Photodynamic therapy (PDT) has been clinically employed to treat mainly superficial cancer, such as basal cell carcinoma. This approach can eliminate tumors by direct cytotoxicity, tumor ischemia, or by triggering an immune response against tumor cells. Among the immune-related mechanisms of PDT, the induction of immunogenic cell death (ICD) in target cells is to be cited. ICD is an apoptosis modality distinguished by the emission of damage-associated molecular patterns (DAMP). Therefore, this study aimed to analyze the immunogenicity of CT26 and 4T1 treated with PDT mediated by aluminum-phthalocyanine in nanoemulsion (PDT-AlPc-NE). Different PDT-AlPc-NE protocols with varying doses of energy and AlPc concentrations were tested. The death mechanism and the emission of DAMPs–CRT, HSP70, HSP90, HMGB1, and IL-1β–were analyzed in cells treated in vitro with PDT. Then, the immunogenicity of these cells was assessed in an in vivo vaccination-challenge model with BALB/c mice. CT26 and 4T1 cells treated in vitro with PDT mediated by AlPc IC₅₀ and a light dose of 25 J/cm² exhibited the hallmarks of ICD, i.e., these cells died by apoptosis and exposed DAMPs. Mice injected with these IC₅₀ PDT-treated cells showed, in comparison to the control, increased resistance to the development of tumors in a subsequent challenge with viable cells. Mice injected with 4T1 and CT26 cells treated with higher or lower concentrations of photosensitizer and light doses exhibited a significantly lower resistance to tumor development than those injected with IC₅₀ PDT-treated cells. The results presented in this study suggest that both the photosensitizer concentration and light dose affect the immunogenicity of the PDT-treated cells. This event can affect the therapy outcomes in vivo.

The use of photodynamic therapy (PDT) and development of novel photosensitizers (PSs) for cancer treatment have received more and more attention nowadays. In the present work, five benzo[a]phenoxazinium derivatives have been prepared and evaluated for their in vitro anticancer photodynamic activity for the first time. They are red light absorbers and show low fluorescence quantum yield. Of these compounds, PS4 exhibited a higher quantum yield for reactive oxygen species (ROS) generation. The assays with cells in vitro showed that PS1 and PS4 were not significantly toxic in the dark, but was robustly toxic against the murine breast adenocarcinoma cells 4T1 and normal murine fibroblast cells NIH-3T3 upon photoactivation. More interestingly, PS5 was particularly selective towards 4T1 cancer cells and nearly non-phototoxic to non-cancerous NIH-3T3 cells. The results described in this report suggest that these new benzo[a]phenoxazinium derivatives are potential candidates as PSs for anticancer PDT. Further investigation of benzo[a]phenoxaziniums for anticancer PDT is warranted.

Canine leishmaniasis (CanL) is a chronic disease caused by Leishmania infantum, and the limitations of the current treatments have encouraged new alternatives, such as the use of immunomodulatory nutrients. The objective of this study was to determine the serum levels of vitamin A (retinol), vitamin D (25(OH)VD₃), and zinc (Zn) in dogs with CanL and the effect of in vitro supplementation with the respective active forms ATRA, 1,25(OH)₂VD₃, and SZn on spleen leukocyte cultures. Serum retinol, 25(OH)VD₃, and Zn were determined by HPLC, ELISA, and ICP-MS, respectively. Spleen leukocyte cultures were used for the detection of NO and ROS by flow cytometry; the IFN-γ, TNF-α, and IL-10 levels were determined by ELISA; and the parasite load was determined by microscopy. We detected low serum levels of retinol and Zn and high levels of 25(OH)VD₃ in the CanL group. The in vitro supplementation of CanL spleen leukocytes with ATRA, 1,25(OH)₂VD₃, and SZn, in addition to a soluble leishmania antigen (SLA) treatment, increased the NO and ROS levels, while the treatments with only ATRA and SZn increased the TNF-a levels. Increased IL-10 and IFN-g levels were observed with the addition of SLA to the medium, although the addition of the three nutrients led to a reduction of the IL-10 levels, and the addition of 1,25(OH)₂VD₃ and SZn led to a reduction of IFN-g. A supplementation with 1,25(OH)₂VD₃ and SZn reduced the parasite load but only in the absence of SLA. We suggest that the nutrients we tested are involved in the leishmanicidal mechanism, showing a potential for investigation in future studies.