Application of Argon Plasma Technology for the Synthesis of Anti-Infective Copper Nanoparticles.

The synthesis of copper nanoparticles (CuNPs) was accomplished by using a rapid, green, and versatile argon plasma reduction method that involves solvent extraction. With this method, a plasma-solid state interaction forms and CuNPs can be synthesized from copper(II) sulfate using a low-pressure, low-temperature argon plasma. Characterization studies of the CuNPs revealed that when a metal precursor is treated under optimal experimental conditions of 80 W of argon plasma for 300 s, brown CuNPs are synthesized. However, when those same brown CuNPs are placed in Milli-Q water for a period of 10 days, oxidation occurs and green CuNPs are formed. Confirmation of the chemical identity of the CuNPs was performed by using X-ray photoelectron spectroscopy. The results reveal that the brown CuNPs are predominantly Cu0 or what we refer to as CuNPs, while the green CuNPs are a mixture of Cu0 and Cu(OH)2 NPs. Upon further characterization of both brown and green CuNPs with scanning electron microscopy (SEM), the results depict brown CuNPs with a rod-like shape and approximate dimensions of 40 nm × 160 nm, while the green CuNPs were smaller in size, with dimensions of 40-80 nm, and more of a round shape. When testing the antibacterial activity of both brown and green CuNPs, our findings demonstrate the effectiveness of both CuNPs against Escherichia coli and Staphylococcus aureus bacteria at a concentration of 17 µg/mL. The inactivation of S. aureus and E. coli 7-day-old biofilms required CuNP concentrations of 99 µg/mL. SEM images of treated 7-day-old S. aureus and E. coli biofilms depict cell membranes that are completely damaged, suggesting a physical killing mechanism. In addition, when the same concentration of CuNPs used to inactivate biofilms were tested with human fibroblasts, both brown and green CuNPs were found to be biocompatible.

Small molecular exogenous modulators of active forms of MMPs.

Matrix metalloproteinases (MMPs) are endopeptidases, and their activity depends on calcium and zinc metal ions. These enzymes are expressed originally in zymogenic form, where the active site of proteins is closed by a prodomain which is removed during activation. A homeostatic balance of their activity is primarily regulated by a 'cysteine switch' located on a consensus sequence of the prodomain and natural endogenous inhibitors, called tissue inhibitors of metalloproteinases (TIMPs). Breakage of this homeostasis may lead to various pathological conditions, which may require further activation and/or inhibition of these enzymes to regenerate that balance. Here, we report four modulators, more specifically, three inhibitors (I1, I2 and I3), and one exogenous activator (L) of the active form of human collagenase MMP-1 (without prodomain). The results were confirmed by binding studies using fluorescence-based enzyme assays.

Rapid Synthesis of Metal Nanoparticles Using Low-Temperature, Low-Pressure Argon Plasma Chemistry and Self-Assembly.

The synthesis of metal nanoparticles has become a priority for the advancement of nanotechnology. In attempts to create these nanoparticles, several different methods: chemistry, physics, and biology, have all been used. In this study, we report the reduction of cations using argon plasma chemistry to produce nanoparticles of gold (AuNPs), silver (AgNPs), and copper (CuNPs). Although other groups have used plasma-reduction methods to synthesize metal nanoparticles from their cation counterparts, these approaches often require plasma|liquid state interactions, high temperature, specific combinations of gases, and extended treatment times (>10 minutes), for which only specific cations (noble or non-noble) may be reduced. As a result, we have developed a non-thermal, low-pressure argon-plasma|solid state approach for the reduction of both noble and non-noble cations. More specifically, when 50-µL droplets of 2-mM solutions of gold(III) chloride, silver nitrate, or copper(II) sulfate are exposed to vacuum, they undergo an evaporation process. As the pressure in the chamber decreases to 220 mtorr, the droplets become completely evaporated, leaving behind a metal precursor. Nucleation and growth studies reveal that when the metal precursors of gold(III) chloride, silver nitrate, and copper(II) sulfate are treated with 80 watts of argon plasma for 5, 60, and 150 seconds, respectively, nanoparticles could be synthesized with efficiency rates of upwards of 98%. The size of nanoparticles synthesized in this work was studied using Scanning Electron Microscopy, and the scattering properties of the nanoparticles was studied using UV/Vis spectroscopy. Transmission Electron Microscopy with corresponding elemental analysis was also very useful in confirming the identity of the synthesized nanoparticles. The results from this study reveal that we have synthesized metal nanoparticles with distinct chemical and physical properties. Scanning Electron Microscopy depicts AgNPs with a round-shape and diameters from 40 - 80 nm, while AuNPs were hexagonal, with sizes from 40 - 80 nm, and CuNPs were rod-shaped, with dimensions 40 by 160 nm. Our findings demonstrate that the argon plasma approach used in this study is a rapid, green, and versatile reduction method for the synthesis of both noble and non-noble metal nanoparticles.

