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Low Temperature Plasma for Applications in Medicine

Periodic Reporting for period 1 - LTPAM (Low Temperature Plasma for Applications in Medicine)

Reporting period: 2017-05-01 to 2019-04-30

Atmospheric pressure low temperature plasmas (LTPs) are attracting a lot of attention due to their vast biomedical potential. Specifically, the emerging field of LTP employment in anti-cancer research and clinical studies is of high importance. Plasmas generate a variety of molecular, ionic and radical reactive oxygen and nitrogen species (RONS) upon interaction with ambient gas and aqueous environment of biological substrates. Understanding how LTPs interact with their surroundings and thus being able to tailor the species cocktail and the respective effect is crucial for successful medicinal applications.
Despite plasma therapy being a very promising anti-cancer modality, it meets a lot of scepticism and hindrance from researchers, medical bodies, and the general public. Reasons for this lay in incomplete understanding of even the fundamental LTP properties, such as the reactivity of its various compartments, its interaction with components of biological media and tissues, etc.
By elucidating and explaining the effects of LTP in various environments and on different targets, we succeeded in bringing LTP closer to wider clinical applications, contributing to a healthier society.
We used the RF COST plasma jet, which was created as a ‘standard’ plasma jet. Firstly, we designed and built a reactor which allowed studying the sources of RONS induced in aqueous media by our plasma jet using various analytical techniques (electron paramagnetic and nuclear magnetic resonance spectroscopy, and UV-Vis spectrophotometry). We also developed a 3D fluid dynamics model and a 0D chemical kinetics model to study the gas phase phenomena in the plasma effluent. We then compared the experimentally obtained trends of RONS (H2O2, OH, H) concentrations with those predicted by the developed models. Our results have shown that unlike other types of plasma jets and devices, parallel field plasma jets, such as the RF COST jet, generate all RONS in the discharge region inside the plasma jet, with no extra species generated from ambient components in the effluent.
Furthermore, we developed analytical methods for the detection of the secondary species created in solutions upon interaction with plasma RONS. We demonstrated that chloride anions in aqueous media interact with LTP-induced O atoms, yielding hypochlorite anions ClO-.
Third, we studied the interaction of LTP with gel-like and tissue-like structures. 3D tumour models (spheroids) were generated, and the effects of LTP exposure on them was studied. We also showed that the short-lived LTP RONS (such as •OH radicals) were necessary to reduce the tumour size and prevent further re-growth. Additionally, we studied the interaction of LTP with gelatinous substrates: solutions of polymers (used to create nanofibers for applications in medicine) were studied with respect to the generation of short-lived RONS during LTP exposure. and their effect on the resulting nanofibers.
Finally, we studied the production and effects of peroxynitrite in LTP treatment of cancer. The goal was to investigate the possibility of ONOO- production using pulsed plasma sources. We developed a method to monitor the stability of ONOO- in aqueous media, and generated various RONS including peroxynitrite and studied their effects on tumours. We then identified which RONS were responsible for immunogenic cell death (ICD, a desired anti-tumour effect) in cancer cells. Overall, we showed that peroxynitrite can be efficiently used for decontamination, but not for ICD.
Throughout the project, we used different dissemination channels of our research. The non-industrial nature of the project implied that the main dissemination method was publication in scientific journals and participation in conferences. I published 7 peer-reviewed journal papers and 1 book chapter (of which 4 as the first author), with another submitted manuscript currently under revision. We succeeded in publishing both in specific journals targeting the plasma community (Plasma Processes and Polymers), and in journals of wider scientific interest (Physical Chemistry Chemical Physics, Analytical Chemistry, Cancers, Scientific Reports, Advanced Science). The aim of this was to bring more attention to the state-of-the-art of plasma research from different fields of science, including wide chemistry, biology and engineering readership. Our results were also presented to the scientific community in the form of 6 conference contributions at the major domestic and international conferences in the plasma domain.
To increase the impact of our work, a YouTube channel was created. We produced and published a video to communicate cold plasma medicine in general and my work in particular to a wide audience, not associated with science. I also participated in several popular science events aimed to reach the general public in different countries (Kurilka Gutenberga, Russia; Pint of Science, Belgium; invited public lecture at the Institute Cayetano Heredia, Peru). These efforts were very fruitful in introducing the LTP phenomenon to audiences with different backgrounds in different countries.
Our work with the RONS sources of the RF COST jet was the first study clearly stating this fundamental property of such plasma jets, emphasising their ‘standard’ nature and enabling their use in different environments comparably.
We also provided the first direct evidence of the reaction between Cl- anions and O atoms in liquid solutions. It has many implications on the use of plasma-treated media (PTM), and on the direct LTP application to biological samples, both of which are rich in chloride.
The research on gelatinous solutions of polymers unambiguously demonstrated that a combination of chemical changes to the solution and to the polymer are required for enhanced nanofiber production.
In terms of biological effects, we produced many results that had one extremely important message. Often even the very necessity of LTP is questioned. One of the methods of plasma application is pre-exposure of a liquid medium to LTP, yielding PTM. Convenient production-wise, the use of PTM is thus preferred by many researchers in studies on LTP cancer treatment. However, our results clearly showed that short-lived RONS (only available during direct LTP exposure) are paramount to efficiently treat 3D cancer tissues, and to induce ICD to create anti-tumour response in a patient’s body.
Overall, our work within the LTPAM project presents a great interest for both the general public and the scientific community. It provided many important insights into the fundamental actions of cold plasma, and made it understandable for the lay audience. Cold plasma therapy is one of the promising novel techniques to fight cancer, which can improve the overall health status in Europe and the world.