RESUMO
The combination of fluorogenic probes (fluorogens) and self-labeling protein tags represent a promising tool for imaging biological processes with high specificity but it requires the adequation between the fluorogen and its target to ensure a good activation of its fluorescence. In this work, we report a strategy to develop molecular rotors that specifically target HaloTag with a strong enhancement of their fluorescence. The divergent design facilitates the diversification of the structures to tune the photophysical and cellular properties. Four bright fluorogens with emissions ranging from green to red were identified and applied in wash-free live cell imaging experiments with good contrast and selectivity. A HaloTag mutant adapted from previous literature reports was also tested and shown to further improve the brightness and reaction rate of the most promising fluorogen of the series both inâ vitro and in cells. This work opens new possibilities to develop bright chemogenetic reporters with diverse photophysical and biological properties by exploring a potentially large chemical space of simple dipolar fluorophores in combination with protein engineering.
Assuntos
Corantes Fluorescentes , Engenharia de Proteínas , Corantes Fluorescentes/química , HumanosRESUMO
We have designed a new Bodipy scaffold for efficient in vivo photoacoustic (PA) imaging of nanoparticles commonly used as drug nanovectors. The new dye has an optimized absorption band in the near-infrared window in biological tissue and a low fluorescence quantum yield that leads to a good photoacoustic generation efficiency. After Bodipy-initiated ring-opening polymerization of lactide, the polylactide-Bodipy was formulated into PEGylated nanoparticles (NPs) by mixing with PLA-PEG at different concentrations. Formulated NPs around 100 nm exhibit excellent PA properties: an absorption band at 760 nm and a molar absorption coefficient in between that of molecular PA absorbers and gold NPs. Highly improved photostability compared to cyanine-labeled PLA NPs as well as innocuity in cultured macrophages were demonstrated. After intravenous injection in healthy animals, NPs were easily detected using a commercial PA imaging system and spectral unmixing, opening the way to their use as theranostic agents.