The Antitumor Efficacy of Redox-Active Inorganic Nanoparticles/Menadione Complexes in 2D and 3D Models of L929 Fibrosarcoma Cells

The oxidoreductase-like activity of nanoparticles, attributed to their electronic properties, offers the potential to target key processes in cancer cells, including ROS generation, low molecular weight antioxidant exchange, and energy substrate metabolism. Evidence suggests that the antitumor effects of nanoparticles (NPs) are enhanced when used in combination with redox-active organic compounds. In this study, cholesterol-based complexes of ultra-small (1-2 nm) inorganic orthovanadate nanoparticles (GdYVO4/Eu ${ }^{3+} \mathrm{N P s)}$ or cerium oxide nanoparticles ($\mathrm{CeO}_{2} \mathrm{NPs}_{\text { }}$) with menadione were developed to specifically disrupt the oxidative balance and metabolism of cancer cells. Given that the combination of ascorbic acid with menadione is known to induce autoschizis (a form of cancer cell death), the effects of menadione-containing complexes were also evaluated in the presence of vitamin C. The antitumor efficacy of these complexes was assessed using L929 mouse fibrosarcoma cell lines in both 2D and 3D models. Complexes composed of nanoparticles: cholesterol: menadione in a ratio of 1:0.02:0.4 were employed. In the 2D model, L929 cells ($\sim 2 \times 10^{4}$ cells $/ \mathrm{ml}$) were incubated with the complexes (NPs - $10 \mu \mathrm{~g} / \mathrm{ml} /$ menadione - $20 \mu \mathrm{~mol}$) in DMEM/F12 with 3% FBS under a humidified atmosphere with $5 \% \mathrm{CO}_{2}$, with or without the addition of vitamin C(2 mmol). In the 3D model, 7-day-old spheroids of $\mathrm{L 9 2 9}$ cells were used. The effects were assessed using fluorescent microscopy with DHE, JC-1, PI, acridine orange staining, and MTT assays. In 2D experiments, complexes based on CeO<inf xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</inf> NPs more effectively inhibited cellular functional activity in the presence of vitamin C, resulting in cell shrinkage (indicative of autoschizis), reduced mitochondrial potential, and superoxide radical formation. The timing and intensity of the destructive effect were influenced by both the concentrations of FBS and the complexes. In the 3D model, achieving this effect required a three-fold increase in the concentrations of both the complexes and vitamin C. Over three weeks, experiments on the 3D model demonstrated partial inhibition of cell viability at $\mathrm{1 x}$ concentrations and complete destruction of the 3D structure at $\mathrm{3 x}$ concentrations. Therefore, unlike in the 2D model, at least a three-fold increase in active components was necessary in the 3D model, which should be considered when evaluating antitumor efficacy and developing treatment protocols.