The present study introduces a TiO2 nanoparticle-based (TiO2-NP) system for the generation of •OΗ and O2•− upon visible photo-excitation, in order to be used for high oxidative stress biological simulations in vitro. The main novelties of TiO2-NP system are: It is set to produce •OΗ and O2•− alone or both (in contrast to •OΗ-based common use of TiO2), as these options cover all possible generation means of these radicals in biological systems in vivo. Moreover, the known non-specific electrostatic interactions of TiO2-NP with H2O and various biological systems (e.g., cells, membrane proteins, even drugs) simulate direct/distant interactions of any in vivo•OΗ, O2•− source with extra/intracellular biological targets taking place in a densely packed biomolecular environment. The TiO2-NP system can use any commercially available TiO2-NP source (dispersion, nanopowder, crystal type), as long as TiO2-NPs’ concentrations in use meet the critical criterion to produce •OΗ levels linearly proportional to irradiation time, set for a given simulation study. The TiO2-NP system is calibrated by a standardized protocol developed to be applicable to most biological systems, offering the option of TiO2-NP removal via coagulation when needed. The production rates of •OΗ and O2•− by the TiO2-NP system are specifically calibrated with the respective specific probes terephthalic acid (TPA) and hydroethidine (HE), and tested in comparison to the •OΗ-producing Fenton system. The reaction kinetics of •OΗ and O2•− with TPA and HE is found to be in competition with their generating source, the TiO2-NP system. Similar ROS source competition phenomena with biological targets (simulated by TPA/HE) are very common in biological systems. In contrast, the Fenton system is shown not to exhibit such competition kinetics. The TiO2-NP system can be used to study •OΗ/O2•− dose–response-depended oxidative modifications in biological simulations. This stems from the fact that •OΗ and O2•− linear production rates (60 min and up to 8 min, respectively) can be controlled by varying (i) TiO2 concentration, (ii) light-source photon emission energy (decreasing from 370 to 410 nm), and (iii) light intensity (as a function of the inverse of squared distance from the irradiated sample). In contrast, •OΗ production by the Fenton system reaches steady state in ∼5 s regardless of varying Fe-II concentration, rendering it inappropriate for •OΗ simulation studies on biological systems. The biological simulating potential of the TiO2-NP system, as producer of both •OΗ and O2•−, is also experimentally verified on indicative biological examples selected to structurally represent most biological systems: BSA, a model hydrophilic protein; LDL, structurally resembling most of the biological systems (cells, membranes, organelles, lipoproteins, proteins, lipids). The TiO2-NP system causes a linear increase of all the tested oxidative modifications on both BSA and LDL for irradiation exposure 20 to 40 min, which strongly suggests that they are mainly •OΗ dose-proportional. In contrast, the Fenton system does not display •OΗ dose-associated oxidative modifications on BSA.
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