Stealth and Bright Monomolecular Fluorescent Organic Nanoparticles Based on Folded Amphiphilic Polymer

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 nonspecific interactions in biological media. Herein, we propose a concept of monomolecular fluorescent organic nanoparticles 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 nonspecific interactions with proteins and 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, a short medium-polar linker favoring location of the dyes at NPs interface ensures good 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 2500000 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 their small size and stealth properties, are artificial analogues of fluorescent proteins, surpassing the latter >50-fold in terms of brightness.