Inflammatory responses of a human keratinocyte cell line to 10 nm citrate- and PEG-coated silver nanoparticles

Silver nanoparticles (AgNPs) are among the most commonly used engineered NPs and various commercially available products are designed to come in direct contact with the skin (wound dressings, textiles, creams, among others). Currently, there is limited understanding of the influence of coatings on the toxicity of AgNPs and in particular their ability to impact on AgNP’s mediated inflammatory responses. As AgNPs are often stabilized by different coatings, including citrate and polyethyleneglycol (PEG), in this study we investigate the influence of citrate (Cit10) or PEG (PEG10) coatings to 10 nm AgNP on skin, using human HaCaT keratinocytes. AgNPs cytotoxicity and inflammatory response (nuclear factor (NF)-κB induction and cytokine production) of HaCaT were assessed after in vitro exposure to 10 and 40 µg/mL after 4, 24, and 48 h. Results showed that although both types of coated AgNPs decreased cell proliferation and viability, Cit10 AgNPs were more toxic. NF-κB inhibition was observed for the highest concentration (40 µg/mL) of PEG10 AgNPs, and the putative link to early apoptotic pathways observed in these cells is discussed. No production of IL-1β, IL-6, IL-10, and TNFα was stimulated by AgNPs. Furthermore, Cit10 and PEG10 AgNPs decreased the release of MCP-1 by HaCaT cells after 48 h of exposure. As cytokines are vital for the immunologic regulation in the human body, and it is demonstrated that they may interfere with NPs, more research is needed to understand how different AgNPs affect the immune system.     

Static Magnetic Field Dictates Protein Corona Formation on the Surface of Glutamine‐Modified Superparamagnetic Iron Oxide Nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs) have become important tools for the imaging and detecting of prevalent diseases for many years. Scientists usually harness their attraction to a static magnetic field (SMF) to increase targeting efficiency and minimize side effects. To prolong blood circulation time and minimize reticuloendothelial system clearance, SPIONs are increasingly designed with a negatively charged surface. Understanding how a SMF affects the SPIONs with a negative surface charge is fundamental to any potential downstream applications of SPIONs as drug delivery carriers and bio‐separation nanoparticles. The goal of our study is to investigate the effect of SMF treatment (204 mT) on the in vitro and in vivo protein corona formed on negatively charged SPIONs. The results reveal that the amount of protein and the composition of protein corona is directly related to the SMF treatment. Compared with the in vivo protein corona, SMF treatment exercises considerable influence on the composition of the in vitro protein corona. The in vitro protein corona formed on SPIONs modulates the secretion of inflammatory cytokines from cells. To the best of our knowledge, this report describes the first demonstration of a SMF as an influencing factor on protein corona formation in vivo. Our results help to elucidate the biological mechanisms of SPIONs with SMF treatment and suggest that the protein corona effect should be considered during the development of a magnetic target.     

Tuning SERS for living erythrocytes: Focus on nanoparticle size and plasmon resonance position

Surface‐enhanced Raman spectroscopy (SERS) is a unique technique to study submembrane hemoglobin (Hb_sm) in erythrocytes. We report the detailed design of SERS experiments on living erythrocytes to estimate dependence of the enhancemen t factor for main Raman bands of Hb_sm on silver nanoparticle (AgNP) properties. We demonstrate that the enhancement factor for ν 4/A1g, ν 10/B1g and A2g Raman bands of Hb_sm varies from 10⁵ to 10⁷ under proposed experimental conditions with 473 nm laser excitation. For the first time we show that the enhancement of Raman scattering increases with the increase in the relative amount of small NPs in colloids, with the decrease in AgNP size and with plasmon resonance shift to the shorter wavelength region. Obtained results can be explained by the ability of smaller AgNPs to get deeper into nano‐invaginations of the plasma membrane than larger AgNPs. This shortens the distance between small AgNPs and Hb_sm and, consequently, leads to the higher enhancement of Raman scattering of Hb_sm. The enhancement of higher wavenumber bands ν 10/B1g and A2g is more sensitive to AgNPs’ size and the relative amount of small AgNPs than the enhancement of the lower wavenumber band ν 4/A1g. This can be used for AgNP‐controlled enhancement of the desired Raman bands and should be taken into account in biomedical SERS experiments. Copyright © 2013 John Wiley & Sons, Ltd.     

