Assessing reactive oxygen species generation during sonophotodynamic activity using chemical probes

Sonophotodynamic therapy (SPDT) is a non-invasive anticancer therapeutic technique that relies on the interaction of two external energy sources, low-intensity ultrasound, and visible light, with a sonophotoactive molecule and the molecular oxygen present in the medium. This interaction induces both mechanical effects (e.g., generation of microjets, and shock waves) and chemical effects (e.g., generation of reactive oxygen species (ROS)), leading to antitumor responses in deeper body regions. Protoporphyrin IX (PpIX), Methylene Blue (MB), and Chlorin e6 (Ce6) are well-known organic molecules that can be activated with light and ultrasound, making them promising candidates as sonophotosensitizers for SPDT. However, there are few studies about the ROS promoted by such sensitizers during a sonophotodynamic activity. So, this study aims to assess the singlet oxygen and hydroxyl radical production for photodynamic, sonodynamic, and sonophotodynamic activity employing PpIX, MB, and Ce6 as sensitizers. For this purpose, DPBF and APF, chemical probes highly sensitive to ¹O₂ and •OH, respectively, were used. The probe+sensitizer solutions were exposed to light (630nm, 12mW/cm2), ultrasound (1Mhz, 1.5W/cm2, 50%) and a combination of both sources. The generation of ¹O₂ and •OH was detected by monitoring the absorption spectrum of the DPBF+sensitizer solution and the fluorescence spectrum of the APF+sensitizer solution, respectively, during the irradiation time. The quantification of ¹O₂ and •OH generation was carried out by calculating the DPBF decay constant and the fluorescence rate constant of fluorescein (fluorescent product generated by the interaction of APF with •OH), respectively. Contrary to studies with light, it was observed that DPBF was degraded, and fluorescein generation increased with ultrasound irradiation, even in the absence of the sensitizer. This has been attributed to a sonochemical reaction whereby •OH molecules can be generated. Therefore, this effect was subtracted from the ¹O₂ quantification calculations. The DPBF decay constant promoted by a sonophotodynamic activity using PpIX was slightly higher than photodynamic activity. The fluorescein fluorescence constant induced by the three molecules was significantly higher under the sonophotodynamic activity. These results suggest that PpIX is an efficient sonophotosensitizer for ¹O₂ generation, while the three studied molecules were efficient for •OH generation under the sonophotodynamic activity. Also, the •OH generation could be the predominant action mechanism during a sonophotodynamic activity. However, further research is needed to thoroughly understand these results.