Complexos polipiridinícos de rutênio(II) como fotossensibilizadores na terapia fotodinâmica: avaliação química, fotoquímica e em cultura celular

Abstract

Cancer has become a disease of great concern worldwide, accounting for almost 10 million deaths in 2020 and ranked among the “top 10” causes of death according to the World Health Organization (WHO). Considering the difficulties in conventional cancer treatments (chemotherapy, radiotherapy and surgery), such as tumor resistance and side effects, Photodynamic Therapy (PDT) is a procedure that has received special attention in medicine, mainly due to its minimally invasive procedure, no side effects and insignificant resistance to photosensitizers (PS). Among the PS classes investigated for PDT, Ruthenium(II) polypyridine complexes have interesting chemical, photophysical and biological properties, including high production of reactive oxygen species (ROS) and photostability. This work studied the interaction of cis-[Ru(dmbpy)2Cl(bpy)](PF6) and cis-[Ru(dmbpy)2Cl(bpe)](PF6) complexes with biomolecules such as protein (represented by BSA) and lipids (represented by liposomes, DMPC), estimated the amount of incorporation and toxicity (cytotoxicity and phototoxicity) in cervical cancer (HeLa) and breast cancer (MDA-MB-231) cells. The interaction studies of the compounds with protein (represented by BSA) carried out in Tris buffer had the results affected by the exchange of the Cl- ligand by Tris, making the interpretation of the data inviable. Therefore, the BSA-complex interaction studies were conducted in PBS buffer, whose binding constant (Kb) values were around 105 for both compounds (Rubpy ≈ 3x105 M-1 and Rubpe ≈ 2x105 M-1), indicating strong interaction between complex and BSA protein. The evaluation of photoinduced oxidation of BSA by Ru complexes was monitored by the decay time of albumin fluorescence intensity. The decay time (t2) was around 2 to 4 minutes in the presence of complex, and ~55 minutes in the absence of them, indicating that photoinduced oxidation by complexes (Rubpy and Rubpe) accelerates BSA photodegradation circa of 10x. Note that there was no significant difference in Kb or t2 values between the Rubpy and Rubpe compounds studied, suggesting that the addition of an ethylene group to one of the Rubpe ligands is not sufficient to increase the interaction and/or rate of protein photooxidation (represented by BSA). On interaction studies of the complexes with lipids (represented by DMPC liposomes), both Ru complexes studied showed a high binding constant value (Kb > 105 M-1), also indicating a strong interaction of these compounds with the lipid bilayer. To assess the rate of lipid oxidation photoinduced by Ruthenium(II) complexes, it was used an assay that monitors the leakage of a fluorescent probe (carboxyfluorescein, CF) to the outside of liposome vesicles. However, the results were inconclusive due to overlapping in the spectrum of the absorption band of the compounds and the emission of the CF probe, making data analysis unfeasible. Regards to % uptake of compounds in HeLa and MDA-MB-231 cells, the % uptake of the complexes was approximately ≈ 50% in HeLa and ≈ 37% in MDA-MB-231, with no statistically significant difference in the incorporation rate between the Rubpy and Rubpe. Regarding the toxicity of the compounds in tumor cells, [Ru(dmbpy)2Cl(bpe)](PF6) has an IC50 value approximately 2.4 times lower than [Ru(dmbpy)2Cl(bpy)](PF6) for both tumor cell lines (HeLa and MDA-MB-231), therefore Rubpe is more cytotoxic than Rubpy. Phototoxicity was measured by the photoinhibition factor (PIF), showing that only [Ru(dmbpy)2Cl(bpy)](PF6) in MDA-MB-231 has probable phototoxicity.