Non-thermal Plasma Exposure Research Articles

Preceding treatment of non-thermal plasma (NTP) assisted the bactericidal effect of ultrasound on Staphylococcus aureus

Ultrasound is a promising non-thermal inactivation technique. However, ultrasound treatment alone is not very effective. In this study, combined applications of ultrasound and non-thermal plasma (NTP) were assessed for their inactivation efficacy and the physiological change on Staphylococcus aureus cells. The lethal and sublethal injury induced by individual ultrasound, NTP, ultrasound-NTP (UP) and NTP-ultrasound treatments was determined by plate count method. Then, we applied fluorescent technology to demonstrate the physiological variations of S. aureus during various treatments. NTP exposure followed by ultrasound treatment exhibited the highest inactivation rate of S. aureus. Prior NTP helped to provide enough reactive oxygen species (ROS) dissolved in the medium, and the subsequent ultrasound assisted in the injection of ROS into S. aureus cells. This accelerated the reaction between ROS and intracellular biomolecules, which led to the rapid death of the microbes. On the contrary, S. aureus cells treated with ultrasound first were more likely to develop and enhance oxidative response, allowing S. aureus to resist toward the following NTP stressor. Therefore, the findings of this study may be used to the optimization of hurdle technologies of ultrasound and NTP in practice.

Effect of preliminary stresses on the resistance of Escherichia coli and Staphylococcus aureus toward non-thermal plasma (NTP) challenge

As the development of hurdle technology, cross-protection of various stresses for pathogens posed the potential risk to food safety and public health. This study tried to explore various preliminary stresses including acidity, osmosis, oxidation, heat and cold on the resistance of microbial cells toward the non-thermal plasma (NTP) exposure. The results indicated that short-term (4h) exposure of Staphylococcus aureus and Escherichia coli to acidity, osmosis, oxidation, heat and cold stresses did not lead to the resistance to the subsequent NTP treatment. On the contrary, acidity, osmosis and heat preadaptation increased the vulnerability of E. coli cells to NTP treatment. After exposing S. aureus to osmosis, oxidation, heat and cold stress for longer period (24h), the reduction level showed significantly (P<0.05) higher. Interestingly, long-term (24h) preliminary exposure of acidic stress exhibited protective effect for S. aureus against the following NTP exposure with less damage in cell membrane integrity, membrane potential and intracellular enzyme activity. It might be due to the protein production for oxidative stress response during preliminary acidic adaptation. In general, the obtained result helped to grasp better understanding of the microbial stress response to NTP treatment and provided insight for the future research in order to accelerate the development of NTP technology in food industry.

Non-thermal plasma induces mitochondria-mediated apoptotic signaling pathway via ROS generation in HeLa cells

Non-thermal plasma (NTP) has been proposed as a novel therapeutic method for anticancer treatment. Although increasing evidence suggests that NTP selectively induces apoptosis in some types of tumor cells, the molecular mechanisms underlying this phenomenon remain unclear. In this study, we further investigated possible molecular mechanisms for NTP-induced apoptosis of HeLa cells. The results showed that NTP exposure significantly inhibited the growth and viability of HeLa cells. Morphological observation and flow cytometry analysis demonstrated that NTP exposure induced HeLa cell apoptosis. NTP exposure also activated caspase-9 and caspase-3, which subsequently cleaved poly (ADP- ribose) polymerase. Furthermore, NTP exposure suppressed Bcl-2 expression, enhanced Bax expression and translocation to mitochondria, activated mitochondria-mediated apoptotic pathway, followed by the release of cytochrome c. Further studies showed that NTP treatment led to ROS generation, whereas blockade of ROS generation by N-acetyl-l-cysteine (NAC, ROS scavengers) significantly prevented NTP-induced mitochondrial alteration and subsequent apoptosis of HeLa cells via suppressing Bax translocation, cytochrome c and caspase-3 activation. Taken together, our results indicated that NTP exposure induced mitochondria-mediated intrinsic apoptosis of HeLa cells was activated by ROS generation. These findings provide insights to the therapeutic potential and clinical research of NTP as a novel tool in cervical cancer treatment.

