Advances on photodynamic therapy through new pyridine-fused diphenylchlorins as photosensitizers for melanoma treatment

Aim: Assessment of cytotoxicity of four new photosensitizers intended for photodynamic therapy (PDT) against melanoma cells (A375 cells).

Introduction: Melanoma is the rarest form of skin cancer. PDT combines a photosensitizer with light culminating in the production of reactive oxygen species leading to cellular death. A new type of stable 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused tetraphenylchlorins1, proved to be very active as photodynamic agents. Thus, looking for a new generation photosensitizers with optimized properties for PDT we synthesized new diphenylchlorins.

Methods: The human melanoma cell line A375 was seeded in 48 well plates. The photosensitizers NAMP103A, NAMP103B (the tetraphenylchlorins monoester), NAMP263A and NAMP263B (the tetraphenylchlorins alcohol) were administered ranging 5 nM to 10 μM. Irradiation was performed after 24 h (λ < 560 nm). MTT and SRB assays as well as flow-cytometry were performed 24 h after the PDT.

Results: MTT assay results allowed to obtain dose-response curves and to calculate the concentration that inhibits the cultures by 50% (IC50). Phototoxicity (10J) was dependent on the chlorines concentration. Moreover, NAMP263B was significantly more cytotoxic than NAMP103A (p = 0.037) and NAMP103B (p = 0.042). From SRB assay we verified that with a 125 nM concentration the NAMP103A, NAMP103B, NAMP263A and NAMP263B produce a cellular viability of 36.9%; 33.2%; 18.3% and 18.8%, respectively. Flow cytometry studies confirmed the decrease of viability associated with cell death by apoptosis and necrosis. Loss of mitochondrial membrane potential, apoptosis hallmark, was also observed. An imbalance of ROS, namely superoxide anion and peroxides, was observed for all photosensitizers studied with an exhaustion of antioxidative intracellular defenses (GSH).

At low PS concentrations (5 nM), metabolic activity was variable with light energy (5 J, 10 J and 20 J) with lower values for higher fluence. Dark toxicity studies revealed photosensitizer dependence of irradiation.

Conclusion: We hereby conclude that the photosensitizers are indeed very promising, which rouses plans for following proceedings to verify in vivo outcome.

Acknowledgements: FCT Portugal: Project PTDC/QEQ-MED/0262/2014, Strategic Project Pest CNC.IBILI UID/NEU/04539/2013 and Strategic Project Coimbra Chemistry Centre UID/QUI/00313/2013, COMPETE-FEDER.^1,2