Photodynamic therapy (PDT) combines light, a photosensitizing chemical substance, and molecular oxygen to elicit cell death and is employed in the treatment of a variety of diseases, including cancer. The development of PDT treatment strategies requires in vitro assays to develop new photosensitizers. One such assay is the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide developed in 1983 and widely used in PDT studies. Despite the exponential growth in the number of publications, a uniform MTT protocol for use in the PDT area is lacking. Herein, we list and standardize the conditions to evaluate the photosensitizer methylene blue (MB) in glioblastoma and neuroblastoma cell lines. In addition, we review technical pitfalls and identify several variables that must be taken into consideration in order to provide accurate results with MTT. We conclude that for each cell line we must have a dose-response curve using the MTT assay and good controls for the standardization. Additionally, the optimal values of the time and cell density must be in the linear range of the curve to avoid errors. We describe all relevant points and outline the best normalization techniques to observe the differences between treatments.

The development of X-ray-absorbing scintillating nanoparticles is of high interest for solving the short penetration depth problem of visible and infrared light in photodynamic therapy (PDT). Thus, these nanoparticles are considered a promising treatment for several types of cancer. Herein, gadolinium oxide nanoparticles doped with europium ions (Gd₂O₃:Eu³⁺) were obtained by using polyvinyl alcohol as a capping agent. Hybrid silica nanoparticles decorated with europium-doped gadolinium oxide (SiO₂-Gd₂O₃:Eu³⁺) were also prepared through the impregnation method. The synthesized nanoparticles were structurally characterized and tested to analyze their biocompatibility. X-ray diffraction, scanning electron microscopy, and transmission electron microscopy confirmed the high crystallinity and purity of the Gd₂O₃:Eu³⁺ particles and the homogeneous distribution of nanostructured rare earth oxides throughout the fumed silica matrix for SiO₂-Gd₂O₃:Eu³⁺. Both nanoparticles displayed stable negative ζ-potentials. The photoluminescence properties of the materials were obtained using a Xe lamp as an excitation source, and they exhibited characteristic Eu³⁺ bands, including at 610 nm, which is the most intense transition band of this ion. Cytotoxicity studies on mouse glioblastoma GL261 cells indicated that these materials appear to be nontoxic from 10 to 500 μg·mL⁻¹ and show a small reduction in viability in non-tumor cell lines. All these findings demonstrate their possible use as alternative materials in PDT.

Photodynamic therapy (PDT) used in combination with sonodynamic therapy (SDT) is a new approach that aims to increase the effectiveness of tumor treatment when compared to the effect of each independent therapy. PDT is based on stimulating sensitizers with photons, while the most accepted theory for SDT is that sensitizers are stimulated by the sonoluminescence phenomenon. However, after the excitation of the sensitizer, both therapies follow a common path, leading to the generation of free radicals and inducing cell death. One of the positive aspects of this combination is the augmentation of anti-tumor activity with fewer side effects, since cell death may be induced using lower sensitizer concentrations or less exposure to ultrasound or light. Another benefit of combining PDT and SDT, especially with the use of low-frequency ultrasound is the induction of sonophoresis. For instance, on the skin, it may facilitate the absorption of the sensitizer. However, research involving both PDT and SDT exhibit many variants, including differences in irradiation sources and their intensities, among others. These aspects contribute to a lack of standardization, leading to result variations, hindering assessment on the real contribution that these combined therapies can offer in tumor treatment. Thus, further research in the pre-clinical and clinical areas are crucial.