Comparison of the effects of photodynamic exposure with the use of chlorine E6 on glioblastoma cells of the U251 line and human embryonic kidney cells of the HEK293 line in vitro

Abstract

Malignant gliomas of the brain are a global medical and social problem with a trend toward a steady increase in morbidity and mortality rates. A method that enables the visual identification of tumor tissue and simultaneously selectively destroys it is photodynamic therapy, which involves the introduction of a photosensitizer (PS) followed by its activation at a certain wavelength of light. The selectivity of the accumulation of PS in the tumor tissue of the malignant gliomas is one of the key issues in the problem of increasing the effectiveness of photodynamic therapy.

Objective: to compare the effects of photodynamic exposure using PS chlorin E6 on human glioblastoma (GB) cells of the U251 line and non-malignant human embryonic kidney cells of the HEK293 line.

Material and methods. Groups of cell cultures were formed depending on the conditions of cultivation and exogenous influence: 1) control - cultivated in a standard nutrient medium (Modified Eagle's Medium (MEM)) with L-glutamine, 1 mmol of sodium pyruvate, 10% fetal bovine serum) and experimental: 2) cultivated under the conditions of adding chlorin E6 (concentrations 1.0 and 2.0 μg/ml); 3) cultivated on a nutrient medium without the addition of PS and exposed to laser irradiation (LI) (λ=660 nm, power in the range 0.4-0.6 W, dose in the range 10-75 J/cm², continuous or pulse mode); 4) cultured under conditions of chlorin E6 addition and subsequent exposure to LI (power in the range 0.4-0.6 W, dose in the range 10-75 J/cm², continuous or pulse mode). After exposure to the specified experimental factors, dynamic observation with microphotographic registration was performed for 24 h, followed by microscopic and micrometric studies (number of viable cells, total number of cells, mitotic index (MI,%)).

Results. PS chlorin E6 is incorporated into the cytoplasm of cells of U251 and HEK293 cell lines, the intensity of fluorescence is comparable. Upon exposure to chlorin E6 (1.0 and 2.0 μg/ml), cytodestructive and antimitotic effects are increased in a dose-dependent manner in the culture of human GB cells of the U251 line. The cytodestructive effect of chlorin E6 on cell cultures of the HEK293 line is less pronounced, but the antimitotic effect is comparable in both types of cell cultures. Under the influence of LI, cytodestructive and antimitotic effects increase in a dose-dependent manner in the culture of human GB cells of the U251 line. The level of cytodestructive and antimitotic effects is significantly lower in the cultures of non-neoplastic HEK293 cells. The most significant drop in the mitotic activity of GB U251 cells (~100%) was recorded at the lowest LI dose of 25 J/cm², power of 0.6 W in pulse mode. For HEK293 cells, the most significant decrease in mitotic activity (~80%) was recorded at LI with a power of 0.6 W and dose of 75 J/cm² in continuous mode. Under the combined effect of chlorin E6 (1 and 2 μg/ml, pre-incubation of 4 h) and LI in different modes, the viability of tumor cells in U251 culture decreases in a dose-dependent manner; the smallest dose of LI to achieve the maximum cytotoxic effect is 25 J/cm², with a power of 0.6 W in pulse mode when using chlorin E6 at a concentration of 2 μg/ml. The specified characteristics of photodynamic exposure do not cause irreversible effects in HEK293 cultures (reference cells).

Conclusions. An effective mode of photodynamic exposure to achieve a cytodestructive and antimitotic effect in the culture of human GB cells of the U251 line, which is relatively safe for non-malignant cells, has been established: the combined application of a laser irradiation dose of 25 J/cm², with a power of 0.6 W in pulse mode during the preliminary incubation of the cell culture with chlorin E6 at a concentration of 2 μg/ml for 4 h.