Plasma Technology Increases the Efficacy of Prothioconazole against Fusarium graminearum and Fusarium proliferatum Contamination of Maize (Zea mays) Seedlings.

The contamination of maize by Fusarium species able to produce mycotoxins raises great concern worldwide since they can accumulate these toxic metabolites in field crop products. Furthermore, little information exists today on the ability of Fusarium proliferatum and Fusarium graminearum, two well know mycotoxigenic species, to translocate from the seeds to the plants up to the kernels. Marketing seeds coated with fungicide molecules is a common practice; however, since there is a growing need for reducing chemicals in agriculture, new eco-friendly strategies are increasingly tested. Technologies based on ionized gases, known as plasmas, have been used for decades, with newer material surfaces, products, and approaches developed continuously. In this research, we tested a plasma-generated bilayer coating for encapsulating prothioconazole at the surface of maize seeds, to protect them from F. graminearum and F. proliferatum infection. A minimum amount of chemical was used, in direct contact with the seeds, with no dispersion in the soil. The ability of F. graminearum and F. proliferatum species to translocate from seeds to seedlings of maize has been clearly proven in our in vitro experiments. As for the use of plasma technology, the combined use of the plasma-generated coating with embedded prothioconazole was the most efficient approach, with a higher reduction of the infection of the maize seminal root system and stems. The debated capability of the two Fusarium species to translocate from seeds to seedlings has been demonstrated. The plasma-generated coating with embedded prothioconazole resulted in a promising sustainable approach for the protection of maize seedlings.

A Synergistic Effect of Reactive Oxygen and Reactive Nitrogen Species in Plasma Activated Liquid Media Triggers Astrocyte Wound Healing.

Astrocyte proliferation and migration toward injured Central Nervous System (CNS) areas are key features of astrogliosis and glial scar formation. Even though it is known that intracellular and environmental Reactive Oxygen and Nitrogen Species (RONS) affect astrocyte behaviour in physiological and pathophysiological conditions, their effects on the migration and growth of astrocytes are still unclear. Plasma-technologies are emerging in medicine as a tool to generate RONS for treating cells directly or through Plasma Activated Liquid Media (PALM). In this paper, we show for the first time how the use of PALM can modulate both astrocyte growth and migration as a function of active species produced by plasma in liquids. Our results show that PALM, generated by means of cold atmospheric pressure plasmas fed with N2, air or O2, can modulate astrocyte behaviour depending on the content of hydrogen peroxide and nitrite in the liquid. In particular, H2O2 enriched PALM induced a negative effect on cell growth associated with the mild wound healing improvement of primary astrocytes, in a scratch assay. Nitrite enriched PALM induced a selective effect on the wound healing without affecting cell growth. PALM containing a more balanced level of H2O2 and NO2- were able to affect cell growth, as well as significantly ameliorate wound healing. None of the PALM investigated induced upregulation of the gliotic inflammatory marker glial fibrillary acidic protein (GFAP), or of the astrocyte markers Aquaporin-4 (AQP4) and Connexin-43 (Cx-43) analysed by Western blot. Finally, immunofluorescence analysis revealed the presence of NO2- able to induce elongated protrusions at the front end of wounded astrocytes in the direction of cell migration. With our study we believe to have shown that PALM offer a novel tool to modulate astrocyte behaviour and that they are promising candidates for controlling astrogliosis in the case of CNS injuries.

Suppression of Hydrophobic Recovery by Plasma Polymer Films with Vertical Chemical Gradients.

Vertical chemical gradients extending over a few nanometers were explored. The gradients are based on plasma-polymerized oxygen-containing ethylene (ppOEt) films. Using plasma conditions with low CO2/C2H4 ratio and high energy input, cross-linked films were deposited as base layer, while increasing CO2 and lowering energy input resulted in less cross-linked yet highly functional films as applied as top layer. Aging studies indicate that, in particular, for very thin gradient structures, the cross-linked subsurface zone effectively hinders reorientation of the surface functional groups, thus restricting hydrophobic recovery and oxidation effects.

