Cell density and extracellular matrix composition mitigate bacterial biofilm sensitivity to UV-C LED irradiation.

Ultraviolet-C light-emitting diodes (UV-C LEDs) are an emerging technology for decontamination applications in different sectors. In this study, the inactivation of bacterial biofilms was investigated by applying an UV-C LED emitting at 280 nm and by measuring both the influence of the initial cell density (load) and presence of an extracellular matrix (biofilm). Two bacterial strains exposing diverging matrix structures and biochemical compositions were used: Pseudomonas aeruginosa and Leuconostoc citreum. UV-C LED irradiation was applied at three UV doses (171 to 684 mJ/cm2) on both surface-spread cells and on 24-h biofilms and under controlled cell loads, and bacterial survival was determined. All surface-spread bacteria, between 105 and 109 CFU/cm2, and biofilms at 108 CFU/cm2 showed that bacterial response to irradiation was dose-dependent. The treatment efficacy decreased significantly for L. citreum surface-spread cells when the initial cell load was high, while no load effect was observed for P. aeruginosa. Inactivation was also reduced when bacteria were grown under a biofilm form, especially for P. aeruginosa: a protective effect could be attributed to abundant extracellular DNA and proteins in the matrix of P. aeruginosa biofilms, as revealed by Confocal Laser Scanning Microscopy observations. This study showed that initial cell load and exopolymeric substances are major factors influencing UV-C LED antibiofilm treatment efficacy. KEY POINTS: • Bacterial cell load (CFU/cm2) could impact UV-C LED irradiation efficiency • Characteristics of the biofilm matrix have a paramount importance on inactivation • The dose to be applied can be predicted based on biofilm properties.

Cold Atmospheric Plasma Jet Treatment Improves Human Keratinocyte Migration and Wound Closure Capacity without Causing Cellular Oxidative Stress.

Cold Atmospheric Plasma (CAP) is an emerging technology with great potential for biomedical applications such as sterilizing equipment and antitumor strategies. CAP has also been shown to improve skin wound healing in vivo, but the biological mechanisms involved are not well known. Our study assessed a possible effect of a direct helium jet CAP treatment on keratinocytes, in both the immortalized N/TERT-1 human cell line and primary keratinocytes obtained from human skin samples. The cells were covered with 200 µL of phosphate buffered saline and exposed to the helium plasma jet for 10−120 s. In our experimental conditions, micromolar concentrations of hydrogen peroxide, nitrite and nitrate were produced. We showed that long-time CAP treatments (≥60 s) were cytotoxic, reduced keratinocyte migration, upregulated the expression of heat shock protein 27 (HSP27) and induced oxidative cell stress. In contrast, short-term CAP treatments (<60 s) were not cytotoxic, did not affect keratinocyte proliferation and differentiation, and did not induce any changes in mitochondria, but they did accelerate wound closure in vitro by improving keratinocyte migration. In conclusion, these results suggest that helium-based CAP treatments improve wound healing by stimulating keratinocyte migration. The study confirms that CAP could be a novel therapeutic method to treat recalcitrant wounds.

Cold helium plasma jet does not stimulate collagen remodeling in a 3D human dermal substitute.

Cold Atmospheric Plasma (CAP) is an emerging physical approach displaying encouraging antitumor and wound healing effects both in vitro and in vivo. In this study, we assessed the potential of direct CAP to remodel skin collagens using an original tissue-engineered human dermal substitute model rich in endogenous extracellular matrix (ECM) covered with 600 µl of culture medium and treated with CAP for 30 and 120 s. Our results indicated that Reactive Oxygen and Nitrogen Species (RONS) such as H2O2, NO3- and NO2- were produced in the medium during treatment. It appeared that in the CAP-treated dermal substitutes 1) cell viability was not altered, 2) pro-collagen I secretion was not modified over 48 h of culture after treatment, 3) global activity of matrix metalloproteinases MMPs was not modulated over 48 h after treatment, and 4) no change in hydroxyproline content was observed over 5 days after treatment. In order to confirm the efficiency of our device, we showed that the plasma-activated culture medium induced cell apoptosis and growth delay using a 3D human tumor spheroid model. In conclusion, no effect of direct CAP treatment was monitored on dermal ECM production and degradation, indicating that CAP does not stimulate collagen remodeling at the tissue scale.