Combined bactericidal activity of silver nanoparticles and hexadecylpyridinium salicylate ionic liquid

Recently, ionic liquids have been used as dispersing agents for silver nanoparticle (AgNP) preparation. In this paper, we have shown a simple method to prepare AgNP in aqueous media using an ionic liquid called hexadecylpyridinium salicylate (HDPSal) as dispersing agent. The dispersions were produced by the chemical reduction of silver ions in aqueous media with different concentrations of HDPSal and tetrabutylammonium borohydride as reducing agent. The UV–Visible electronic spectra showed the characteristic plasmonic resonance band around 420 nm, confirming the formation of AgNPs. The TEM images confirmed the formation of spherical particles with diameters lower than 10 nm. The charge of these particles was determined by Zeta potential and they were around +50 mV, indicating that the HDP cations are surrounding the AgNPs, avoiding their agglomeration. Most of the dispersions remained stable for at least 1 month. Microbiological assays showed that the combination of AgNP with HDPSal results in wider range of antimicrobial effect.     

Action of corona discharges on bacteria and spores

We have measured the E.coli bactericidal efficiency of DC and AC point-to-plane coronas in air and in argon in the corona modes positive glow, positive streamer, negative Trichel pulse and negative glow. Negative Trichel pulse and positive streamer coronas are the most efficient for production of bactericidal agents in air. Less than 5 minute exposure to the products from 30 A of these coronas will sterilize the E.coli covered agar surfaces in a 3l volume to a survival ratio of 10^–5. Positive glow coronas are two orders of magnitude less efficient. 50 Hz AC coronas seem to mainly work during the negative half cycles. The bactericidal agent(s) produced by the coronas have not all been identified, but ozone O₃ with concentration 35–40 ppm is probably the most important one. We show that charged particles and photons can play only a minor role, so the agent(s) must be neutral particles. The main part of them must have a lifetime in the vessel of more than 3 minutes. The much smaller, and volume/distance dependent, efficiency of coronas in humid argon indicates that metastable neutrals or radicals from H₂O can play only a minor role. A preliminary test showed negligible effects of the corona treatment on the spores of Bacillus subtilis.     

Surface spin disorder and spin-glass-like behaviour in manganese-substituted cobalt ferrite nanoparticles

Due to the increasing use of silver nanoparticles (AgNPs) in consumer products, it is essential to understand how variables, such as light exposure, may change the physical and chemical characteristics of AgNP suspensions. To this end, the effect of 300?nm ultraviolet (UV) light on (20, 40, 60 and 80)?nm citrate-capped AgNP suspensions has been investigated. As a consequence of irradiation, the initial yellow hue of the AgNP suspensions is transformed towards a near colorless solution due to the loss of the surface plasmon resonance (SPR) absorbance. The decrease in SPR absorbance followed a first-order decay process for all particle sizes with a rate constant that increased linearly with the AgNP specific surface area and non-linearly with light intensity. The rate of loss of the SPR absorbance decreased with increasing citrate concentration, suggesting a surface-mediated transformation. Absorbance, atomic force microscopy, and dynamic light scattering results all indicated that AgNP photolysis was accompanied by a diameter decrease and occasional aggregation. Furthermore, in situ transmission electron microscopy imaging using a specialized liquid cell also showed a decrease in the particle size and the formation of a core?Cshell structure in UV-exposed AgNPs. X-ray photoelectron spectroscopy analysis suggested that this shell consisted of oxidized silver. The SPR in UV-exposed AgNP suspensions could be regenerated by addition of a strong reducing agent (NaBH₄), supporting the idea that oxidized silver is present after photolysis. Evidence for UV-enhanced dissolution and the production of silver ions was obtained with the Donnan membrane technique. This study reveals that the physico-chemical properties of aqueous AgNP suspensions will change significantly upon exposure to UV light, with implications for environmental health and safety risk assessments.     