Highly Sensitive and Practical Detection of Plant Viruses via Electrical Impedance of Droplets on Textured Silicon-Based Devices.

Early diagnosis of plant virus infections before the disease symptoms appearance may represent a significant benefit in limiting disease spread by a prompt application of appropriate containment steps. We propose a label-free procedure applied on a device structure where the electrical signal transduction is evaluated via impedance spectroscopy techniques. The device consists of a droplet suspension embedding two representative purified plant viruses i.e., Tomato mosaic virus and Turnip yellow mosaic virus, put in contact with a highly hydrophobic plasma textured silicon surface. Results show a high sensitivity of the system towards the virus particles with an interestingly low detection limit, from tens to hundreds of attomolar corresponding to pg/mL of sap, which refers, in the infection time-scale, to a concentration of virus particles in still-symptomless plants. Such a threshold limit, together with an envisaged engineering of an easily manageable device, compared to more sophisticated apparatuses, may contribute in simplifying the in-field plant virus diagnostics.

Anticancer Effects of Plasma-Treated Water Solutions from Clinically Approved Infusion Liquids Supplemented with Organic Molecules.

Water solutions treated by cold atmospheric plasmas (CAPs) currently stand out in the field of cancer treatment as sources of exogenous blends of reactive oxygen and nitrogen species (RONS). It is well known that the balance of RONS inside both eukaryotic and prokaryotic cells is directly involved in physiological as well as pathological pathways. Also, organic molecules including phenols could exert promising anticancer effects, mostly attributed to their pro-oxidant ability in vitro and in vivo to generate RONS like O2-, H2O2, and a mixture of potentially cytotoxic compounds. By our vision of combining the efficacy of plasma-produced RONS and the use of organic molecules, we could synergistically attack cancer cells; yet, so far, this combination, to the best of our knowledge, has been completely unexplored. In this study, l-tyrosine, an amino acid with a phenolic side chain, is added to a physiological solution, often used in clinical practice (SIII) to be exposed to plasma. The efficacy of the gas plasma-oxidized SIII solution, containing tyrosine, was evaluated on four cancer cell lines selected from among tumors with poor prognosis (SHSY-5Y, MCF-7, HT-29, and SW-480). The aim was to induce tumor toxicity and trigger apoptosis pathways. The results clearly indicate that the plasma-treated water solution (PTWS) reduced cell viability and oxygen uptake due to an increase in intracellular ROS levels and activation of apoptosis pathways in all investigated cancer cells, which may be related to the activation of the mitochondrial-mediated and p-JNK/caspase-3 signaling pathways. This research offers improved knowledge about the physiological mechanisms underlying cancer treatment and a valid method to set up a prompt, adequate, and effective cancer treatment in the clinic.

Tuning Surface Properties via Plasma Treatments for the Improved Capture of MicroRNA Biomarkers.

Advanced materials could bring about fundamental improvements in the evolution of innovative analytical devices, i.e., biosensors or lab-on-a-chip devices, in particular in the context of liquid biopsies. Here, plasma deposition processes were tested for the introduction of primary amines on silicon surfaces by tuning the amounts and availability of amino-charged residues. Different binary (CH4/NH3) and ternary (CH4/NH3/H2 and CH4/NH3/N2) mixtures of gases were used as feeds for the plasma treatments. The obtained surfaces were fully characterized for their chemical and physical properties before their use as capture materials in a functional test. Synthetic and fluorescently conjugated microRNA-21 (miR-21) was selected as the target molecule. The capture of miR-21 increased linearly with the increase in amino nitrogen measured on surfaces. The surface showing the most promising performance was further analyzed in different conditions, i.e., varying pH and time of incubation, incubation with different microRNAs, and possible elution of captured microRNAs. The apparent pH range of primary amines present on the surfaces was around 3.5-4. Positively charged surfaces prepared via PE-CVD were, therefore, demonstrated as being suitable materials for the capture of microRNA biomarkers, paving the way for their inclusion in biomedical devices for the purification and analysis of circulating biomarkers.

