Study On The Synergistic Sterilization Effect Of Cold Atmospheric Plasma Combined With Pulsed Electric Field And Its Effect On Subcellular Structures

Cold atmospheric plasma(CAP)can act on biological samples at normal temperature and pressure,and is rich in a large number of active species,which has the advantages of safety,high efficiency,green environmental protection and no residue after reaction.It has shown great application potential in food microbial inactivation,induction of cancer cell apoptosis,wound healing and so on.In order to meet the sterilization requirements of 5 log value set by the FDA for the food processing industry,the single sterilization technology has some limitations in practical production and application.For example,high-dose CAP treatment can easily lead to lipid peroxidation of high-fat foods,discoloration of fruits and vegetables,decrease of hardness,increase of acidity and decrease of nutrients(such as anthocyanins).As another non-thermal sterilization technology based on irreversible electroporation,pulsed electric field(PEF)requires higher electric field intensity(30 kV/cm)to meet the needs of practical application,but increasing PEF intensity will affect the flavor characteristics and nutritional value of food,and even lead to the production of substances harmful to human body,such as hypochlorite.In recent years,the related research on CAP combined with other sterilization methods has been paid close attention by more and more researchers.Using CAP combined with PEF to sterilize and explore whether it can produce synergistic effect is of great guiding significance to the practical application of non-thermophysical sterilization technology.In addition,because of its selective killing of tumor cells and no obvious side effects,CAP has been proved to have obvious inhibitory effect on many kinds of tumors at the cellular and in vivo level.It is an urgent problem for plasma medicine to fully reveal the anti-tumor mechanism of CAP.Super high resolution optical microscopic imaging technology can dynamically show the effect of plasma on subcellular structure and provide important information for further understanding the anti-cancer mechanism of CAP.Aiming at the above scientific problems,on the one hand,using Staphylococcus aureus(ATCC 6538)and Escherichia coli(ATCC 25922)as biological models,the sterilization effect of CAP combined with PEF was studied systematically,and the damage mechanism behind the synergistic effect was discussed.At the same time,the key factors in the synergistic process were excavated deeply.On the other hand,using osteosarcoma cells(U2OS)as a biological model,the effects of CAP on the morphology and movement of mitochondria and vesicle system were explored by using structured light super-resolution microscopy(SIM).The specific results are as follows:1.Study on the feasibility and effectiveness of low temperature CAP combined with PEF sterilization.When the parameters of CAP(kINPen,5 SLM,13 min)and PEF(15 kV/cm,100 μs,100-300 X)were treated separately,the killing effect on S.aureus and E.coli was not complete(2-3 log),which was much lower than the 5-log value required by FDA.In this paper,through the colony formation(CFU)experiment and combination index(CI)analysis,it was found that the combined use of CAP and PEF produced a significant synergistic bacteriostatic effect,and the sterilization effect of the treatment sequence of plasma followed by PEF was better.The combination of CAP(3 min)followed by PEF(15 kV/cm,100 X)could kill S.aureus and E.coli at 5.98±0.52 and 5.82±0.12 log,respectively.In addition,the subsequent fluorescent apoptosis double staining of Annexin-V and PI also confirmed the synergistic sterilization effect of CAP combined with PEF.This study confirmed the feasibility of synergistic bacteriostasis of CAP combined with PEF,and provided the necessary basis for follow-up research.2.Study on the biological damage mechanism of CAP combined with PEF in synergistic sterilization.In this study,the damage mechanism was studied from three aspects:cell membrane,intracellular antioxidant system and genetic DNA substance.After PEF was sensitized by CAP,the membrane permeability of S.aureus and E.coli increased significantly,the leakage of intracellular nucleic acid and protein increased,the conformation of membrane protein changed significantly,and the microenvironment of Phenylalanine(Phe),Tryptophan(Trp)and Tyrosine(Tyr)residues became more hydrophilic.And through scanning electron microscope(SEM)observation,bacterial morphology suffered more serious damage,there is a large area of cell membrane shrinkage,rupture and even collapse.In addition,the bacteria sensitized by plasma showed higher levels of intracellular ROS accumulation and more serious lipid peroxidation.The results showed that the activities of superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GPx)were significantly inhibited after this combined treatment.Through the double-stranded DNA specific probe(SYBR Green I)experiment,it was found that plasma combined with PEF induced more double-stranded DNA breaks.This study provides a comprehensive description of the mechanism of PEF sterilization sensitized by low temperature plasma.3.Study on different inactivation mechanisms of Gram-positive bacteria and Gram-negative bacteria by combined treatment.Gram-positive bacteria(S.aureus)and gram-negative bacteria(E.coli)depend on two different inactivation mechanisms for CAP combined with PEF treatment.E.coli with thinner cell membrane is mainly due to the death caused by irreversible damage to cell membrane after combined treatment,such as the increase of cell membrane permeability,the exudation of cell contents and so on.For S.aureus with thick peptide polysaccharide layer,the inactivation of S.aureus depends more on the oxidative damage of intracellular substances(lipids and DNA)induced by the accumulation of a large amount of intracellular ROS.4.Study on the key effect molecules in the process of synergistic sterilization by CAP combined with PEF.Combined with the results of emission spectrum diagnosis(OES),the components of RONS in plasma and PEF treated liquid phase were quantitatively measured in this paper.It is proved that the content of exogenous RONS mainly depends on the production of CAP,while the role of PEF is negligible.Through RONS inhibitors,it was found that different strains showed different sensitivity to RONS.The effects on S.aureus and E.coli were ·OH>O2->H2O2>NO and ·OH>H2O2>O2->NO respectively.In vitro experiments,it was found that the synergistic action of plasma-derived long-lived(H2O2、NO2-、NO3-)and short-lived(-OH、O2-)free radicals significantly enhanced the efficacy of subsequent PEF sterilization.Pearson correlation coefficient analysis shows that the acidification of liquid medium caused by plasma also plays an important role.This study proved the key role of short-lived OH groups induced by CAP and low pH value of solution in the process of sensitized PEF sterilization,which provided a theoretical basis for regulating plasma sensitization therapy.5.Study on the effect of CAP on the morphology and movement of vesicles and mitochondria of cancer cells.In this study,the effects of CAP on cancer cell vesicles and mitochondria were analyzed in real-time and in situ by structural illumination super-resolution microscopic imaging technique.The results showed that the cluster structure of vesicles in osteosarcoma U2OS cells treated with 20s CAP gradually disintegrated,the vesicles changed from round to circular,and the membrane structure of vesicles became blurred.After 20 seconds of CAP treatment,the average velocity of vesicles decreased from 0.140 to 0.037 μm,and the average maximum displacement phase decreased from 7.76 to 3.26 μm within 60s.In addition,through the Transwell co-culture model,it was found that CAP treatment significantly inhibited the vesicle transport between U2OS and normal pulmonary epithelial cell BEAS-2B.On the other hand,CAP treatment for 20 s caused morphological damage and movement disturbance of mitochondria.It is mainly characterized by the imbalance between mitochondrial fusion and division and changes in the state of movement,which is characterized by an increase in the proportion of mitochondria that gradually break from linear breakage to dots and perform rapid and long-distance directional movements,which is accompanied by a decrease in mitochondrial membrane potential(ΔΨm)and an increase in mitochondrial ROS(mtROS)levels.In all the above studies,the possibility of interference of working gas(Ar)to the experimental results was eliminated by setting up a control group.This study provides a possible new perspective and idea for a better understanding of the anti-cancer mechanism of plasma.