Intracellular NO measurement

The NO release from the B@SP–C NPs-treated HUVECs was measured using the commercial NO fluorescent probe, DAF-FM DA. DAF-FM DA can cross the plasma membrane and be cleaved by esterases to generate intracellular DAF-FM, which is then oxidized by NO to a triazole product with increased fluorescence. Typically, HUVECs were cultured in complete EGM-2 medium at 37 ^oC in a CO₂ incubator. The HUVECs were seeded in confocal cell culture dishes. After 24 h, the cells were incubated with B@SP–C NPs (0.1 mg mL^‒1 based on PTIIG) for 12 h. Then cells were washed three times with PBS buffer. As followed, the NO probe DAF-FM DA (the final concentration was 5 μM) was added and incubated at 37 ^oC for 30 min, and washed three times with PBS. The cells were irradiated with a 1064 nm laser (1 W cm^‒2) for 5 min to trigger the NO release. Finally, the generation of NO from B@SP–C NPs-treated HUVECs was detected by imaging cells using confocal fluorescence microscopy (LSM 800 with Airyscan, ZEISS, Germany) at excitation and emission wavelengths of 495 and 515 nm, respectively. Statistical analysis and graphing were done with ZEN 2012 (Version 1.1.2.0).