Fig. 8: In vitro photothermal efficacy and photoinduced cytotoxicity.

a Photothermal heating curves of H4-PEG-PT with different concentrations (200 μL) at a laser power of 1.6 W cm⁻². b H4-PEG-PT (75 μM, 200 μL) over several ON/OFF cycles involving irradiation with an 808 nm laser (2.0 W cm⁻²) for 80 min followed by passive cooling. c Cell viability of 143B cells after incubation with various concentrations of H4-PEG-PT and exposure to different laser power densities (n = 3 biologically independent samples). d Cellular ATP production of 143B cells treated with H4-PEG-PT w/o 808 nm laser irradiation, CCCP was the positive control (n = 4 or 5 biologically independent samples). The ATP level of control was 100%. e Mitochondrial membrane potential quantified by JC-1 fluorescence assay of 143B cells treated with H4-PEG-PT w/o 808 nm laser irradiation, CCCP was the positive control (n = 5 or 6 biologically independent samples). f Mitochondrial permeability transition (MPT) study of H4-PEG-PT after 808 nm laser irradiation using calcein-cobalt assay. H₂O₂ treatment is the positive control (n = 4 biologically independent samples). g–i Caspase-3/8/9 activities measured by caspase multiplex activity assay kit in different groups (n = 3 biologically independent samples). j Representative western blot analysis of AIF, EndoG, and Smac/Diablo in different groups. k Representative western blot analysis of Caspase-9, Caspase-3, cleaved Caspase-3 (or active Caspase-3), and cleaved Caspase-9 (or active Caspase-9) after photothermal therapy under 808 nm excitation for H4-PEG and H4-PEG-PT, respectively. l Relative absorption intensity at 410 nm of DPBF as a functional of 808 nm light radiation time (DPBF alone, incubated with H4-PEG-PT with different concentrations, respectively). m–p Absorption spectra of DPBF solution (DPBF alone, or incubated with H4-PEG-PT with different concentrations) under 808 nm irradiation for different time. q Confocal images of 143B cells at 12 h after 3k-PEG, H4-PEG, and H4-PEG-PT treatments in combination with 808 nm laser irradiation. Mitochondria were stained with MitoTracker Green, whereas Cyto c (red fluorescence) was detected using immunofluorescence technique. Scale bar: 10 μm. r Confocal fluorescence microscopic images of 143B cells incubated with H4-PEG-PT and MitoSOX according to the treatment variables. Scale bar: 10 μm. s, t Apoptosis or necrosis of 143B cells determined by (s) Annexin V-FITC/PI staining or (t) Hochest/PI staining (scale bar: 50 μm). Statistical significance was calculated with two-tailed Student’s t test (c–i). Data are presented as mean values ± SD (c–i). Results in (j), (k), (q–t) are representative of three independent experiments.