Cell adhesion on nanotextured slippery superhydrophobic substrates.

In this work, the response of Saos2 cells to polymeric surfaces with different roughness/density of nanometric dots produced by a tailored plasma-etching process has been studied. Topographical features have been evaluated by atomic force microscopy, while wetting behavior, in terms of water-surface adhesion energy, has been evaluated by measurements of drop sliding angle. Saos2 cytocompatibility has been investigated by scanning electron microscopy, fluorescent microscopy, and optical microscopy. The similarity in outer chemical composition has allowed isolation of the impact of the topographical features on cellular behavior. The results indicate that Saos2 cells respond differently to surfaces with different nanoscale topographical features, clearly showing a certain inhibition in cell adhesion when the nanoscale is particularly small. This effect appears to be attenuated in surfaces with relatively bigger nanofeatures, though these express a more pronounced slippery/dry wetting character.

Osteoblast-like cell behavior on plasma deposited micro/nanopatterned coatings.

The behavior of cells in terms of cell-substrate and cell-cell interaction is dramatically affected by topographical characteristics as shape, height, and distance, encountered in their physiological environment. The combination of chemistry and topography of a biomaterial surface influences in turns, important biological responses as inflammatory events at tissue-implant interface, angiogenesis, and differentiation of cells. By disentangling the effect of material chemistry from the topographical one, the possibility of controlling the cell behavior can be provided. In this paper, surfaces with different roughness and morphology were produced by radiofrequency (RF, 13.56 MHz) glow discharges, fed with hexafluoropropylene oxide (C(3)F(6)O), in a single process. Coatings with different micro/nanopatterns and the same uppermost chemical composition were produced by combining two plasma deposition processes, with C(3)F(6)O and tetrafluoroethylene (C(2)F(4)), respectively. The behavior of osteoblast-like cells toward these substrates clearly shows a strict dependence of cell adhesion and proliferation on surface roughness and morphology.

Plasma Treated Water Solutions in Cancer Treatments: The Contrasting Role of RNS.

Plasma Treated Water Solutions (PTWS) recently emerged as a novel tool for the generation of Reactive Oxygen and Nitrogen Species (ROS and RNS) in liquids. The presence of ROS with a strong oxidative power, like hydrogen peroxide (H2O2), has been proposed as the main effector for the cancer-killing properties of PTWS. A protective role has been postulated for RNS, with nitric oxide (NO) being involved in the activation of antioxidant responses and cell survival. However, recent evidences proved that NO-derivatives in proper mixtures with ROS in PTWS could enhance rather than reduce the selectivity of PTWS-induced cancer cell death through the inhibition of specific antioxidant cancer defenses. In this paper we discuss the formation of RNS in different liquids with a Dielectric Barrier Discharge (DBD), to show that NO is absent in PTWS of complex composition like plasma treated (PT)-cell culture media used for in vitro experiments, as well as its supposed protective role. Nitrite anions (NO2-) instead, present in our PTWS, were found to improve the selective death of Saos2 cancer cells compared to EA.hy926 cells by decreasing the cytotoxic threshold of H2O2 to non-toxic values for the endothelial cell line.

Nanometric self-assembling peptide layers maintain adult hepatocyte phenotype in sandwich cultures.

