Mechanism of photodynamic therapy on Leishmania amazonensis parasites using Soranjidiol and white LED

DIMMER JESICA; MARIONI JULIANA; RIVAROLA HECTOR WALTER; NUÑEZ MONTOYA SUSANA

Rosario

Otro; 7ma edición de Ricifa 2023; 2023

Ricifa

Soranjidiol (Sor) is a natural compound obtained from Heterophyllaea genus and has demonstrated photosensitizing properties. Previously, Sor exhibited activity against promastigotes and amastigotes of Leishmania amazonensis, even reducing the lesion size in infected Balb/c mice. The photodynamic therapy involves the use of a photosensitizer (PS), proper light, and oxygen to produce reactive oxygen species (ROS) to induce cellular death. The aim of this work was to study the production of superoxide anion (O2.-) and reactive nitrogen species (RNS), the activation of superoxide dismutase (SOD), and their correlation to cellular death mechanisms (apoptosis/necrosis).A suspension of 5x106 parasites/mL was incubated with the following conditions in darkness: negative control (NC, treated with PBS), Sor 5 and 10 µM in PBS. Amphotericin B 0.5 µM was used as positive control. Irradiated samples include negative control of light (NCL) and Sor at the same concentrations (Sor-L5 and Sor-L10). Solutions were mixed with the parasites, and 10 min later they were irradiated with a light dose of 11 J/cm2 for 15 min. After irradiation, the trypan blue dye exclusion method was used to measure cell viability (CV), the production of O2.- and RNS were measured by NBT assay and Griess reaction respectively, and the SOD activation was determined based on inhibition of NBT reduction. Finally, the death mechanism was established by flow cytometry, using FICT-Annexin/IP. All these assays were repeated 24 h after treatment. Photoactivation of Sor reduced parasite burden immediately post-PDT, showing a decrease of 50 % at the highest concentration tested. However, 24 h after the treatment, the effect of PDT evidenced a decrease in cell viability in a dose-dependent manner: 70% for Sor-L5 and more than 90% for Sor-L10. These findings strongly correlate with ROS and RNS production and SOD activity, which were highly increased 24 h post-treatment. The production of O2.- was increased 264 times for Sor-L5 and 615 times for Sor-L10 regarding NC (p<0,001). Due to the elevated amounts of ROS produced and as detoxification mechanisms, it was observed on the one hand a high production of RNS (4.7 times and 11.6 times, respectively, regarding NC p<0,001), and for the other a SOD activation at both concentrations (3 and 10.5 times, respectively, regarding NC p<0.001). Under darkness, CV was not reduced by Sor treatment at both concentrations. Therefore, no ROS, RNS, or SOD activation was detected in the dark, confirming that PDT causes oxidative and nitrosative stress in the parasite. On the other hand, evaluation of cell death by Annexin-V (A-V) and propidium iodide (PI) stain revealed apoptosis for Sor-L10 immediately post-PDT (A-V: 3,6%), which correlates with low ROS and RNS production and could probably be a direct effect of singlet oxygen (1O2) generation by Sor irradiated. In addition, 24 h after treatment, it was observed apoptosis for Sor-L5 (A-V: 9%; A-V/IP: 3.2%) and necrosis for Sor-L10 (IP: 15.3 %) probably due to the activation cascade of ROS and RNS. These results also evidence that the death mechanism is significantly influenced by PS concentration.In conclusion, we postulate that immediately post-PDT parasites death could be mediated by 1O2 (PDT-type II mechanism) inducing apoptosis, and we confirm that 24 h after PDT, ROS and RNS (PDT-type I mechanism) induce cell death by apoptosis or necrosis in a dose-dependent manner.