Targeted Single Particle Tracking with Upconverting Nanoparticles.

Single particle tracking (SPT) is a powerful technique for real-time microscopic visualization of the movement of individual biomolecules within or on the surface of living cells. However, SPT often suffers from the suboptimal performance of the photon-emitting labels used to tag the biomolecules of interest. For example, fluorescent dyes have poor photostability, while quantum dots suffer from blinking that hampers track acquisition and interpretation. Upconverting nanoparticles (UCNPs) have recently emerged as a promising anti-Stokes luminescent label for SPT. In this work, we demonstrated targeted SPT using UCNPs. For this, we synthesized 30 nm diameter doped UCNPs and coated them with amphiphilic polymers decorated with polyethylene glycol chains to make them water-dispersible and minimize their nonspecific interactions with cells. Coated UCNPs highly homogeneous in brightness (as confirmed by a single particle investigation) were functionalized by immunoglobulin E (IgE) using a biotin-streptavidin strategy. Using these IgE-UCNP SPT labels, we tracked high-affinity IgE receptors (FcεRI) on the membrane of living RBL-2H3 mast cells at 37 °C in the presence and absence of antigen and obtained good agreement with the literature. Moreover, we used the FcεRI-IgE receptor-antibody system to directly compare the performance of UCNP-based SPT labels to organic dyes (AlexaFluor647) and quantum dots (QD655). Due to their photostability as well as their backgroundless and continuous luminescence, SPT trajectories obtained with UCNP labels are no longer limited by the photophysics of the label but only by the dynamics of the system and, in particular, the movement of the label out of the field of view and/or focal plane.

Fluorescent labeling of biocompatible block copolymers: synthetic strategies and applications in bioimaging.

Among biocompatible materials, block copolymers (BCPs) possess several advantages due to the control of their chemistry and the possibility of combining various blocks with defined properties. Consequently, BCPs drew considerable attention as biocompatible materials in the fields of drug delivery, medicine and bioimaging. Fluorescent labeling of BCPs quickly appeared to be a method of choice to image and track these materials in order to better understand the nature of their interactions with biological media. However, incorporating fluorescent markers (FM) into BCPs can appear tricky; we thus intend to help chemists in this endeavor by reviewing recent advances made in the last 10 years. With the choice of the FM being of prior importance, we first reviewed their photophysical properties and functionalities for optimal labeling and imaging. In the second part the different chemical approaches that have been used in the literature to fluorescently label BCPs have been reviewed. We also report and discuss relevant applications of fluorescent BCPs in bioimaging.

Ultrabright Green-Emitting Nanoemulsions Based on Natural Lipids-BODIPY Conjugates.

Nanoemulsions (NEs) are water-dispersed oil droplets that constitute stealth biocompatible nanomaterials. NEs can reach an impressive degree of fluorescent brightness owing to their oily core that can encapsulate a large number of fluorophores on the condition the latter are sufficiently hydrophobic and oil-soluble. BODIPYs are among the brightest green emitting fluorophores and as neutral molecules possess high lipophilicity. Herein, we synthesized three different natural lipid-BODIPY conjugates by esterification of an acidic BODIPY by natural lipids, namely: α-tocopherol (vitamin E), cholesterol, and stearyl alcohol. The new BODIPY conjugates were characterized in solvents and oils before being encapsulated in NEs at various concentrations. The physical (size, stability over time, leakage) and photophysical properties (absorption and emission wavelength, brightness, photostability) are reported and showed that the nature of the lipid anchor and the nature of the oil used for emulsification greatly influence the properties of the bright NEs.

Tunable functionalization of nano-emulsions using amphiphilic polymers.

Nano-emulsions are defined as stable oil droplets sizing below 300 nm. Their singular particularity lies in the loading capabilities of their oily core, much higher than other kinds of carrier. On the other hand, functionalizing the dynamic oil/water interface, to date, has remained a challenge. To ensure the best anchoring of the reactive functions onto the surface of the droplets, we have designed specific amphiphilic polymers (APs) based on poly(maleic anhydride-alt-1-octadecene), stabilizing the nano-emulsions instead of surfactants. Aliphatic C18 chains of the APs are anchored in the droplet core, while the hydrophilic parts of the APs are poly(ethylene glycol) (PEG) chains. In addition, PEG chains are terminated with reactive (i) azide functions in order to prove the concept of the droplet decoration with clickable rhodamine (Rh-DBCO, specifically synthesized for this study), or (ii) biotin functions to verify the potential droplet functionalization with fluorescent streptavidin (streptavidin-AF-488). This study describes AP synthesis, physico-chemical characterization of the functional droplets (electron microscopy), and finally fluorescence labeling and droplet decoration. To conclude, these APs constitute an interesting solution for the stable functionalization of nano-emulsion droplets, paving a new way for the applications of nano-emulsions in targeting drug delivery.

