Titanium dioxide nanoparticle-based hydroxyl and superoxide radical production for oxidative stress biological simulations

The present study introduces a TiO2 nanoparticle-based (TiO2-NP) system for the generation of (OH)-O-center dot and O-2(center dot-) 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 (OH)-O-center dot and O-2(center dot-) alone or both (in contrast to (OH)-O-center dot-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 (OH)-O-center dot, O-2(center dot-) 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 (OH)-O-center dot 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 (OH)-O-center dot and O-2(center dot-) 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 (OH)-O-center dot-producing Fenton system. The reaction kinetics of (OH)-O-center dot and O-2(center dot-) 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 (OH)-O-center dot/O-2(center dot-) dose-response-depended oxidative modifications in biological simulations. This stems from the fact that (OH)-O-center dot and O-2(center dot-) 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, (OH)-O-center dot production by the Fenton system reaches steady state in similar to 5 s regardless of varying Fe-II concentration, rendering it inappropriate for (OH)-O-center dot simulation studies on biological systems. The biological simulating potential of the TiO2-NP system, as producer of both (OH)-O-center dot and O-2(center dot-), 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 (OH)-O-center dot dose-proportional. In contrast, the Fenton system does not display (OH)-O-center dot dose-associated oxidative modifications on BSA.

Automated summary

This study introduces a TiO2 nanoparticle-based system for high oxidative stress biological simulations. The system can produce (OH)-O center dot and O-2(center dot-) and simulate interactions with biological targets. It allows control of the production rates of these radicals and can be used for studying oxidative modifications in biological simulations.