Fig. 1. Synthesis of IR780 nanoplatform and its application for NIR-II fluorescence/PA bimodal imaging and image-guided surgery/adjuvant PTT combined therapy.

Fig. 2. (a) Representative TEM image and TEM size distribution of IR780 loaded liposomes. (b) DLS analysis of IR780 loaded liposomes. (c) UV-Vis spectra of IR780 ethanol solution, IR780 loaded liposomes, and blank liposomes, respectively. The inset shows the corresponding photographs of these samples. (d) NIR-II fluorescence images (1000 nm long-pass filter, exposure time 10 ms) of EP tubes filled with ethanol solution (bottom) and liposomes (top) of IR780. The color bar ranges from -498.7 to 4248.1. (e) NIR-II fluorescence spectra of IR780 dispersed in ethanol and liposomes. (f) Temperature curves of IR780 loaded liposomes at different concentrations under irradiating at 808 nm laser (1 w/cm2). (g) Viabilities of A431 and NIH3T3 cells treated with IR780 liposomes at different concentrations. (h) H&E-stained slices of major organs from mice treated with PBS (top) and IR780 liposomes (bottom), where signals from hematoxylin (blue) and eosin (red) were composited (Scale bar: 100 μm). Means ± SD, n = 3.

Fig. 3. In vivo NIR-II fluorescent imaging and photoacoustic imaging. (a), (c) NIR-II fluorescent images (1000 nm long-pass filter, exposure time 10 ms) and photoacoustic images of mice with A431 tumors injected with IR780 liposomes by tail vein under 808 nm laser excitation. (b), (d) Quantitative analysis of fluorescent and photoacoustic intensity from the tumor site at predetermined time points, respectively. Means ± SD, n = 3.

Fig. 4. (a) Representative in vivo NIR-II fluorescent images (1000 nm long-pass filter, exposure time 10 ms) of nude mice bearing A431 tumors after intravenous injection of IR780 liposomes under 808 nm laser excitation. The color bar ranges from 0 to 30000 and corresponds to NIR-II images at 0.17-48 h. The white circle indicates the tumor site, and the white rectangle points out the small intestine site. (b) NIR-II fluorescence average intensities of tumor, liver, and background regions presented in panel (a) during the imaging period. (c) T/B and L/B fluorescence intensity ratios in mice bearing A431 tumor after injection of IR780 liposomes under 808 nm laser excitation (*** p < 0.001). (d) Representative ex vivo NIR-II fluorescence and white light images of tumors and organs from nude mice bearing A431 tumor at 48 h post-injection of IR780 liposomes. The color bar ranges from 0 to 35000, and the scale bar represents 2 mm. (e) Quantitative fluorescence intensities corresponding to tumors and organs shown in panel (d) (*** p < 0.001). Means ± SD, n = 3.

Fig. 5. Intraoperative NIR-II fluorescence imaging (1000 nm long-pass filter, exposure time 10 ms) guided surgery at 48 h post the intravenous injection of IR780 liposomes. (a) White light image and (b) NIR-II fluorescence image of the mice bearing A431 tumor before tumor resection. The white circle indicates the tumor site. (c) Quantitative analysis of the TNR of signals from white light image and NIR-II fluorescence image, respectively. (d) Step-wise resection of the tumor under the guidance of NIR-II fluorescence imaging resulting in complete removal of the tumor. The color bar ranges from -716 to 46758. (e) NIR-II fluorescence signal in the tumor region decreased dramatically after resection. (f) Quantitative analysis of TNR and SBR from NIR-II fluorescence imaging (*** p < 0.001). Means ± SD, n=3.

Fig. 6. In vivo combination anti-tumor effect of IR780 liposomes (160 μL, 250 μg/mL IR780, tail vein injection). The power density of the laser at 808 nm was 1 w/cm2 for 5 min per mouse. (a) In vivo IR thermal images and (b) temperature curves of A431 xenografts with different treatments (surgery only, Surgery+PTT, or PTT only) under laser irradiation for 5 min. (c) Typical decay-corrected whole-body PET/CT images of nude mice with A431 tumor before and after different treatments. The transaxial (bottom) and coronal (top) images are overlaid with CT (gray) and PET (golden) images, and the tumors are circled by the white dashed line. The recurrent tumor is also pointed out with white arrows. (d) Tumor region-calculated biodistribution of 18F-FDG in mice at the 2nd, 6th, 15th, and 23rd-day post-therapy. (e) The metabolically active tumor volume calculated with PET imaging on mice with different treatments on the 2nd, 6th, 15th, and 23rd-day post-therapy. Means ± SD, n = 3.