Lipid nanocarriers are considered as promising candidates for drug delivery and cancer targeting because of their low toxicity, biodegradability and capacity to encapsulate drugs and/or contrasting agents. However, their biomedical applications are currently limited because of a poor understanding of their integrity in vivo. To address this problem, we report on fluorescent nano-emulsion droplets of 100 nm size encapsulating lipophilic near-infrared cyanine 5.5 and 7.5 dyes with a help of bulky hydrophobic counterion tetraphenylborate. Excellent brightness and efficient Förster Resonance Energy Transfer (FRET) inside lipid NCs enabled for the first time quantitative fluorescence ratiometric imaging of NCs integrity directly in the blood circulation, liver and tumor xenografts of living mice using a whole-animal imaging set-up. This unique methodology revealed that the integrity of our FRET NCs in the blood circulation of healthy mice is preserved at 93% at 6 h of post-administration, while it drops to 66% in the liver (half-life is 8.2 h). Moreover, these NCs show fast and efficient accumulation in tumors, where they enter in nearly intact form (77% integrity at 2 h) before losing their integrity to 40% at 6 h (half-life is 4.4 h). Thus, we propose a simple and robust methodology based on ratiometric FRET imaging in vivo to evaluate quantitatively nanocarrier integrity in small animals. We also demonstrate that nano-emulsion droplets are remarkably stable nano-objects that remain nearly intact in the blood circulation and release their content mainly after entering tumors.

Fluorescent nanoparticles (NPs), owing to their superior brightness, are an attractive alternative to organic dyes. However, their cellular applications remain limited because of their large size, poor homogeneity and non-specific interactions in biological media. Herein, we propose a concept of monomolecular fluorescent organic nanoparticle of high brightness and very small size (10-14 nm) built of a single amphiphilic polymer bearing specially designed fluorescent dyes. We found that high PEGylation of poly(maleic anhydride-alt-1-octadecene (PMAO) favors a single-chain polymer folding into monomolecular stealth NPs with highly reduced non-specific interactions with live cells. To ensure high stability of our NPs, the fluorophores (BODIPYs) are covalently linked to the polymer through an optimized linker. Among tested linkers of different lengths and polarity, short medium-polar linker favoring location of the dyes at NPs interface ensures good 2 fluorescence quantum yield and small particle size. The fluorescence brightness of these NPs has been dramatically enhanced by increasing the bulkiness of the BODIPY dyes that prevents their H-aggregation, reaching 2,500,000 M -1 cm -1 (extinction coefficient × quantum yield).Fluorescence microscopy revealed that the single-particle brightness of these NPs is ~5-fold higher than that of QDot-585 using the same excitation wavelength (532 nm). Finally, when microinjected inside cells, these small and stealth NPs (10-nm diameter) distribute more evenly than 20-nm QDots inside the cytosol, showing similar spreading as a fluorescent protein. Thus, the developed monomolecular NPs, owing to small size and stealth properties, are artificial analogues of fluorescent proteins, surpassing the latter >50-fold in terms of brightness.