Photodynamic Therapy Using Toluidine Blue O (TBO) Dye as a Photosensitizer against Leishmania major

Abstract

Background: Photodynamic therapy (PDT) is alternative treatment of cutaneous leishmaniasis (CL), and phenolthiazine dyes such as Toluidine Blue O (TBO) have the potential role in PDT and notably affect parasites inactivation. This study aimed to evaluate the effectiveness of PDT by using TBO and a light-emitting diode (LED) in the treatment of zoonotic CL (ZCL).

Methods: The study was conducted in Iran University of Medical Sciences, Tehran, Iran in 2018-2020. Different concentration (7.8 μg/mL up to 500 μg/mL) of TBO as a photosensitizer and a 630 nm LED light as a source of light were used for antileishmanial activity against both forms of Leishmania major promastigotes and intracellular amastigotes. Effective concentration (EC₅₀) and cell cytotoxicity (CC₅₀) were calculated in both infected and non-infected J774.A1 macrophages, respectively. As well as inhibitory concentration (IC₅₀) was quantified in L. major promastigotes for 2 h, 24 h, and 48 h after incubation using a MTT colorimetric assay.

Results: TBO dye in combination with the PDT significantly decreases the L. major promastigotes and intra-cellular amastigotes viability when compared with TBO alone. Both TBO dye in combination with the PDT and TBO alone had no toxic effects on the mice macrophages; however, it significantly killed the entered parasites inside the cells. Our results in the current study established satisfactory findings in clearing intracellular L. major parasites in in-vitro conditions.

Conclusion: TBO dye in combination with the PDT can be considered as a harmless, effective and importantly perfect treatment against L. major, causative agent of ZCL, in an in-vitro situation without any negative toxicity to the mice macrophages.

Keywords: In vitro; Leishmania major; Photodynamic therapy (PDT); Toluidine blue O (TBO).

Fig. 1:

Cytotoxicity assay (CC₅₀) of several concentrations (7.8 μg/ml up to 500 μg/ml) of TBO on J774A.1 macrophages cell line with and without illumination after 2 h (A), 24 h (B), and 48 h (C) using MTT method. All data have been reported as the mean ± SD of triple repeated experiments. CC₅₀ μg/ml was calculated for each groups by using dose response curve (Prism 8 software)

Fig. 2:

Inhibitory concentration (IC₅₀) of TBO (7.8 μg/ml up to 500 μg/ml) on promastigotes of L. major parasite with and without illumination after 2 h (A), 24 h (B), and 48 h (C) using MTT method. All data have been reported as the mean ± SD of triple repeated experiments. IC₅₀ μg/ml was calculated for each groups by using dose response curve (Prism 8 software)

Fig. 3:

Effective concentration (EC ₅₀) of several concentrations (7.8 μg/ml up to 500 μg/ml) of TBO on amastigotes of L. major parasite inside the J774A.1 macrophages with and without illumination after 2 h (A), 24 h (B), and 48 h (C) using MTT method. All data have been reported as the mean ± SD of triple repeated experiments. IC₅₀ μg/ml was calculated for each groups by using dose response curve (Prism 8 software)