SILVER-NANO FACE MASK
NO FILTER REQUIRED
Cloth Face Masks – a sustainable choice
With the spread of the coronavirus worldwide, the consumption of disposable face masks has seen an incredible spike.
Whilst wearing a mask had previously only been recommended to those that already showed symptoms, the situation has now changed. In fact, there is increasing evidence that the Covid-19 can be spread by pre-symptomatic and asymptomatic carriers. For this reason, many Governments and health organisations , like the CDC, are recommending people consider wearing cloth face coverings in public settings where other social distancing measures are difficult to maintain.
Is it always better to wear a mask?
Although there is still much to be learned about the novel coronavirus, it appears that many people who are infected are shedding the virus – through coughs, sneezes and other respiratory droplets – for 48 hours before they start feeling sick. And others who have the virus – up to 25%, according to Centers for Disease Control and Prevention Director Dr. Robert Redfield — may never feel symptoms but may still play a role in transmitting it.
That’s why wearing a mask even if you don’t feel sick can be advisable for the general population.
Are there sustainable alternatives?
Now that everyone is concerned about protective equipment to stay safe, face masks are a must to prevent the contagion.
However, some consumers criticized the use of disposable masks because this does not represent an environmentally-friendly choice.
Are there sustainable alternatives for those who do not want to use surgical masks or respirators?
Can these consumers opt for masks that they can use again and stay safe?
Cloth Masks – are they effective against coronavirus?
In particular, our suppliers are developing 4-PLY Droplet Resistant Antibacterial Masks and Nano Silver Antibacterial Masks to help combat the virus.
Nano Silver Masks.
These masks are composed by 4 integrated layers.
In particular, the one in the middle is treated with Silver Nano technology that works as a safety filter. In fact, it eliminates small particles of bacteria that pass through the outer layer of the mask.
Nano Silver technology is an antimicrobial colloid composed of silver nanoparticles stabilized by a polymer that exhibits excellent antimicrobial efficacy across a wide spectrum of microorganisms.
While Silver inhibits the oxygen exchange in bacteria and kills it, this technology has been demonstrated to enhance that effect.
Looking for Cotton Masks? We can help!
We are currently working with our partners to provide a range of Cloth Masks to meet the world’s needs and to combat this deadly virus. From a sourcing perspective, the current regulatory landscape in China is very complex on these products so please make sure if you are buying direct, you are doing the appropriate due diligence and checks. For all enquiries, please contact us for more information at officelife4you@yahoo.com
Disclaimer. According to the World Health Organisation, masks alone will not prevent people from catching the coronavirus, and must be used in combination with normal hygiene methods, such as handwashing with soap and water.
SCIENCE BEHIND THE MASK
Natural Silver works against bacteria only in ion form — it must lose an electron to become positively charged. Silver ions pierce through the external surfaces of the cells and damage their DNA, as well as the protein structure, halting their metabolism and reproduction until they die.
Nanozid Nano-Coating Kills SARS Virus – New Product
Only last spring the SARS epidemic sent a shiver of fear around the globe. Now scientists working at the Institute of Virology at the University Hospital of Philipps University Marburg discovered that the viruses responsible for the severe acute respiratory syndrome (SARS) lose their terror on surfaces coated with special nanoparticles. These nanoparticles, only a few millionths of a millimeter in diameter, consist of titanium dioxide encased in a nano-thin layer of silver. In laboratory tests they proved capable of inactivating the coronaviruses which cause SARS in addition to bacteria and fungi. The Marburg researchers found that, after four hours of contact with the coating, the infectiousness of the viruses had decreased by more than two log steps. The nanoparticles have an especially large specific surface of around 200 sqm per gram. Owing to their silver coating, the particles release silver ions over long periods of time; these ions effectively deactivate pathogenic bacteria, fungi and viruses but are harmless for human beings. The effectiveness of the coating against SARS was confirmed during a study commissioned by ItN Nanovation GmbH, a nanotechnology company located in Saarbrücken. The objective of the study, which was carried out by the well known German research institute in Marburg, was to find additional applications for ItN’s microbicidal nanoparticles. The nano-powder with the brand name Nanozid is added to paints and lacquers used to coat operating tables, door knobs and door handles in hospitals (e.g. “clean rooms”) and surfaces in sanitary facilities. In addition to their obvious utility in the clinical sector, biocidal coatings have numerous applications in the food and beverage industries and in HVAC systems, which frequently serve as vehicles for the spread of infectious diseases. |
Posted 17th November 2003 |
https://www.azonano.com/article.aspx?ArticleID=417
Application of Nano Silver Particles on Textile Materials for Improvement of Antibacterial Finishes
Application of Nano Silver Particles on Textile Materials for Improvement of Antibacterial Finishes
Abstract
The article reviews the role of nano silver particles in improving the antibacterial properties of textile materials. Efforts have been directed at the extracellular synthesis of highly stable silver nano particles for the development of nanosafe textile using the extracts of yellow papaya peel. Owing to their potent antibacterial activity, papaya peel derived silver nanoparticles can be incorporated into fabrics and the textile producers can make textiles free from spoilage from micro organisms. Silver nano particles have been synthesized through biological approach using natural extracts of Acalypha indica and applied onto cotton fabrics. Even a mere 5% treatment with herbal extracts showed superior antibacterial activity indicating usage in medical and infection prevention applications. Silver nano particles have been synthesized by the reduction of silver nitrate with sodium borohydride in an aqueous medium. The silk protein sericin extracted from the silk worm cocoons has been used as effective capping agent. The silver nanoparticles exhibit antimicrobial properties when applied onto silk fabric without significantly changing its color. In yet other interesting research nano chitosan particles have been coupled with nano silver colloid to improve the antibacterial properties of cotton fabric.
Keywords: Antibacterial property; Silver nano particles; Cotton; Silk; Nano Chitosan; Herbal extract
Gokarneshan N* and Velumani K
Department of Textile Technology, Park College of Engineering and Technology, India
Submission: April 1, 2017; Published: May 31, 2017
*Corresponding author: Gokarneshan N, Department of Textile Technology, Park College of engineering and technology, India; Email:
Introduction
Nano particles have a wide range of applications such as electronics, catalysis, chemistry energy and medicine and have thus gained commercial acceptance [1]. Besides improving their functionality, the use of nano technology can result in the production of textiles with completely novel properties or the combination of various functions [2]. Such multifunctional textiles include antistatic textiles, reinforced textiles, antibacterial, self cleaning textiles, bleaching resistant, etc. andpaves the way for the use of its products in other fields outside the traditional industries [3-5]. Silver nano particles due to their strong and wide spectrum of antimicrobial activities have gained major focus among all nano particles. In order to protect against microbial contamination, silver has been incorporated into various forms of plastics such as catheters, dental material, medical devices, implants and burn dressings. These nano particles have also been used for durable finish on fabrics. As bactericides, the silver nano particles may help in solving the serious antibiotic resistance problem.
Papaya peel derived silver nano particles
Of the various techniques of nano particle synthesis available, the green synthesis technique holds advantage in
controlling particle size and morphology very effectively. When compared with other conventional techniques this technique is convenient and fast. Due to their potential antibacterial activity, papaya peel derived silver nano particles can be incorporated into fabrics and the manufacturers can make textiles free fromspoilage by microorganisms [6]. The significant reduction in reaction time with fruit peel extract is an important result and will enable nanoparticle biosynthesis methods to compete with other routes for the formation of nanoparticles that are currently much more rapid and reproducible.
Several strategies have been employed for the synthesis of silver nano particles including chemical techniques, physical techniques and recently, via biological techniques [7]. Biological techniques have received much attention as a viable alternative for the development of metal nanoparticles [8]. Many bacterial as well as fungal species have been used for silver nano particle synthesis [9,10]. But most of them are reported to accumulate silver nanoparticles intracellularly. On the contrary, plant extract mediated synthesis, i.e., green synthesis always takes place extracellularly, and the reaction times remain very short as compared to microbial synthesis. Extracts of many plants and weeds have shown the potential of reducing silver nitrate
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for the formation of silver nanoparticles without any chemical ingredients [11,12]. Various parts of the papaya plant have been used including flower and fruits are used for the synthesis of silver nano particles. Some reports relating to the use of silver nano particles using peel extracts are available. For the first time the potential of the peels of yellow papaya as non toxic biological systems for the biosynthesis of green silver nano particles have been used.
Application of nano chitosan coupled with nano silver colloid
\Ionotropic gelatin with pentasodium tripolyphosphate is employed for the preparation of nano chitosan dispersion. Nano chitosan together with nano silver treatment when applied on cotton fabrics shows enhanced antibacterial activity [13]. An enhanced antibacterial property is observed with the reduction of nano chitosan particles and when coupled with nano silver colloid.
Chitosan holds promise in varied application such as biomedical, waste water treatment, cosmetics, dentrifices, food, agriculture, pulp and paper, and textile industries [14- 16]. This is because it has many valuable inherent properties like antibacterial, antifungal, antiviral, antacid, non toxic, total biodegradability, biocompatibility with animal and plant tissues as well as film formation, fibre formation and hydrogel fibre formation properties. By virtue of its bacteria impedingproperty, chitosan can prevent garments develop bad odour [17-22]. It is found that complete inhibition of Eschirichia coli and Hay Bacillus bacteria is possible by treatment of cotton with 0.5gpL chitosan concentration [23].
Silver nano coating on cotton fabric adopting green approach
Biosynthesized sliver nanoparticles using A. indica leaf extract has been coated over cotton fabric through in-situ chemical reaction. SEM image, UV spectra proved the formation of sliver nanoparticles. The biological approach is a cost effective method as compared with the chemical synthesis [24]. Further it is proved that the sliver nanoparticles coated cotton fabric exhibit effective antimicrobial effect against microorganism. Finally the 5g A. indica leaf extract produces highest antimicrobial effect and release properties as compared with 3g and 4g leaf extracts. Due to the highest control release properties of this coating utilized for wound healing dressing.
Over the last few decades, various research work was happening around the world made to produce antibacterial coated textile materials due to the enormous growth of microbial infections via textile surfaces [25,26]. Attempts have been made to develop a non-toxic, cost effective and eco- friendly source of antimicrobial finishing textiles for health care application. Cotton fibres are mostly utilized as raw material towards medical and healthcare products [27]. However the moisture absorbability of cotton fibres is very high, which makes
them more prone to microbial attack under certain conditions of humidity and temperature. Cotton may acts as a nutrient, becoming suitable medium for bacterial and fungal growth [28]. Therefore, cotton fibres are treated with numerous chemicals to get better antimicrobial cotton textiles [29-31]. Among the various antimicrobial treatments, nano material based treatment is very effective. Silver nano particles (AgNPs) have shown strong inhibitory and antibacterial effects [32].
It has been reported that strong toxicity of silver nanoparticle against wide range of microorganisms is well known [33]. Further the antimicrobial activity of silver nanoparticles against Escherichia colli’s a model of Gram-negative bacteria has been studied. Chemical reducing method is one of the important technique followed in synthesis of sliver nano particles, which is normally associated with environmental toxicity [34].Therefore the development of sliver nano particle through natural extract is consider as most important method. Biosynthesis of silver nanoparticles using bacteria, fungi, yeast and plants were well documented.
Mechanism of reaction
Disc diffusion technique has been used to assess the antibacterial activity of A. indica silver nano particles against both against Gram positive and Gram negative pathogenic microorganisms. The antimicrobial activity of the coated silver nano particles on cotton fabrics has been measured as zone of inhibition. Silver nanoparticles displayed almost similar range of antimicrobial activity against studied pathogens, which was understood through diameter of inhibition zone. Many studies have concluded the biocidal properties of silver nano particles against microorganisms. It is believed that the silver nano particles attach the negatively charged cell surface, then change its physical and chemical properties of the cell membranes and the cell wall and disturb the permeability and osmoregulation, electron transport and respiration [35]. Secondly the silver nanoparticle produces further damage by permeating into the cell, interact with the DNA [36]. Thirdly, the silver nanoparticle releases the silver ions producing higher biocidal effect on the microorganisms.
Use of silk sericin in the synthesis of silver nanoparticles
Sericin extracted by a simple procedure from the cocoons of silk worms has been used effectively in the preparation of silver nanoparticles as a capping agent. Sericin effectively prevents coagulation of the silver nano particles and keeps them in a moderately stabilized condition in an aqueous medium [37]. The spherical particles of average size 15nm are fairly monodispersed. Further, the sericin-capped silver nano particles when applied to silk fabric impart antimicrobial properties to it. As silver nano particles are very small, have high surface energy and are unstable, capping agents should be used to capture them in the early stage of reaction and these include pvp, gelatin,
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How to cite this article: Gokarneshan N, Velumani K. Application of Nano Silver Particles on Textile Materials for Improvement of Antibacterial Finishes. Glob J Nano. 2017; 2(3) : 555586. DOI: 10.19080/GJN.2017.02.555586.
polyglutamic acid, sophorolipids, mixtures of different agents, and pva [38-43].
Earlier, during the preparation of silver nano particles, silk protein fibroin has been used as reducing and capping agent [44]. Recently sericin has attracted attention since it has useful properties and has many areas of applications [45,46]. Attempts have been made to prepare even smaller sized silver nano particles using sericin as the capping agent. After application of the nano particles on silk fabrics the antimicrobial efficiency of the nano particles has been confirmed. The treated silk fabric exhibits no significant change in colour, despite the yellow colour of the solution, and this has been found to be an improvement over the earlier method [47].
Conclusion
Among the various types of metal oxide nano particles used in textile finishing, silver oxide has assumed more prominence, more particularly for its very good antimicrobial property. Papaya peel derived silver nano particles, owing to its potent antibacterial activity can be incorporated into fabrics and the manufacturers can render textiles free from ruination by microorganisms An important finding is that there is a considerable decrease in reaction time with fruit peel extract which would help in nanoparticle biosynthesis methods to compete with other methods of formation of nano particles which are presently far more rapid and reproducible. Biological approach involving biosynthesized silver nano particles using A. indica leaf extract proves to be a cost effective method in comparison with the chemical synthesis method.
Also, the silver the cotton fabrics coated with silver nano particles show effective antimicrobial effect against microorganism. This coating is being used in would healing dressing owing to the highest control release properties. Nano chitosans have been synthesized by ionic gelation of pentasodium tripolyphosphate and chitosan. The cotton fabric have been pretreated with normal and nano chitosan solutions by pad dry cure technique. An enhanced antibacterial property is observed with the reduction of nano chitosan particles and when coupled with nano silver colloid. Silver nanoparticles have been synthesized by the reduction of silver nitrate with sodium borohydride in an aqueous medium. The silk protein sericin, extracted from the cocoons of Bombyx mori silkworms, has been used as an effective capping agent. Such particles also show antimicrobial properties when applied onto silk fabric without significantly changing the colour of the fabric.
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24. kumar SB (2016) Study On Antimicrobial Effectiveness Of Sliver Nano Coating Over Cotton Fabric Through Green Approach. IJPSR 7(9): 363- 368.
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Nanosilver particles in medical applications: synthesis, performance, and toxicity
INTRODUCTION
Nanosilver particles (NSPs) generally present at 1 to 100 nm in size in at least one dimension.1–4 As particle size decreases, the surface area-to-volume ratio of NSPs increases dramatically, which leads to significant changes in their physical, chemical, and biological properties. NSPs have been among the most commonly used nano-materials in our health care system for hundreds of years. Recently, NSPs have become of intense interest in biomedical applications (Figure 1), because of their antibacterial, antifungal, antiviral, and anti-inflammatory activity.5,6
Biomedical applications of nanosilver particles in human health care.