BACKGROUND: Isolated hepatocytes removed from their microenvironment soon lose their hepatospecific functions when cultured. Normally hepatocytes are commonly maintained under limited culture medium supply as well as scaffold thickness. Thus, the cells are forced into metabolic stress that degenerate liver specific functions. This study aims to improve hepatospecific activity by creating a platform based on classical collagen sandwich cultures. RESULTS: The modified sandwich cultures replace collagen with self-assembling peptide, RAD16-I, combined with functional peptide motifs such as the integrin-binding sequence RGD and the laminin receptor binding sequence YIG to create a cell-instructive scaffold. In this work, we show that a plasma-deposited coating can be used to obtain a peptide layer thickness in the nanometric range, which in combination with the incorporation of functional peptide motifs have a positive effect on the expression of adult hepatocyte markers including albumin, CYP3A2 and HNF4-alpha. CONCLUSIONS: This study demonstrates the capacity of sandwich cultures with modified instructive self-assembling peptides to promote cell-matrix interaction and the importance of thinner scaffold layers to overcome mass transfer problems. We believe that this bioengineered platform improves the existing hepatocyte culture methods to be used for predictive toxicology and eventually for hepatic assist technologies and future artificial organs.

Initiated Chemical Vapor Deposition of Crosslinked Organic Coatings for Controlling Gentamicin Delivery.

A coating consisting of a copolymer of methacrylic acid and ethylene glycol dimethacrylate was deposited over a gentamicin film by initiated chemical vapor deposition with the aim of controlling the drug release. Gentamicin release in water was monitored by means of conductance measurements and of UV-vis Fluorescence Spectroscopy. The influence of the polymer chemical composition, specifically of its crosslinking density, has been investigated as a tool to control the swelling behavior of the initiated chemical vapor deposition (iCVD) coating in water, and therefore its ability to release the drug. Agar diffusion test and microbroth dilution assays against Staphylococcus aureus and Pseudomonas aeruginosa on cellulose coated substrates confirmed that the antibacterial activity of the drug released by the coating was retained, though the release of gentamicin was not complete.

Plasma-activated medium triggers cell death and the presentation of immune activating danger signals in melanoma and pancreatic cancer cells.

Over the past decade, cold atmospheric plasmas have shown promising application in cancer therapy. The therapeutic use of plasma-activated media is a topic addressed in an emerging field known as plasma pharmacy. In oncology, plasma-activated media are  used to harness the therapeutic effects of oxidant species when they come in contact with cancer cells. Among several factors that contribute to the anticancer effect of plasma-activated liquid media (PALM), H2O2 and NO derivatives likely play a key role in the apoptotic pathway. Despite the significant amount of literature produced in recent years, a full understanding of the mechanisms by which PALM exert their activity against cancer cells is limited. In this paper, a sealed dielectric-barrier discharge was used to disentangle the effect of reactive nitrogen species (RNS) from that of reactive oxygen species (ROS) on cancer cells. Two cancers characterized by poor prognosis have been investigated: metastatic melanoma and pancreatic cancer. Both tumour models exposed to PALM rich in H2O2 showed a reduction in proliferation and an increase in calreticulin exposure and ATP release, suggesting the potential use of activated media as an inducer of immunogenic cell death via activation of the innate immune system.

Plasma-Deposited Nanocapsules Containing Coatings for Drug Delivery Applications.

Coatings consisting in gentamicin-containing nanocapsules have been synthetized by means of an aerosol-assisted atmospheric pressure plasma deposition process. The influence of different parameters affecting the process has been extensively investigated by means of a morphological and chemical characterization of the coatings. Scanning electron microscopy highlighted the presence of nanocapsules whose size and abundance depend on power input and deposition time. A detailed analysis carried out with matrix-assisted laser desorption ionization coupled to high-resolution mass spectrometry allowed to detect and identify the presence of gentamicin embedded in the coatings and its rearrangement, as a result of the interaction with the plasma. The release of gentamicin in water has been monitored by means of UV-vis fluorescence spectroscopy, and its biological activity has been evaluated as well by the disk diffusion assay against Staphylococcus aureus and Pseudomonas aeruginosa. It is confirmed that the antibacterial activity of gentamicin is preserved in the plasma-deposited coatings. Preliminary cytocompatibility investigations indicated that eukaryotic cells well tolerate the release of gentamicin from the coatings.

