Development of Ultrasound-Switchable Fluorescence Imaging Contrast Agents Based on Thermosensitive Polymers and Nanoparticles

In this paper, we first introduced a recently developed high-resolution, deep-tissue imaging technique, ultrasound-switchable fluorescence (USF). The imaging principles based on two types of USF contrast agents were reviewed. To improve USF imaging techniques further, excellent USF contrast agents were developed based on high-performance thermoresponsive polymers and environment-sensitive fluorophores. Herein, such contrast agents were synthesized and characterized with five key parameters: 1) peak excitation and emission wavelengths (λ<sub>ex</sub> and λ<sub>em</sub>); 2) the fluorescence intensity ratio between on- and off-states (I<sub>On</sub>/I<sub>Off</sub>); 3) the fluorescence lifetime ratio between on- and off-states (τ<sub>On</sub>/τ<sub>Off</sub>); 4) the temperature threshold to switch on fluorophores (T<sub>th</sub>); and 5) the temperature transition bandwidth (T<sub>BW</sub>). We mainly investigated fluorescence intensity and lifetime changes of four environment-sensitive dyes [7-(2-Aminoethylamino)-N,N-dimethyl-4-benzofurazansulfonamide (DBD-ED), St633, Sq660, and St700] as a function of temperature, while the dye was attached to poly(N-isopropylacrylamide) linear polymers or encapsulated in nanoparticles. Six fluorescence resonance energy transfer systems were invented in which both the donor (DBD-ED or ST425) and the acceptor (Sq660) were adopted. Our results indicate that three Förster resonance energy transfer systems, where both I<sub>On</sub>/I<sub>Off</sub> and τ<sub>On</sub>/τ<sub>Off</sub> are larger than 2.5, are promising for application in future surface tissue bioimaging by the USF technique.

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