Photodynamic therapy (PDT) is one of the modern methods of oncotherapy widely used in clinical practice. In brief, the photodynamic effect is mediated by the sequential energy transfer from a photosensitizer (PS), excited by the light of a specific wavelength, to the oxygen molecule. This induces generation of a high concentration the reactive oxygen species (ROS), leading to damage, cell death by necrosis and apoptosis, vascular damage and activation of the antitumor immune response. In order for PDT to be effective the PS should have the following characteristics: (1) selective accumulation at the tumor tissue, (2) low dark toxicity, (3) high phototoxicity, (4) high absorption coefficients in the spectrum region of 700–800 nm, (5) chemical stability both in darkness and during irradiation, (6) chemical purity and accessibility [1], [2], [3]. Currently, a number of PDT agents are undergoing clinical trials or have already used in clinics for treating the tumors of skin [4], [5], [6], head and neck [7], [8], [9], bladder [10], lung [11,12], prostate [13], and other localizations [14].

Despite the success of PDT, new compounds are still being searched to improve the effective use of photodynamic therapy in clinical oncology. The derivatives of synthetic bacteriochlorin are promising PS characterized by enhanced selective accumulation in cancer cells, low dark cytotoxicity, and high photocytotoxicity [15]. They have characteristic three-band absorption spectra, the longest wavelength absorption band lying within the near-infrared region (700–800 nm). In this spectral range, also known as the “tissue optical window”, the intrinsic absorption of native biological tissue is minimal [15,16]. This enables deeper light penetration into a tissue and allows for the treatment of tumors with clinically relevant sizes and deep-seated tumors [17]. Furthermore, they exhibit lower tendency to aggregate and can be produced at a low cost [15].

To date, spheroids have proven to be one of the most appropriate models for testing antitumor drugs since they can better recapitulate the architecture and microenvironment of in vivo tumors [18], [19], [20]. In particular, the internal environment of spheroids, especially large ones, is heterogeneous and is developed due to the uneven distribution of oxygen, nutrients and metabolites. At the periphery, the spheroids contain a thin layer of actively proliferating cells, followed by a hypoxic cell layer and a necrotic central core [21,22]. Hypoxia in progressive solid tumors leads to heterogeneity forming cancer cell populations that are more resistant to ROS-dependent radiation [23]. The oxygen-dependent mechanism of underlying PDT cytotoxicity serves as an extra argument in favor of selecting spheroids for predicting the effects of PDT therapy in vivo. For example, Pereira at al. [24] showed that significantly more ROS is generated in the monolayer culture compared to spheroids and, thus, the phototoxicity of porphyrin strongly depended on the choice of the model.

Previously, we have shown that PS based on polycationic derivatives of the synthetic bacteriochlorin exert a high phototoxicity towards A549 cells due to the induction of necrosis and apoptosis in cancer cells including cancer cells with signs of stemness, accompanied by a decrease in mitotic and proliferative activity [15,25]. In this study, we compared the kinetics of the uptake and phototoxicity of the tetracationic bacteriochlorin derivative between the monolayer culture and spheroids derived from human hepatocellular carcinoma cells Huh7 and colon cancer cells HCT-116. We also investigated the effects of PDT on morphology and the mechanism of cell death in spheroids.