Effects of Dielectric Barrier Ambient Air Plasma on Two Brassicaceae Seeds: Arabidopsis thaliana and Camelina sativa.

We investigated low-temperature plasma effects on two Brassicaceae seeds (A. thaliana and C. sativa) using dielectric barrier discharge in air. Comparisons of plasma treatments on seeds showed distinct responses on germination rate and speed. Optimal treatment time giving optimal germination is 15 min for A. thaliana with 85% increase compared to control after 48 h of germination and 1 min for C. sativa with 75% increase compared to control after 32 h of germination. Such germination increases are associated with morphological changes shown by SEM of seed surface. For better understanding at the biochemical level, seed surfaces were analyzed using gas chromatography-mass spectrometry which underlined changes of lipidic composition. For both treated seeds, there is a decrease of saturated (palmitic and stearic) fatty acids while treated C. sativa showed a decrease of unsaturated (oleic and linoleic) acids and treated A. thaliana an increase of unsaturated ones. Such lipid changes, specifically a decrease of hydrophobic saturated fatty acids, are coherent with the other analyses (SEM, water uptake and contact angle). Moreover, an increase in A. thaliana of unsaturated acids (very reactive) probably neutralizes plasma RONS effects thus needing longer plasma exposure time (15 min) to reach optimal germination. For C. sativa, 1 min is enough because unsaturated linoleic acid becomes lower in treated C. sativa (1.2 × 107) compared to treated A. thaliana (3.7 × 107).

Response of Controlled Cell Load Biofilms to Cold Atmospheric Plasma Jet: Evidence of Extracellular Matrix Contribution.

AIM: Study of the biocidal effect of a cold atmospheric-pressure plasma in ambient air on single-species bacterial biofilms with controlled cell density, characterized by different extracellular matrices. METHODS AND RESULTS: Two bacterial strains were chosen to present different Gram properties and contrasted extracellular matrices: Pseudomonas aeruginosa ATCC 15442 (Gram-negative), and Leuconostoc citreum NRRL B-1299 (Gram-positive). P. aeruginosa biofilm exhibits a complex matrix, rich in proteins while L. citreum presents the specificity to produce glucan-type exopolysaccharides when grown in the presence of sucrose. Plasma was applied on both surface-spread cells and 24-h grown biofilms with controlled cell loads over 5, 10, or 20 min. Surface-spread bacteria showed a time dependent response, with a maximal bacterial reduction of 2.5 log after 20 min of treatment. On the other hand, in our experimental conditions, no bactericidal effect could be observed when treating biofilms of P. aeruginosa and glucan-rich L. citreum. CONCLUSIONS: For biofilms presenting equivalent cell loads, the response to plasma treatment seemed to depend on the properties of the extracellular matrix characterized by infrared spectroscopy, scanning electron microscopy, or dry weight. SIGNIFICANCE AND IMPACT OF STUDY: Both cell load standardization and biofilm characterization are paramount factors to consider the biocide effect of plasma treatments. The extracellular matrix could affect the plasma efficacy by physical and/or chemical protective effects.

Anti-Cancer Potential of Two Plasma-Activated Liquids: Implication of Long-Lived Reactive Oxygen and Nitrogen Species.

Cold atmospheric plasma-exposed culture medium may efficiently kill cancer cells in vitro. Due to the complexity of the medium obtained after plasma exposure, less complex physiological liquids, such as saline solutions and saline buffers, are gathering momentum. Among the plethora of reactive oxygen and nitrogen species (RONS) that are produced in these plasma-activated liquids, hydrogen peroxide, nitrite and nitrate appear to be mainly responsible for cytotoxic and genotoxic effects. Here, we evaluated the anti-cancer potential of plasma-activated phosphate-buffered saline (P-A PBS) and sodium chloride 0.9% (P-A NaCl), using a three-dimensional tumor model. Two epithelial cancer cell lines were used to evaluate cellular effects of either P-A PBS or P-A NaCl. Human colorectal cancer cells HCT 116 and human ovarian carcinoma, SKOV-3 were used to investigate the manner by which different cell types respond to different plasma-activated liquids treatments. Our investigations indicate that P-A PBS is more efficient than P-A NaCl mainly because RONS are produced in larger quantities. Indeed, we show that the cytotoxicity of these liquids directly correlates with the concentration of hydrogen peroxide and nitrite. Moreover, P-A PBS induced a faster-occurring and more pronounced cell death, which arose within deeper layers of the 3D multicellular spheroid models.