303 - Chemical reaction mechanism in non-thermal plasma from the viewpoint of oxidative stress toward clinical cancer applications

Non-thermal plasma (NTP) is a recently developed, non-thermal technique with many biomedical applications. Although NTP reportedly generates reactive oxygen/nitrogen species, electrons and ultraviolet radiation, few research projects have been conducted to merge this technique with the preexisting free radical biology. Recently, NTP device with a high electron density was developed. Here electron spin resonance revealed hydroxyl radicals as a major product. To merge novel NTP biology with the preexisting free radical biology, we evaluated lipid peroxidation and DNA modifications in various in vitro and ex vivo experiments. Among these, direct exposure to rat liver in medium produced immunohistochemical evidence of 4-hydroxy-2-nonenal- and acrolein-modified proteins. Exposure to plasmid DNA induced dose-dependent single/double strand breaks and increased the amounts of 8-hydroxy-2’-deoxyguanosine and cyclobutane pyrimidine dimers. These results indicate that oxidative biomolecular damage by NTP is dose-dependent and thus can be controlled in a site-specific manner. Simultaneous oxidative and UV-specific DNA damage may be useful in the treatment of wounds, endometriosis as well as certain cancers. Plasma-activated medium (PAM) is another advancement, which is relatively stable and is advantageous for medical applications. We further show catalytic iron as a major regulating factor and autophagy as cellular response in NTP exposure.

Non-thermal plasma prevents progression of endometriosis in mice

Endometriosis is observed in ∼10% of reproductive age women. Ovarian endometriosis not only causes dysmenorrhea but also causes infertility and a high risk of adenocarcinoma. Due to its scattered nature, complete surgical resection is difficult. Endometriosis consists of glandular and stromal cells. Previously, we showed that endometrial stromal cells (ESCs) play a role in the protection against pathologic events caused by monthly repeated hemorrhage. Here, we undertook a preclinical study of non-thermal plasma (NTP) as a surgical treatment of endometriosis. Epithelial cells were most sensitive to NTP-activated medium in vitro, whereas ectopic ESCs were most resistant. We then transplanted excised uteruses into BALB/c mice from donors of the same strain with estradiol supplementation. Four weeks after the transplantation, we exposed NTP to each endometriotic lesion after laparotomy. Immunohistochemical analysis revealed that immediately after NTP exposure, epithelial cells exhibited significantly higher levels of nuclear immunostaining for 8-hydroxy-2′-deoxyguanosine than did stromal cells. Four weeks after NTP exposure, the total surface area consisting of endometriotic cysts was significantly smaller with less epithelial proliferative activity than the helium-exposed control, whereas the number of endometriotic lesions had not changed. Therefore, NTP exposure may be useful to prevent the progression and recurrence of endometriosis.

Non-thermal Plasma Exposure Rapidly Attenuates Bacterial AHL-Dependent Quorum Sensing and Virulence.

The antimicrobial activity of atmospheric pressure non-thermal plasma has been exhaustively characterised, however elucidation of the interactions between biomolecules produced and utilised by bacteria and short plasma exposures are required for optimisation and clinical translation of cold plasma technology. This study characterizes the effects of non-thermal plasma exposure on acyl homoserine lactone (AHL)-dependent quorum sensing (QS). Plasma exposure of AHLs reduced the ability of such molecules to elicit a QS response in bacterial reporter strains in a dose-dependent manner. Short exposures (30–60 s) produce of a series of secondary compounds capable of eliciting a QS response, followed by the complete loss of AHL-dependent signalling following longer exposures. UPLC-MS analysis confirmed the time-dependent degradation of AHL molecules and their conversion into a series of by-products. FT-IR analysis of plasma-exposed AHLs highlighted the appearance of an OH group. In vivo assessment of the exposure of AHLs to plasma was examined using a standard in vivo model. Lettuce leaves injected with the rhlI/lasI mutant PAO-MW1 alongside plasma treated N-butyryl-homoserine lactone and n-(3-oxo-dodecanoyl)-homoserine lactone, exhibited marked attenuation of virulence. This study highlights the capacity of atmospheric pressure non-thermal plasma to modify and degrade AHL autoinducers thereby attenuating QS-dependent virulence in P. aeruginosa.

Haem-assisted dityrosine-cross-linking of fibrinogen under non-thermal plasma exposure: one important mechanism of facilitated blood coagulation.

Although blood coagulation facilitated by non-thermal plasma has been reported several years ago, the insight to the involved mechanisms is still rather limited. In this work, we report our discovery of a new mechanism for the haem-promoted blood-coagulation caused by non-thermal plasma treatment. The reason for the haem role is due to that its oxidized form, namely, hematin, can promote the dityrosine cross-linking of fibrinogen, the most important coagulation protein, to form a membrane-like layer on the surface of the treated blood with plasma exposure. Both haem and non-thermal-plasma generated hydrogen peroxide are requisite for the cross-linking process. We confirmed that fibrinogen can coordinate with the haem iron to form a protein-haem complex which shows pseudo-peroxidase activity, and in the presence of hydrogen peroxide, the complex can induce the dityrosine formation between fibrinogen molecules, leading to the fibrin network necessary for the blood coagulation. Understanding of such an underlying mechanism can be useful to guide more efficient application of non-thermal plasma in the management of hemostasis, thrombosis and etc.