Near infrared fluorogenic probe as a prodrug model for evaluating cargo release by nanoemulsions.

Nanoemulsions (NEs) are biocompatible and stealth nanodroplets that can efficiently encapsulate hydrophobic cytoactive drugs in their oily core. NEs were shown to accumulate in tumors by enhanced permeability and retention (EPR) effect and thus display appealing features as nanocarriers to selectively deliver drugs to the tumors. However, to ensure efficient encapsulation with minimal early release, drugs must possess a high degree of lipophilicity. To circumvent this limitation, the latter could be transformed into prodrugs with enhanced hydrophobicity. In return, once delivered in the cell, the prodrug must be efficiently transformed into its active drug form. Herein we chemically and reversibly modified a near infrared Huda dye (HD) into pro-fluorophore (Pro-HD), a non-fluorescent and lipophilic prodrug model that was efficiently loaded in NEs. Thanks to the fluorogenecity of the system (fluorescence enhancement of 35-fold at 723 nm), we demonstrated that Pro-HD did not leak out of NEs, was efficiently delivered into cancer cells and was transformed in cellulo into HD. This proof of concept demonstrates the high potential of lipophilic "pro-fluorophore" approach for visualizing delivery of cargos using NEs as nanocarriers.

Lipid-core/polymer-shell hybrid nanoparticles: synthesis and characterization by fluorescence labeling and electrophoresis.

Among the lipid nanoparticles, lipid polymer hybrid nanoparticles (HNPs) composed of an oily core and a polymeric shell display interesting features as efficient drug carriers due to the high loading capability of the oil phase and the stability and surface functionalization of the polymer shell. Herein, we formulated lipid-core/polymer-shell hybrid nanoparticles (HNPs) using a simple nanoprecipitation method involving Vitamin E Acetate (VEA) as the oily core and a tailor-made amphiphilic polymer as a wrapping shell. The fluorescence labeling of the oil, using a newly developed green fluorogenic BODIPY tracker, and of the polymer using a covalent attachment of a red emitting rhodamine was done to assess the formation, the composition and the stability of these new hybrid nanoparticles using dual color electrophoresis gel analysis. This technique, combined to conventional DLS and electronic microscopy analysis, allowed us to quickly determine that 20 wt% of the polymer was an optimal ratio for obtaining stable HNPs by nanoprecipiation. Finally, we showed that using different polymeric shells, various HNPs can be obtained and finely discriminated using a combined approach of electrophoresis and two-color labeling.

Probing Polarity and Heterogeneity of Lipid Droplets in Live Cells Using a Push-Pull Fluorophore.

Lipid droplets (LDs) are organelles composed of a lipid core surrounded by a phospholipid monolayer. Lately, LDs have attracted considerable attention due to recent studies demonstrating their role in a variety of physiological processes as well as diseases. Herein we synthesized a push-pull molecule named DAF (Dimethyl Aniline Furaldehyde) that possesses a strong positive solvatochromism in emission of 119 nm from toluene to methanol. Its impressive fluorogenic properties from water to oil (2000-fold) as well as its high quantum yields (up to 0.97) led us to investigate its ability to sense the distribution of polarity in live cells by fluorescence ratiometric imaging. When added to live cells and excited at 405 nm, DAF immediately and brightly stain lipid droplets using a blue channel (410-500 nm) and cytoplasm in a red channel (500-600 nm). DAF also proved to be compatible with fixation thus allowing 3D imaging of LDs in their cytoplasm environment. Taking advantage of DAF emission in two distinct channels, ratiometric imaging was successfully performed and led to the polarity mapping of the cell unraveling some heterogeneity in polarity within LDs of the same cell.