Silver nanoparticles in complex biological media: assessment of colloidal stability and protein corona formation

Engineered silver nanoparticles (AgNPs) are among the most used nanomaterials in consumer products, therefore concerns are raised about their potential for adverse effects in humans and environment. Although an increasing number of studies in vitro and in vivo are being reported on the toxicity of AgNPs, most of them suffer from incomplete characterization of AgNPs in the tested biological media. As a consequence, the comparison of toxicological data is troublesome and the toxicity evaluation still remains an open critical issue. The development of a reliable protocol to evaluate interactions of AgNPs with surrounding proteins as well as to assess their colloidal stability is therefore required. In this regard, it is of importance not only to use multiple, easy-to-access and simple techniques but also to understand limitations of each characterization methods. In this work, the morphological and structural behaviour of AgNPs has been studied in two relevant biological media, namely 10 % FBS and MP. Three different techniques (Dynamic Light Scattering, Transmission Electron Microscopy, UV–Vis spectroscopy) were tested for their suitability in detecting AgNPs of three different sizes (10, 40 and 100 nm) coated with either citrate or polyvinylpyrrolidone. Results showed that UV–Vis spectroscopy is the most versatile and informative technique to gain information about interaction between AgNPs and surrounding proteins and to determine their colloidal stability in the tested biological media. These findings are expected to provide useful insights in characterizing AgNPs before performing any further in vitro/in vivo experiment.     

Influence of the corona-wire diameter and length on corona discharge characteristics of a cylindrical tri-axial charger

In this paper, the corona discharge characterization in terms of current–voltage relationships of a unipolar cylindrical tri-axial charger on the effects of the corona wire diameter and length have been experimentally studied and discussed. A commercial computational fluid dynamics software package, COMSOL Multiphysics™, was used to predict the electric field distribution in the ion generation and charging zones of the charger and the ion penetration through the perforated screen opening on the inner electrode of the charger. It was found from experimental results that both positive and negative charging currents in the charging zone of the charger increased with increasing corona and ion-driving voltages. At the same corona and ion-driving voltages, both positive and negative coronas were decreased with increasing diameter of the corona-wire. Compared with the corona-wire of 22 mm in length, the magnitude of both positive and negative charging currents were markedly higher for corona-wire of 11 mm in length at the same corona voltage. It was found that the charging currents for negative coronas were about 1.2 times higher than those positive coronas at the same corona and ion-driving voltages. Numerical results of the electric field distribution and the ion and charged particles migrations in the discharge and charging zones of the charger is correlated to have the same direction with the experimental results of the current–voltage relationships. Also, this can be used to guidance in describing the electric field distribution and the behavior of ion and charged particle trajectories that cannot be seen from experiments in order to improve the applicably design and refinement of a unipolar cylindrical tri-axial charger.     

Compared ionic wind measurements on multi-tip corona and DBD plasma actuators

Two sets of actuators with triangular tips on their active electrodes (13 corona and 15 DBD) are studied in the laboratory. Far field ionic wind velocity, mass flow and efficiency are measured for all the actuators. The best electrode shape as function of tip sharpness and tips number/unit length is determined for each of the above measurements. The gas velocity increases downstream of the tips in all the actuators, but the DBD flow has a three-dimensional structure more complicated than for coronas. The tips improve the efficiency of all the actuators, and the stability of coronas is remarkably improved.     

Multiple layer formation of bovine serum albumin on silver nanoparticles revealed by dynamic light scattering and spectroscopic techniques

It is well known that when nanoparticles (NPs) are exposed to biological fluid, it results into formation of nanoparticle protein corona, which has been the subject of extensive studies for the development of targeted drug delivery. In this work, we demonstrated the dynamic light scattering, fluorescence, and UV-visible spectroscopy as quantitative and qualitative tools to monitor adsorption of BSA protein onto silver nanoparticles (AgNPs). The adsorption resulted in significant gradual increase in average hydrodynamic radius of BSA-AgNP corona from 24 to 35 nm and its attainment of equilibrium point (saturation) that correlated with albumin concentration enables condition for bound and unbound protein adsorption to be interpreted. Using DLS, the dissociation constant (K_D) was obtained for soft corona to be 2.09?±?0.30 μM. The UV-visible and fluorescence spectroscopy results were correlated with DLS. Loss of percent helicity in secondary structure of adsorbed BSA was monitored in both coronas as compared to native protein. Both coronas were found to be biocompatible with RBC membrane. Further, the results of adsorption isotherm model were used to validate the multilayer formation of albumin protein on silver nanoparticles. The obtained results would be relevant in the drug design development for tumor-targeted therapy.

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Graphical abstract ?