NSPs have been widely used for diagnosis,7 treatment,8 drug delivery,9 medical device coating,10 wound dressings,11 medical textiles,12 and contraceptive devices.13 As the use of nanosilver products is continually increasing, a better understanding of nanosilver biological interactions and their toxicity becomes necessary. This review critically discusses NSP synthesis methods, properties, and current and emerging medical NSP applications. Finally, recent advances concerning NSP potential toxicity will also be described.
NSP SYNTHESIS
Different synthetic NSP routes lead to variable sizes, shapes, morphology, and even stability. Generally, these methods can be classified into three broad categories: physical, chemical, and biological (or green) synthesis.
Physical synthesis
Evaporation/condensation and laser ablation are the main physical techniques for deriving nanosilver from metal samples. The evaporation/condensation technique uses a furnace tube under atmospheric pressure to produce NSPs; however, conventional furnace tubes have several drawbacks, such as high energy consumption, and require a long time to achieve thermal stability. Jung et al used a small ceramic heater with a local heating area, thus the evaporated vapor could cool at a suitable rate and a high concentration of nanosilver could be obtained.14 Laser synthesis employs the laser ablation of metals in solution without chemical reagents, which leads to pure nanosilver colloids.15 The concentration and morphology of nanosilver are affected by laser fluence and the number of laser shots. Greater laser fluence and amount of time, lead to larger particle size and higher particle concentration.16 Recently, Tien et al reported a novel arc-discharge method of producing silver suspension in pure water without any surfactants or stabilizers.17In their research, silver wires were utilized as positive and negative electrodes and etched in pure water. During discharge, the surface layer of the silver wires was evaporated and condensed in the water, thus stable and well-dispersed NSPs of 20–30 nm in size were obtained.17
Chemical synthesis
Chemical reduction is the most frequent method of nanosilver synthesis, and uses silver salt, reductants, and a stabilizer or capping agents as three main components to control NSP growth (Figure 2). Among these, silver nitrate is a silver salt that is often used for NSPs, due to its low cost and chemical stability compared to the other available silver salts.17 The reductants include borohydride,18 citrate,19ascorbate,20 and hydrogen gas.11
Chemical synthesis of nanosilver particles.
Abbreviation: Ag, silver.
Borohydride is a strong reducing agent that can result in small particles with a faster reduction rate, because borohydride can also act as an NSP stabilizer and avoid aggregation of NSPs during its decomposition.11 It is hard to obtain high concentrations of NSPs because of their aggregative instability. Using a stabilizer in preparation is a common strategy. The stabilizers include surfactants and ligands or polymers that contain functional groups such as polyvinylpyrrolidone, poly(ethylene glycol), poly(methacrylic acid), poly(methyl methacrylate), and others. Furthermore, temperature-sensitive polymers such as poly(N-isopropylacrylamide) and collagen can also serve as stabilizers, and nanosilver capped by those chemicals allows for novel thermal switching applications.1
NSPs can also be synthesized in a two-phase water-organic system. This method produces uniform and controllable nanoparticles. In this system, metal precursor and reducing agent are separated in two phases, thus the rate of interaction can be controlled by the intensity of interphase transport between aqueous and oil phases; however, large amounts of surfactant and organic solvent may contaminate the surface of formed NSPs, and the removal of surfactant and organic solvent is also time-consuming and expensive.
Biological synthesis
Biosynthesis (green synthesis) of nanosilver has received extensive attention due to the growing need for environmentally friendly synthesis methods that use eco-friendly reducing and capping agents, such as protein;21 peptides;22 carbohydrate;23 various species of bacteria,24 fungi,25 and yeast;26 and algae and plants.27 For example, Naik et al synthesized NSPs of 60–150 nm in size using silver-binding peptides identified from a combinatorial phage-display peptide library. The peptides were placed in an aqueous solution of 0.1 mM silver nitrate for 24–48 hours at room temperature.21 Thomas et al developed an economical, fascicled, and in situ approach to prepare large-scale chitosan–nanosilver (400 nm) films using chitosan as a chelating and stabilizing agent; the films demonstrated excellent antibacterial action against Escherichia coli and Bacillus.28 Sintubin et al reviewed different biological synthesis methods using microorganisms or plants for nanosilver synthesis.26
In biological synthesis, as the reducing agents and stabilizers are molecules produced by protein, carbohydrate, bacteria, fungi, yeasts, algae, or plants, organic solvents and toxic reagents are avoided. The possible mechanism of biological synthesis includes enzymatic and non-enzymatic reduction (Figure 3). Nicotinamide adenine dinucleotide phosphate-dependent reductase can produce NSPs by enzymatic reduction; however, the enzymatic reduction rate is often slow (between 24 and 120 hours).29 The non-enzymatic reduction of silver is similar to chemical reduction, but the reducing and stabilizing agents are microorganisms or plants. Non-enzymatic reduction is usually fast, often completed within a few minutes, and can handle extreme parameters, such as high pH or high temperature, that accelerate the synthesis.24
Biological (or green) synthesis of nanosilver particles.
Abbreviations: NADP+, nicotinamide adenine dinucleotide phosphate (oxidized form); NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); Ag, silver.
The main advantage of biogenic synthesis over other methods is that the green synthesis avoids organic solvents and toxic reagents. Thus, biosynthesized NSPs are more stable than those that are chemically produced, and they can remain stable over a long period of time.30 In addition, biological synthesis makes it possible to produce NSPs under a nontoxic silver nitrate concentration because microbial cells can continue to multiply;31 however, the biosynthesis drawback is that the purification process may lead to pathogenic bacteria and the potential bacteria may cause contamination, which should be a reason for exercising caution in medical application.26
NSP PERFORMANCE
Antibacterial properties
NSPs have a broad antibacterial effect on a range of Gram-negative and Gram-positive bacteria and antibiotic-resistant bacteria strains.32 Antimicrobial efficacy of NSPs depends on their size and concentration. Normally, a high concentration leads to more effective antimicrobial activity, while particles of small sizes can kill bacteria at a lower concentration. Apart from size and concentration, shape also influences the antimicrobial efficiency of NSPs. Sadeghi et al investigated the antimicrobial activity of different nanosilver shapes, which included silver nanoplates, silver nanorods, and silver nanoparticles, on Staphylococcus aureus and E. coli. They found that silver nanoplates had the best antimicrobial activity.33It has also been reported that NSPs combined with various antibiotics have better antimicrobial effects than NSPs or antibiotics alone. Li et al, for example, found a greater antibacterial effect on E. coli when amoxicillin and silver nanoparticles were combined than when they were applied separately.34
Although the antimicrobial effect of nanosilver has been widely studied, the exact mechanism of NSPs is still elusive. It is widely accepted that NSPs can anchor to and subsequently penetrate the bacterial cell wall, thereby causing structural change of the cell membrane and increasing cell permeability, leading to cell death (Figure 4).35 The formation of free radicals and subsequent free radical-induced membrane damage is another potential mechanism, which has been investigated by Kim et al.32 It has also been found that NSPs can release silver ions and interact with the thiol groups of many vital enzymes and phosphorus-containing bases, thus inhibiting some functions in cells, such as preventing cell division and DNA replication.36 In addition, NSPs may modulate signal transduction through changing the phosphotyrosine profile of bacterial peptides for the potential antibacterial mechanism (Figure 4).37
Antibacterial mechanism of nanosilver particles.
Abbreviation: DNA, deoxyribonucleic acid.
Antifungal properties
Nanosilver is an effective antifungal agent against a broad spectrum of common fungi. Kim et al investigated NSP antifungal properties on a total of 44 strains of six fungal species, and found that NSPs can inhibit the growth of Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, and Trichophyton mentagrophytes effectively.38 Nasrollahi et al39 and Kim et al40 observed that NSPs can disrupt cellular membrane and inhibit the normal budding process; however, the exact mechanisms of action of nanosilver against fungi are still not clear.
Antiviral properties
NSPs are also an antiviral agent against HIV-1,41 hepatitis B virus,42 respiratory syncytial virus,43 herpes simplex virus type 1,44 and monkeypox virus.45 It has been observed that NSPs have higher antiviral activity than silver ions, due to species difference as they dissolve to release Ag0 (atomic) and Ag+ (ionic) clusters, whereas silver salts release Ag+ only.46 Lara found that the anti-HIV mechanism of nanosilver is based on the inhibition of the initial stages of the HIV-1 cycle.47 NSPs can bind to glycoprotein (gp)120, thus inhibit cluster of differentiation (CD) 4-dependent binding, fusion, and infectivity. They act as an effective virucidal agent to block HIV-1 cell-free and cell-associated infection. Furthermore, NSPs inhibit post-entry stages of the HIV-1 life cycle.47 Although the mechanism underlying their viral-inhibitory activity is not yet fully understood, NSPs could be considered to be a broad-spectrum agent against a variety of viral strains and are not prone to developing resistance.
Anti-inflammatory properties
NSPs show anti-inflammatory properties in both animal models and in clinic. For example, in the swine model with contact dermatitis induced by topically applying 1,2-dinitrochlorobenzene, nanosilvers altered the expression of proinflammatory cytokines transforming growth factor-β and tumor necrosis factor-α.48Shin and Ye found that NSPs attenuated nasal symptoms in allergic rhinitis mice and inhibited OVA-specific immunoglobulin E, IL-4, and interleukin-10, and that inflammatory cell infiltration and goblet cell hyperplasia were inhibited by nanosilvers.49 In a human clinical study, wound dressing containing NSPs promoted the healing of chronic leg ulcers by not only reducing bacteria numbers in the wound bed, but by decreasing inflammatory response as well.8 NSPs’ ability to reduce cytokine release and matrix metalloproteinases,8,50 decrease lymphocyte and mast cell infiltration,48 and induce apoptosis in inflammatory cells8,49 may explain their anti-inflammatory mechanisms.
MEDICAL NSP APPLICATIONS
Wound dressings
Robert Burrell developed the world’s first commercially available nanosilver product (Acticoat™; Smith and Nephew, London, UK) to treat various wounds in clinic, including burns, chronic ulcers, toxic epidermal necrolysis, and pemphigus.51 Huang et al observed that NSP-loaded wound dressings significantly reduced the healing time by an average of 3.35 days and increased bacterial clearance from infected wounds compared to silver sulfadiazine, with no adverse effects;52 however, Chen et al showed that nanosilver-loaded wound dressings could enhance healing in superficial burn wounds but made no difference in deep burn wounds, compared with 1% silver sulfadiazine.53 This suggests that NSPs accelerate reepithelialization but not angiogenesis.
Currently, new dressings are being fabricated with the aim of increasing antibacterial efficacy and promotion of wound healing. For example, Lu et al developed a wound dressing composed of NSPs and chitosan, and found that it significantly increased wound healing during treatment of deep partial-thickness wounds and inhibited infection, as well as diminished the risk of silver absorption, compared with 1% silver sulfadiazine dressings.54
Cardiovascular implants
The first cardiovascular medical device containing silver in clinic was a prosthetic silicone heart valve coated with silver element, which was designed to prevent bacterial infection on the silicone valve and to reduce inflammation response;55 however, metal silver may cause hypersensitivity, inhibits normal fibroblast function, and leads to paravalvular leakage in patients.56 NSPs are safe and nontoxic in medical devices, unlike metal silver. Therefore, Andara et al synthesized a new nanocomposite with NSPs and diamond-like carbon as a surface coating for heart valves and stents, and found that the surface of the nanocomposite showed antithrombogenic and antibacterial properties.57 In addition, Ghanbari et al58 and Fu et al59 also constructed antibacterial multilayer films containing NSPs, and investigated their antibacterial, mechanical, and hemodynamic properties in vitro for use in cardiovascular implant coating.
Catheters
Much research has been conducted to investigate NSPs as antibacterial materials for coating catheters, including central venous catheters and neurosurgical catheters. Silverline (Spiegelberg GmbH and Co. KG, Hamburg, Germany) and ON-Q Silver Soaker™ (I-Flow Corporation, CA, USA) are two commercially available medical catheters containing NSPs to prevent catheter-associated infections.51 Medical catheters are prone to bacterial infection, which can rapidly spread to the wound and its surrounding, and lead to serious complications. Because of their superior antibacterial properties and lack of observed toxicity, NSPs can decrease the incidence of bacterial infection and complications after surgery, thus they have been widely accepted for use in medical catheters. Andara et al found that plastic catheter tubes coated with nanosilver could inhibit bacterium growth in vitro for at least 72 hours, with no significant toxicity, in an animal model.57 In a pilot clinical study, 19 patients who received a nanosilver catheter did not show catheter-associated ventriculitis, and all cerebrospinal fluid cultures were negative, while five patients were positive for catheter-associated ventriculitis in the control group (20 patients).60
Bone cement
Alt et al evaluated antibacterial activity of plain poly(methyl methacrylate) bone cement loaded with different NSP concentrations in vitro, and found that bone cement-loaded 1% nanosilver completely inhibited the proliferation of Staphylococcus epidermidis, methicillin-resistant S. epidermidis, and methicillin-resistant S. aureus, with no significant difference between the nanosilver bone cement and the nontoxic control group in quantitative and qualitative cytotoxicity tests.61 NSPs were also added to ultra-high-molecular-weight polyethylene for fabricating inserts for total joint replacement components, and it was found that NSPs drastically reduced the wear and tear of the polymer.62
Dental materials
NSPs also have applications in dental instruments and bandages. Yoshida et al showed that a resin composite incorporated with NSP-containing materials had a long-term inhibitory effect against Streptococcus mutans.63 Yamamoto et al also showed that a resin composite containing silver ion-implanted fillers released silver ions with antibacterial effects on oral streptococci.64 In addition, Magalhães et al showed that incorporating NSPs in endodontic filling materials provided a significantly enhanced anti-bactericidal effect against Streptococcus milleri, S. aureus, and Enterococcus faecalis.65NSPs in dental adhesives are also very effective against streptococci without affecting the adhesive mechanical properties, thus enabling their use in orthodontic treatments.66
Biodiagnosis
NSPs can be used for bio-diagnosis, where plasmonic properties of NSPs strongly depend on size, shape, and dielectric medium that surrounds it.67 Zhou et al developed a silver nanoparticle array biosensor for clinical detection of serum p53 in head and neck squamous cell carcinoma.68 NSPs are also employed to produce dual-imaging/therapy-immunotargeted nanoshells to locate cancer cells and can absorb light and selectively destroy targeted cancer cells through photothermal therapy.69 In addition, NSPs can detect the interaction between amyloid β-derived diffusible ligands (ADDL) and the anti-ADDL antibody, which are related to the development of Alzheimer’s disease;70 however, silver is easily oxidized and forms plasmonically unattractive compounds such as halides in biological solutions, which deteriorates the plasmonic performance of NSPs.18
Other medical applications
NSPs have applications in the diagnosis and treatment of cancer,71 and are drug carriers that can deliver therapeutic agents,72 which are used in eye care for coating contact lenses.17 In addition, the use of nanosilver in combination with vanadium oxide in battery cell components is one example of advanced silver nanotechnology improving battery performance in next-generation active implantable medical devices.73
NSP TOXICITY
NSPs may have potential toxicities at some concentrations and can cause various health problems if used improperly. Thus, it is necessary to address the biosafety of NSPs in human health.