New insights of low-temperature plasma effects on germination of three genotypes of Arabidopsis thaliana seeds under osmotic and saline stresses.

In order to investigate the effects of low temperature plasmas on germination of Arabidopsis thaliana seeds, a dielectric barrier discharge device generating the plasma in ambient air was used. To highlight the different plasma effects on the seed surface, saline and osmotic stresses were considered in the case of reference Col-0 seeds and two further seed coat mutants gl2 and gpat5 to better analyse the seed surface changes and their consequences on germination. The GL2 gene encode a transcription factor controlling the balance between the biosynthesis of fatty acids in the embryo and the production of mucilage and flavonoid pigments in the seed coat. The GPAT5 gene encode for an acyltransferase necessary for the accumulation of suberin in the seed coat which is essential for the embryo protection. The testa and endosperm ruptures are identified to note the germination stage. An increasing of germination rate, possibly due to the modification of mantle layers structure, is observed in most of cases, even in presence of saline or osmotic stress, after plasma treatment. Furthermore, we demonstrated that the germination rate of the gl2 mutant seeds is increased by at most 47% after plasma treatment, contrariwise, the germination of gpat5 mutant being initially lower is inhibited by the same plasma treatment. The scanning electron microscopy pictures and confocal microscopy fluorescence both showed changes of the exterior aspects of the seeds after plasma treatment. Considering these results, we assumed that lipid compounds can be found on the surface. To validate this hypothesis, permeability tests were performed, and it was clearly shown that a permeability decrease is induced by the low temperature plasma treatment.

Pulsed Electric Field Treatment Enhances the Cytotoxicity of Plasma-Activated Liquids in a Three-Dimensional Human Colorectal Cancer Cell Model.

Cold atmospheric plasma and more recently, plasma-activated liquids (culture media, water or buffered solutions previously exposed to plasma), are gathering momentum in cancer cells treatment. Nevertheless, in vitro tests show that this novel approach is sometimes less efficient than expected. We here evaluate the mechanisms of action of the plasma-activated PBS and suggest to use electropermeabilization (EP) in combination with the plasma-activated phosphate-buffered saline (PBS), in order to potentiate the cytotoxic effect of the plasma activated liquid. Human multicellular tumor spheroids (MCTS), a three-dimensional cell model, which resembles small avascular tumors, was used to define the optimal treatment conditions for single and dual-mode treatments. MCTS growth, viability, and global morphological changes were assessed by live cell video-microscopy. In addition, the induction of caspases activation, the appearance of DNA damages, and cell membrane permeabilization, as well as the early modifications in the cellular ultrastructure, were examined by immunofluorescence, propidium iodide staining, confocal fluorescence microscopy and transmission electron microscopy, respectively. Altogether, our results show that a combined treatment resulted in an earlier onset of DNA damage and caspases activation, which completely abolished MCTS growth. This report is a proof of concept study evidencing that electropermeabilization greatly potentiates the cytotoxic effect of plasma-activated PBS in vitro in a three-dimensional cancer cell model.

Elucidation of in vitro cellular steps induced by antitumor treatment with plasma-activated medium.

Numerous studies have reported cold atmospheric plasma cytotoxic activities in various cancer cell lines, either by direct exposure to non-thermal plasma or indirectly by activating a medium (plasma-activated medium, PAM) prior to cell treatment. We suggested the use of in vitro 3D tumor model spheroids to determine the potential of PAM for cancer therapy at the tissue scale, especially in human tumor tissue. This work aimed to better understand the effect of PAM on human colorectal tumor spheroids by describing the in vitro-induced-cell death kinetics and associated mechanisms to further improve its therapeutic potential. Tumor spheroid growth was delayed depending on contact time with PAM. Medium osmolarity was increased by activation with low temperature Helium plasma jet but it did not fully explain the observed growth delay. PAM impaired tumor cell viability through intracellular ATP depletion, leading within hours to both cell apoptosis and necrosis as well as mitochondrial oxidative stress. When successive treatments were spaced over time, cumulative effects on the growth delay of spheroids were observed. Taken together, these results demonstrated that plasma-activated liquids may represent a novel and efficient therapeutic method for the treatment of tumors, especially when successive treatments are applied.