Plasma-initiated graft polymerization of carbon nanoparticles as nano-based drug delivery systems.

Plasma-initiated free radical polymerization was used to engineer carbon nanoparticles (CNPs) with tailored chemical and physical properties. Following surface modification, CNPs were loaded with a highly effective anti-infection agent called metal-free Russian propolis ethanol extract (MFRPEE), thus, creating nano-based drug delivery systems (NBDDSs). The loading of MFRPEE onto grafted CNPs occurred naturally through both electrostatic interactions and hydrogen bonding. When constructed under optimal experimental conditions, the NBDDSs were stable under physiologic conditions, and demonstrated enhanced anti-biofilm activity when compared with free MFRPEE. Mechanistic studies revealed that the enhanced anti-infectious activity of the NBDDSs was attributed to the modified surface chemistry of grafted CNPs. More specifically, the overall positive surface charge on grafted CNPs, which stems from quaternary ammonium polymer brushes covalently bound to the CNPs, provides NBDDSs with the ability to specifically target negatively charged components of biofilms. When studying the release profile of MFRPEE from the modified CNPs, acidic components produced by a biofilm triggered the release of MFRPEE bound to the NBDDS. Once in its free form, the anti-infectious properties of MFRPEE became activated and damaged the extracellular polymeric matrix (EPM) of the biofilm. Once the architecture of the biofilm became compromised, the EPM was no longer capable of protecting the bacteria encapsulated within the biofilm from the anti-infectious agent. Consequently, exposure of bacteria to MFRPEE led to bacterial cell death and biofilm inactivation. The results obtained from this study begin to examine the potential application of NBDDSs for the treatment of healthcare-associated infections (HCAIs).

Plasma-Initiated Graft Polymerization of Acrylic Acid onto Fluorine-Doped Tin Oxide as a Platform for Immobilization of Water-Oxidation Catalysts.

The discovery of new and versatile strategies for the immobilization of molecular water-oxidation catalysts (WOCs) is crucial for developing clean energy conversion devices [e.g., (photo)electrocatalytic cells for water splitting]. The traditional approach for surface attachment to transparent conductive oxides [e.g., fluorine doped tin oxide (FTO)] is via synthetic modification of the ligand architecture to incorporate functional groups such as carboxylic acids (-COOH) or phosphonates (-PO3H2) prior to immobilization. However, challenges arising from desorption and the cumbersome derivatizations steps have limited the scope and applications of surface-bound WOCs. Herein, we report the successful immobilization of underivatized Ru(II)-based WOCs (Ru-Cat1 = [Ru(tpy) (bpy) (H2O)]2+ (tpy = 2,2':6'2″-terpyridine and bpy = 2,2'-bipyridine) and Ru-Cat2 = [Ru(Mebimpy) (bpy) (H2O)]2+ (Mebimpy = 2,6-bis(1-methylbenzimidazol-2-yl) pyridine)) and the Ru(II) polypyridyl chromophore Ru-C3 = [Ru(bpy)3]2+ onto a FTO plasma-grafted poly(acrylic acid) surface (PAA|FTO). Various characterization techniques such as attenuated total reflectance Fourier transform infrared spectroscopy, scanning electron microscopy, atomic force microscopy, and cyclic voltammetry measurements provide evidence for the plasma-induced grafted PAA|FTO film and immobilization. Surface stability and electrocatalytic properties of these new hybrid composite films upon cycling were investigated at different pH values. Immobilized Ru-Cat1 and Ru-Cat2 onto PAA|FTO displayed pH-dependent (RuIII/RuII) couples and onset potentials indicative of PCET (proton-coupled electron transfer) reactions. Based on cyclic voltammetry results and spectroscopic monitoring, the immobilized WOCs Ru-Cat1 and Ru-Cat2 exhibited a higher surface stability in neutral aqueous solutions relative to Ru-C3 upon electrochemical oxidation. We attribute the surface PCET and stability to the presence of a water ligand in the coordination sphere of immobilized Ru-Cat1 and Ru-Cat2 which can H-bond with negatively charged carboxylate groups of the cross-linked PAA brushes. Our findings demonstrate that the plasma-grafted polymeric network onto FTO offers a versatile platform to directly anchor unmodified homogeneous WOCs or chromophores for potential applications in solar-to-fuel energy conversion.

Plasma-initiated graft polymerization as an immobilization platform for metal free Russian propolis ethanol extracts designed specifically for biomaterials.

