Background: Microvessels in tumor tissue play a crucial role in meeting the metabolic needs of transformed cells, controlling the entry of xenobiotics into tumor tissue, and regulating local inflammation that promotes metastasis. Methylene blue has photosensitizing properties and can also affect dysfunctional mitochondria. Methods: The study was performed on the primary culture of CECs. The cells underwent photodynamic treatment through 660 nm laser irradiation at a power density of 300 mW/cm². The MTT, TMRE, and TUNEL assays were used to assess the survival, redox metabolism, mitochondrial activity, and apoptosis of CECs. Additionally, the metabolic activity of cells was evaluated using FLIM by measuring the fluorescence lifetime of NADH and FAD. Results: When CECs were incubated with MB, there was an increase in mitochondrial activity that was dependent on the concentration of MB. Additionally, mitochondrial activity increased when the CECs were exposed to 660 nm laser irradiation at an energy dose of up to 5 J/cm². Following PDT, a slight shift towards oxidative phosphorylation was observed. Conclusions: In vitro application of MB accumulation or laser irradiation causes a shift in the redox status of CECs towards increased reducing activity, without causing any cell damage. However, the combined action of PS and laser radiation has the opposite effect on the redox status of cells, resulting in an increase in the oxidized form of FAD.

Despite advances in cancer treatment, head and neck squamous cell carcinoma (HNSCC) remains a serious clinical problem due to tumor aggressiveness, tumor resistance to therapy, and treatment toxicity. The combination of photodynamic therapy (PDT) with chemotherapy is a promising approach to improve efficacy while reducing side effects. For the first time, the possibility and antitumor effect of the combined use of PDT and chemotherapy with intra-arterial administration of chlorin e6 (Ce6) and cisplatin in patients with HNSCC were assessed. Two patients with locally advanced HNSCC received intra-arterial administration of Ce6 (at a dose of 0.5 mg/kg) and cisplatin (at a dose of 50 mg/m²) via a catheter into the tumor-feeding artery followed by laser irradiation. Ce6 distribution, tumor response, and treatment efficacy were assessed by fluorescence diagnostics, confocal microscopy, and histopathological analysis. Intra-arterial administration of the photosensitizer (PS) and chemotherapeutic agent ensured high selectivity of their tumor accumulation. Fluorescence diagnostics showed rapid and selective Ce6 accumulation in the tumor and PS photobleaching after PDT. For a patient with three PDT sessions, there is a significant acceleration of the Ce6 spread from the tumor-feeding artery throughout the tumor bed with each therapy session. This is a good sign of a tumor stroma density decrease. The combined use of PDT and chemotherapy with intra-arterial administration of Ce6 and cisplatin is safe and feasible, with preliminary evidence of local cytotoxicity treatment for HNSCC, allowing targeted drug delivery to the tumor. This is the first report of the combined use of PDT and chemotherapy with selective intra-arterial administration of a PS and a chemotherapeutic drug for the treatment of cancer.

In this work, a new approach was tested to assess the cellular composition of tissues by time-resolved methods of fluorescence analysis of exogenous and endogenous fluorophores. First of all, the differences in fluorescence kinetics of endogenous fluorophores (coenzymes NADH and FAD) in tumour and immunocompetent cells were determined. After that, differences in fluorescence kinetics of photosensitizer 5 ALA-induced protoporphyrin IX were established due to its different metabolism in cells of different phenotypes. Kinetics of photoluminescence of NADH and FAD coenzymes as well as photosensitizer were studied by means of two different methods: time-resolved spectroscopy based on a streak-camera and fibre optic neuroscopy, which served to perform process monitoring and regular fluorescence diagnosis of the probed region. Time-resolved fluorescence microscopy (FLIM) was used as a control technique. Time-resolved spectroscopic fluorescence lifetime analysis was performed on sexually mature female rats induced with glioma C6 brain tumour under in vivo conditions; thus, under conditions where the immune system actively intervenes in the process of oncogenesis. In this regard, the aim of the study was to recognize the cellular composition of the brain tumour tissue, namely the ratio of cancer and immunocompetent cells and their mutual localization. Understanding the role of the immune system thus provides new ways and approaches for further diagnosis and therapy, making tumour-associated immune cells a prime target for modern therapies.

In this work, we tested a new approach to assess the presence of inflammatory process in the implant area using spectral methods and the technique of fiber fluorescence analysis of photosensitizers in nanoform. First of all, the spectral characteristics of the photosensitizer when interacting with the porous surface of the implant, based on hydroxyapatite under in vitro and in vivo conditions, were determined. Thus, it was shown that spectral characteristics of photosensitizers can be used for judgement on the process of inflammation in the implant area and thus on the local presence of the immunocompetent cells. The analysis was performed at a sufficient depth in the biotissue by using the near-infrared spectral region, as well as two different methods: fiber-based laser spectroscopy and fiber-optic neuroscopy, which served to monitor the process and regular fluorescence diagnosis of the studied area. Fluorescence spectroscopic analysis was performed on experimental animals in vivo, i.e., under conditions of active immune system intervention, as well as on cell cultures in vitro in order to judge the role of the immune system in the interaction with the implant in comparison. Thus, the aim of the study was to determine the relationship between the fluorescence signal of nanophotosensitizers in the near infrared spectral region and its parameters with the level of inflammation and the type of surface with which the photosensitizer interacts in the implant area. Thus, fiber-optic control opens up new approaches for further diagnosis and therapy in the implant area, making immune cells a prime target for advanced therapies.