Use of cold-atmospheric plasma in oncology: a concise systematic review.

BACKGROUND: Cold-atmospheric plasma (CAP) is an ionized gas produced at an atmospheric pressure. The aim of this systematic review is to map the use of CAP in oncology and the implemented methodologies (cell targets, physical parameters, direct or indirect therapies). METHODS: PubMed, the International Clinical Trials Registry Platform and Google Scholar were explored until 31 December 2017 for studies regarding the use of plasma treatment in oncology (in vitro, in vivo, clinical trials). RESULTS: 190 original articles were included. Plasma jets are the most-used production systems (72.1%). Helium alone was the most-used gas (35.8%), followed by air (26.3%) and argon (22.1%). Studies were mostly in vitro (94.7%) and concerned direct plasma treatments (84.2%). The most targeted cancer cell lines are human cell lines (87.4%), in particular, in brain cancer (16.3%). CONCLUSIONS: This study highlights the multiplicity of means of production and clinical applications of the CAP in oncology. While some devices may be used directly at the bedside, others open the way for the development of new pharmaceutical products that could be generated at an industrial scale. However, its clinical use strongly needs the development of standardized reliable protocols, to determine the more efficient type of plasma for each type of cancer, and its combination with conventional treatments.

Effects of low temperature plasmas and plasma activated waters on Arabidopsis thaliana germination and growth.

Two plasma devices at atmospheric pressure (air dielectric barrier discharge and helium plasma jet) have been used to study the early germination of Arabidopsis thaliana seeds during the first days. Then, plasma activated waters are used during the later stage of plant development and growth until 42 days. The effects on both testa and endospserm ruptures during the germination stage are significant in the case of air plasma due to its higher energy and efficiency of producing reactive oxygen species than the case of helium plasma. The latter has shown distinct effects only for testa rupture. Analysis of germination stimulations are based on specific stainings for reactive oxygen species production, peroxidase activity and also membrane permeability tests. Furthermore, scanning electron microscopy (SEM) has shown a smoother seed surface for air plasma treated seeds that can explain the plasma induced-germination. During the growth stage, plants were watered using 4 kinds of water (tap and deionized waters activated or not by the low temperature plasma jet). With regards to other water kinds, the characterization of the tap water has shown a larger conductivity, acidity and concentration of reactive nitrogen and oxygen species. Only the tap water activated by the plasma jet has shown a significant effect on the plant growth. This effect could be correlated to reactive nitrogen species such as nitrite/nitrate species present in plasma activated tap water.

Effects of Plasma Activated Medium on Head and Neck FaDu Cancerous Cells: Comparison of 3D and 2D Response.

OBJECTIVE: The aim of this work is to investigate the inhibitory effect of Plasma Activated Medium (PAM) on Head and Neck cancerous cells (FaDu). The response of FaDu cells in monolayer cultures and Multi Cellular Tumor Spheroids (MCTS) after treatment with different PAMs will be compared. BACKGROUND: Head and Neck squamous cell carcinoma is a widespread cancer that responds poorly to anticancer treatments such as chemotherapy and radiotherapy. Nowadays there is a growing interest in cold plasmas and their applications in cancer therapy. METHODS: A homemade helium plasma jet is used to produce PAM. The effects of PAM and hydrogen peroxide H2O2 on FaDu 2D cells cultures and MCTS were characterized by evaluating the cell viability with PrestoBlue test and by measuring the size of MCTS. RESULTS: One treatment with PAM induce cell detachment from MCTS since the first day in a PAM exposure dependent manner. This is due to the presence of H2O2 in PAM. However, a rapid spheroids regrowth is observed attributed to a resistance of FaDu cells to H2O2. After multiple treatments of MCTS with PAM we obtained an inhibition of cell growth. MCTS are brought out when comparing PAM effect on 2D versus MCTS. Inversely, PAM induces cell death in the case of 2D cell culture. CONCLUSION: PAM may be considered as a potentially efficient agent in the therapy of head and neck cancer. We also point out that MCTS is a more valuable model than 2D cell culture for the evaluation of the anti-cancer activity of PAM.

