Laboratoire de Bioimagerie et Pathologies, UMR 7021 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch, France.
ACS Nano. 2020 Oct 27;14(10):13924-13937. doi: 10.1021/acsnano.0c06348. Epub 2020 Oct 6.
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--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 -aggregation, reaching 2500000 M cm (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.
荧光纳米粒子（NPs）由于其亮度高，是有机染料的一种有吸引力的替代品。然而，由于其尺寸大、均一性差以及在生物介质中的非特异性相互作用，它们的细胞应用仍然受到限制。在此，我们提出了一个概念，即构建由单个两亲聚合物组成的高亮度和非常小尺寸（10-14nm）的单分子荧光有机纳米粒子，该聚合物带有专门设计的荧光染料。我们发现，聚（马来酸酐-1-十八烯（PMAO）的高度聚乙二醇化有利于单链聚合物折叠成单分子隐形纳米粒子，与蛋白质和活细胞的非特异性相互作用大大降低。为了确保我们的 NPs 具有高稳定性，荧光团（BODIPYs）通过优化的连接子共价连接到聚合物上。在所测试的不同长度和极性的连接子中，短的中等极性连接子有利于将染料定位在 NPs 界面上，从而确保良好的荧光量子产率和较小的颗粒尺寸。通过增加 BODIPY 染料的体积，这些 NPs 的荧光亮度得到了显著增强，达到了 2500000 M cm（消光系数×量子产率）。荧光显微镜显示，这些 NPs 的单颗粒亮度比使用相同激发波长（532nm）的 QDot-585 高约 5 倍。最后，当这些小而隐形的 NPs（10nm 直径）被微注射到细胞内时，它们在细胞质内的分布比 20nm QDot 更均匀，扩散程度与荧光蛋白相似。因此，开发的单分子 NPs 由于其尺寸小和隐形特性，是荧光蛋白的人工类似物，其亮度比后者高 50 多倍。