     

Effect of needle cone angle and air flow rate on electrostatic discharge characteristics of a corona-needle ionizer

In this study, the corona-needle ionizer was designed, constructed, and characterized. Experimental characterizations of the electrostatic discharge in terms of current–voltage relationships of the corona ionizer, including the effects of discharge electrode cone angle and air flow rate were presented. It was found that the charging current and ion concentration in the charging zone increased monotonically with corona voltage. Conversely, discharge currents decreased with increasing angle of the needle cone. The negative corona was found to have higher current than the positive corona. At higher air flow rates, the ion current and concentration were found to be relatively high for the same corona voltage. The effect of air flow rate was more pronounced than the corona voltage. It was also shown that the ion penetration through the ionizer decreased with increasing corona voltage, and increased with increasing air flow rate. The highest ion penetration through the ionizer of the 10° needle cone angle was found to be about 93.7 and 7.7% for positive and negative coronas, respectively. The highest ion penetration for the needle cone angle of 20° was found to be 96.6 and 6.1% for positive and negative coronas, respectively.     

Investigating mechanical properties of polymethylmethacrylate/silver nanoparticle composites by molecular dynamics simulation

The molecular dynamics simulation (MD) was carried out to investigate the mechanical properties of pristine polymethylmethacrylate (PMMA) and the composites of PMMA mixed with the silver nanoparticles (PMMA/AgNPs) at two AgNP weight fractions at 0.60 and 1.77 wt%. From the stress–strain profiles by the tensile process, it can be seen that the improvement on Young’s modulus is insignificant at these lower AgNP fractions. The tensile strength of pristine PMMA can be slightly improved by the embedded AgNPs at 1.77 wt%, because the local density and strength of PMMA in the vicinity of AgNP surface within about 8.2 Å are improved. For the temperature effect on the mechanical properties of pristine PMMA and PMMA/AgNP composite, the Young’s moduli and strength of pristine PMMA and PMMA/AgNP composite significantly decrease at temperatures of 450 and 550 K, which are close to the predicted melting temperature of pristine PMMA about 460 K. At these temperatures, the PMMA materials become more ductile and the AgNPs within the PMMA matrix display higher mobility than those at 300 K. When the tensile strain increases, the AgNPs tend to get closer and the fracture appears at the PMMA part, leading to the close values of Young’s modulus and ultimate strength for pristine PMMA and PMMA/AgNP composite at 450 and 550 K.

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Graphical abstract Stress–strain curves of pristine PMMA, polymethylmethacrylate (PMMA)/silver nanoparticles (AgNP) (0.60%), and PMMA/AgNP (1.77%). Inset images: local shear strain of pristine PMMA (red) and PMMA/AgNP (1.77%) (green).

     

Fe3O4 nanoparticle loaded paclitaxel induce multiple myeloma apoptosis by cell cycle arrest and increase cleavage of caspases in vitro

We experimentally and theoretically characterize back-scattering and extinction of Ag nanoparticle (AgNP) arrays on both Si wafer substrates and optically-thick Ag substrates with and without organic poly(3-hexylthiophene):[6,6]-phenyl C61-butyric acid methyl ester (P3HT:PCBM) bulk-heterojunction thin film coatings. A strong red-shift in back-scattered light wavelength occurs from AgNP arrays on Si as a function of increasing mean nanoparticle diameter (ranging from 30 to 90 nm). Back-scattering from the AgNP array is notably quenched in the wavelength range of strong P3HT absorption when the organic layer is applied. However, back-scattering is enhanced to a degree relative to the uncoated AgNP array on Si at wavelengths greater than the absorption band edge of P3HT. For comparison, the optical properties of AgNPs on an optically-thick Ag substrate are reported with and without P3HT:PCBM thin film coatings. On the reflective Ag substrates, a significant enhancement (by a factor of 7.5) and red-shift of back-scattered light occurred upon coating of the AgNPs with the P3HT:PCBM layer. Additionally, red-edge extinction was enhanced in the P3HT:PCBM layer with the presence of the AgNPs compared to the planar case. Theoretical electromagnetic simulations were carried out to help validate and explain the scattering and extinction changes observed in experiment. Both increasing nanoparticle size and an increasing degree of contact with the Si substrate (i.e., effective index of the nanoparticle environment) are shown to play a role in increasing back- and forward-scattering intensity and wavelength, and in increasing absorption enhancements in both the organic and Si layers. AgNPs placed at the P3HT:PCBM/Si interface give rise to absorption increases in P3HT of up to 18 %, and only enhance Si absorption at wavelengths longer than the absorption band edge of P3HT (by almost 90 % in the 660–1,200 nm wavelength range). These results provide insight into how metal nanoparticles placed near an organic/inorganic interface can be employed for light management in tandem or hybrid organic/inorganic thin-film semiconductor configurations for solar energy harvesting applications or light detection applications.     