The antibacterial and anti-biofilm activities of propolis have been intensively reported. However, the application of this folk remedy as a means to prevent biomedical implant contamination has yet to be completely evaluated. In response to the significant resistant and infectious attributes of biofilms, biomaterials engineered to possess specific chemical and physical properties were immobilized with metal free Russian propolis ethanol extracts (MFRPEE), a known antibacterial agent. The results obtained from this study begin to examine the application of MFRPEE as a novel alternative method for the prevention of medical and biomedical implant infections. When constructed under specific experimental conditions, immobilized biomaterials showed excellent stability when subjected to simulated body fluid and fetal bovine serum. The ability of immobilized biomaterials to specifically target pathogens (both Gram-positive and Gram-negative biofilm forming bacteria), while promoting tissue cell growth, renders these biomaterials as potential candidates for clinical applications.

Anti-infection silver nanoparticle immobilized biomaterials facilitated by argon plasma grafting technology.

Many research groups have attained slow, persistent, continuous release of silver ions through careful experimental design using existing methods. Such methods effectively kill planktonic bacteria and therefore prevent surface adhesion of pathogens. However, the resultant modified coatings cannot provide long-term antibacterial efficacy due to sustained anti-microbial release. In this study, the anti-infection activity of AgNP immobilized biomaterials was evaluated, facilitated by argon plasma grafting technology and activated by bacterial colonization. The modified materials generated in this study showed excellent specificity and were active against both Gram-positive and Gram-negative biofilm forming bacteria, including methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli. The anti-infection biomaterials developed in this study demonstrate several attractive advantages in comparison to traditional anti-bacterial surfaces loaded with antibiotics or other types of antibacterial agents and include (1) broad spectrum of activity against antibiotic resistant bacteria, (2) the unlikelihood of bacterial resistance, (3) specificity, (4) biocompatibility, and (5) stability.

Are Russian propolis ethanol extracts the future for the prevention of medical and biomedical implant contaminations?

BACKGROUND: Most studies reveal that the mechanism of action of propolis against bacteria is functional rather than structural and is attributed to a synergism between the compounds in the extracts. HYPOTHESIS/PURPOSE: Propolis is said to inhibit bacterial adherence, division, inhibition of water-insoluble glucan formation, and protein synthesis. However, it has been shown that the mechanism of action of Russian propolis ethanol extracts is structural rather than functional and may be attributed to the metals found in propolis. If the metals found in propolis are removed, cell lysis still occurs and these modified extracts may be used in the prevention of medical and biomedical implant contaminations. STUDY DESIGN: The antibacterial activity of metal-free Russian propolis ethanol extracts (MFRPEE) on two biofilm forming bacteria: penicillin-resistant Staphylococcus aureus and Escherichia coli was evaluated using MTT and a Live/Dead staining technique. Toxicity studies were conducted on mouse osteoblast (MC-3T3) cells using the same viability assays. METHODS: In the MTT assay, biofilms were incubated with MTT at 37°C for 30min. After washing, the purple formazan formed inside the bacterial cells was dissolved by SDS and then measured using a microplate reader by setting the detecting and reference wavelengths at 570nm and 630nm, respectively. Live and dead distributions of cells were studied by confocal laser scanning microscopy. RESULTS: Complete biofilm inactivation was observed when biofilms were treated for 40h with 2µg/ml of MFRPEE. Results indicate that the metals present in propolis possess antibacterial activity, but do not have an essential role in the antibacterial mechanism of action. Additionally, the same concentration of metals found in propolis samples, were toxic to tissue cells. Comparable to samples with metals, metal free samples caused damage to the cell membrane structures of both bacterial species, resulting in cell lysis. CONCLUSION: Results suggest that the structural mechanism of action of Russian propolis ethanol extracts stem predominate from the organic compounds. Further studies revealed drastically reduced toxicity to mammalian cells when metals were removed from Russian propolis ethanol extracts, suggesting a potential for medical and biomedical applications.

The inactivation of Staphylococcus aureus biofilms using low-power argon plasma in a layer-by-layer approach.

The direct application of low power argon plasma for the decontamination of pre-formed Staphylococcus aureus biofilms on various surfaces was examined. Distinct chemical/physical properties of reactive species found in argon plasmas generated at different wattages all demonstrated very potent but very different anti-biofilm mechanisms of action. An in-depth analysis of the results showed that: (1) the different reactive species produced in each plasma demonstrated specific antibacterial and/or anti-biofilm activity; and (2) the commonly associated etching effect could be manipulated and even controlled, depending on the experimental conditions. Under optimal experimental parameters, bacterial cells in S. aureus biofilms were killed (> 99.9%) by plasmas within 10 min of exposure and no bacteria nor biofilm regrowth from argon discharge gas treated biofilms was observed for 150 h. The decontamination ability of plasmas for the treatment of biofilm related contaminations on various materials was confirmed and an entirely novel layer-by-layer decontamination approach was designed and examined.

