrating NEs. Thanks to their impressive brightness and photostability, NEs displaying different charge surfaces were microinjected in HeLa cells and were individually tracked in the cytosol in order to study their relative velocity.