Near-infrared fluorescence imaging has emerged as a noninvasive, inexpensive, and ionizing-radiation-free monitoring tool for assessing tumor growth and treatment efficacy. In particular, ultrasound switchable fluorescence (USF) imaging has been explored with improved imaging sensitivity and spatial resolution in centimeter-deep tissues. This study achieved the size control of polymer-based and indocyanine green (ICG) encapsulated USF contrast agents, capable of accumulating in the tumor after intravenous injections. These nanoprobes varied in size from 58 to 321 nm. The bioimaging profiles demonstrated that the proposed nanoparticles can efficiently eliminate the background light from normal tissue and show a tumor-specific fluorescence enhancement in the BxPC-3 tumor-bearing mice models possibly via the enhanced permeability and retention effect. In vivo tumor USF imaging further demonstrated that these nanoprobes can effectively be switched “ON” with enhanced fluorescence in response to a focused ultrasound stimulation in the tumor microenvironment, contributing to the high-resolution USF images. Therefore, our findings suggest that ICG-encapsulated nanoparticles are good candidates for USF imaging of tumors in live animals, indicating their great potential in optical tumor imaging in deep tissue.

One of the thorny problems currently impeding the applications of the fluorescence imaging technique is the poor spatial resolution in deep tissue. Ultrasound-switchable fluorescence (USF) imaging is a novel imaging tool that has recently been explored to possibly surmount the above-mentioned bottleneck. Herein, a β-cyclodextrin/indocyanine green (ICG) complex-encapsulated poly(N-isopropylacrylamide) (PNIPAM) nanogel was synthesized and studied for ex vivo/in vivo deep tissue/high-resolution near infrared USF (NIR-USF) imaging. To be specific, our results revealed that the average diameter of the as-prepared nanogels was significantly decreased to ~ 32 nm from ~ 335 nm compared to the reported ICG-PNIPAM nanoparticles. Additionally, the excitation/ emission characteristics of the ICG itself in present nanogels were almost completely retained, and the resultant nanogel exhibited high physiological stability and positive biocompatibility. In particular, the signal-to-noise ratio of the USF image for the PNIPAM/ β-cyclodextrin/ICG nanogel (33.01 ± 2.42 dB) was prominently higher than that of the ICG-PNIPAM nanoparticles (18.73 ± 0.33 dB) in 1.5-cm-thick chicken breast tissues. The NIR-USF imaging in 3.5-cm-thick chicken breast tissues was achieved using this new probe. The ex vivo NIR-USF imaging of the mouse liver was also successfully obtained. Animal experiments showed that the present nanogels were able to be effectively accumulated into U87 tumor-bearing mice via enhanced permeability and retention effects, and the high-resolution NIR-USF imaging of in vivo tumor was efficiently acquired. The metabolism and in vivo biodistribution of the nanogels were evaluated. Overall, the results suggest that the current nanogel is a highly promising NIR-USF probe for deep tissue and high-resolution USF imaging.