CC BY
30B-P-17
A study of activated macrophages in the accumulation of different photosensitizers and the PDT effect on their metabolic changes
Klementeva M.V.1'2, Sadykova E.Z. Skobeltcin A.S.1'2, Romanishkin I.D.2, Pominova D.V.1'2, Loschenov V.B.1'2, Ryabova A.V.1'2
1-National Research Nuclear University, MEPhI (Moscow Engineering Physics Institute), Moscow, Russia 2- Prokhorov General Physics Institute, Russian Academy of Sciences, Vavilov Str., 38, Moscow, 119991, Russia
klementevamv@yandex. ru
Introduction Antitumor immunity induced by photodynamic therapy (PDT) plays an essential role in the outcome of treatment, and it depends on the degree of inflammation. In this regard, understanding how immune cells such as macrophages, which are caused by inflammation, interact with different photosensitizer (PS) is great of interest. Here, we investigated accumulation of various PS, such as 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PplX), Photosens, Chlorin e6 and methylene blue (MB) in LPS-induced macrophages. Also, we examined the effects of PDT mediated by MB to detect phenotypic changes in M0, Ml and M2 macrophages.
Experiments The LPS-induced macrophages were derived from THP-1 monocytes cell line by addition 1 ^g/ml of LPS from Escherichia coli O55:B5 [1]. The cells were incubated with PS at next doses: 5-ALA -20 mg/kg, Photosens - 5 mg/kg, Chlorin e6 - 10 mg/kg and MB - 6 mg/kg. PS accumulation was estimated using a confocal microscopy (LSM-710, Carl Zeiss, Germany). M0 polarized macrophages were derived from THP-1 monocytes using PMA (Phorbol 12-myristate 13-acetate, 20nM). To obtain M2 polarization M0 macrophages were incubated with IL-13 20 ng/ml. To obtain M1 polarization M0 macrophages were incubated with IFN-gamma (20ng/ml) and LPS (100ng/ml) [2]. For PDT cells were irradiated with a laser 675 nm at doses 5 J/cm2 and 50 J/cm2. Changes in cell viability were analyzed with using Annexin V and propidium iodide staining. For macrophages phenotype changes staining CD206/CD86 was applied. Also, for all experiments fluorescence lifetime data was received using FLIM (Becker&Hickl, Germany, with 2P excitation at770 nm).
Results Significant differences in dye accumulation between LPS-activated and non-activated monocytes were demonstrated for PpIX. The intensity of the PpIX was higher in LPS-activated cells, while MB and Chlorine e6 showed higher values for cells without activation. A high dose of PDT led to a depolarization of all macrophages to the M2 type. For low-dose (5 J/cm2) PDT M0 macrophages turned to Ml phenotype, in other cases, the M2 type predominated slightly. For M1 macrophages, the apoptotic type of cell death prevails during PDT treatment with a dose of 5 J/cm2. For a dose of 50 J/cm2, necrosis predominates in all macrophage types.
Conclusion Low dose PDT can change macrophages polarization. Found, that 5-ALA induced PpIX accumulations in macrophages is the most sensitive to their polarization.
This work was supported by RFBR, № 20-02-00928.
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