Fig. 4: Synergistic therapeutic mechanism of IIT and 5-ALA-PDT.

Cytotoxicity of different NPs in a 293T cells and b 4T1 cells after co-incubation with CCP/CCPC/CCPA/CCPCA NPs for 24 h. The dark toxicity and phototoxicity of CCPA and CCPCA NPs with c A375 cells and d U87MG cells. Data represent the means ± SD (a–c, n = 3, d, n = 5). Statistical significance was calculated via two-tailed Student’s t test. e Annexin V-FITC/PI apoptosis analyses (FACS gating strategy) of A375 cells after the indicated treatments. Images are representative of three biologically independent replicates (n = 3). Q1, dead cells; Q2, late apoptotic cells; Q3, early apoptotic cells; Q4, live cells. The symbols “+” indicate laser irradiation. f Immunofluorescence images and corresponding quantitative FL intensity analysis of g anti-caspase-3 (green) and h anti-HIF-1α (red) in A375 cells after different treatments under hypoxic (1% O₂, 5% CO₂, and 94% N₂) conditions. A375 cells were incubated with blank DMEM, 5-ALA, CCPA, and CCPCA NPs for 24 h and then irradiation with a 635 nm laser (200 mW cm⁻², 5 min), scale bar = 50 μm. Images are representative of five independent replicates. Data represent the means ± SD (n = 5). Statistical significance was calculated via one-way ANOVA with Tukey’s multiple comparisons test. i The GSH level in A375 cells after incubation with free 5-ALA (5.4 µg mL⁻¹) and different NPs (80 µg mL⁻¹) for 24 h. Statistical significance was calculated via one-way ANOVA with Tukey’s multiple comparisons test. n = 3 independent experiments. j Intracellular FL images of ROS stained with DCFH-DA and k corresponding quantification, l JC-1 and m corresponding quantification after treatment with blank medium, 5-ALA, CCPA and CCPCA NPs (equivalent 5.4 µg mL⁻¹ 5-ALA) for 24 h and then irradiation with a 635 nm laser (200 mW cm⁻², 5 min), scale bar = 50 μm. Data represent the means ± SD (k, n = 5, m, n = 3). Statistical significance was calculated via one-way ANOVA with Tukey’s multiple comparisons test. n Synergistic mechanism of IIT and 5-ALA-PDT. Source data are provided as a Source data file.