Positive point-to-plane corona discharge forms in O2-N2 mixtures

An extensive survey has been made of positive point-to-plane corona discharges with 0–100% O₂ in O₂-N₂ mixtures in the pressure range 0.5–100 kPa. Selected results are presented in the form of pressure-voltage diagrams. They show the regions of existence for the different point-plane discharge forms observed: burst pulses, glow corona with oscillations, streamers, sparks, and a pulse- and oscillationless glow-spike corona. The latter positive corona discharge form has formerly only been observed in very pure nitrogen. The main aim of the paper is to give a short account of these different forms of positive coronas and their stability or oscillations, and to guide experimenters in establishing experimental conditions for future studies of these phenomena. Dedicated to Prof. Jan Janča on the occasion of his 60th birthday.     

Copolymer Particles with Incorporated Gold and Silver Nanoparticles to Absorb Short‐Wavelength Scattering in Full‐Color Photonic Glasses

Photonic glasses (PGs) have fascinated researchers because the structural colors that are reflected from PGs are angle independent. However, low color saturation, especially for colors at longer wavelengths, is an obstacle for realizing full‐color PGs. It is reported that the form‐factor‐(FF)‐derived reflections that degrade the color saturation of PGs can be removed by embedding Ag and Au nanoparticles (NPs) into copolymers. Styrene and 4‐vinylpyridne copolymer particles are prepared and used as nanotemplates to synthesize AgNPs and AuNPs. The wavelength of the FF‐derived reflections in relation to the diameter of the particles is estimated. NPs with an absorption spectrum corresponding to the calculated values are then selectively embedded in the copolymer particles. PGs fabricated with AgNP‐ and AuNP‐embedded copolymer particles exhibit fewer FF‐derived reflections owing to the surface‐plasmon‐resonance‐induced absorption of the embedded NPs. Using these FF‐reduced PGs, angle‐independent full‐color PG pigments are realized.     

High-performance nonenzymatic electrochemical glucose biosensor based on AgNP-decorated MoS2 microflowers

A facile hydrothermal route was used to synthesize silver nanoparticle (AgNP)-decorated microflower molybdenum disulfide (MoS₂-MF) for bio-electrochemical platform fabrication to detect nonenzymatic glucose concentration. The morphologies of the materials were studied by scanning electron microscopy, and their structural characteristics were analyzed by X-ray diffractometry and energy-dispersive X-ray spectroscopy. The electrochemical characteristics of the AgNPs/MoS₂-MF/PtE biosensor were studied by cyclic voltammetry. The obtained data indicated that the developed nonenzymatic glucose sensor has a large linear response between 1.0 and 15.0 mM, a limit of detection of as low as 1.0 mM, and a sensitivity of 46.5 μA nM⁻¹ cm⁻². The biosensor also displayed outstanding selectivity, stability, reproducibility, and repeatability. Additionally, the AgNPs/MoS₂-MF/PtE biosensor was utilized to detect glucose concentration in real sample and showed practical application potential for glucose detection.     

Catalytic properties of gold nanoparticles immobilized on the surfaces of nanocarriers

Gold nanoparticles are immobilized in the hydrophilic coronas of spherical micelle carriers for high catalytic activity. The micelle is formed by self-assembly of block copolymer, polystyrene-b-poly (acrylic acid), in basic aqueous solution (pH 10) and has a polystyrene core and a poly (acrylic acid) corona. The gold nanoparticles are anchored into the poly (acrylic acid) corona by in situ reduction of the mixture of HAuCl₄ and micelle with NaBH₄. The sizes of the gold nanoparticles can be adjusted by changing the content of the HAuCl₄. In the process of catalyzing p-nitrophenol to p-aminophenol, the reaction shows one-order kinetics, furthermore, the reaction rate increases with the concentration of composites as well as reaction temperature. Comparing the composites with polystyrene as core and poly (4-vinylpyridine)/Au as corona, the catalytic activity of the present composites is higher, which is ascribed to their hydrophilic corona structure.     