In vitro toxicity
NSPs have been reported to be cytotoxic to several types of cells, including human peripheral blood mononuclear cells,74 human alveolar epithelial cell line (A549)75, murine and human alveolar macrophage cell line,76 neuroendocrine cells,77 rat liver cell line,78 and mouse germline cells.79 Alt et al, however, found that bone cement containing 1.0% nanosilver did not lead to significant cytotoxicity in mouse fibroblasts (L929) and human osteoblast cell line.61 Although the details of the toxic mechanism are unclear, it suggests that NSPs are ionized in the cells, which leads to activate ion channels and changes the permeability of the cell membrane to both potassium and sodium,80 interaction with mitochondria,81and induction of the apoptosis pathway via the production of reactive oxygen species,82 which leads to cell death.
In vivo toxicity
Chen and Schluesener have reviewed biodistribution, organ accumulation, degradation, possible adverse effects, and toxicity associated with the medical use of nanosilver.13 Respiratory tract, gastrointestinal tract, skin, and female genital tract are the main entry portals of nanosilver into the human body through direct substance exchange with the environment. Additionally, systemic administration is also a potential route of entry, since colloidal silver nanoparticles have been exploited for diagnostic imaging or therapeutic purposes. Inhalation and instillation experiments in rats showed that low concentration, but detectable, ultrafine silver (14.6±1.0 nm) appeared in the lung and was subsequently distributed to the blood and other organs, such as heart, liver, kidney, and even brain.83 In a recent oral toxicity study of rats, Kim et al also found that silver nanoparticles accumulated in blood, liver, lungs, kidneys, stomach, testes, and brain, but NSPs showed no significant genotoxicity after oral administration of silver nanoparticles of 60 nm average size for 28 days at different doses.84 Lee et al showed that NSPs less than 12 nm in size affected early development of fish embryos, caused chromosomal aberrations and DNA damage, and induced proliferation arrest in cell lines of zebrafish;85 however, Lansdown found that silver was not a cause of neurotoxic damage, even though silver deposits have been identified in the region of cutaneous nerves,86 and Ji et al found that NSPs did not affect respiratory system in a 28-day in vivo study.87
Animal and human studies indicate that it is difficult to remove silver completely once it has been deposited in the body; however, nanosilver can be excreted through the hair, urine, and feces.88 There is no consensus on nanosilver’s toxicity to humans, and most toxicity investigations of silver nanoparticles are based on in vitro cellular experiments and relatively short-term animal experiments.
CONCLUSION
NSPs represent a prominent nanoproduct and are already widely used in medical applications, including wound dressing, diagnosis, and pharmacological treatment. Since the shape, size, and composition of NSPs can have significant effects on their function and possible risks to human health, extensive research is needed to fully understand their synthesis, characterization, and possible toxicity. In this review, we first gave an overview of NSP synthesis, then reviewed applications of NSPs in the field of biomedicine. Finally, possible toxicology was discussed.
There is a limited number of well-controlled studies on the potential toxicities of nanosilver, though these studies tend to suggest that NSPs can induce toxicity in living beings. It should be noted that in vitro conditions are drastically different from in vivo conditions; however, longer-term studies and assessment of NSP toxicity must be conducted so that NSP exposure does not exceed toxic levels.
ACKNOWLEDGMENTS
This work was supported by International Science and Technology Cooperation Program of China (S2013ZR0398), Chongqing Basic Scientific Research Grant (cstc2013jcyjC80001), Chongqing Agriculture Development Grant (14408, 12402), the NSERC Discovery Grant, Manitoba Health Research Council, Dr Moorehouse Fellowship, Manitoba Institute of Child Health and China 863 Project (Grant 2012AA020504).
REFERENCES
Medical textiles with silver/nanosilver and their potential application for the prevention and control of healthcare-associated infections
Silver nanoparticles as a medical device in healthcare settings
1. BACKGROUND
The production of auto-sanitizing products for healthcare is highly desirable given the increasing incidence of healthcare-associated infection (HCAIs) [1]. The use of nanoparticles as active components in composite materials in place of conventional chemical products such as ethanol or bleach can guarantee long lasting bactericidal effects while not being toxic to the human body. Silver nanoparticles (Ag NPs) are known for their antimicrobial applications in common/household items, and their use in commercial products is increasing. In fact, they are already widely found as antiseptic additives in packaging, fabric, and are also ideal candidates as additives for tile coatings [2]. The global Ag NP market is indeed expected to reach $2.45 billion by 2022, with increasing demand for antimicrobial materials in healthcare applications [3]. Healthcare is the largest sector of that market, accounting for over 30% of the global Ag NP market revenue in 2014. With the pressing need to prevent HCAIs, it is expected that the use of Ag NPs in medical devices, equipment and textiles will further expand. The increased usage of nanoparticle-based medical devices has raised the attention of the European and International regulators and occupational safety community [4–6], leading to new guidelines for safety assessment of nanotechnology-enabled medical devices [7]. Ag NPs are thought to exert their antimicrobial effect through the release of free metal Ag+ ions. Indeed, silver ions are powerful antimicrobials themselves, but they are easily sequestered by chloride, phosphate, proteins and other cellular components. However, Ag NPs are less susceptible to sequestration and are thus a more effective delivery method [8]. The biological effect of the nanoparticles is largely unproven, but recent results supported the theory that the cytotoxic effects of nanosilver are a combination of precipitated silver complexes and organic silver compounds rather than free silver ions [9]. It is suggested that the antibacterial activity is owing to the generation of silver ions in the aqueous solution binding with the proteins on the bacteria cell membrane and inhibiting cell respiration and reproduction [10–12]. Particles size, free surface area, shape and charge will affect the bioavailability of ions in terms of dissolution or transport and interaction with biological targets [13–15]. Toxicity refers to any deleterious effects on an organism upon exposure to silver. Obviously, if the practical intent is to disinfect or sterilize a specific type of organism, then toxicity may be interpreted as a positive outcome (e.g. antibacterial, antiviral, etc.). However, if the same material exerts unintended or undesired impacts to other organisms, then such toxicity may be interpreted as a potential hazard [1–16]. An ideal antimicrobial candidate, therefore, needs to be selectively toxic, i.e. it is antibacterial at a given concentration but not toxic to humans. For a realistic evaluation of risk/benefit ratio, the comparison between human toxicity and antimicrobial effect has to be considered in terms of exposure times which may differ considerably, [14] e.g. a treated inanimate surface (such as a wall) is unlikely to have a long human exposure time (contact) while the antiseptic effect can be evaluated over a longer period. Concerns have been raised currently regarding the potential toxicities of Ag NPs [17]. For example, the Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR committees) [18] highlighted the importance of considering the different forms of silver used in consumer and medical products, because Ag NPs undergo several transformations as aggregation, agglomeration, dissolution and subsequent speciation. The chemical species that are actually present determine the bioavailability and toxicity of silver in the environment. Focusing on medical devices, the ‘Guidance on the Determination of Potential Health Effects of Nanomaterials Used in Medical Devices’, where nanosilver is widely mentioned, addresses the specific aspects that need to be considered in the safety evaluation of nanomaterials. This guidance highlights the need for special considerations in relation to the safety evaluation of nanomaterials, in view of the possible distinct properties, interactions, and effects that may differ from conventional forms of the same materials [7]. It recommends a stepwise approach where the first step is the chemical identification and characterization of nanomaterials used in the production of a medical device [19]. Relevant methods for nanomaterial characterization may include size separation and extraction and chemical analysis/detection by spectroscopic or mass spectrometric techniques [7]. The European NanoSafety Cluster has also come forward by suggesting a multi-step approach based on the implementation of the three classes of characterization techniques: imaging-based, light scattering-based and separation-based. Such platform should be able to measure nanoparticle primary size, the size distribution in complex matrices, while providing information on different populations present and their surface properties [20]. To address all these issues and identify the purpose-specific applicability window of Ag NPs we propose a five-step approach based first on separation (through flow-field flow fractionation, FlFFF) and characterization of particles, obtaining information about the dimension, the shape and the effective coating of particles, and quantifying the initial free ion presence. FlFFF has been used widely to characterize, concentrate and quantify ion release of engineered nanoparticles, especially when combined with inductively coupled plasma-mass spectrometry (ICP-MS) [21] and has been also employed to evaluate protein corona onto Ag NPs [22,23]. By exploiting hollow fibre flow-field flow fractionation (HF5), the commercial miniaturized version of FlFFF, it has been also possible to collect fractions of purified particles, without destroying their colloidal properties as more traditional ultrafiltration systems do. Secondly, to rapidly and effectively study the antimicrobial effect of the particles over time, luminescent bacteria were used: luminescence is directly proportional to viability thanks to a plasmid modification thus reducing analysis time [24]. The third step involved in vitro toxicity tests on skin models. Further to that isolated particles have been tested for their antimicrobial activity and cellular toxicity to quantify particle-specific contribution. Finally, all the parameters were gathered together for each preparation of Ag NPs, in order to select the best antimicrobial candidate and the set of physicochemical properties required of silver nanomaterials to be used within medical devices in healthcare settings.
2. MATERIAL AND METHODS
2.1. Reagents
Foetal bovine serum (FBS) and culture media were purchased from Sigma-Aldrich (Dublin, Ireland). Calcein was purchased from Molecular Probes, Invitrogen (Dublin, Ireland). ThermoFisher (Dublin, Ireland) was the source of all the other chemicals, whenever not specified otherwise.
2.2. Silver nanoparticles
For this study, four different Ag NP suspensions have been employed. Ag Pristine (0.02 wt% silver concentration) was provided by Colorobbia SpA (Italy). Polyvinylpyrrolidone-coated sample (Ag PVP, 0.02 wt% silver concentration), citrate-coated sample (Ag CIT, 0.02 wt% silver concentration) and hydroxyethyl cellulose-coated sample (Ag HEC, 0.02 wt% silver concentration) were synthesized in ISTEC-CNR (Faenza, Italy). Pristine and Ag PVP were obtained within the same process on a different scale (industrial scale for the Pristine, laboratory scale for the PVP coated material) using the same reducing (glucose) and capping (polyvinylpyrrolidone) agents but an excess of polyvinylpyrrolidone for the industrial scale synthesis [25]. Ag CIT was obtained using sodium citrate (Sigma-Aldrich) both as reducing agent and as a stabilizer, starting from AgNO3 solution. The synthesis reaction occurred in basic environmental and at 70°C by microwave heating, which enables homogeneous heating and rapid achievement of the desired temperatures [26]. The Ag HEC was synthesized, at room temperature, reducing a solution of AgNO3 by hydroxyethyl cellulose, which was also used as the capping agent. The reduction synthesis was catalysed by NaOH (Sigma-Aldrich) [27].
2.3. Hollow-fibre flow-field flow fractionation (HF5)
HF5 analyses were performed using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) complete with degasser, autosampler, isocratic pump and an Agilent 1100 diode array detector (DAD) UV/Vis spectrophotometer combined with an Eclipse® DUALTEC separation system (Wyatt Technology Europe, WTE, Dernbach, Germany). The HF5 channel (Wyatt Technology Europe) consisted of the commercial PES fibre and cartridge provided by WTE. Detailed description of the system was reported in a previous work published by some of the co-authors [28]. The software ChemStation version B.04.02 (Agilent Technologies) and Wyatt Eclipse @ ChemStation version 3.5.02 (Wyatt Technology Europe) were used to handle the separation parameters. An 18-angle multi-angle light scattering (MALS) detector model DAWN HELEOS (Wyatt Technology Corporation, Santa Barbara, CA, USA) operating at a wavelength of 658 nm, was used to measure compute the Rg of particles in solution and was handled with ASTRA® software v. 5.3.2.14 (Wyatt Technology Corporation). An HF5 method is composed of four steps: focus, focus–injection, elution and elution–injection. During focus the mobile phase is split into two different streams entering from the fibre’s inlet and outlet; during focus–injection, the sample is introduced through the inlet and focalized in a narrow band. During the elution step, the inlet flow splits in two: the longitudinal flow (Vc, going to the detectors) and the crossflow (Vx, determining the applied field). Lastly, the crossflow is released and the stream of mobile phase passes through the injection line to clean it before the next injection. The flow conditions for the different HF5 analysis are shown in table 1. A volume of Ag NPs of 5 µl was injected for the characterization of the sample in order to avoid saturation of the scattering signal, while a volume of Ag NPs of 100 µl was injected to collect both the ionic fraction and the isolated nanoparticles. When particles travelling in a parabolic flow profile are subjected to a crossflow, they localize at different points of the flow profile, according to their diffusion coefficient D (proportional to their hydrodynamic radius Rh). Smaller particles experience a higher flow rate and faster elution (normal mode). The Rh can be obtained from direct calculation or through calibration with standards of known size. The FlFFF theory behind this work is described in the previous literature [7–29]. When the analysed particles are not spherical, Rh is an estimation of the equivalent radius of a sphere with the same coefficient of diffusion D. On the other hand, the Rg value provided by the MALS gives information about the compactness on the particles: two particles with same hydrodynamic radius (Rh), but with different Rgvalues, may have a different mass distribution, and thus, different shapes. Combining the two sizing techniques via Rg/Rh ratios, a shape factor is obtained, reflecting the compactness and shape of the particles. For example, this corresponds to a value of 0.77 for a compact sphere and increases to about 4 for needle-like conformation, or decreases to about 0.6/0.5 for particles presenting a hard core and a soft shell/coating.
Table 1.
Flow conditions for HF5 analyses. (Vc, longitudinal flow; Vx, cross/focus flow.)
| steps (versus) method | focus (ml min−1) | focus–injection (ml min−1) | elution (ml min−1) | elution–injection (ml min−1) |
|---|---|---|---|---|
| particle characterization | Vc = 0.35 | Vc = 0.35 | Vc = 0.35 | Vc = 0.35 |
| Vx = 0.80 | Vx = 0.80 | Vx = 0.1 | Vx = 0.0 | |
| time= 2 min | time = 3 min | time = 10 min | time = 3 min | |
| cationic Ag collection and fraction collection | Vc = 0.35 | Vc = 1.0 | Vc = 0.35 | Vc = 0.35 |
| Vx = 0.80 | Vx = 0.8 | Vx = 0.1 | Vx = 0.0 | |
| time = 0.5 min | time = 20 min | time = 10 min | time = 3 min |
2.4. Ag+/Ag determination
The Ag+/Ag ratio was determined in Milli-Q water, analysing samples through an ad hoc analytical method listed in table 2, able to retain Ag NPs and filtrate away Ag+ ions. The proof of concept of the efficacy of this method has already been demonstrated in a previous work [30] employing flame absorption atomic spectroscopy (FAAS). The subsequent quantification of ionic Ag with FAAS was used to estimate the Ag+/Ag ratio. This is a non-destructive method, allowing for the collection of filtered nanoparticles that can be, therefore, tested individually to investigate particle-specific activity. In this work, because the preliminary determination of Ag+ through FAAS showed a very low concentration of silver, graphite furnace was used instead. To quantify the ionic fraction a volume of 10 ml was collected, 1 ml of concentrated HNO3 was added and the concentration of Ag+ was determined with atomic absorption spectroscopy by interpolation on a standard calibration curve (LoD = 0.2 ppb) following opportune dilution. Pd and Mn(NO3)2 were used as modifiers to prevent analyte loss. Each quantitative analysis was repeated three times and the ionic silver amount, expressed as percentage on the total silver content, is listed in table 2. By subtraction, the amount of ‘nano’ silver present in the collected nanoparticles was obtained and by correlating it with the fraction volume the subsequent concentration was determined. The scheme of such a procedure divided in steps is further detailed in the electronic supplementary information.
