Envision 2015

To assess changes in cytoplasmic calcium concentration, we used Fluo-4 AM (Beyotime, #S1060, China). We seeded 150,000 PC-12 cells in 1 mL of cell culture medium with 10% FBS in a 24-well plate (growth area 1.9 cm²) and incubated them overnight. The cells were then exposed to different concentrations of VCNCs, CNCs, VNCs, 5-HT, CAT, and HSA for 3 h, as specified in Additional file 1: Table S5. After exposure, we washed the PC-12 cells twice with 1 mL of PBS and stained them in 0.5 mL of FBS-free DMEM medium containing 0.2 μM Fluo-4 AM for 0.5 h. The cells were then washed twice with PBS and kept stained for another 0.5 h. The fluorescence of Fluo-4 was detected using a microplate reader (Envision@2015, PerkinElmer, USA) at excitation and emission wavelengths of 488 nm and 520 nm, respectively.

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.

The aMMP-8 level from mouth rinse samples was determined by a time-resolved immunofluorescence assay (IFMA) as described by Öztürk et al. [40 (link)]. Briefly, aMMP-8-specific monoclonal antibodies 8708 and 8706 (Actim Oy, Espoo, Finland) were used in the analysis as a catching antibody and a tracer antibody, respectively. In this protocol, the diluted samples were allowed to incubate for 1 h with the Europium labelled tracer antibody. The fluorescence was measured using an EnVision 2015 multimode reader (PerkinElmer, Turku, Finland).