(a) Chemical structure of the PDT photosynthesizer TLD1433. (b) Jablonski diagram showing the formation of singlet oxygen (¹O₂) by irradiation of TLD1433 via initial population of metal-to-ligand charge transfer (MLCT) states and intersystem crossing to intra-ligand (IL, ILCT) states.

Cell viability after TLD1433 treatment of eight tumour cell lines (CRMM1, CRMM2, CM2005.1, OMM1, OMM2.5, MEL270, A431, A375). The green line shows TLD1433 activated by 520 nm light, 21 mW/cm², 19 J.cm⁻² (light-induced toxicity). The dark line shows TLD1433 treatment without light irradiation (dark toxicity). The tumour cells were treated with TLD1433 for 24 h with concentrations ranging from 0.001 µM to 5 µM and kept in the dark, or ranging from 0.0001 µM to 0.025 µM and illuminated with a light dose of 21 mW/cm², 19 J/cm². SRB assay was carried out at 48 h after light irradiation. The absorbance of Sulforhodamine B in solution was measured at 520 nm. Results are presented as means ± SD from three independent experiments with 95% confidence intervals.

Green light irradiation of TLD1433 induces apoptosis and necrosis in CRMM1 and CRMM2 cells. (A) CRMM1 and (C) CRMM2 were stained with Annexin-V-FITC and Propidium Iodide. The percentages of live, early apoptotic, later apoptotic and necrotic cells in CRMM1 (B) and CRMM2 (D) were counted by FACS. Results are presented as means ± SD from three independent experiments.

Light toxicity in zebrafish embryos. 2 dpf embryos (n = 30) were exposed to green light (21 mW/cm², 520 nm) for 0, 3, 6, or 12 h. (A) Transmitted light images of the embryos after light irradiation. (B–D) The percentage of mortality, malformation and fish length after various time of light exposure. Results represents the means ± SD from three independent experiments.

The maximum tolerated dose of TLD1433 in wild type zebrafish embryos administered through three different routes. (A) Schedule of TLD1433 treatment in wild type zebrafish. WA: TLD1433 (2.3 nM, 4.6 nM, 9.2 nM, 11.5 nM, 23 nM) were added to the water containing 10 embryos per well at 2.5, 3.5, 4.5, 5.5 dpf, for 12 h (yellow box). After these treatments, the drug was removed and replaced by egg water followed by 90 min green light irradiation (21 mW/cm², 114J.cm⁻², 520 nm), depicted as a green lightning bolt. IV or RO: 1 nL of TLD1433 (1.15 mM, 2.3 mM, 4.6 mM, 9.2 mM, 11.5 mM) were injected into the embryos at 3 dpf to 6 dpf every morning, followed by 60 min drug-to-light interval (yellow box) and 90 min green light irradiation (21 mW/cm², 114 J.cm⁻², 520 nm), depicted as a green lightning bolt. (B) WA, (C) IV, (D) RO. (B–D) Images were made of irradiated (light) and non-irradiated (dark) embryos (n = 30) at 6dpf and the percentages of mortality, malformation and fish length were calculated (shown as means ± SD from three independent experiments). Representative images of embryos under dark and light conditions are shown.

The maximum tolerated dose of TLD1433 in wild type zebrafish embryos administered through three different routes. (A) Schedule of TLD1433 treatment in wild type zebrafish. WA: TLD1433 (2.3 nM, 4.6 nM, 9.2 nM, 11.5 nM, 23 nM) were added to the water containing 10 embryos per well at 2.5, 3.5, 4.5, 5.5 dpf, for 12 h (yellow box). After these treatments, the drug was removed and replaced by egg water followed by 90 min green light irradiation (21 mW/cm², 114J.cm⁻², 520 nm), depicted as a green lightning bolt. IV or RO: 1 nL of TLD1433 (1.15 mM, 2.3 mM, 4.6 mM, 9.2 mM, 11.5 mM) were injected into the embryos at 3 dpf to 6 dpf every morning, followed by 60 min drug-to-light interval (yellow box) and 90 min green light irradiation (21 mW/cm², 114 J.cm⁻², 520 nm), depicted as a green lightning bolt. (B) WA, (C) IV, (D) RO. (B–D) Images were made of irradiated (light) and non-irradiated (dark) embryos (n = 30) at 6dpf and the percentages of mortality, malformation and fish length were calculated (shown as means ± SD from three independent experiments). Representative images of embryos under dark and light conditions are shown.