Table 2.
Physico-chemical properties of Ag NPs. (Rh, hydrodynamic radius determined by NTA; Rg, radius of gyration determined by MALS; ζ, zeta potential.)
| measured parameters (versus) Ag NPs | Rh(nm) | Rg (nm) | shape factor Rg/Rh | ζH2O(mV) | ζDMEM(mV) | % Ag+(w/w) |
|---|---|---|---|---|---|---|
| Pristine | 33.0 ± 2.5 | 18.5 ± 0.15 | 0.55 | −13.0 ± 9.0 | −7.61 ± 0.05 | 3.87 ± 0.08 |
| Ag PVP | 19.0 ± 3.6 | 13.5 ± 0.39 | 0.73 | −25.0 ± 9.0 | −6.99 ± 0.05 | 3.67 ± 0.09 |
| Ag CIT | 28.0 ± 2.9 | 13.6 ± 0.25 | 0.49 | −29.0 ± 12.4 | −22.00 ± 0.05 | 0.53 ± 0.03 |
| Ag HEC | 29.0 ± 4.0 | 25.0 ± 0.4 | 0.86 | +4.4 ± 4.3 | +0.06 ± 0.05 | 0.01 ± 0.02 |
2.5. Nanoparticle tracking analysis
The average hydrodynamic radius of Ag NPs in water was characterized using nanoparticle tracking analysis (NTA) developed by Malvern Instruments Limited (Wiltshire, UK). This technique uses the properties of light scattering and Brownian motion to obtain particle size distributions of samples in liquid suspension [31]. A NS500 instrument, equipped with a 405 nm laser in conjunction with software version NTA 3.1, was used for the purpose of this study. Ag NPs at the concentration of 200 µg ml−1 in Milli-Q water were vortexed for 5 s to disperse the particles and then diluted at 0.2 µg ml−1. The four different dispersions were then analysed via NTA for the measurement of hydrodynamic diameter at room temperature. All measurements were carried out three times in Milli-Q water to match the Rgdetermination. Results are reported as average mode ± standard deviation.
2.6. Zeta potential
Zeta potential of Ag NPs (200 µg ml−1), diluted 10-fold in Milli-Q water and Dulbecco’s Modified Eagle Medium (DMEM), were evaluated using a Zetasizer Nano Z (ZEN5600, Malvern Instruments, UK). Three zeta potential measurements were taken for each sample, each made of 20 accumulations. Measurements were carried out at 25°C, and elaborated using a Smoluchowski model.
2.7. Transmission electron microscopy
A Jeol 2100 transmission electron microscope (TEM; USA) was used to image the Ag NPs, with sizes of the Ag NPs being calculated using Image J software. A droplet of each preparation was deposited on a glass slide and left for 30 min. Then a grid was dragged onto the droplet surface to collect the nanoparticles, which accumulate on the droplet surface because of surface tension. Each grid was left to dry and then analysed.
2.8. Incubation with the silver nanoparticles
Ag NPs dispersed in a stock solution at a concentration of 200 µg ml−1 in Milli-Q water were diluted in the following way: for the cell treatments they were diluted in medium to reach the desired range of concentrations (2.5–100 µg ml−1), whereas for the antibacterial activity they were diluted in Milli-Q water to reach a range of concentrations from 0.625 to 100 µg ml−1.
2.9. Antibacterial activity testing
The antibacterial effect of the nanoparticles was tested against Escherichia coli. Escherichia coli strain TOP10 or CFT073 harbouring plasmid pGen-Lux [32] were incubated with various concentrations of nanoparticles over a time course of up to seven days. Plasmid pGen-Lux encodes the lux operon from Photorhabdus, the gene products of this operon imparts bioluminescence specifically on viable bacteria. One relative light unit (RLU) is approximately equal to 100 viable bacteria. Dead or non-viable bacteria are non-luminescent. All nanoparticles were tested up to a concentration of 100 µg ml−1 and were found to be non-luminescent, thus the effects of the particles on bacterial viability were amenable to measurement by luminometry. Bacteria were cultured at 37°C with shaking at 200 r.p.m. in L broth (Sigma, St Louis, MO, USA) to mid-logarithmic phase. Fifty microlitre aliquots of mid-logarithmic cultures (equivalent to approx. 106 cells) were incubated with an equal volume of the requisite nanoparticles in Lumitrac 200 96-well plates (Greiner). Milli-Q water was used as a negative control. Luminescence was read in a Thermofisher Luminoskan™ ascent microplate luminometer (Dublin, Ireland). Each concentration was tested in duplicate, and the experiment was repeated three times. The RLUs of untreated samples were normalized to 100% and treated samples were adjusted accordingly.
2.10. Cell culture and experimental treatments
The viability tests after exposure to Ag NP preparations were performed onto A431 (human epidermoid carcinoma) and HaCaT (human keratinocytes) cell lines representative of human skin models. A431 cells were obtained from ATCC (LGC Standard, UK) and cultured in Dulbecco’s modified Eagle’s medium (DMEM High Glucose) supplemented with 10% FBS, 2 mM L-glutamine, streptomycin (100 µg ml−1) and penicillin (100 U ml−1). HaCaT cells, obtained from ATCC (LGC Standard, UK), were cultured in DMEM (Dulbecco’s modified Eagle’s medium with Low Glucose) supplemented with 10% FBS, 2 mM L-glutamine, streptomycin (0.01 µg ml−1) and penicillin (0.01 U ml−1). Cells were routinely cultured in a humidified atmosphere of 5% CO2 in air in T75 cell culture flasks (Nunc, Fisher Scientific, Dublin, Ireland) For cytotoxicity experiments and ELISA assay, cells were seeded in complete growth medium on Nunc-96-well multiwell plates, at a density of 10 × 103cells well−1 and 20 × 103cells well−1, for A431 and HaCaT, respectively. For the recovery experiments cells were seeded in complete growth medium on Nunc-96-well multiwell plates, at a density of 2 × 103cells well−1 and 4 × 103cells well−1, for A431 and HaCaT, respectively. After 24 h, the growth medium of the cells was replaced with Ag NPs prepared as previously described. FBS was not employed for this stage to avoid the formation of artefact through protein corona effects. Doses of NPs were adjusted so as to obtain a silver concentration range from 4 µg cm−2 to 160 µg cm−2 (corresponding to a range from 2.5 to 100 µg ml−1). For the particle specific activity, the doses used were adjusted to obtain a final silver concentration of 5, 10, 20 µg ml−1 for Ag Pristine, 5, 10, 15 µg ml−1 for Ag PVP and 4, 6, 8 µg ml−1 for Ag HEC, corresponding to 3.12, 6.24, 12.48 µg cm−2, 3.12, 6.24, 9.36 µg cm−2 and 2.5, 3.75 and 5 µg cm−2 respectively. Since the fractionation led to dilution of the preparation, lower concentrations of total silver were used to assess toxicity. After 24 h exposure cell viability was assessed. In all the experiments, vehicle (1 : 1, Milli-Q water: DMEM) was added as negative control.
2.11. Calcein assay
Live cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually nonfluorescent cell-permeant calcein AM to the intensely fluorescent calcein. The polyanionic dye calcein is well retained within live cells, producing an intense uniform green fluorescence in live cells. After 24 h of incubation in the presence of Ag NPs, cell viability was tested replacing medium with a solution of calcein (1 mM) in serum-free medium. After 45 min of incubation at room temperature, protected from light, fluorescence was read at 635 nm with an Epoch microplate reader (Epoch, BioTek, UK). Since nanomaterials could interfere with this assay, a preliminary experiment was performed incubating both dyes with Ag NP preparations at the highest concentration used (100 µg ml−1). No fluorescence signal was detected above the background signal.
2.12. Resazurin assay
Resazurin is a substrate that changes colour in response to metabolic activity. It is a nonfluorescent molecule converted by intracellular enzymes in the fluorescent compound resorufin (λem = 590 nm). After 24 h of incubation in the presence of Ag NPs, cell viability was tested replacing medium with a solution of resazurin (44 mΜ) in serum-free medium. After 1 h of incubation, fluorescence was measured at 604 nm with Epoch microplate reader. Also in this case we performed a preliminary experiment to test the interference of Ag NPs with resazurin assay. No fluorescence signal was detected above the background.
2.13. Lactate dehydrogenase cytotoxicity assay
Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many different cell types. Plasma membrane damage releases LDH into the cell culture media. Extracellular LHD in the media can be quantified with Pierce LDH cytotoxicity assay kit (Thermo Scientific, UK). LDH catalyses the conversion of lactate to pyruvate via NAD+ reduction to NADH. Diaphorase then uses NADH to reduce the tetrazolium salt (INT) to a red formazan product that can be measured at 490 nm. The level of formazan formation is directly proportional to the amount of LDH released into the medium, which is indicative of cytotoxicity. In summary, after 24 h of incubation with Ag NPs, 50 µl of medium were transferred to a 96-well plate. Then, 50 µl of reaction mixture was added to each sample and, after 30 min of incubation at room temperature, 50 µl of stop solution was added. The absorbance was read at 490 and at 680 nm. To determinate the LDH activity, the value of absorbance at 680 nm (background) was subtracted from the 490 nm absorbance before calculation of per cent cytotoxicity. Total LDH activity (maximum LDH release control activity) was used as positive control and was performed by adding 10× lysis buffer (contained in the kit) to the cells.
2.14. Cytokine secretion
Details of the Materials, Methods and Results of the Cytokine secretion section are available in the electronic supplementary material.
2.15. Statistics
Statistic evaluation of effects has been performed with one-way ANOVA with a Bonferroni test. Statistics have been performed using GraphPad Prism™ software version 4.00 (GraphPad Software Inc., San Diego, CA). Differences have been considered significant for values of p < 0.05.
3. RESULTS
3.1. Physicochemical properties of nanoparticles: size, shape, surface charge and ionic content
Four different Ag NPs have been tested to correlate the particles antiseptic activity and toxicity to their physicochemical properties: Ag Pristine (commercial sample provided by Colorobbia SpA), Ag PVP, Ag CIT and Ag HEC, (CNR-ISTEC synthesized samples), respectively, coated by polyvynilpyrrolidone, citrate and hydroxyethilcellulose added during sol–gel synthesis as stabilizers. These preparations were characterized in terms of size, shape, charge and ionic content (table 2). By using a soft fractionation technique, the HF5 coupled with MALS, we exploited a hyphenated analytical platform able to in-flow size-separate analyte while calculating the gyration radius (Rg) of the particles, which—correlated to the hydrodynamic one (Rh) gives the shape factor. The absorption profile measured online for each preparation is reported in figure 1(a(i)), where the three dimensional (3D) absorption spectrum—collected during the separation is shown. With the exclusion of Ag Pristine, all the particles present a sharp absorption peak in a range between 395 and 425 nm, in accordance with the expected plasmon resonance of Ag NPs at such dimensions [33]. The broadening of the Ag Pristine absorption peak towards larger wavelengths can represent the influence of a small population of non-spherical particles. However, it is more likely that the lack of a stabilizing coating caused a partial, although minimal, aggregation process and the forming of aggregates are responsible for a red shift. In fact, we previously observed that PVP-stabilized nanoparticles tend to form chain-shaped aggregates when destabilized [30]. It is interesting to note that while TEM images (figure 1) are included as a comparison, the associated HF5-MALS fractograms (see the electronic supplementary material, figure S2) show a mono-modal size distribution, with only one band at each defined retention time. Furthermore, TEM images show a variety of species of different size and shape. Indeed, while drying Ag NPs could rearrange, agglomerate and nucleate into platelets, and cannot reflect the original state of the sample. Additionally, big agglomerates are usually omitted in TEM analysis. It is also difficult to assess aggregation by TEM owing to drying artefacts that can result in NP agglomeration during sample preparation [34]. Hence, all the calculations and the predictions have been made basing onto size/shape assessment performed in suspension. Hydrodynamic radius measurement, performed with NTA, was determined in Milli-Q water to match MALS measurements, in order to estimate particles shape [31]. Compared to dynamic light scattering (DLS), where the analysis is weighted towards larger particle size, and, therefore, tends to overestimate them [35], NTA has a lower concentration detection limit, and analyses NPs on a particle-by-particle basis. Among the four preparations, Ag PVP particles were the smallest whereas the other three were of similar dimensions (table 2). After the determination of the different radii, we considered the shape factor. This simple comparison of measurable dimensional parameters can in fact provide valuable information about the conformation/shape of particles in solution [36]. Observing the reported Rg/Rh ratios (table 2), ranging from 0.49 to 0.86, the particles appear to be spherical with a solid core and a less dense coating. In particular, even though the synthesis method is the same for Ag Pristine and PVP, both the difference in absorption spectra and Rg/Rh forecast a different behaviour between the two. Pristine nanoparticles have a smaller core compared to the coating, which is wider than that of Ag PVP particles, as confirmed by the low Rg/Rh ratio; indeed even commercial metal nanosols are stabilized by high amount of organic capping agents. Ag PVP, Ag CIT and Ag HEC have a spherical and coated shape as well, whereas Ag HEC shows a very compact nature and, therefore, a very thin coating. The zeta potential of the Ag PVP particles was found to be negative in pure water owing to the interaction of the surface with gluconate residue (generated during synthesis), but it was at least partially neutralized in medium (table 2). Ag Pristine particles undergo the same effect even though the decrease is less drastic. Ag CIT particles showed the expected negative charge, while Ag HEC had a neutral/weakly positive potential. The lower absolute values of zeta potential measured in medium, predict a low electrostatic repulsion even though from a visual observation of dispersed samples, the steric component of the coating seems to prevent the solid coagulation, segregation and sedimentation. In fact, a layer of ligands creates a repulsive potential to counteract the attractive van der Waals force [37]. This repulsion can be of steric nature (coating with polymers, such as PVP or uncharged molecules) or of electrostatic nature (coating with charged ligands, such as citrate). One direct consequence is that an increase in the ionic strength of the solution will shield the electrostatic repulsive potential, and lead to the aggregation of the nanoparticles, or their heterocoagulation onto living membranes [38]. The ionic content of Ag Pristine and Ag PVP is similar to predictable, because they were obtained through similar synthesis routes. Differences in activity and toxicity then can be dependent on the nature of the particles, in particular we expected Ag Pristine to be more active and toxic because the hard silver-based core is smaller. Citrate and HEC-coated particles have a considerably lower amount of free ions in the solution owing to the more precise stoichiometry applied to the synthesis (table 2). Being that the ionic percentage of Ag CIT particles is higher by an order of magnitude compared to Ag HEC, a consequent higher activity would have been expected if one considers the ionic bioavailability as the only mechanism of action. Nevertheless, the results we obtained showed an opposite trend, which makes necessary taking into account surface charge/particle specific activity. All the Ag NPs tested, are of a spherical shape; hence this parameter is kept constant. However, size, coating, charge and ion release vary for the four preparations and have been evaluated individually and as a combination. In this work, all the physical and chemical characterization of Ag NPs used has been made in suspension (water or cell medium), to allow us to predict the ‘real’ activity of nanoparticles in vitro for each preparation and to justify differences in antiseptic/toxic behaviour between similar preparations. In fact, the properties of Ag NPs can change during the life cycle of a nanomaterial and are partly depending on interactions with the surrounding environment, which may lead to a different behaviour of nanomaterials in different situations [39].
