Rosup reagent

To assess the anti-inflammatory properties of nanocapsules after passing through bEnd.3 cells, we investigated their ability to scavenge ROS. 2ʹ,7ʹ –dichlorofluorescein diacetate (DCFH-DA) is commonly used to detect intracellular ROS levels. To induce intracellular ROS production, we added the ROSUP reagent (1:1000, Beyotime, #S0033S, China) to PC-12 cells in serum-supplemented medium for 0.5 h. The cells were pre-incubated with bEnd.3 cells containing nanocapsules in the transwell filter for t_exp + t_inc = 3 h + 24 h. The PC-12 cells were then washed twice with PBS and incubated with 0.5 mL of 0.1% DCFH-DA (Beyotime, #S0033S, China) diluted in serum-free DMEM medium for 0.5 h at 37 °C. The cells were then washed twice with PBS and exposed to 0.5 mL of FBS-free DMEM medium. The fluorescence of DCFH was measured using a microplate reader (Envision@2015, PerkinElmer, USA) and an inverted fluorescence microscope (Eclipse Ti2, Nikon, Japan) at excitation and emission wavelengths of 488 nm and 520 nm, respectively. To investigate whether phenolic hydroxyl groups in 5-HT polymerization influences ROS concentration, we performed additional experiments using the same methods as described above. Further details of these experiments are provided in the supporting information.

To investigate the cellular release of 5-HT monomers from nanocapsules, PC-12 cells were seeded in 24-well plates with a surface area of 1.9 cm² at a density of 150,000 cells per well in a volume of 1 mL. The following day, pH 6.4 medium was prepared by mixing 1 M HCl and serum-supplemented medium to obtain the medium at pH 6.4. The ROSUP medium was prepared by diluting 1 μL of ROSUP reagent (Beyotime, #S0033S, China) with 1 mL of complete DMEM medium. Cells were incubated with VCNCs, CNCs, and VNCs at a dose of C_5-HT = 25 μg/mL in complete DMEM media, pH 6.4 media, ROSUP media (1:1000), and pH 6.4 + ROSUP media for 3 h. After incubation, the supernatant above the cells was collected, and the cells were washed twice with PBS. The cells were detached from the 24-well plate using 0.05% trypsin/EDTA in a volume of 0.1 mL. After aspirating the trypsin, 0.3 mL of medium was added to collect the cell pellets, which were then centrifuged at 400g for 8 min and washed twice with PBS to obtain the cell pellet. Each sample was added to 0.1 mL of PBS, and a handheld homogenizer was used to crumb the cell samples. The supernatant and cell samples were then analyzed using a 5-HT Elisa Kit (Elabscience, #E-EL-0033c, China), following the manufacturer's instructions.