Investigations into novel metal-incorporated THPP photosensitisers for photodynamic therapy efficiency against HT-29 Colorectal Adenocarcinoma cells

Abstract

Background: Colorectal cancer is one of the most lethal cancers globally, requiring urgent attention. Research is moving away from current conventional anti-cancer therapeutics with highly toxic side effects that diminish the long-term quality of life towards targeted therapies that only deal with the tumour. Photodynamic therapy has gained attraction as a minimally invasive and tumour-selective phototherapy that utilises a photosensitising agent, oxygen, and specific light irradiance to produce reactive oxygen species and induce tumour cytotoxicity.

Aim: Our study aimed at investigating the coordination of Pd(II) and Cu(II) metals into the free base H2THPP core for photosensitiser efficiency in PDT when targeting colorectal cancer cells.

Methods: Pd(II)-THPP and Cu(II)-THPP were synthesised and characterised using UV-Vis and 1H NMR spectroscopy. Successful coordination of these metals led to the treatment of cultured HT-29 colorectal adenocarcinoma with the photosensitisers to investigate dark and irradiation toxicity. The cells were then subjected to Pd(II)- and Cu(II)-THPP PDT to determine IC50 and IC80 concentrations. Cellular homeostatic conditions and potential stress were investigated using alamarBlue, Trypan Blue, ATP and LDH assays. ROS production, mode of cell death and cell cycle were assessed using flow cytometric analyses.

Results: Pd(II)- and Cu(II)-THPP presented no toxicity in the dark nor irradiation toxicity. However, Cu(II)-THPP PDT had a stimulatory effect in HT-29 colorectal cancer and was discontinued, while Pd(II)-THPP IC50 and IC80 concentrations were determined to be 0.35 μM/mL and 0.9 μM/mL, respectively. Morphological and nuclear apoptotic and necrotic characteristics were observed microscopically and with the Hoechst-33258 stain. PDT produced ROS that disrupted the mitochondrial membrane, reducing cell viability and ATP production dose-dependently. Compromised membranes led to increased LDH release in the medium and phosphatidylserine presentation. Flow cytometric analysis showed induction of apoptosis and necrosis, and the formation of a sub-G1 indicating cell cycle arrest confirmed cell stress.

Conclusion: Cu(II)-THPP could not induce cytotoxicity in PDT treated colorectal carcinoma cells but can help gain future insight into the resistive mechanisms set in motion in cancer cells during PDT. On the other hand, Pd (II)-THPP is a potent inducer of cell death when combined with blue light irradiation and can be used in PDT as a substitute therapy to treat colorectal and other cancers.