3D absorption spectra acquired in flow during HF5 characterization and NPs collection and representative TEM images of Ag Pristine (a), Ag PVP (b), AG CIT (c) and Ag HEC (d). Scale bar of TEM images represents 40 nm. For 3D spectra, horizontal axis represents wavelength (nm), depth axis represents time (min), height axis represent absorption intensity (mAU). Ag Pristine: min: 330 nm, max 450 nm. Ag PVP: min 325 nm, max 425 nm. Ag CIT: min 327 nm, max 420 nm. Ag HEC: min 320, max 410 nm.
3.2. Antimicrobial activity of silver nanoparticles over time and under re-contamination
We set different time points for bactericidal activity to simulate ageing of the medical device. Then, we designed a way to test the ability of Ag NPs to display a long lasting antiseptic effect. Lastly, the purified nanoparticles obtained through HF5 were tested to identify particle-specific effects. An important step is the choice of an appropriate model. Escherichia coli is a good model to test antibacterial activity of nanoparticles [40], however it is prudent to measure the activity of nanoparticles against pathogenic strains, because these are more likely encountered in a clinical situation. Bacterial strains TOP10 (a K-12 isolate) and CFT073 were chosen for analysis. Strain CFT073 is a uropathogenic strain that can form biofilms and cause urinary tract infections [41]. It belongs to sequence type 73 (ST73) of pathogenic E. coli and is one of the most frequent causes of E. coli extraintestinal infection. To maximize the information obtainable, the luminescence reads were performed until the negative control displayed a decrease in intensity when compared to the initial value. In this way, every experiment is balanced on the strain tested and the effect of Ag NPs can be evaluated over the longest period possible, accounting for strain-to-strain variability. Escherichia coli TOP10 was incubated with the nanoparticles over a time course of up to 72 h, while CFT073 allowed us to carry on the experiments up to 96 h without significant loss of luminescence on the untreated control (figure 2). At 24 h of exposure (red lines) the E. coli was not viable at concentrations ≥40 µg ml−1 for Ag Pristine and Ag PVP (figure 2a–c), while Ag CIT and Ag HEC displayed a smaller effect. Indeed, at the concentration of 100 µg ml−1 of Ag HEC viability dropped to 30% (figure 2g). After 72 h of exposure, a similar trend was registered for Ag Pristine and Ag PVP, with a decrease of viability to 75% and 65%, for Ag Pristine and Ag PVP respectively, even at the lowest concentration (figure 2a–c). However, Ag HEC induced a clear-cut killing effect for E. coli, with a complete loss of viability at concentrations ≥60 µg ml−1. On the contrary, Ag CIT was the least effective, with a decrease of viability to 50% for the highest concentration used (figure 2e). The particles were then tested against the clinical isolate CFT073 and all these agents displayed a bactericidal effect. Comparing these results to those obtained with E. coli TOP10 we observed a reduced sensitivity to all the preparations. CFT073 showed a higher resistance to Ag Pristine and Ag PVP, and the complete loss of viability was only observed at a concentration of ≥60 µg ml−1 (figure 2b–d), whereas Ag CIT displayed a mediocre effect (figure 2f). None of the three preparations showed a time-dependent antimicrobial effect. Instead, Ag HEC showed a time and dose dependent toxicity, with a complete loss of viability at doses ≥80 µg ml−1, thus maintaining its activity even towards a more resilient strain. This observation is in contrast with what could be expected when only applying the direct relationship between free ions and toxicant activity, which confirmed the presence of particle-specific interaction with living organisms. To test the ability of Ag NPs to maintain their antiseptic effect, an alternative experimental strategy was used. The used plates from the previous experiment with CFT073 were left to dry, the lack of remaining living bacteria was confirmed and another inoculation of viable luminescent bacteria was performed (figure 3). CFT073 viability was affected in a time-dependent manner when bacteria were treated with Ag NPs that had previously been used to test killing. The overall antiseptic effect was lower in comparison to that of freshly prepared nanoparticles, and one of the concurring factors is that particles were not uniformly distributed in the wells, because they were only left to dry after the previous experiment. After 24 h, particles are ineffective (Ag CIT) or only effective for the highest concentrations: this lack of acute toxicity (when compared to the previous experiment) finds explanation in the lack of starting free Ag+. This is also confirmed by the fact that Ag HEC displayed an almost unvaried dose–response pattern (figures 2h and and33d). All the preparations except for Ag CIT caused a time-dependent decrease in bacterial viability, supporting the hypothesis of the particles being interacting with bacteria.
Viability of bacterial strains E. coli Top10 and CFT073 after 24 h (red line) and 72 or 96 h (green or blue line). Bacteria were cultured at 37°C with shaking at 200 r.p.m. in L broth to mid-logarithmic phase. Of note, 50 µl aliquots of mid-logarithmic cultures (equivalent to approx. 106 cells) were incubated with an equal volume of Ag NPs (from 0.62 to 100 µg ml−1 final concentration) in 96-well plates. Milli-Q water was used as a negative control and ethanol as a positive control. After 24, 72 and 96 h luminescence was read and the RLUs of untreated samples were normalized to 100% (see Methods). (a,c,e,g) Escherichia coli TOP10; (b,d,f,h) E. coli CFT073. Data are means of three independent determinations ± s.d. Ag Pristine (a,b) Ag PVP (c,d) Ag CIT (e,f) and Ag HEC (g,h).
Viability of bacterial strain CFT073 after 24 h (red line) and 96 h (blue line) against reused Ag NPs. Bacteria were cultured at 37°C with shaking at 200 r.p.m. in L broth to mid-logarithmic phase. Of note, 50 µl aliquots of mid-logarithmic cultures (equivalent to approx. 106 cells) were incubated with an equal volume of water in the 96-well plates containing the previously employed nanoparticles at different concentrations (from 0.62 to 100 µg ml−1final concentration). The same well employed for the previous experiment was used as a negative control while ethanol was added instead of water as a positive control. After 24 and 96 h luminescence was read and the RLUs of untreated samples were normalized to 100% (see Material and methods). (a–d): CFT073. Data are means of three independent determinations ± s.d. Ag Pristine (a), Ag PVP (b), Ag CIT (c) and Ag HEC (d).
3.3. Cytotoxicity assessment of silver nanoparticles with two relevant cell models: A431 and HaCaT cells
To determine the safety of the Ag NP candidates for use as medical device we individuated the most relevant exposure scenario according to the SCENIHR guidelines [19]. Their use is meant to be as a surface coating, and can be then defined ‘surface contacting’, it can interact with consumers (workers, patients) through contact thus ‘facing/interacting with skin tissue’, and is not meant for topical use hence the scenario is of ‘limited contact’ (= or less than 24 h) [7]. Investigating the effect of Ag NPs on two human skin models is useful because they represent different skin layers. A431 are representative of the outer skin layer while HaCaT (keratinocytes), also part of the stratum granulosus of the skin, can simulate in vitro the first effects of penetration of the nanoparticles [29,42–45]. Different mechanisms of cytotoxicity, by carrying out three in vitro assays where the toxic effects of Ag NPs towards human skin cells were assessed (figures 4 and and5)5) in terms of presence of viable cells (calcein assay), damaging of cell membranes (LDH assay), or cellular metabolism (resazurin assay). Moreover, the cytotoxicity results obtained allowed us to calculate the IC50 for each preparation of Ag NPs and to compare the values of IC50 obtained between the different assays (table 3). A431 and HaCaT cells were exposed to the four preparations of Ag NPs and after 24 h the cell viability was assessed (figures 4 and and5,5, respectively). A431 and HaCaT cells reacted similarly to a 24 h exposure to Ag Pristine. Indeed this preparation induced an evident decrease in cell viability with calcein assay (figures 4a and and55a), whereas resazurin assay showed a dose-dependent decrease in viability (figures 4b and and55b). Moreover, LDH assay showed, for both cell lines, a cytotoxic effect starting from 20 µg ml−1, with an increase in LDH activity of 60–70% for the highest dose used (100 µg ml−1) (figures 4c and and55c). Ag PVP induced a similar cytotoxic effect onto A431 and HaCaT cells when compared to Ag Pristine. Both calcein and resazurin assay showed a dose-dependent decrease in cell viability (figures 4d,e and and55d,e). LDH assay confirmed the obtained results (figures 4f and and55f). On the contrary, Ag CIT, in both cell lines, did not show any cytotoxic effect (figure 4g–i and and55g–i) and the values of IC50 were greater than 100 µg ml−1 both for calcein and resazurin assays. Lastly, Ag HEC showed a moderate dose-dependent toxicity in both cell lines (figures 4j–l and and55j–l). However, no complete loss of viability was observed even at the highest concentrations used. In regard to the sensitivity of the assays, in our study we observed that calcein assay appears to be less sensitive compared with resazurin in detecting decrease of cell viability upon exposure to Ag NPs. The cytotoxic effect of Ag NPs on skin cell models was also investigated through the quantification of the cytokines TNF-α, IL-6, IL-8 and IL-1β secreted upon exposure (electronic supplementary material, figure S3). Ag HEC displayed a lack of acute inflammatory response, which can be a promising feature for surface treating applications. Even though skin contact is a possible pathway for contamination, a long-term effect for human health is avoided.
Cytotoxicity of Ag NPs towards A431 cell line. Cells, grown for 24 h in complete growth medium, were treated with different concentrations of Ag NPs or with ethanol (80%), used as a positive control. After 24 h of exposure cell viability was assessed using calcein assay, resazurin assay or LDH assay (see Material and methods). (a,d,g,j) Calcein assay; (b,e,h,k) resazurin assay; (c,f,i,l) LDH assay. Data are means of three independent determinations ± s.d. Ag Pristine (a–c), Ag PVP (d–f), Ag CIT (g–i) and Ag HEC (j–l).
Cytotoxicity of Ag NPs towards HaCaT cells. Cells, grown for 24 h in complete growth medium, were treated with different concentrations of Ag NPs or with ethanol (80%), used as a positive control. After 24 h of exposure cell viability was assessed using calcein assay, or resazurin assay, or LDH assay (see Material and methods). (a,d,g,j) Calcein assay; (b,e,h,k) resazurin assay; (c,f,i,l) LDH assay. Data are means of three independent determinations ± s.d. Ag Pristine (a–c), Ag PVP (d–f), Ag CIT (g–i) and Ag HEC (j–l).
Table 3.
IC50 values (µg ml−1) of A431 and HaCaT cells exposed for 24 h to Ag NPs.
| A431 | HaCaT | |||
|---|---|---|---|---|
| IC50 values (µg ml−1) | calcein | resazurin | calcein | resazurin |
| Ag Pristine | 21.87 | 9.33 | 10.00 | 7.58 |
| Ag PVP | 18.62 | 8.31 | 12.02 | 10.96 |
| Ag CIT | >100 | >100 | >100 | >100 |
| Ag HEC | 45.7 | 21.87 | 28.84 | 29.51 |
To investigate the effect of long-lasting toxicity of Ag NPs, A431 and HaCaT cells were exposed for 24 h to the four preparations of Ag NPs. Then, the cells were rinsed and allowed to recover in a complete growth medium for additional 6 days (figure 6). After the recovery period of 6 days, an increase in viability (assessed with calcein assay) of both cell lines treated with Ag NPs was observed, especially for Ag Pristine, Ag PVP and Ag HEC at the higher concentrations used (60–80–100 µg ml−1), as shown in figure 6. Moreover, the recovery experiment confirmed the lower cytotoxicity of Ag CIT (figure 6e,f). In summary, the cellular toxicity followed the same pattern we observed for bacterial strains, with a higher toxicity for Ag Pristine and Ag PVP, and a lower and moderate toxicity for Ag CIT and Ag HEC, respectively.
Recovery A431 and HaCaT cells after exposure to Ag NPs. Cells, grown for 24 h in complete growth medium, were treated with different concentrations of Ag NPs or with ethanol (80%), used as a positive control. After 24 h of exposure cell medium was replaced with full growth medium and cells were cultured for six additional days. On the seventh day viability was assessed using calcein assay. (a,c,e,g) A431; (b,d,f,h) HaCaT. Data are means of three independent determinations ± s.d. *p < 0.05, **p < 0.01 and ***p < 0.001 versus untreated, control cells. Ag Pristine (a,b), Ag PVP (c,d), Ag CIT (e,f) and Ag HEC (g,h).
3.4. Specific toxicity and antimicrobial activity of flow-field flow fractionated silver nanoparticles
In order to assess the toxic and antiseptic effects ascribable to nanoparticles themselves, isolated from the suspension of each Ag NP sample and from the starting concentration of Ag+, we treated both bacteria and cell lines with the FlFFF fractionated nanoparticles, according to the FlFFF process described in the Material and methods section. The concentrations used for the specific exposure are calculated, keeping in consideration the dilution factor occurred during fractionation and the measured initial Ag+ amount. These are 5, 10, 20 µg ml−1 for Ag Pristine, 5, 10, 15 µg ml−1 for Ag PVP and 4, 6, 8 µg ml−1 for Ag HEC. On these, viability of bacterial strain CFT073 after 24 and 96 h and cytotoxicity has been measured for the three relevant AgNPs and results are shown in figure 7. Of note, citrate-coated particles have not been screened in this further study because their antimicrobial effect was found to be negligible (as shown in figures 2 and and3).3). Interestingly, Pristine and PVP-coated nanoparticles exerted a similar effect on the pathogenic strain CFT073, at both time points. The antimicrobial activity is preserved and increased with respect to unfractionated samples (e.g. for fractionated Ag PVP we observed a loss of viability of 80% at 15 µg ml−1 as opposed to 60% for the unfractionated sample). The acute effect of these two preparations could reflect the fact that nanoparticles establish a new equilibrium by releasing ions in the new medium, and, therefore, a certain (and equal) amount of Ag+ is present both for Ag Pristine and Ag PVP. Ag HEC nanoparticles did not show a strong antimicrobial effect after 24 h, which is linked to their initial lesser amount of free Ag+ ions; we however observed a dose-dependent response. Nevertheless, after 96 h, viability of CFT073 was reduced to 27% even at the very low concentrations used (4, 6, 8 µg ml−1) (figure 7e). Fractionated Ag Pristine and Ag PVP determined a higher decrease in viability of A431 and HaCaT cells than the unfractionated samples (figure 7b–d). Ag HEC did not produce any significant toxic effect in A431 cells, and HaCaT viability remained well over 50% when compared to the untreated cells (figure 7f).
