| (1) Multiplicity of plasma sources | | Dozens of different plasma sources have been used for biomedical research, differing in the electrode configuration and principle of plasma generation, the power input, frequency and waveform, the type and flow rate of the working gas used (if any), geometry, and distance between source and target, ultimately determining ROS output. There is no current standard proposed in the field of plasma medicine yet, e.g., plasma source, lead ROS entity, standard assays, and nomenclature, making the comparison of experimental or clinical results challenging. The argument that, from a biological point of view, the type of plasma source with its specific ROS pattern and output may be irrelevant (as all of them simply confer oxidation) is not in line with findings in the field of redox biology that specify ROS entities can confer specific biological effects. This is further complicated by the multicomponent nature of cold physical plasmas. | | (2) Multicomponent and multi-ROS systems | | Plasmas are multicomponent systems comprised not only of ROS but also of electric fields; UV, visible, and NIR light emissions; electrons; and gas ions, as well as neutral particles. While ROS seem to dominate biological effects, the specific role of the other components is technically challenging to investigate. This includes potential synergistic or additive effects in the treatment of tissues, in which individual cells are more difficult to manipulate and to monitor (e.g., use of antioxidants and multiple components of microenvironment). Moreover, the ROS component of plasmas is extremely diverse, with hundreds of chemical reactions taking place on short time scales, in the interdependence of the type of species and concentration present, and with additional dynamics in the presence of organic molecules, as always the case in biomedical research. | | (3) Time scales of primary plasma effects are short, while the biological processes continue on longer time scales | | Similar to other physical technologies in medicine, such as ionizing radiation, pulsed electric fields, and photodynamic therapy, the primary plasma effect is only active as long as the target is exposed to plasma (usually seconds to minutes). Once the plasma is switched off, further impact of the treatment is determined by the cellular signalling pathways interpreting the exposure and translating it into biological responses. This implies that the key events of plasma medicine are taking effect during the treatment of the target, which is challenging to investigate due to short time scales. This is especially different from drugs that are usually continuously perfused into patients or added to cell cultures over several days and act unremittingly. | | (4) Lack of tools for spatiotemporal resolution of plasma-derived ROS in cells and tissues. | | Plasma medicine faces similar challenges as other fields in redox biology concerning the lack of research tools allowing a spatial and temporal resolution of ideally different types of ROS separately in cells and tissues. Most redox-sensitive fluorescent dyes are nonspecific in biological systems, and the action of ROS is usually identified indirectly via their modification of proteins and lipids. Reporter assay systems engrafted into animal models are needed to identify the specific contribution of individual ROS in specific (pathological) conditions in order to accelerate the knowledge of the field that would allow disease-specific tailoring of plasma sources. |
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