Fig. 3: In vitro characterization of FFs-ICG nanoNETs in antibacterial activity and macrophage polarization.

a Cytocompatibility assays (n = 6) of lysozyme monomers, FFs, and RFs using methyl thiazolyl tetrazolium (MTT) assay. b Quantification (n = 3) of released monomers from lysozyme FFs and RFs at 20 ^oC and 60 ^oC. RFs 20 ^oC vs. RFs 60^oC (p = 0.3398), FFs 20 ^oC vs. FFs 60^oC (p = 0.0196). Lysozyme activity assays (n = 3) of FFs-ICG at different concentrations after NIR irradiation (c) and RFs/FFs before and after NIR irradiation (d). RFs vs. FFs (p = 0.0583), RFs-ICG vs. FFs-ICG (p = 0.0031). e Schematic illustration for RFs/FFs bioactivity and thermally-induced disassembly. f Antibacterial efficiency assays (n = 3) of Lys, RFs, FFs, RFs-ICG and FFs-ICG against MRSA using CFU counting methods. Lys (p = 0.0001), RFs (p = 0.0013), FFs (p < 0.0001), ICG (p < 0.0001), RFs-ICG (p = 0.0004), FFs-ICG (p < 0.0001) vs. PBS control. SEM images (scale bar: 1 μm) (g) and live/dead bacteria staining confocal images (scale bar: 100 μm) (h) of MRSA strains after different treatments. i Inductively-coupled plasma mass spectrometry (ICP-MS) assays (n = 3) of released Mg²⁺ concentration before and after heating treatments. RFs 20 ^oC vs. FFs 20 ^oC (p = 0.0056), RFs 60 ^oC vs. FFs 60 ^oC (p = 0.0017). Flow cytometry analysis (n = 3) (j, k) and confocal microscopic images (scale bar: 50 μm) (l) of Raw 264.7 cells cultured with RFs, FFs, RFs-ICG, and FFs-ICG, with PBS and IL-4 as the negative and positive control, respectively. IL-4 (p = 0.0149), RFs (p = 0.0475), FFs (p = 0.1702), RFs-ICG (p = 0.0036), FFs-ICG (p = 0.0063) vs. PBS control. The data were expressed as mean ± standard deviation (S.D). Statistical significance was analyzed by one-way ANOVA using GraphPad Prism 8, followed by Tukey’s post hoc test for pairwise comparisons. Statistical significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001. Source data are provided as a Source Data file.