Development of a new conjunctival melanoma orthotopic tumour model in ZF. (A) Location of CM cell injection, (B) red fluorescent CRMM1 and (C) CRMM2 cells were injected RO into 2 dpf tg(Fli:GFP/Casper) (n = 10) and imaged by fluorescence microscopy at 1 and 4 days post injection (dpi). Relative tumour burden was calculated as fluorescent intensity and area of tumour cells by Image J. Results are means ± SD of three independent experiments.

Development of a new conjunctival melanoma orthotopic tumour model in ZF. (A) Location of CM cell injection, (B) red fluorescent CRMM1 and (C) CRMM2 cells were injected RO into 2 dpf tg(Fli:GFP/Casper) (n = 10) and imaged by fluorescence microscopy at 1 and 4 days post injection (dpi). Relative tumour burden was calculated as fluorescent intensity and area of tumour cells by Image J. Results are means ± SD of three independent experiments.

Treatment of zebrafish ectopic and orthotopic CM models with TLD1433 through WA. (A) Schedule of tumour injection and TLD1433 administration in zebrafish embryos. Fluorescent CRMM1 and CRMM2 cells were injected at 2 dpf into the Duct of Cuvier (ectopic model) and behind the eye (orthotopic model) and TLD1433 was administered with or without a light treatment following the schedule presented in A. Relative tumour burden estimated as fluorescence intensity and tumour area was calculated by Image J. (B) CRMM1 tumour burden in ectopic model (n ≈ 30). (C) CRMM1 tumour burden in orthotopic model (n ≈ 15). (D) CRMM2 tumour burden in ectopic model (n ≈ 30). (E) CRMM2 tumour burden in orthotopic model (n ≈ 15). Results are presented as means ± SD from three independent experiments. Representative images show CM tumour burden in the head and tail regions in the ectopic model and a localised tumour in the orthotopic model.

TLD1433 treatment by IV administration in the zebrafish ectopic and orthotopic CM model. (A) Schedule of tumour injection and TLD1433 administration in zebrafish embryos. Relative tumour burden was calculated as described in Figure 7. (B) CRMM1 tumour burden in the ectopic model (n ≈ 30). (C) CRMM1 tumour burden in the orthotopic model (n ≈ 15). (D) CRMM2 tumour burden in the ectopic model (n ≈ 30). (E) CRMM2 tumour burden in the orthotopic model (n ≈ 15). Results are presented as means ± SD from three independent experiments. Representative images show CM tumour burden in the head and tail regions in ectopic model and localised tumours in the orthotopic model.

TLD1433 treatment by RO administration in the zebrafish ectopic and orthotopic CM model. (A) Schedule of tumour injection and TLD1433 administration in zebrafish embryos. Relative tumour burdens were calculated as described in Figure 7. (B) CRMM1 tumour burden in the ectopic model (n ≈ 30). (C) CRMM1 tumour burden in the orthotopic model (n ≈ 15). (D) CRMM2 tumour burden in the ectopic model (n ≈ 30). (E) CRMM2 tumour burden in the orthotopic model (n ≈ 15). Results are presented as means ± SD from three independent experiments. Representative images show CM tumour burden in the head and tail regions in ectopic model and localised tumour in orthotopic model.

TUNEL assay of in CRMM1 and CRMM2 orthotopic model after RO of TLD1433. Red fluorescent CRMM1 (A) and CRMM2 (B) cells were injected at 2dpf behind the ZF eye and divided into four groups for drug treatment. RO administration of vehicle control and TLD1433 was performed as described in Figure 9C,E. After dark or light exposure (21 mW/cm², 114 J.cm⁻², 520 nm) embryos were fixed and TUNEL staining was performed. Representative images of embryos are shown in this figure. (A,B) In TLD1433 light groups nuclear DNA fragmentation by nucleases is detected by co-localization of green (DNA fragments) and red signal of engrafted CM cells, depicted as yellow signal and marked by white arrows. In control dark, control light, TLD1433 dark, there are no positive green apoptotic tumour cells observed. Background green signal in TLD1433 light groups, does not co-localized with cytosolic red signal, which is diminished in degraded cells and TUNEL stains only the DNA breaks in these CM apoptotic cells. Experiment was performed 3 times with a group size of 10 embryos.

Time line for the SRB assay.