Analysis of reactive oxygen and nitrogen species generated in three liquid media by low temperature helium plasma jet.

In order to identify aqueous species formed in Plasma activated media (PAM), quantitative investigations of reactive oxygen and nitrogen species (ROS, RNS) were performed and compared to Milli-Q water and culture media without and with Fetal Calf Serum. Electron paramagnetic resonance, fluorometric and colorimetric analysis were used to identify and quantify free radicals generated by helium plasma jet in these liquids. Results clearly show the formation of ROS such as hydroxyl radical, superoxide anion radical and singlet oxygen in order of the micromolar range of concentrations. Nitric oxide, hydrogen peroxide and nitrite-nitrate anions (in range of several hundred micromolars) are the major species observed in PAM. The composition of the medium has a major impact on the pH of the solution during plasma treatment, on the stability of the different RONS that are produced and on their reactivity with biomolecules. To emphasize the interactions of plasma with a complex medium, amino acid degradation by means of mass spectrometry was also investigated using methionine, tyrosine, tryptophan and arginine. All of these components such as long lifetime RONS and oxidized biological compounds may contribute to the cytotoxic effect of PAM. This study provides mechanistic insights into the mechanisms involved in cell death after treatment with PAM.

Short and long time effects of low temperature Plasma Activated Media on 3D multicellular tumor spheroids.

This work investigates the regionalized antiproliferative effects of plasma-activated medium (PAM) on colon adenocarcinoma multicellular tumor spheroid (MCTS), a model that mimics 3D organization and regionalization of a microtumor region. PAM was generated by dielectric barrier plasma jet setup crossed by helium carrier gas. MCTS were transferred in PAM at various times after plasma exposure up to 48 hours and effect on MCTS growth and DNA damage were evaluated. We report the impact of plasma exposure duration and delay before transfer on MCTS growth and DNA damage. Local accumulation of DNA damage revealed by histone H2AX phosphorylation is observed on outermost layers and is dependent on plasma exposure. DNA damage is completely reverted by catalase addition indicating that H2O2 plays major role in observed genotoxic effect while growth inhibitory effect is maintained suggesting that it is due to others reactive species. SOD and D-mannitol scavengers also reduced DNA damage by 30% indicating that O(2)(-)* and OH* are involved in H2O2 formation. Finally, PAM is able to retain its cytotoxic and genotoxic activity upon storage at +4 °C or -80 °C. These results suggest that plasma activated media may be a promising new antitumor strategy for colorectal cancer tumors.

Low-temperature plasmas at atmospheric pressure: toward new pharmaceutical treatments in medicine.

This article concerns a new field covered by low-temperature plasmas at atmospheric pressure for medical treatments. This is based on the very attractive possibility to tune and design plasmas as possible pharmaceutical products using selectively some active species (charged particles, radicals, atomic and molecular agents, UV radiations) and even electric fields self-generated by the plasma. The delivery of active species occurs at the gaseous level. This means that there is no need for a carrier medium, and the treatment of living tissue or surface is optimal because plasmas can penetrate small pores, spread over rough surfaces, and reach both prokaryotic and eukaryotic cells. The present article gives first a review on the main low-temperature plasma setups potentially usable for medical treatments with an emphasis on the setups as, for instance, plasma jets developed in our laboratory. Then, the present article gives a review of the current state of the art of such plasmas as pharmaceutical products or therapeutic tools in medicine with a light on a selection of forefront researches particularly in the field of chronic wounds, blood coagulation, and cancer treatment.

The use of thermal techniques for the characterization and selection of natural biomaterials.

In this paper we explore the ability of thermal analysis to check elastin and collagen integrity in different biomaterial applications. Differential Scanning Calorimetry (DSC) has been used to analyze the first and second order transitions of the biological macromolecules in the hydrated and dehydrated state. First, we report the characterization of control cardiovascular tissues such as pericardium, aortic wall and valvular leaflet. Their thermal properties are compared to pure elastin and pure collagen. Second, we present results obtained on two collagen rich tissues: pericardia with different chemical treatments and collagen with physical treatments. Finally, more complex cardiovascular tissues composed of elastin and collagen are analyzed and the effect of detergent treatment on the physical structure of collagen and elastin is brought to the fore.