Gardiquimod

The TLR7 agonist gardiquimod (Sigma) was dissolved in DMSO (Sigma) at a concentration of 100 μg/ml and stored in aliquots at −20°C. gardiquimod was added to cell cultures at a working concentration of 0.5 μg/ml. Antibodies for western blot include primary antibodies: rabbit anti-phospho-p65 (Ser536) (3033, Cell Signaling Technology), rabbit anti-p65 (4764, Cell Signaling Technology), rabbit anti-TLR7 (ab24184, Abcam), mouse anti-Lamp1 (H4A3, Santa Cruz Biotechnology), rabbit anti-IFN-α (YT5170, Immunoway), rabbit anti-GAPDH (AF1186, Beyotime) and secondary antibodies: HRP conjugated goat anti-rabbit IgG (H+L) (A0208, Beyotime), Alexa Fluor 594 goat anti-rabbit IgG (H+L) (A-11012, Thermo Fisher Scientific) and Alexa Fluor 488 goat anti-mouse IgG (H+L) (A-11017, Thermo Fisher Scientific). Antibodies for flow cytometry analysis were listed in Supplementary Table 1.

5 mg of gardiquimod (Sigma, St. Louis, MO, USA) with 60 mg of PLGA (Sigma) in 2 ml of dichloromythelene (DCM) was added to 10 ml of 2.5% poly (vinylalcohol) and sonicated for 2 minutes in a probe sonicator. DCM was allowed to evaporate overnight, by stirring the emulsion at 600 rpm using a magnetic stirrer. The nanoparticle suspension was centrifuged at 4,000 rpm for 3 min and the pellet was discarded to remove large particles. The supernatant was centrifuged at 13,000 rpm for 15 minutes followed by dispersion in 30 ml of distilled water. This process was repeated for three times. Nanoparticles were freeze-dried and stored until further use. For preparation of indocyanine green (ICG, Dongindang Pharm., Siheung, Korea)-encapsulated PLGA nanoparticles, 5 mg of ICG was added in the first step, and followed the same procedure as mentioned above. DMXAA solution was prepared by dissolving 5 mg of DMXAA (Sigma) in 1 ml of 5% sodium bicarbonate (Sigma) solution.