Viability of bacterial strain CFT073 after 24 and 96 h (a,c,e) and skin cells (b,d,f) after 24 h when treated with fractionated Ag NPs. (a–e) Bacteria were cultured at 37°C with shaking at 200 r.p.m. in L broth to mid-logarithmic phase. Of note, 50 µl aliquots of mid-logarithmic cultures (equivalent to approx. 106 cells) were incubated with an equal volume of Ag NPs (5, 10, 20 µg ml−1 for Ag Pristine, 5, 10, 15 µg ml−1 for Ag PVP and 4, 6, 8 µg ml−1 for Ag HEC) in 96-well plates. Milli-Q water was used as a negative control and ethanol as a positive control. After 24 and 96 h luminescence was read and the RLUs of untreated samples were normalized to 100% (see Material and methods). (a,c,e): CFT073. Data are means of three independent determinations ± s.d. *p < 0.05, **p < 0.01 and ***p < 0.001 versus untreated, control bacterial cells. # p < 0.05 and ## p < 0.01 versus 5 µg ml−1 Ag Pristine; ; $ p < 0.05 versus 10 µg ml−1 Ag Pristine;: ## p < 0.01 versus 5 µg ml−1 Ag PVP; : # p < 0.05 and ### p < 0.001 versus 4 µg ml−1 Ag HEC; versus 4 µg ml−1. (b,d,f) Cells, A431 and HaCaT, grown in complete growth medium for 24 h, were treated with different concentrations of Ag NPs (5, 10, 20 µg ml−1 for Ag Pristine, 5, 10, 15 µg ml−1 for Ag PVP and 4, 6, 8 µg ml−1 for Ag HEC) or with ethanol (80%) used as a positive control. After 24 h of exposure cell viability was assessed using calcein assay. Data are means of three independent determinations ± s.d. *p < 0.05, **p < 0.01 and ***p < 0.001 versus untreated, control bacterial cells. # p < 0.05 and ### p < 0.001 versus 5 µg ml−1 Ag Pristine; $ p < 0.05 and $$ p < 0.01 versus 10 µg ml−1 Ag Pristine; ## p < 0.01 versus 5 µg ml−1 Ag PVP; $ p < 0.05 versus 10 µg ml−1 Ag PVP. Ag Pristine (a,b), Ag PVP (c,d) and Ag HEC (e,f).
4. DISCUSSION
Our work intends to address the lack of existing platforms to obtain coherent information pertaining the Ag NP samples, and to provide more than simple characterization. In fact, we choose to characterize Ag NPs based on a separation step (achieved with FlFFF) followed by characterization through light scattering techniques and fraction collection, to assess together the antibacterial activity and the toxic response of human cells to different Ag NPs. HF5 is a miniaturized and disposable field-flow fractionation device, with the advantage of a lower channel volume, flow rates and, therefore, lower dilution factors. Both narrow peaks and low dilution contribute to increase detectability and sensitivity [46,47]. Short analysis time and high throughput, ease of use and minimum downtime are determining factors for a productive analytical tool; moreover the sample is analysed in suspension thus not creating artefacts through handling (e.g. drying). The advantage of using characterization techniques that can process samples in suspension, e.g. NTA together with FlFFF, UV–vis detector and MALS, is that realistic size and shape can be determined while the orthogonal data provides the information on the particles’ activity [48]. By using a DAD as a concentration detector we were also able to acquire the absorption spectra of the nanoparticles, thus providing a contemporary surface evaluation in terms of different coatings. In fact, when acquired during a separation, an absorption spectrum consists of a 3D plot, where one dimension is time, one is wavelength and the third is intensity of absorption. Last, by coupling graphite FAAS to quantify the collected free ions, a simultaneous quantification of free Ag+ and nanoparticles characterization and collection allowed for a direct calculation of ‘nano’ silver, to understand the amount of nano-dimensioned metal with respect to the total [49]. Indeed another advantage of using a miniaturized device such as HF5, is that the collected cross-flow resulted in being concentrated enough to allow for low-cost techniques such as graphite FAAS to be employed, as opposed to ICP-MS. The nature of the separation, and the simultaneous quantification of free ions, allowed for the recollection of separated Ag NPs that could be selectively tested for toxicity and activity. This is a new feature that overcomes the a priori calculation of the contributions of ions, medium, and other contaminants and provides a direct quantification of particle-specific effect. In this setting, our approach for the evaluation of Ag NPs was based on a five-step procedure (figure 8), able to accomplish: (i) characterization of the particles in suspension to match in vitro tests, (ii) testing of the nanoparticles to quantify their antibacterial response (acute and in a life cycle scenario), (iii) in vitro test to assess toxic response upon contact (skin model), (iv) testing of collected, purified nanoparticles to assess particle-specific activity, and (v) correlation of relevant properties and nanoparticles activity (antiseptic/toxic). The ranking and evaluation of the most suitable Ag NPs across the four under investigation to be used as an antimicrobial agent with reduced acute toxicity was performed by taking into account all the parameters involved from the physicochemical properties (PCP) to the in vitro and antiseptic response. Our results reported a highest toxicity (assessed with viability assays and the determination of cytokines secretion) towards human skin cells and antiseptic activity on bacteria, respectively, for both Ag Pristine and Ag PVP, suggesting that the higher content of Ag+ plays a crucial role in determining the toxicity of Ag NPs (figures 2–5) [50]. However, the effect on the viability of CFT073, A431 and HaCaT cells was evaluated also using the fractionated and collected nanoparticles where the only available silver was the solid phase contained in the nanostructure and the ionic phase adsorbed on it. The results showed a higher toxic/antiseptic effect compared to that of the entire sol (figure 7). These results seem to correlate with the hypothesis of a particle size-dependent activity/toxicity. Particle size plays an important role in toxicity and antimicrobial activity; many studies suggest that smaller particles have a higher chance to interact with the cell membrane and are, therefore, more toxic [11,51,52]. A direct prediction and comparison in activity (versus bacteria) and toxicity (versus human cell lines) is possible between Ag Pristine and Ag PVP, where size was the main distinguishing factor, as reported in table 2. Ag Pristine and Ag PVP derived from similar sol–gel synthesis route [25] were obtained with different reagents concentration and this led to both different dimension and a different absorption spectrum. We predicted that the different Rg/Rh ratio—hence the different coating thickness—together with the bigger size, could lead to a lesser activity of Ag Pristine, because a lower ratio is indicative of a thicker layer of polymer onto the surface of the particles, making the active surface less available for interactions. Nevertheless, the silver ion content, which has been quantified through flow field flow fractionation and atomic absorption, is almost the same between the two preparations (table 2). Fractionated Ag PVP, as showed in figure 7a–c, is more active/toxic than fractionated Ag Pristine, and this is coherent with the lower size of Ag PVP particles. In fact, free silver ions can be sequestrated by the medium and made less available, while the particles—being stable—maintain their potential. Moreover, the increase observed for fractionated samples of Ag Pristine and Ag PVP can also be partially owing to a destabilizing effect caused by dilution and subsequent different equilibrium achieved between Ag NPs and free ions, compared to the initial preparations. However, when diluted to obtain stock concentration for the previous experiments the nanoparticles did not show this deviation from linearity, and the dilution effect can be taken out from the concurring parameters to cause destabilization and hence increased toxicity. Purification of nanoparticles through FlFFF is also able to remove the impurities without affecting the characteristics of the nanoparticles. Those impurities present in Ag NPs suspensions do not only include Ag+ but also the residual reducing and stabilizing agents from the synthesis process that could hinder or modify the overall activity. Ag CIT and Ag HEC were expected to be both less toxic and less antiseptic, because the acute effect mediated by ion release is minor. Indeed, they are both less toxic than Ag Pristine and Ag PVP, either to bacteria strains or to the cell lines. For Ag HEC the difference between the activity of unfractionated and fractionated samples was very low (figures 2g,h, ,33d, ,44j–l, ,55j–l, ,77e,f), and this can be explained by the fact that the free ion concentration in the starting material was low enough not to interfere particularly with the particle-specific activity. Moreover, our work reported that Ag HEC is more effective in terms of activity and toxicity (viability assays) than Ag CIT, suggesting that other parameters than ionic concentration become relevant in this case. By simply following the theory of silver ions being the active element, Ag CIT should have been toxic (even though less than Ag Pristine and Ag PVP) and Ag HEC should have had a negligible activity, because the difference in ionic content of the two sols is more than one order of magnitude (table 2). However, these two preparations have the same size but we reported opposite surface charge (Ag CIT is negatively charged and Ag HEC is neutral/weakly positive). When considering surface charge of the nanoparticles, one should also consider the counterpart’s one, and both cells and bacteria have a negatively charged membrane. Similarly charged particles tend to repulse each other in proportion to the magnitude of the (negative) potential. Therefore, by taking in account surface charge then the lowest activity found in Ag CIT finds explanation and suggests that for a low ionic content (less than 1%), the main role is played by attractive forces [14,50]. As found in previous studies [14], on differently charged particles, as the absolute value of the negative potential decreases, the electrostatic barrier between membranes is reduced and the chance of cell-particle interaction increases, determining a higher toxicity. Repulsion turns to attraction when cells and bacteria are exposed to more positively charged particles like Ag HEC. Hence, when screening for the best Ag NP candidate, particle activity is a relevant aspect to be considered: differently charged materials exert a relevant influence on the overall activity, especially when longer exposure is considered. Therefore, particles cannot be considered only as Ag ion-release devices. When focusing on a real-life scenario, a further step is necessary and it involves accounting for the time of action of the candidate compounds. Indeed, if the 24 h exposure is sufficient to describe skin contact, the antimicrobial potential needs to be monitored over a longer amount of time, and a specific experimental design has to be set up for the scope. In fact, nanoparticles can have a long-term effect and measuring their activity over too short a span can lead to a biased evaluation. This long-term activity is also linked to the fact that bacteria can be selected through the previous use of silver ion-based antiseptics, widely used, that can increase resistance and cancel the acute toxicity determined by free ions [12]. On the other hand, nanoparticles need time to exert their particle-specific interaction and could have a delayed bacterial toxicity. By re-infusing living bacteria on dried plates containing used nanoparticles, the long-lasting antiseptic potential of Ag Pristine, Ag PVP, Ag CIT and Ag HEC was confirmed and so was the particle-specific activity: the dose-dependent effect is similar to that of newly diluted nanoparticles and ion-dependent effect is to be considered negligible (figure 4). The applicability of these nanoparticles, or of similar engineered ones, is desirable as they have shown to be good candidates for surface treatment when the correct time points were considered [53]. In fact the lack of individuation of long-term antiseptic effect would have disqualified Ag HEC, which instead seems to be the most interesting candidate because we did not report an inflammatory effect on human skin cells and, moreover, after the recovery experiment cell viability is above 50% for all the concentration used (figure 6). To summarize the results in a compact view we rationalized the property-effect relationships between the evaluated parameters, establishing a basis to categorize the key parameters needed to predict nanoparticle activity. Figure 9 shows how the different PCP of the Ag NPs can impact toxicity on skin cells and antiseptic activity. By exploiting this multi-step approach, based on characterization, toxicity assessment and activity evaluation, it is possible to extrapolate which combination of PCP is more effective. This result can be expanded over the four candidates screened and represents a process for the selection of the required PCP set needed for a successful antiseptic medical device. This selection is visualized in figure 10. As shown in figures 9 and and10,10, small particles with a negative surface charge and a higher ionic content like Ag PVP are acutely toxic and, even though antiseptic, have a small window of concentrations that can be exploited for surface treatment because the two trends go accordingly. Even bigger particles, such as Ag Pristine, that possess the same charge and ion content parameters, display a similar behaviour. Strongly negatively charged particles like Ag CIT do not show remarkable effects and are not good candidates. Instead, the combination of a more positive surface charge, a very low amount of free silver ions, and a size above 20 nm leads to the best candidate, represented in this work by Ag HEC. In fact, this preparation showed a low acute toxicity, a good cellular recovery after a 24 h exposure, and a remarkable long-term antiseptic activity. This is especially true for purified particles, where the decrease in bacterial viability was 80% (at 96 h of exposure) even for very low concentrations of nanoparticles.
Schematic representation of the multi-step approach used. (i) Characterization of the particles in suspension to match in vitro tests, (ii) testing of the nanoparticles to quantify their antibacterial response (acute and in a life-cycle scenario), (iii) in vitro test to assess toxic response upon contact (skin model), (iv) testing of collected, purified nanoparticle to assess particle-specific activity, and (v) correlation of relevant properties and nanoparticles activity (antiseptic/toxic and particle-specific).
Summary of physico-chemical properties of the four Ag NPs preparations and their impact on skin toxicity and antiseptic activity.
Qualitative representation of positive outcomes and correlated physico-chemical properties of the four candidates. Colours refer to toxicity towards human skin cells/bacteria. Red, high toxicity; orange, low toxicity; yellow, very low toxicity and green, non-toxic.
5. CONCLUSION
In our work, we developed and performed a five-step approach to assess and identify the purpose-specific applicability window of candidate Ag NPs, as antimicrobials in healthcare settings while also protecting consumer safety upon occasional or unintentional exposure.
FlFFF, coupled online with UV and MALS detectors, and offline with atomic absorption, provided together with NTA all the needed information to evaluate each nanoparticle descriptor in a realistic medium, allowing for the determination of the key parameters for the safe development of antiseptic nanoparticles. Moreover, the separation step provided purified particles for individual testing. We evaluated efficacy aspects by monitoring the long-term effect of nanoparticles onto luminescent strains of E. coli and of CFT073, a pathogenic strain present in hospitals and responsible for urinary tract infections. We addressed safety aspects by studying toxicity, inflammatory response and cellular recovery upon exposure of skin models to Ag NPs. Lastly, the design of experiments to verify preservation of antiseptic activity and particle-specific effects, led us to a realistic evaluation of the best candidate materials as coating agents, in correlation with their physicochemical requirements.
ACKNOWLEDGEMENTS
G. Clarke of the Department of Clinical Medicine, Trinity College Dublin is acknowledged for technical assistance in generating the TEM data. S. Casolari of the Department of Chemistry, University of Bologna, is acknowledged for the technical support regarding atomic absorption measurements.
DATA ACCESSIBILITY
The datasets supporting the results presented in this article are uploaded as part of the electronic supplementary material in this submission and are available online under the Data Dryad archive (http://dx.doi.org/10.5061/dryad.36j5d) [54] and under the Trinity College Access to Research Archive system (http://hdl.handle.net/2262/81721) under A. P.-M. research publication. This work was also included as part of V.Ms PhD thesis, as a result of her two internships and collaborative work with the co-authors of this manuscript carried out at Trinity College Dublin, Ireland in 2015 and 2016 (supervised by A.P.-M.). (http://dx.doi.org/10.6092/unibo/amsdottorato/7995).
AUTHORS’ CONTRIBUTIONS
A.P.-M., S.G. J.S. and L.D.C. conceived this study and designed the experiments, with V.M., and structured the paper, V.M. and L.D.C. performed the experiments, analysed the data, and carried out the statistical analysis. A.P-M., L.D.C. and V.M., drafted the paper. A.L.C, M.B. and S.O. synthesized the Ag NPs. V.M., L.D.C., S.G.J.S., A.L.C., P.R., Y.V. and A.P.-M. revised the paper. L.D.C. and A.P.-M. finalized the paper.
FUNDING
The research leading to these results has received partial funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) through the projects SANOWORK (EC-GA no. 280716) (A.L.C., M.B., S.O.), MARINA (EC-GA no. 263215) (L.D.C., A.L.C., M.B., S.O.), NANoREG (EC-GA no. 310584) (A.P.-M., L.D.C.) and the Erasmus +/UNIPHARMA project (V.M.).
REFERENCES
Conductive, antibacterial, and electromagnetic shielding silver‐plated cotton fabrics activated by dopamine.
