In Vitro Study On The Safety Issue Of Non-equilibrium Atmospheric Pressure Plasma Jets In Biomedicine

Non-equilibrium atmospheric pressure plasma jets(N-APPJ)are able to generate a mixture of biologically active elements including reactive oxygen and nitrogen species(RONS),electrons and ions while maintaining its gas temperature close to room temperature.These features enable N-APPJ to have exciting application prospects in biomedical field.There are two ways of using N-APPJ for biomedical applications,i.e.,direct treatment and indirect treatment like plasma activated water(PAW)and plasma activated medium(PAM).However,the biomedical effects of N-APPJ have a dose-dependent manner,it will induce many pathological effects when the concentrations of RONS generated by N-APPJ are too high in the human body.Therefore,a systematic research on the safety issue of N-APPJ in biomedicine is of great significance for accurately assessing and minimizing the potential risks during the applications of N-APPJ.At present,the formation pathways of various RONS during the interaction between N-APPJ and living organisms,the specific roles of various RONS in the biomedical effects of N-APPJ have not yet been fully understood.The safe concentration ranges of various RONS to living organisms,whether RONS solutions made by chemical method can also selectively kill cancer cells,whether the biological effects of direct plasma treatment and indirect treatment are always the same in all cases,what potential toxic and side effects N-APPJ may have on normal cells?These questions that closely related to the biomedical safety of N-APPJ are still unclear.Therefore,in this dissertation,two kind N-APPJ devices that can be touched by human body were used to investigate the questions bellow,and the in vitro experimental research works were carried out in the following aspects:1.Because of most of RONS are general reaction products generated through interactions between plasma and liquids,the composition of distilled water is relatively simple and easy to analyze.Therefore,the main long-lived species(H₂O₂,NO₂^-and NO₃^-)and short-lived species(ONOOH,?OH and ¹O₂)in PAW were diagnosed firstly,then the generation pathways,chemical characteristics and roles in the bactericidal process of PAW were analyzed.The experimental results indicated that the acidified H₂O₂,NO₂^-or NO₃^-whose concentration close to the same kind species in PAW alone have little contribution to the bactericidal effects of PAW.The contribution of?OH can also be ignored.For the first time,it was found that the concentration of ONOOH in PAW was too low to directly realize complete bactericidal effects of PAW.Actually,ONOOH further interacted with H₂O₂ to generate peroxynitric acid(O₂NOOH),which eventually decomposed into?O₂^-and ¹O₂;these two species enhanced the bactericidal effects of PAW substantially.2.Human keratinocytes(HaCaT cells)and melanoma cells(A875)were chosen as in vitro models to compare the cytotoxicity of RONS solutions made by chemical meatod on human non-malignant and cancer cells.It was found that even the concentrations of NO₂^-and NO₃^-are one order of magnitude higher than those in the application of N-APPJ,NO₂^-,NO₃^-or their combination almost had no toxicity on both cell while the combination of H₂O₂ and NO₂^-had stong toxicity on both cells.More importantly,exogenous RONS solutions exhibited more toxicity on normal HaCaT cells than A875 cells under the same conditions.This is mainly because A875 cells have stronger antioxidation ability than HaCaT cells.3.HaCaT cells and A875 cells were chosen as in vitro models to compare the cytotoxicity of direct plasma treatment and PAM on human non-malignant and cancer cells.It was found that the survival rate of A875 cells were higher than HaCaT cells when PAM was used while direct plasma treatment can selectively kill A875 cells when the treatment time was well controlled.These results indicated that the biological effects of direct plasma treatment and PAM treatment are not similar in all cases.The intrinsic characteristics of the cell lines and the short-lived reactive species may be the two important reasons for the selectivity of direct plasma treatment to A875 cells.4.In order to confirm the long-term safety of N-APPJ,normal human liver cell line L02was chosen as an in vitro model to investigate the potential genotoxic and mutagenic properties of the direct treatment of N-APPJ.It was found that long time N-APPJ treatment can not only cause normal cell death,chromosome damage also existed in the surviving cells,the micronucleus frequency of surviving cells increased obviously.However,the cell damage induced by N-APPJ can be repaired in the progeny of surviving cells through sufficient cell division,and no delayed genomic instability like delayed reproductive cell death and micronucleus formation was found in the progeny cells.When the progeny cells were exposed to the plasma for the second time,the biological endpoints detected above still had no statistical difference compared to the results of the corresponding time during the first plasma treatment.Besides,there are still no significant differences between treated cells and untreated cells in terms of HPRT gene mutation frequency indicating that once or twice treatment with the plasma jet will not induce increased HPRT mutation frequency in L02 cells.