Optimizing the Fluorescence Properties of Nanoemulsions for Single Particle Tracking in Live Cells

Abstract

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 30×10 6 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 polymer-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.

Dye‐Loaded Nanoemulsions: Biomimetic Fluorescent Nanocarriers for Bioimaging and Nanomedicine

“…This dye showed a good molar extinction coefficient of 120 000 m −1 cm −1 (two times higher than Nile Red) and an increased quantum yield of 0.98 (similar to Nile Red). [63] The brightness of DBS fluorophores in oil was enhanced due to higher viscosity, [63] a phenomenon similar to that observed earlier by Texier et al using cyanines. [114] The developed DBS dyes showed highest brightness (quantum yield and molar extinction coefficient) in castor oil (with the highest viscosity) compared with other oils (e.g., MCT and VEA).…”

“…Recently, a novel dioxaborine dye named DBS-C 8 was developed and encapsulated into NEs with vitamin E acetate as liquid core (around 40 nm) at high concentration of 37 ×10 −3 m in oil without obvious aggregation caused quenching issue and a high quantum yield of 0.44. [63] Moreover, in comparison to NR668, it showed significantly improved photostability and single-particle brightness. These improved optical properties of fluorescent NEs enabled their single-particle tracking as well as determination of their trajectory and velocity in the cytoplasm (Figure 9).…”

“…[59] Oils based on D--tocopherol (vitamin E) have also been extensively used in the formulation of NEs. [60][61][62][63] Oleic acid and ethyl oleate have been used in oral, topical and parenteral NEs. [64,65] Alternatively, synthetic oils are also widely used, which include long-chain triglycerides (LCT), medium-chain triglycerides (MCT), or short-chain triglycerides (SCT) are used either alone or in combination for formulation of NEs.…”

“…[198][199][200] However, this scanning technique is slower than wide-field single-photon excitation which can easily provide video rate imaging of single molecules and NPs. [63,112,201] The key approach to improve signal to noise ratio is to use NPs of highest possible brightness. Up to now, most of particle tracking experiments in cells have been done using quantum dots, because they are 10-100 times brighter than single organic dyes and feature high photostability, which allows robust continuous recording of single-particle traces.…”

Further insights into release mechanisms from nano-emulsions, assessed by a simple fluorescence-based method

“…Compared to other types of nano-carriers, NEs have a real advantage to be a liquid reservoir for lipophilic active pharmaceutical ingredients (APIs), and thus, a priori, lipophilic fluorescent probes as models. This original feature has recently allowed the encapsulation of an outstanding number of fluorophores in a single droplet, making ultrabright nano-droplets enabling new possibilities such as single droplet tracking in cells or in small laboratory animals [6][7][8]. Although a large number of literature reports has shown that NE droplets themselves are intrinsically very stable, shown experimentally [9,10], and explained by the fact that droplets cannot enter in contact each other for steric reasons [11][12][13], the important question of the leakage of encapsulated APIs is still not clearly addressed.…”