Background: The use of nanoradiosensitizers is a promising strategy for the precision enhancement of tumor tissue damage during radiotherapy. Methods: Here, we propose a novel biocompatible theranostic agent based on gadolinium fluoride doped with cerium and terbium (Gd_0.7Ce_0.2Tb_0.1F₃ NPs), which showed pronounced radiocatalytic activity when exposed to photon or proton beam irradiation, as well as remarkable MRI contrast ability. A scheme for the production of biocompatible colloidally stable Gd_0.7Ce_0.2Tb_0.1F₃ NPs was developed. Comprehensive physicochemical characterization of these NPs was carried out, including TEM, SEM, XRD, DLS, and EDX analyses, as well as UV–vis spectroscopy and MRI relaxation assays. Results: Cytotoxicity analysis of Gd_0.7Ce_0.2Tb_0.1F₃ NPs in vitro and in vivo revealed a high level of biocompatibility. It was shown that Gd_0.7Ce_0.2Tb_0.1F₃ NPs effectively accumulate in MCF-7 tumor cells. A study of their radiosensitizing activity demonstrated that the combined effect of Gd_0.7Ce_0.2Tb_0.1F₃ NPs and X-ray irradiation leads to a dose-dependent decrease in mitochondrial membrane potential, a sharp increase in the level of intracellular ROS, and the subsequent development of radiation-induced apoptosis. Conclusions: This outstanding radiosensitizing effect is explained by the radiocatalytic generation of reactive oxygen species by the nanoparticles, which goes beyond direct physical dose enhancement. It emphasizes the importance of evaluating the molecular mechanisms underlying the sensitizing effectiveness of potential nanoradiosensitizers before choosing conditions for their testing in in vivo models.

The use of various nanoformulations for cancer radiotherapy has great potential due to their increased effectiveness and safety. At the stage of preclinical evaluation of such nanoformulations in vitro, they are tested on monolayers of tumor cell cultures, which is an unrealistic representation of tumors in vivo, which leads to an inaccurate selection of suitable drugs. To solve this problem, 3D cellular spheroids are becoming an additional tool for confirming the effectiveness of new therapeutic nanoformulations. Here we show the possibility of using citrate-stabilized cerium oxide nanoparticles (nanoceria, CeO2 NPs) as a nanoradiosensitizer in an experimental 3D model of tumor spheroids formed from 4T1 human breast cancer cells. It has been shown that ultra-small size (about 1-2 nm) CeO2 NPs have pH-sensitive radiocatalytic activity, increasing the generation of ROS under irradiation conditions. It has been shown that preloading of CeO2 NPs into a cellular spheroid at a micromolar concentrations does not adversely affect the rate of spheroid formation and its final size. However, such preloading has a pronounced inhibitory effect on the rate of cell migration from the spheroid after X-ray irradiation and also reduces their clonogenic activity. Thus, CeO2 NPs can be considered as a promising nanoradiosensitizer for radiotherapy of tumors.