Biphasic effects of l-ascorbate on the tumoricidal activity of non-thermal plasma against malignant mesothelioma cells

Non-thermal plasma (NTP) is a recently developed technology that elicits a variety of biological effects. This includes cancer cell-specific cytotoxicity, which is mainly attributed to the regional generation of reactive oxygen species (ROS). We studied the effects of NTP on malignant mesothelioma (MM) and its modulation by l-ascorbate. l-ascorbate is a major water-soluble anti-oxidant in vivo, but its pro-oxidant activity in vitro has been well recognized. Thus, the effects of ascorbate on the efficacy of NTP is important to examine. NTP exposure dose-dependently killed MM cells, whereas MM cells tolerated 1 mM l-ascorbate. However, brief pre-treatment with a pharmacological dose (250–750 μM) of l-ascorbate immediately prior to NTP exposure significantly increased its cytotoxicity in a dose-dependent manner, which was inhibited by the iron chelator, deferoxamine. However, paradoxically, this potentiating effect of l-ascorbate was completely abolished by a prolonged 4 h pre-incubation with l-ascorbate (500 μM). MM cytotoxicity induced by NTP was associated with immediate oxidative stress evaluated by 2′,7′-dichlorodihydrofluorecein diacetate, which was followed by an increase in the expression of the autophagosome marker, LC3B-II. In conclusion, MM can be a target for NTP treatment and l-ascorbate can increase or decrease its efficacy depending on the length of the pre-incubation period.

Non-thermal plasma-induced apoptosis is modulated by ATR- and PARP1-mediated DNA damage responses and circadian clock.

Non-thermal plasma (NTP) has been emerging as a potential cancer therapeutic. However, the practical use of NTP as a cancer therapy requires a better understanding of the precise mechanisms underlying NTP-induced DNA damage responses in order to achieve optimal efficacy. It has been shown that the addition of oxygen gas flow during NTP treatment (NTPO), when compared to NTP exposure alone, can induce a 2–3 fold greater generation of intracellular reactive oxygen species (ROS) in A549 cells. Here, we examined NTPO-induced DNA damage responses and found that NTPO generated a substantial number of genomic DNA lesions and breaks that activated ATR-mediated cell-cycle checkpoints. In addition, we discovered that NTPO-induced DNA lesions were primarily removed by base excision repair (BER) rather than by nucleotide excision repair (NER). Therefore, the inhibition of the BER pathway using a PARP1 inhibitor drastically induced the phosphorylation of γH2AX, and was followed by the programmed cell death of cancer cells. However, the knock-down of XPA, which inhibited the NER pathway, had no effect on NTPO-induced phosphorylation of γH2AX. Finally, in agreement with a recent report, we found a circadian rhythm of PARP1 activity in normal mouse embryonic fibroblasts that needed for cell viability upon NTPO treatment. Taken together, our findings provided an advanced NTP regimen for cancer treatment by combining NTPO treatment with chemical adjuvants for the inhibition of ATR- and PARP1-activated DNA damage responses, and circadian timing of treatment.

Non-thermal plasma inhibits human cervical cancer HeLa cells invasiveness by suppressing the MAPK pathway and decreasing matrix metalloproteinase-9 expression.

Non-thermal plasma (NTP) has been proposed as a novel therapeutic method for anticancer treatment. However, the mechanism underlying its biological effects remains unclear. In this study, we investigated the inhibitory effect of NTP on the invasion of HeLa cells, and explored the possible mechanism. Our results showed that NTP exposure for 20 or 40 s significantly suppressed the migration and invasion of HeLa cells on the basis of matrigel invasion assay and wound healing assay, respectively. Moreover, NTP reduced the activity and protein expression of the matrix metalloproteinase (MMP)-9 enzyme. Western blot analysis indicated that NTP exposure effectively decreased phosphorylation level of both ERK1/2 and JNK, but not p38 MAPK. Furthermore, treatment with MAPK signal pathway inhibitors or NTP all exhibited significant depression of HeLa cells migration and MMP-9 expression. The result showed that NTP synergistically suppressed migration and MMP-9 expression in the presence of ERK1/2 inhibitor and JNK inhibitor, but not p38 MAPK inhibitor. Taken together, these findings suggested that NTP exposure inhibited the migration and invasion of HeLa cells via down-regulating MMP-9 expression in ERK1/2 and JNK signaling pathways dependent manner. These findings provide hints to the potential clinical research and therapy of NTP on cervical cancer metastasis.

