## 4 Discussion  
The features of NIR-excited dyes are also effectively used in photodynamic protocols, where this cyanine fits perfectly into the therapeutic window of biological tissues (700-900 nm) [19]. NIRF-based imaging is an effective tool that enables early cancer detection and treatment [20; 21]. Recently, NIRF dyes are also incorporated for multifunctional protocols that are combined with chemotherapy [22], photodynamic therapy [23], or photothermal therapy [19]. In this study, we have demonstrated for the first time the usage of indocyanine green (ICG) with electrochemotherapy (ECT) with bleomycin (BLM). The idea behind a certain decision was influenced by the lack of control over the distribution of drugs during ECT treatment. ECT-based protocols mostly use non-fluorescent chemotherapuits such as bleomycin or cisplatin [24; 25; 26]. In our study, various models in vitro and in vivo were used. Fibrosarcoma (used in vitro) and mast cell tumor (MCT) (feline case) are both malignant tumors that can originate from connective tissue, so it is possible to compare them to some extent. Moreover, in vitro validation allows you to check the toxicity of the therapy before applying it to the patient. Our results in vitro reveal that ICG can be safely combined with bleomycin and does not affect the efficacy of BLM. The decision on additional ECT in this patient's case was based on inconclusive cytology results, the clinical appearance of the mass, and the owner's capability for only one-time treatment. Additionally, the presented case study on the domestic cat confirms the safety and effectiveness of the ICG-BLM-ECT protocol. The advantage of combining BLM with ICG is spatial and temporal visualization of the electroporation field. ICG has several clinically valuable properties, including a good signal-to-noise ratio and operation in optical tissue windows, enabling deep imaging [13]. Both BLM and ICG are characterized by good solubility in water as well as low toxicity. ICG dose--60 to 80 mg/kg, caused 50% mortality (LD\({}_{50}\)) in mice following intravenous administration [27], whereas LD\({}_{50}\) of intraperitoneally administered bleomycin ranged from 72 to 520 mg/kg in rats [9; 10]. In the context of ECT, some (bio)chemical differences between ICG and BLM may influence their behavior during the electroporation procedure. ICG molecular weight is 774.97 g/mol, with an average size from 3 to 300 nm [28], whereas bleomycin, a complex mixture of related glycolipopeptides with a larger molecular weight (1415.56 g/mol), can also range from a few nanometers to tens of nanometers, depending on factors such as its conformation and hydration state. It was demonstrated that electric pulses induced an increased cell permeability and might augment the cytotoxicity of bleomycin by several 100-fold [29], which suggests that the compound size is not the  
Figure 6: ICG distribution in tumor tissue and lymph node of the feline, where lymph nodes were not exposed to ECT (unfixed tissue, 24 h after resection) ICG—(indocyanine green, 20 mg/mL). critical aspect of ECT efficacy. Due to its hydrophilic character, bleomycin easily penetrates through pores created during electroporation. ICG on the other hand possesses both hydrophilic and lipophilic properties and may interact with cellular phospholipids [30] and proteins [31]. The ability of ICG to bind to serum proteins was used by [PERSON] et al. who aimed to detect ovarian cancer metastases based on a resulting enhanced permeability and retention of ICG [32]. Unfortunately, this approach appeared ineffective due to low specificity--all malignant lesions were detected, and a high-false positive signal was also present. In our study, we expected the tumor penetration by ICG to be similar or worse compared to BLM. Thus, we mixed both drugs and our results indicate an increased ICG uptake after electroporation in cells and in the tumor tissue. The most interesting was the detectable fluorescent ICG signal in lymph nodes, which were not electroporated.