Fetuin-A gene expression, synthesis and release in primary human hepatocytes cultured in a galactosylated membrane bioreactor.

This paper reports on human hepatocytes cultured in a galactosylated membrane bioreactor in order to explore the modulation of the effects of a pro-inflammatory cytokine, Interleukin-6 (IL-6) on the liver cells at molecular level. In particular the role of IL-6 on gene expression and production of a glycoprotein, fetuin-A produced by hepatocytes, was investigated by culturing hepatocytes in the membrane bioreactor, both in the absence and presence of IL-6 (300 pg/ml). IL-6 modulated the fetuin-A gene expression, synthesis and release by primary human hepatocytes cultured in the bioreactor. A 75% IL-6-induced reduction of fetuin-A concentration in the medium was associated with a 60% increase of C-reactive protein in the same samples. Real-time-PCR demonstrated an 8-fold IL-6-induced reduction of fetuin-A gene expression. These results demonstrate that the hepatocyte galactosylated membrane bioreactor is a valuable tool to study IL-6 effects and gave evidence, for the first time, that IL-6 down-regulates the gene expression and synthesis of fetuin-A by primary human hepatocytes. The human hepatocyte bioreactor behaves like the in vivo liver, reproducing the same hepatic acute-phase response that occurs during the inflammatory process.

Novel membranes and surface modification able to activate specific cellular responses.

The design of new polymeric biomaterials together with new strategies to modify membrane surface are crucial to optimise cell-biomaterial interactions in vivo and in vitro biohybrid systems. In this study we report on the novel semipermeable membranes synthesised from a polymeric blend of modified polyetheretherketone and polyurethane able to support the long-term maintenance and differentiation of human liver cells and on the surface modification of polyethersulfone membranes by plasma polymerisation of acrylic acid monomers and by immobilization of arginine-glycine-aspartic acid (RGD) peptide through a hydrophilic "spacer arm" molecule. The performance of the modified and unmodified membranes was tested by evaluation of the liver function expression of primary human hepatocytes in terms of albumin production, protein secretion and drug biotransformation.

Improving Internal Cell Colonization of Porous Scaffolds with Chemical Gradients Produced by Plasma Assisted Approaches.

Cell colonization of the surrounding environment is a very significant process in both physiological and pathological events. In order to understand the tissue regeneration process and thereby provide guidance principles for designing new biomaterials, it is of paramount importance to study the cell colonization in the presence of physical, chemical, and biological cues. Flat "gradient" materials are generally used with this purpose. Three dimensional gradient scaffolds mimicking more precisely the situation in vivo are somewhat more complex to fabricate and characterize. Scaffolds for Tissue Engineering (TE) made of hydrophobic synthetic polymers do not allow good cell colonization: far from their periphery, in fact, internal cell colonization is usually low. In this research poly-ε caprolactone (PCL) scaffolds have been "decorated" with chemical gradients both on top and along their thickness by means of cold plasma processes, in order to improve cell colonization of their core. Plasma treatments with a mixture of argon and oxygen (Ar/O2), as well as plasma deposition of differently cross-linked poly(ethylene oxide) (PEO)-like coatings, have been performed. This study establishes that cross-linked PEO-like domains interspaced with native PCL ones deposited only on top of the scaffold (i.e., coating that penetrates less than 300 µm inside the scaffold) are more effective in promoting cell colonization across the scaffolds than the other tested materials including superhydrophilic samples and that ones produced by tested double step approaches. Last but not least, one result of this research is that, in the case of plasma coatings with low deposition rates and porous materials with a low pore interconnectivity, it is possible to improve penetration of low pressure plasma active species inside the scaffold's core thorough a pretreatment of the porous materials (i.e., penetration up to 4500 mm far from topside).

Towards highly stable aqueous dispersions of multi-walled carbon nanotubes: the effect of oxygen plasma functionalization.

