Femtosecond pulsed laser photodynamic therapy activates melanin and eradicates malignant melanoma

Abstract

Photodynamic therapy (PDT) relies on a series of photophysical and photochemical reactions leading to cell death. While effective for various cancers, PDT has been less successful in treating pigmented melanoma due to high light absorption by melanin. Here, this limitation is addressed by 2-photon excitation of the photosensitizer (2p-PDT) using ~100 fs pulses of near-infrared laser light. A critical role of melanin in enabling rather than hindering 2p-PDT is elucidated using pigmented and non-pigmented murine melanoma clonal cell lines in vitro. The photocytotoxicities were compared between a clinical photosensitizer (Visudyne) and a porphyrin dimer (Oxdime) with ~600-fold higher σ_2p value. Unexpectedly, while the 1p-PDT responses are similar in both cell lines, 2p activation is much more effective in killing pigmented than non-pigmented cells, suggesting a dominant role of melanin 2p-PDT. The potential for clinical translational is demonstrated in a conjunctival melanoma model in vivo, where complete eradication of small tumors was achieved. This work elucidates the melanin contribution in multi-photon PDT enabling significant advancement of light-based treatments that have previously been considered unsuitable in pigmented tumors.

Keywords: melanin; melanoma; multiphoton; ocular melanoma; photodynamic therapy.

Fig. 1.

Pulsed-laser PDT responses. (A and B) Micrographs of pigmented and non-pigmented cells treated using Visudyne. (C–E) Normalized cell viability measured by cell morphology (means ± 1 SD, n = 9) for Visudyne (2.5 µM, 3 h incubation), Oxdime (10 µM, 1 h incubation), and corresponding light-only controls. (F) Fluences (Jcm⁻²) for 50% cell kill.

Fig. 2.

(A and B) Spectral fluorescence imaging of B16F10 cells after fs pulsed irradiation, showing variable inter- and intra-cellular melanin emission. Panel C shows the averaged spectra over five different individual cells. The overlap of the melanin fluorescence emission and the photosensitizer absorption spectra is evident in D and E.

Fig. 3.

Representative log–log plots of fluorescence emission as a function of pulsed laser power for individual cells. (A) B16F10 cells without Visudyne (melanin fluorescence only). (B) B16F10 cells with Visudyne (3 h incubation, 2.5 µM). (C) B78H1 cells with Visudyne (3 h incubation, 2.5 µM). The slopes (±1 SD) for the linear regression fits are listed for each cell.

Fig. 4.

Photosensitizer photobleaching (A–C and G–I) and ROS generation (D–F and G–I) during multi-photon PDT using 3 different average laser powers of 29, 42, and 55 mW at 865 nm for Visudyne (A–F) and 25, 36, and 48 mW for Oxdime (G–I). Each plot is normalized to the value following the first laser scan at a given power. The photobleaching rates (β, cm² J⁻¹) were calculated by fitting to a single exponential form, I = I₀ e^−βE. Error bars correspond to SD, n = 4 each over 50 cells.

Fig. 5.

H&E-stained sections of smaller pigmented tumors at 48 h post-treatment. Each row shows different sections for the same eye and black arrows indicate the tumor. (A) light-only, (B) 2p-PDT with 0.5 mg/kg Visudyne, (C) with 0.8 mg/kg Visudyne. Although some residual tumor is still present in A and B, complete ablation is seen at the highest photosensitizer concentration (C). (Scale bar indicates 2 mm.)