Silver release from nanocomposite Ag/alginate hydrogels in the presence of chloride ions: experimental results and mathematical modeling

A stepwise experimental and mathematical modeling approach was used to assess silver release from nanocomposite Ag/alginate microbeads in wet and dried forms into water and into normal saline solution chosen as a simplified model for certain biological fluids (e.g., blood plasma, wound exudates, sweat, etc). Three phenomena were connected and mathematically described: diffusion of silver nanoparticles (AgNPs) within the alginate hydrogel, AgNP oxidation/dissolution and reaction with chloride ions, and diffusion of the resultant silver-chloride species. Mathematical modeling results agreed well with the experimental data with the AgNP diffusion coefficient estimated as 1.3 × 10^?18 m² s^?1, while the first-order kinetic rate constant of AgNP oxidation/dissolution and diffusivity of silver-chloride species were shown to be inversely related. In specific, rapid rehydration and swelling of dry Ag/alginate microbeads induced fast AgNP oxidation/dissolution reaction with Cl^? and AgCl precipitation within the microbeads with the lowest diffusivity of silver-chloride species compared to wet microbeads in normal saline. The proposed mathematical model provided an insight into the phenomena related to silver release from nanocomposite Ca-alginate hydrogels relevant for use of antimicrobial devices and established, at the same time, a basis for further in-depth studies of AgNP interactions in hydrogels in the presence of chloride ions.     

Laser-induced synthesis of silver nanoparticles and their conjugation with protein

Partially oxidized spherical silver nanoparticles (AgNPs) of different size are prepared by pulsed laser ablation in water and directly conjugated to protein S-ovalbumin for the first time and characterized by various optical techniques. UV–Visible spectrum of AgNPs showed localized surface plasmon resonance (LSPR) peak at 396 nm which red shift after protein addition. Further the increased concentration of AgNPs resulted a decrease in intensity and broadening of S-ovalbumin peak (278 nm), which can be related to the formation of protein NPs complex caused by the partial adsorption of S-ovalbumin on the surface of AgNPs. The red shift in LSPR peak of AgNPs after mixing with S-ovalbumin and decrease in protein-characteristic peak with increased silver loading confirmed the formation of protein–AgNPs bioconjugates. The effect of laser fluence on the size of AgNPs and nanoparticle–protein conjugation in the size range 5–38 nm is systematically studied. Raman spectra reveal broken disulphide bonds in the conjugated protein and formation of Ag–S bonds on the nanoparticle surface. Fluorescence spectroscopy showed quenching in fluorescence emission intensity of tryptophan residue of S-ovalbumin due to energy transfer from tryptophan moieties of albumin to AgNPs. Besides this, small blue shift in emission peak is also noticed in presence of AgNPs, which might be due to complex formation between protein and nanoparticles. The binding constant (K) and the number of binding sites (n) between AgNPs and S-ovalbumin have been found to be 0.006 M^?1 and 7.11, respectively.     

The effect of the deposition parameters on size, distribution and antimicrobial properties of photoinduced silver nanoparticles on titania coatings

Controlled photodeposition of silver nanoparticles (AgNP) on titania coatings using two different sources of UV light is described. Titania (anatase) thin films were prepared by the sol-gel dip-coating method on silicon wafers. AgNPs were grown on the titania surface as a result of UV illumination of titania films immersed in aqueous solutions of silver nitrate. UV xenon lamp or excimer laser, both operating at the wavelength 351 ± 5 nm, was used as illumination sources. The AFM topography of AgNP/TiO₂ nanocomposites revealed that silver nanoparticles could be synthesized by both sources of illumination, however the photocatalysis carried out by UV light from xenon lamp illumination leads to larger AgNP than those synthesized using the laser beam. It was found that the increasing concentration of silver ions in the initial solution increases the number of Ag nanoparticles on the titania surface, while longer time of irradiation results the growth of larger size nanoparticles. Antibacterial tests performed on TiO₂ covered by Ag nanoparticles revealed that increasing density of nanoparticles enhances the inhibition of bacterial growth. It was also found that antibacterial activity drops by only 10-15% after 6 cycles compared to the initial use.