Bacteria responsive antibacterial surfaces for indwelling device infections.

Indwelling device infections now represent life-threatening circumstances as a result of the biofilms' tolerance to antibiotic treatments. Current antibiotic impregnation approaches through sustained antibiotic release have some unsolved problems which include short life-span, narrowed antibacterial spectrum, ineffectiveness towards resistant mutants, and the potential to hasten the antibiotic resistance process. In this study, bacteria responsive anti-biofilm surfaces were developed using bioactive peptides with proved activity to antibiotic resistant bacteria and biofilms. Resulting surfaces were stable under physiological conditions and in the presence of high concentrations of salts (0.5M NaCl) and biomacromolcules (1.0% DNA and 2.0% alginate), and thus showed good biocompatibility to various tissue cells. However, lytic peptide immobilized surfaces could sense bacteria adhesion and kill attached bacteria effectively and specifically, so biofilms were unable to develop on the lytic peptide immobilized surfaces. Bacteria responsive catheters remained biofilm free for up to a week. Therefore, the bacteria responsive antibacterial surfaces developed in this study represent new opportunities for indwelling device infections.

Insights into discharge argon-mediated biofilm inactivation.

Formation of bacterial biofilms at solid-liquid interfaces creates numerous problems in biomedical sciences. Conventional sterilization and decontamination methods are not suitable for new and more sophisticated biomaterials. In this paper, the efficiency and effectiveness of gas discharges in the inactivation and removal of biofilms on biomaterials were studied. It was found that although discharge oxygen, nitrogen and argon all demonstrated excellent antibacterial and antibiofilm activity, gases with distinct chemical/physical properties underwent different mechanisms of action. Discharge oxygen- and nitrogen-mediated decontamination was associated with strong etching effects, which can cause live bacteria to relocate thus spreading contamination. On the contrary, although discharge argon at low powers maintained excellent antibacterial ability, it had negligible etching effects. Based on these results, an effective decontamination approach using discharge argon was established in which bacteria and biofilms were killed in situ and then removed from the contaminated biomaterials. This novel procedure is applicable for a wide range of biomaterials and biomedical devices in an in vivo and clinical setting.

Low power gas discharge plasma mediated inactivation and removal of biofilms formed on biomaterials.

The antibacterial activity of gas discharge plasma has been studied for quiet some time. However, high biofilm inactivation activity of plasma was only recently reported. Studies indicate that the etching effect associated with plasmas generated represent an undesired effect, which may cause live bacteria relocation and thus contamination spreading. Meanwhile, the strong etching effects from these high power plasmas may also alter the surface chemistry and affect the biocompatibility of biomaterials. In this study, we examined the efficiency and effectiveness of low power gas discharge plasma for biofilm inactivation and removal. Among the three tested gases, oxygen, nitrogen, and argon, discharge oxygen demonstrated the best anti-biofilm activity because of its excellent ability in killing bacteria in biofilms and mild etching effects. Low power discharge oxygen completely killed and then removed the dead bacteria from attached surface but had negligible effects on the biocompatibility of materials. DNA left on the regenerated surface after removal of biofilms did not have any negative impact on tissue cell growth. On the contrary, dramatically increased growth was found for these cells seeded on regenerated surfaces. These results demonstrate the potential applications of low power discharge oxygen in biofilm treatments of biomaterials and indwelling device decontaminations.

Susceptibility of Staphylococcus aureus biofilms to reactive discharge gases.

Formation of bacterial biofilms at solid-liquid interfaces creates numerous problems in both industrial and biomedical sciences. In this study, the susceptibility of Staphylococcus aureus biofilms to discharge gas generated from plasma was tested. It was found that despite distinct chemical/physical properties, discharge gases from oxygen, nitrogen, and argon demonstrated very potent and almost the same anti-biofilm activity. The bacterial cells in S. aureus biofilms were killed (>99.9%) by discharge gas within minutes of exposure. Under optimal experimental conditions, no bacteria and biofilm re-growth from discharge gas treated biofilms was found. Further studies revealed that the anti-biofilm activity of the discharge gas occurred by two distinct mechanisms: (1) killing bacteria in biofilms by causing severe cell membrane damage, and (2) damaging the extracellular polymeric matrix in the architecture of the biofilm to release biofilm from the surface of the solid substratum. Information gathered from this study provides an insight into the anti-biofilm mechanisms of plasma and confirms the applications of discharge gas in the treatment of biofilms and biofilm related bacterial infections.