ABSTRACT
A two‐step method was developed in this article to fabricate conductive silver‐plated cotton fabrics, imparting them with antibacterial and electromagnetic shielding properties. Cotton fabric was firstly functionalized by a spontaneous polymerization of dopamine, which acted as an activation and adsorption layer to initiate the following silver plating through the catechol and indole functional groups. The chemical composition of the functional cotton fabric was investigated by X‐ray photoelectron spectroscopy and the surface morphology of the fabric was observed by scanning electron microscopy. The crystalline structure of the silver‐coated cotton fibers was characterized by power X‐ray diffraction, and thermogravimetric analysis of the fabric was also studied to show thermal stability. The homogeneous silver plating was highly conductive with surface resistance about 23.55 mΩ sq.−1 and shielding effectiveness was about 55~95 dB. It also demonstrated excellent and durable antibacterial property against Staphylococcus aureus and Escherichia coli both with reduction percent of bacteria over 99.99%. All of above features made this silver‐plated cotton fabrics a promising candidate as multifunctional textiles. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 46766.
Reference:
https://onlinelibrary.wiley.com/doi/full/10.1002/app.46766
Cloth Face Masks – a sustainable choice
With the spread of the coronavirus worldwide, the consumption of disposable face masks has seen an incredible spike.
Whilst wearing a mask had previously only been recommended to those that already showed symptoms, the situation has now changed. In fact, there is increasing evidence that the Covid-19 can be spread by pre-symptomatic and asymptomatic carriers. For this reason, many Governments and health organisations , like the CDC, are recommending people consider wearing cloth face coverings in public settings where other social distancing measures are difficult to maintain.
Is it always better to wear a mask?
Although there is still much to be learned about the novel coronavirus, it appears that many people who are infected are shedding the virus – through coughs, sneezes and other respiratory droplets – for 48 hours before they start feeling sick. And others who have the virus – up to 25%, according to Centers for Disease Control and Prevention Director Dr. Robert Redfield — may never feel symptoms but may still play a role in transmitting it.
That’s why wearing a mask even if you don’t feel sick can be advisable for the general population.
Are there sustainable alternatives?
Now that everyone is concerned about protective equipment to stay safe, face masks are a must to prevent the contagion.
However, some consumers criticized the use of disposable masks because this does not represent an environmentally-friendly choice.
Are there sustainable alternatives for those who do not want to use surgical masks or respirators?
Can these consumers opt for masks that they can use again and stay safe?
Cloth Masks – are they effective against coronavirus?
In particular, our suppliers are developing 4-PLY Droplet Resistant Antibacterial Masks and Nano Silver Antibacterial Masks to help combat the virus.
Nano Silver Masks.
These masks are composed by 4 integrated layers.
In particular, the one in the middle is treated with Silver Nano technology that works as a safety filter. In fact, it eliminates small particles of bacteria that pass through the outer layer of the mask.
Nano Silver technology is an antimicrobial colloid composed of silver nanoparticles stabilized by a polymer that exhibits excellent antimicrobial efficacy across a wide spectrum of microorganisms.
While Silver inhibits the oxygen exchange in bacteria and kills it, this technology has been demonstrated to enhance that effect.
Looking for Cotton Masks? We can help!
We are currently working with our partners to provide a range of Cloth Masks to meet the world’s needs and to combat this deadly virus. From a sourcing perspective, the current regulatory landscape in China is very complex on these products so please make sure if you are buying direct, you are doing the appropriate due diligence and checks. For all enquiries, please contact us for more information at officelife4you@yahoo.com
Disclaimer. According to the World Health Organisation, masks alone will not prevent people from catching the coronavirus, and must be used in combination with normal hygiene methods, such as handwashing with soap and water.
SCIENCE BEHIND THE MASK
Natural Silver works against bacteria only in ion form — it must lose an electron to become positively charged. Silver ions pierce through the external surfaces of the cells and damage their DNA, as well as the protein structure, halting their metabolism and reproduction until they die.
Nanozid Nano-Coating Kills SARS Virus – New Product
Only last spring the SARS epidemic sent a shiver of fear around the globe. Now scientists working at the Institute of Virology at the University Hospital of Philipps University Marburg discovered that the viruses responsible for the severe acute respiratory syndrome (SARS) lose their terror on surfaces coated with special nanoparticles. These nanoparticles, only a few millionths of a millimeter in diameter, consist of titanium dioxide encased in a nano-thin layer of silver. In laboratory tests they proved capable of inactivating the coronaviruses which cause SARS in addition to bacteria and fungi. The Marburg researchers found that, after four hours of contact with the coating, the infectiousness of the viruses had decreased by more than two log steps. The nanoparticles have an especially large specific surface of around 200 sqm per gram. Owing to their silver coating, the particles release silver ions over long periods of time; these ions effectively deactivate pathogenic bacteria, fungi and viruses but are harmless for human beings. The effectiveness of the coating against SARS was confirmed during a study commissioned by ItN Nanovation GmbH, a nanotechnology company located in Saarbrücken. The objective of the study, which was carried out by the well known German research institute in Marburg, was to find additional applications for ItN’s microbicidal nanoparticles. The nano-powder with the brand name Nanozid is added to paints and lacquers used to coat operating tables, door knobs and door handles in hospitals (e.g. “clean rooms”) and surfaces in sanitary facilities. In addition to their obvious utility in the clinical sector, biocidal coatings have numerous applications in the food and beverage industries and in HVAC systems, which frequently serve as vehicles for the spread of infectious diseases. |
Posted 17th November 2003 |
https://www.azonano.com/article.aspx?ArticleID=417
Application of Nano Silver Particles on Textile Materials for Improvement of Antibacterial Finishes
Application of Nano Silver Particles on Textile Materials for Improvement of Antibacterial Finishes
Abstract
The article reviews the role of nano silver particles in improving the antibacterial properties of textile materials. Efforts have been directed at the extracellular synthesis of highly stable silver nano particles for the development of nanosafe textile using the extracts of yellow papaya peel. Owing to their potent antibacterial activity, papaya peel derived silver nanoparticles can be incorporated into fabrics and the textile producers can make textiles free from spoilage from micro organisms. Silver nano particles have been synthesized through biological approach using natural extracts of Acalypha indica and applied onto cotton fabrics. Even a mere 5% treatment with herbal extracts showed superior antibacterial activity indicating usage in medical and infection prevention applications. Silver nano particles have been synthesized by the reduction of silver nitrate with sodium borohydride in an aqueous medium. The silk protein sericin extracted from the silk worm cocoons has been used as effective capping agent. The silver nanoparticles exhibit antimicrobial properties when applied onto silk fabric without significantly changing its color. In yet other interesting research nano chitosan particles have been coupled with nano silver colloid to improve the antibacterial properties of cotton fabric.
Keywords: Antibacterial property; Silver nano particles; Cotton; Silk; Nano Chitosan; Herbal extract
Gokarneshan N* and Velumani K
Department of Textile Technology, Park College of Engineering and Technology, India
Submission: April 1, 2017; Published: May 31, 2017
*Corresponding author: Gokarneshan N, Department of Textile Technology, Park College of engineering and technology, India; Email:
Introduction
Nano particles have a wide range of applications such as electronics, catalysis, chemistry energy and medicine and have thus gained commercial acceptance [1]. Besides improving their functionality, the use of nano technology can result in the production of textiles with completely novel properties or the combination of various functions [2]. Such multifunctional textiles include antistatic textiles, reinforced textiles, antibacterial, self cleaning textiles, bleaching resistant, etc. andpaves the way for the use of its products in other fields outside the traditional industries [3-5]. Silver nano particles due to their strong and wide spectrum of antimicrobial activities have gained major focus among all nano particles. In order to protect against microbial contamination, silver has been incorporated into various forms of plastics such as catheters, dental material, medical devices, implants and burn dressings. These nano particles have also been used for durable finish on fabrics. As bactericides, the silver nano particles may help in solving the serious antibiotic resistance problem.
Papaya peel derived silver nano particles
Of the various techniques of nano particle synthesis available, the green synthesis technique holds advantage in
controlling particle size and morphology very effectively. When compared with other conventional techniques this technique is convenient and fast. Due to their potential antibacterial activity, papaya peel derived silver nano particles can be incorporated into fabrics and the manufacturers can make textiles free fromspoilage by microorganisms [6]. The significant reduction in reaction time with fruit peel extract is an important result and will enable nanoparticle biosynthesis methods to compete with other routes for the formation of nanoparticles that are currently much more rapid and reproducible.
Several strategies have been employed for the synthesis of silver nano particles including chemical techniques, physical techniques and recently, via biological techniques [7]. Biological techniques have received much attention as a viable alternative for the development of metal nanoparticles [8]. Many bacterial as well as fungal species have been used for silver nano particle synthesis [9,10]. But most of them are reported to accumulate silver nanoparticles intracellularly. On the contrary, plant extract mediated synthesis, i.e., green synthesis always takes place extracellularly, and the reaction times remain very short as compared to microbial synthesis. Extracts of many plants and weeds have shown the potential of reducing silver nitrate
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for the formation of silver nanoparticles without any chemical ingredients [11,12]. Various parts of the papaya plant have been used including flower and fruits are used for the synthesis of silver nano particles. Some reports relating to the use of silver nano particles using peel extracts are available. For the first time the potential of the peels of yellow papaya as non toxic biological systems for the biosynthesis of green silver nano particles have been used.
Application of nano chitosan coupled with nano silver colloid
\Ionotropic gelatin with pentasodium tripolyphosphate is employed for the preparation of nano chitosan dispersion. Nano chitosan together with nano silver treatment when applied on cotton fabrics shows enhanced antibacterial activity [13]. An enhanced antibacterial property is observed with the reduction of nano chitosan particles and when coupled with nano silver colloid.
Chitosan holds promise in varied application such as biomedical, waste water treatment, cosmetics, dentrifices, food, agriculture, pulp and paper, and textile industries [14- 16]. This is because it has many valuable inherent properties like antibacterial, antifungal, antiviral, antacid, non toxic, total biodegradability, biocompatibility with animal and plant tissues as well as film formation, fibre formation and hydrogel fibre formation properties. By virtue of its bacteria impedingproperty, chitosan can prevent garments develop bad odour [17-22]. It is found that complete inhibition of Eschirichia coli and Hay Bacillus bacteria is possible by treatment of cotton with 0.5gpL chitosan concentration [23].
Silver nano coating on cotton fabric adopting green approach
Biosynthesized sliver nanoparticles using A. indica leaf extract has been coated over cotton fabric through in-situ chemical reaction. SEM image, UV spectra proved the formation of sliver nanoparticles. The biological approach is a cost effective method as compared with the chemical synthesis [24]. Further it is proved that the sliver nanoparticles coated cotton fabric exhibit effective antimicrobial effect against microorganism. Finally the 5g A. indica leaf extract produces highest antimicrobial effect and release properties as compared with 3g and 4g leaf extracts. Due to the highest control release properties of this coating utilized for wound healing dressing.
Over the last few decades, various research work was happening around the world made to produce antibacterial coated textile materials due to the enormous growth of microbial infections via textile surfaces [25,26]. Attempts have been made to develop a non-toxic, cost effective and eco- friendly source of antimicrobial finishing textiles for health care application. Cotton fibres are mostly utilized as raw material towards medical and healthcare products [27]. However the moisture absorbability of cotton fibres is very high, which makes
them more prone to microbial attack under certain conditions of humidity and temperature. Cotton may acts as a nutrient, becoming suitable medium for bacterial and fungal growth [28]. Therefore, cotton fibres are treated with numerous chemicals to get better antimicrobial cotton textiles [29-31]. Among the various antimicrobial treatments, nano material based treatment is very effective. Silver nano particles (AgNPs) have shown strong inhibitory and antibacterial effects [32].
It has been reported that strong toxicity of silver nanoparticle against wide range of microorganisms is well known [33]. Further the antimicrobial activity of silver nanoparticles against Escherichia colli’s a model of Gram-negative bacteria has been studied. Chemical reducing method is one of the important technique followed in synthesis of sliver nano particles, which is normally associated with environmental toxicity [34].Therefore the development of sliver nano particle through natural extract is consider as most important method. Biosynthesis of silver nanoparticles using bacteria, fungi, yeast and plants were well documented.
Mechanism of reaction
Disc diffusion technique has been used to assess the antibacterial activity of A. indica silver nano particles against both against Gram positive and Gram negative pathogenic microorganisms. The antimicrobial activity of the coated silver nano particles on cotton fabrics has been measured as zone of inhibition. Silver nanoparticles displayed almost similar range of antimicrobial activity against studied pathogens, which was understood through diameter of inhibition zone. Many studies have concluded the biocidal properties of silver nano particles against microorganisms. It is believed that the silver nano particles attach the negatively charged cell surface, then change its physical and chemical properties of the cell membranes and the cell wall and disturb the permeability and osmoregulation, electron transport and respiration [35]. Secondly the silver nanoparticle produces further damage by permeating into the cell, interact with the DNA [36]. Thirdly, the silver nanoparticle releases the silver ions producing higher biocidal effect on the microorganisms.
Use of silk sericin in the synthesis of silver nanoparticles
Sericin extracted by a simple procedure from the cocoons of silk worms has been used effectively in the preparation of silver nanoparticles as a capping agent. Sericin effectively prevents coagulation of the silver nano particles and keeps them in a moderately stabilized condition in an aqueous medium [37]. The spherical particles of average size 15nm are fairly monodispersed. Further, the sericin-capped silver nano particles when applied to silk fabric impart antimicrobial properties to it. As silver nano particles are very small, have high surface energy and are unstable, capping agents should be used to capture them in the early stage of reaction and these include pvp, gelatin,
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How to cite this article: Gokarneshan N, Velumani K. Application of Nano Silver Particles on Textile Materials for Improvement of Antibacterial Finishes. Glob J Nano. 2017; 2(3) : 555586. DOI: 10.19080/GJN.2017.02.555586.
polyglutamic acid, sophorolipids, mixtures of different agents, and pva [38-43].
Earlier, during the preparation of silver nano particles, silk protein fibroin has been used as reducing and capping agent [44]. Recently sericin has attracted attention since it has useful properties and has many areas of applications [45,46]. Attempts have been made to prepare even smaller sized silver nano particles using sericin as the capping agent. After application of the nano particles on silk fabrics the antimicrobial efficiency of the nano particles has been confirmed. The treated silk fabric exhibits no significant change in colour, despite the yellow colour of the solution, and this has been found to be an improvement over the earlier method [47].
Conclusion
Among the various types of metal oxide nano particles used in textile finishing, silver oxide has assumed more prominence, more particularly for its very good antimicrobial property. Papaya peel derived silver nano particles, owing to its potent antibacterial activity can be incorporated into fabrics and the manufacturers can render textiles free from ruination by microorganisms An important finding is that there is a considerable decrease in reaction time with fruit peel extract which would help in nanoparticle biosynthesis methods to compete with other methods of formation of nano particles which are presently far more rapid and reproducible. Biological approach involving biosynthesized silver nano particles using A. indica leaf extract proves to be a cost effective method in comparison with the chemical synthesis method.