In order to improve the dispersion of multi-walled carbon nanotubes (MWCNTs) in aqueous media, their surface functionalization was carried out in O2-fed low-pressure plasmas. Differently from what can be found in the literature of this field, homogeneous functionalization was achieved by generating the plasma inside vials containing the nanotube powders properly stirred. Experimental parameters, such as input power, treatment time and pressure, were varied to investigate their influence on the process efficiency. A detailed characterization of the plasma treated nanotubes, dry and in aqueous suspension, was carried out with a multi-diagnostic analytical approach, to evaluate their surface chemical properties, morphology, structural integrity and stability in the colloidal state. The plasma grafting of polar ionizable (e.g. acid) groups has been proved to successfully limit the agglomeration of MWCNTs and to produce nanotubes suspensions that are stable for one month and more in water.

Galactose derivative immobilized glow discharge processed polyethersulfone membranes maintain the liver cell metabolic activity.

New strategies aimed to surface modification of polymeric membranes are crucial to optimise cell-biomaterial interactions in vivo and in vitro biohybrid systems. In this paper, we investigated the surface modification of Polyethersulfone (PES) membranes by plasma polymerisation of acrylic acid monomers (PES-pdAA) and by immobilization of galactonic acid through a hydrophilic "spacer arm" molecule (PES-pdAA-SA-GAL). The modification steps were characterised by high resolution X-ray photoelectron spectroscopy. The performance of modified and unmodified membranes was evaluated by assessing the expression of liver specific biotransformation functions of pig and human hepatocytes. Human liver cells cultured on PES-pdAA-SA-GAL membranes displayed an enhanced albumin production, urea synthesis and protein secretion for 24 days of culture. The immobilisation of galactose derivative units on the membrane allowed specific interactions with hepatocytes biomimicking the cellular microenvironment and produced an improvement of the long-term maintenance and differentiation of human hepatocytes.

Non-Equilibrium Plasma Processing for the Preparation of Antibacterial Surfaces.

Non-equilibrium plasmas offer several strategies for developing antibacterial surfaces that are able to repel and/or to kill bacteria. Due to the variety of devices, implants, and materials in general, as well as of bacteria and applications, plasma assisted antibacterial strategies need to be tailored to each specific surface. Nano-composite coatings containing inorganic (metals and metal oxides) or organic (drugs and biomolecules) compounds can be deposited in one step, and used as drug delivery systems. On the other hand, functional coatings can be plasma-deposited and used to bind antibacterial molecules, for synthesizing surfaces with long lasting antibacterial activity. In addition, non-fouling coatings can be produced to inhibit the adhesion of bacteria and reduce the formation of biofilm. This paper reviews plasma-based strategies aimed to reduce bacterial attachment and proliferation on biomedical materials and devices, but also onto materials used in other fields. Most of the activities described have been developed in the lab of the authors.

Proliferative and re-defferentiative effects of photo-immobilized micro-patterned hyaluronan surfaces on chondrocyte cells.

A photo-immobilisation procedure was utilised to create two different micro-patterned surfaces (tracks 25 and 5 microm wide) of hyaluronan (Hyal) on polyethylene-terephthalate (PET) previously plasma activated. Aim of the study was to investigate the proliferation and re-differentiation capacity of articular chondrocytes cultured on micro-patterned Hyal, compared to homogeneous Hyal and plain plasma-treated (pt-)PET substrates. Cytotoxicity, cell proliferation, activation and differentiation of articular knee cartilage chondrocytes (Mongrel sheep) were evaluated after 14 days of culture. It was found that micro-patterned Hyal surfaces induced the adhesion, migration and alignment of chondrocytes, as shown by light and scanning electron microscopy. Furthermore, the same surfaces induced chondrocyte differentiation, with a significant increase of aggrecan and collagen type II production, while homogeneous Hyal and pt-PET surfaces did not.