Fig. 3: 5-ALA and CAT co-delivery by CCPCA NPs upregulates PpIX accumulation in vitro.

a Increased intracellular O₂ induced PpIX accumulation through a heme negative feedback mechanism. b FL images of RDPP and c corresponding quantitative analysis after incubation with different NPs (CCP, CCPC, CCPA, CCPCA) at equivalent concentrations (80 µg mL⁻¹) for 24 h, scale bar = 100 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. d Quantitative analysis of H₂O₂ levels in A375 cells after viaious treatments. Statistical significance was calculated via one-way ANOVA with Tukey’s multiple comparisons test. n = 3 independent experiments. e FL images and f corresponding quantitative analysis of PpIX in A375 cells after exposure to equivalent amounts of free 5-ALA (5.4 µg mL⁻¹) or 80 µg mL⁻¹ NPs (CCP, CCPC, CCPA, CCPCA), scale bar = 100 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. g Intracellular PpIX content in the lysate after 24 h incubation with free 5-ALA (10.8 µg mL⁻¹) or NPs (160 µg mL⁻¹). Statistical significance was calculated via one-way ANOVA with Tukey’s multiple comparisons test. n = 4 independent experiments. h Real-time FL quantitative analysis of PpIX metabolism after incubation with free 5-ALA (10.8 µg mL⁻¹) or NPs (160 µg mL⁻¹). Data represent the means ± SD (n = 7). Statistical significance was calculated via one-way ANOVA with Tukey’s multiple comparisons test. i HIF-1α, FECH, and ALAS expression in A375 cells determined by western blotting. n = 3 independent experiments. j Immunofluorescence images and k quantitative FL intensity analysis of anti-α-tubulin (green) and anti-HIF-1α (red) in A375 cells after different treatments under hypoxic (1% O₂, 5% CO₂, and 94% N₂) and normoxic (21% O₂, 5% CO₂, and 74% N₂) conditions, respectively. Scale bar = 50 μm. Images are representative of six independent replicates. Data represent the means ± SD (n = 6). Statistical significance was calculated via two-tailed Student’s t test. Western immunoblots analysis of HIF-1α expression levels in A375 cells treated with CCPCA NPs at different l time (treated with 80 µg mL⁻¹ CCPCA NPs) and m concentrations (treated for 24 h). n = 3 independent experiments. Source data are provided as a Source data file.