Abstract 2839: Antitumor activity on colorectal and pancreatic tumors of a new strategy based on ROS generation by non-thermal plasma

Abstract Background. Local treatments of tumors are mainly based on surgical resection and/or treatment such as photodynamic therapy (PDT) or ionizing radiation (IR). Action mechanisms of IR and PDT are based on the generation of ROS in the vicinity of the cells. In this context, cold or Non Thermal Plasma (NTP), a new technology which can generate in situ ROS, is currently under investigations. NTP is a cold (&amp;lt;40°C) ionized gas (air or noble gas) thank to an electric discharge. It contains free charges (electrons, ions), free radicals and excited molecules. NTP can be generated at the end of a catheter (plasma jet) allowing a local treatment that is compatible with usual endoscopes for dysplasia or non resecable tumors. Previous studies have shown an in vitro antitumor activity of NTP on various cells lines and in vivo on subcutaneous xenografts. The aim of this work was to assess the antitumor potential of fibred NTP when applied directly on in situ orthotopic human pancreatic or colorectal carcinoma xenografts. Methods. For in vitro experiments, HCT116-Luc and Mia Paca-Luc cells were seeded in 24-well plate and then treated with a plasma jet device called plasma gun. The effects on cells were assessed by bioluminescence imaging (BLI). For in vivo experiments, HCT116-Luc cells were xenografted in the caecum wall of nude mice and tumors were treated by a single NTP exposure 7 days post-induction. Concerning pancreatic tumors, MIA PaCa2-Luc cells were injected in the pancreas head and NTP treatments were started 4 days after implantation. Two others exposures were performed on D14 and D24. For both models, tumor growth was monitored by BLI once a week. Results and Discussion. NTP treatment showed a significant antitumor effect on HCT116 and Mia Paca cells in vitro and in vivo. In vitro plasma induced a decrease of BLI resulting from a massive cell death. Localized treatments of tumor using plasma gun induced significant tumor stabilization. On colorectal tumors, a BLI difference of 90% between treated and CTRL tumors were observed at D22. This effect was confirmed by tumor weight measurement. Moreover, a significant metastasis decrease was observed. Similar results were observed on pancreatic tumors with also a stabilization of tumor growth and a BLI decrease following each treatment leading finally at D36 to a significant difference of tumor volume (84%). Comparison of NTP with the references drug of these pathologies is ongoing. The results obtained on two different tumor types show the potential antitumor activity of NTP on implanted small tumors. These effects were linked to a high rate generation of ROS in the vicinity of tumors, inducing lethal DNA damages, a multiphase cell cycle arrest and finally apoptosis. Such results demonstrate the interest of NTP for treatment of dysplasia and non-resecable tumors that could be associated to real time per operative near infrared fluorescence imaging via fibro/coelioscopy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2839. doi:1538-7445.AM2012-2839

Rapid Inactivation of Biological Species in the Air using Atmospheric Pressure Nonthermal Plasma

Here, nonthermal plasma generated by a dielectric barrier discharge (DBD) system was applied to inactivating aerosolized Bacillus subtilis cells and Pseudomonas fluorescens as well as indoor and outdoor bioaerosols. The culturability, viability, and diversity losses of the microorganisms in air samples treated by the plasma for 0.06-0.12 s were studied using culturing, DNA stain as well as polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) methods. In addition, the viable fraction of bacterial aerosols with and without the plasma treatment was also quantified using qPCR coupled with ethidium monoazide (EMA). It was shown that less than 2% of B. subtilis aerosols survived the plasma treatment of 0.12 s, while none of the P. fluorescens aerosols survived. Viability tests, EMA-qPCR results, and Scanning Electron Microscopy (SEM) images demonstrated that both bacterial species suffered significant viability loss, membrane, and DNA damages. Exposure of environmental bacterial and fungal aerosols to the plasma for 0.06 s also resulted in their significant inactivations, more than 95% for bacteria and 85-98% for fungal species. PCR-DGGE analysis showed that plasma exposure of 0.06 s resulted in culturable bacterial aerosol diversity loss for both environments, especially pronounced for indoor environment. The results here demonstrate that nonthermal plasma exposure could offer a highly efficient air decontamination technology.