Also, the silver the cotton fabrics coated with silver nano particles show effective antimicrobial effect against microorganism. This coating is being used in would healing dressing owing to the highest control release properties. Nano chitosans have been synthesized by ionic gelation of pentasodium tripolyphosphate and chitosan. The cotton fabric have been pretreated with normal and nano chitosan solutions by pad dry cure technique. An enhanced antibacterial property is observed with the reduction of nano chitosan particles and when coupled with nano silver colloid. Silver nanoparticles have been synthesized by the reduction of silver nitrate with sodium borohydride in an aqueous medium. The silk protein sericin, extracted from the cocoons of Bombyx mori silkworms, has been used as an effective capping agent. Such particles also show antimicrobial properties when applied onto silk fabric without significantly changing the colour of the fabric.
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24. kumar SB (2016) Study On Antimicrobial Effectiveness Of Sliver Nano Coating Over Cotton Fabric Through Green Approach. IJPSR 7(9): 363- 368.
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Nanosilver particles in medical applications: synthesis, performance, and toxicity
INTRODUCTION
Nanosilver particles (NSPs) generally present at 1 to 100 nm in size in at least one dimension.1–4 As particle size decreases, the surface area-to-volume ratio of NSPs increases dramatically, which leads to significant changes in their physical, chemical, and biological properties. NSPs have been among the most commonly used nano-materials in our health care system for hundreds of years. Recently, NSPs have become of intense interest in biomedical applications (Figure 1), because of their antibacterial, antifungal, antiviral, and anti-inflammatory activity.5,6
Biomedical applications of nanosilver particles in human health care.
NSPs have been widely used for diagnosis,7 treatment,8 drug delivery,9 medical device coating,10 wound dressings,11 medical textiles,12 and contraceptive devices.13 As the use of nanosilver products is continually increasing, a better understanding of nanosilver biological interactions and their toxicity becomes necessary. This review critically discusses NSP synthesis methods, properties, and current and emerging medical NSP applications. Finally, recent advances concerning NSP potential toxicity will also be described.
NSP SYNTHESIS
Different synthetic NSP routes lead to variable sizes, shapes, morphology, and even stability. Generally, these methods can be classified into three broad categories: physical, chemical, and biological (or green) synthesis.
Physical synthesis
Evaporation/condensation and laser ablation are the main physical techniques for deriving nanosilver from metal samples. The evaporation/condensation technique uses a furnace tube under atmospheric pressure to produce NSPs; however, conventional furnace tubes have several drawbacks, such as high energy consumption, and require a long time to achieve thermal stability. Jung et al used a small ceramic heater with a local heating area, thus the evaporated vapor could cool at a suitable rate and a high concentration of nanosilver could be obtained.14 Laser synthesis employs the laser ablation of metals in solution without chemical reagents, which leads to pure nanosilver colloids.15 The concentration and morphology of nanosilver are affected by laser fluence and the number of laser shots. Greater laser fluence and amount of time, lead to larger particle size and higher particle concentration.16 Recently, Tien et al reported a novel arc-discharge method of producing silver suspension in pure water without any surfactants or stabilizers.17In their research, silver wires were utilized as positive and negative electrodes and etched in pure water. During discharge, the surface layer of the silver wires was evaporated and condensed in the water, thus stable and well-dispersed NSPs of 20–30 nm in size were obtained.17
Chemical synthesis
Chemical reduction is the most frequent method of nanosilver synthesis, and uses silver salt, reductants, and a stabilizer or capping agents as three main components to control NSP growth (Figure 2). Among these, silver nitrate is a silver salt that is often used for NSPs, due to its low cost and chemical stability compared to the other available silver salts.17 The reductants include borohydride,18 citrate,19ascorbate,20 and hydrogen gas.11
Chemical synthesis of nanosilver particles.
Abbreviation: Ag, silver.
Borohydride is a strong reducing agent that can result in small particles with a faster reduction rate, because borohydride can also act as an NSP stabilizer and avoid aggregation of NSPs during its decomposition.11 It is hard to obtain high concentrations of NSPs because of their aggregative instability. Using a stabilizer in preparation is a common strategy. The stabilizers include surfactants and ligands or polymers that contain functional groups such as polyvinylpyrrolidone, poly(ethylene glycol), poly(methacrylic acid), poly(methyl methacrylate), and others. Furthermore, temperature-sensitive polymers such as poly(N-isopropylacrylamide) and collagen can also serve as stabilizers, and nanosilver capped by those chemicals allows for novel thermal switching applications.1
NSPs can also be synthesized in a two-phase water-organic system. This method produces uniform and controllable nanoparticles. In this system, metal precursor and reducing agent are separated in two phases, thus the rate of interaction can be controlled by the intensity of interphase transport between aqueous and oil phases; however, large amounts of surfactant and organic solvent may contaminate the surface of formed NSPs, and the removal of surfactant and organic solvent is also time-consuming and expensive.
Biological synthesis
Biosynthesis (green synthesis) of nanosilver has received extensive attention due to the growing need for environmentally friendly synthesis methods that use eco-friendly reducing and capping agents, such as protein;21 peptides;22 carbohydrate;23 various species of bacteria,24 fungi,25 and yeast;26 and algae and plants.27 For example, Naik et al synthesized NSPs of 60–150 nm in size using silver-binding peptides identified from a combinatorial phage-display peptide library. The peptides were placed in an aqueous solution of 0.1 mM silver nitrate for 24–48 hours at room temperature.21 Thomas et al developed an economical, fascicled, and in situ approach to prepare large-scale chitosan–nanosilver (400 nm) films using chitosan as a chelating and stabilizing agent; the films demonstrated excellent antibacterial action against Escherichia coli and Bacillus.28 Sintubin et al reviewed different biological synthesis methods using microorganisms or plants for nanosilver synthesis.26
In biological synthesis, as the reducing agents and stabilizers are molecules produced by protein, carbohydrate, bacteria, fungi, yeasts, algae, or plants, organic solvents and toxic reagents are avoided. The possible mechanism of biological synthesis includes enzymatic and non-enzymatic reduction (Figure 3). Nicotinamide adenine dinucleotide phosphate-dependent reductase can produce NSPs by enzymatic reduction; however, the enzymatic reduction rate is often slow (between 24 and 120 hours).29 The non-enzymatic reduction of silver is similar to chemical reduction, but the reducing and stabilizing agents are microorganisms or plants. Non-enzymatic reduction is usually fast, often completed within a few minutes, and can handle extreme parameters, such as high pH or high temperature, that accelerate the synthesis.24
Biological (or green) synthesis of nanosilver particles.
Abbreviations: NADP+, nicotinamide adenine dinucleotide phosphate (oxidized form); NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); Ag, silver.
The main advantage of biogenic synthesis over other methods is that the green synthesis avoids organic solvents and toxic reagents. Thus, biosynthesized NSPs are more stable than those that are chemically produced, and they can remain stable over a long period of time.30 In addition, biological synthesis makes it possible to produce NSPs under a nontoxic silver nitrate concentration because microbial cells can continue to multiply;31 however, the biosynthesis drawback is that the purification process may lead to pathogenic bacteria and the potential bacteria may cause contamination, which should be a reason for exercising caution in medical application.26
NSP PERFORMANCE
Antibacterial properties
NSPs have a broad antibacterial effect on a range of Gram-negative and Gram-positive bacteria and antibiotic-resistant bacteria strains.32 Antimicrobial efficacy of NSPs depends on their size and concentration. Normally, a high concentration leads to more effective antimicrobial activity, while particles of small sizes can kill bacteria at a lower concentration. Apart from size and concentration, shape also influences the antimicrobial efficiency of NSPs. Sadeghi et al investigated the antimicrobial activity of different nanosilver shapes, which included silver nanoplates, silver nanorods, and silver nanoparticles, on Staphylococcus aureus and E. coli. They found that silver nanoplates had the best antimicrobial activity.33It has also been reported that NSPs combined with various antibiotics have better antimicrobial effects than NSPs or antibiotics alone. Li et al, for example, found a greater antibacterial effect on E. coli when amoxicillin and silver nanoparticles were combined than when they were applied separately.34
Although the antimicrobial effect of nanosilver has been widely studied, the exact mechanism of NSPs is still elusive. It is widely accepted that NSPs can anchor to and subsequently penetrate the bacterial cell wall, thereby causing structural change of the cell membrane and increasing cell permeability, leading to cell death (Figure 4).35 The formation of free radicals and subsequent free radical-induced membrane damage is another potential mechanism, which has been investigated by Kim et al.32 It has also been found that NSPs can release silver ions and interact with the thiol groups of many vital enzymes and phosphorus-containing bases, thus inhibiting some functions in cells, such as preventing cell division and DNA replication.36 In addition, NSPs may modulate signal transduction through changing the phosphotyrosine profile of bacterial peptides for the potential antibacterial mechanism (Figure 4).37
Antibacterial mechanism of nanosilver particles.
Abbreviation: DNA, deoxyribonucleic acid.
Antifungal properties
Nanosilver is an effective antifungal agent against a broad spectrum of common fungi. Kim et al investigated NSP antifungal properties on a total of 44 strains of six fungal species, and found that NSPs can inhibit the growth of Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, and Trichophyton mentagrophytes effectively.38 Nasrollahi et al39 and Kim et al40 observed that NSPs can disrupt cellular membrane and inhibit the normal budding process; however, the exact mechanisms of action of nanosilver against fungi are still not clear.
Antiviral properties
NSPs are also an antiviral agent against HIV-1,41 hepatitis B virus,42 respiratory syncytial virus,43 herpes simplex virus type 1,44 and monkeypox virus.45 It has been observed that NSPs have higher antiviral activity than silver ions, due to species difference as they dissolve to release Ag0 (atomic) and Ag+ (ionic) clusters, whereas silver salts release Ag+ only.46 Lara found that the anti-HIV mechanism of nanosilver is based on the inhibition of the initial stages of the HIV-1 cycle.47 NSPs can bind to glycoprotein (gp)120, thus inhibit cluster of differentiation (CD) 4-dependent binding, fusion, and infectivity. They act as an effective virucidal agent to block HIV-1 cell-free and cell-associated infection. Furthermore, NSPs inhibit post-entry stages of the HIV-1 life cycle.47 Although the mechanism underlying their viral-inhibitory activity is not yet fully understood, NSPs could be considered to be a broad-spectrum agent against a variety of viral strains and are not prone to developing resistance.
Anti-inflammatory properties
NSPs show anti-inflammatory properties in both animal models and in clinic. For example, in the swine model with contact dermatitis induced by topically applying 1,2-dinitrochlorobenzene, nanosilvers altered the expression of proinflammatory cytokines transforming growth factor-β and tumor necrosis factor-α.48Shin and Ye found that NSPs attenuated nasal symptoms in allergic rhinitis mice and inhibited OVA-specific immunoglobulin E, IL-4, and interleukin-10, and that inflammatory cell infiltration and goblet cell hyperplasia were inhibited by nanosilvers.49 In a human clinical study, wound dressing containing NSPs promoted the healing of chronic leg ulcers by not only reducing bacteria numbers in the wound bed, but by decreasing inflammatory response as well.8 NSPs’ ability to reduce cytokine release and matrix metalloproteinases,8,50 decrease lymphocyte and mast cell infiltration,48 and induce apoptosis in inflammatory cells8,49 may explain their anti-inflammatory mechanisms.
MEDICAL NSP APPLICATIONS
Wound dressings
Robert Burrell developed the world’s first commercially available nanosilver product (Acticoat™; Smith and Nephew, London, UK) to treat various wounds in clinic, including burns, chronic ulcers, toxic epidermal necrolysis, and pemphigus.51 Huang et al observed that NSP-loaded wound dressings significantly reduced the healing time by an average of 3.35 days and increased bacterial clearance from infected wounds compared to silver sulfadiazine, with no adverse effects;52 however, Chen et al showed that nanosilver-loaded wound dressings could enhance healing in superficial burn wounds but made no difference in deep burn wounds, compared with 1% silver sulfadiazine.53 This suggests that NSPs accelerate reepithelialization but not angiogenesis.
Currently, new dressings are being fabricated with the aim of increasing antibacterial efficacy and promotion of wound healing. For example, Lu et al developed a wound dressing composed of NSPs and chitosan, and found that it significantly increased wound healing during treatment of deep partial-thickness wounds and inhibited infection, as well as diminished the risk of silver absorption, compared with 1% silver sulfadiazine dressings.54
Cardiovascular implants
The first cardiovascular medical device containing silver in clinic was a prosthetic silicone heart valve coated with silver element, which was designed to prevent bacterial infection on the silicone valve and to reduce inflammation response;55 however, metal silver may cause hypersensitivity, inhibits normal fibroblast function, and leads to paravalvular leakage in patients.56 NSPs are safe and nontoxic in medical devices, unlike metal silver. Therefore, Andara et al synthesized a new nanocomposite with NSPs and diamond-like carbon as a surface coating for heart valves and stents, and found that the surface of the nanocomposite showed antithrombogenic and antibacterial properties.57 In addition, Ghanbari et al58 and Fu et al59 also constructed antibacterial multilayer films containing NSPs, and investigated their antibacterial, mechanical, and hemodynamic properties in vitro for use in cardiovascular implant coating.
Catheters
Much research has been conducted to investigate NSPs as antibacterial materials for coating catheters, including central venous catheters and neurosurgical catheters. Silverline (Spiegelberg GmbH and Co. KG, Hamburg, Germany) and ON-Q Silver Soaker™ (I-Flow Corporation, CA, USA) are two commercially available medical catheters containing NSPs to prevent catheter-associated infections.51 Medical catheters are prone to bacterial infection, which can rapidly spread to the wound and its surrounding, and lead to serious complications. Because of their superior antibacterial properties and lack of observed toxicity, NSPs can decrease the incidence of bacterial infection and complications after surgery, thus they have been widely accepted for use in medical catheters. Andara et al found that plastic catheter tubes coated with nanosilver could inhibit bacterium growth in vitro for at least 72 hours, with no significant toxicity, in an animal model.57 In a pilot clinical study, 19 patients who received a nanosilver catheter did not show catheter-associated ventriculitis, and all cerebrospinal fluid cultures were negative, while five patients were positive for catheter-associated ventriculitis in the control group (20 patients).60
Bone cement
Alt et al evaluated antibacterial activity of plain poly(methyl methacrylate) bone cement loaded with different NSP concentrations in vitro, and found that bone cement-loaded 1% nanosilver completely inhibited the proliferation of Staphylococcus epidermidis, methicillin-resistant S. epidermidis, and methicillin-resistant S. aureus, with no significant difference between the nanosilver bone cement and the nontoxic control group in quantitative and qualitative cytotoxicity tests.61 NSPs were also added to ultra-high-molecular-weight polyethylene for fabricating inserts for total joint replacement components, and it was found that NSPs drastically reduced the wear and tear of the polymer.62
Dental materials
NSPs also have applications in dental instruments and bandages. Yoshida et al showed that a resin composite incorporated with NSP-containing materials had a long-term inhibitory effect against Streptococcus mutans.63 Yamamoto et al also showed that a resin composite containing silver ion-implanted fillers released silver ions with antibacterial effects on oral streptococci.64 In addition, Magalhães et al showed that incorporating NSPs in endodontic filling materials provided a significantly enhanced anti-bactericidal effect against Streptococcus milleri, S. aureus, and Enterococcus faecalis.65NSPs in dental adhesives are also very effective against streptococci without affecting the adhesive mechanical properties, thus enabling their use in orthodontic treatments.66
Biodiagnosis
NSPs can be used for bio-diagnosis, where plasmonic properties of NSPs strongly depend on size, shape, and dielectric medium that surrounds it.67 Zhou et al developed a silver nanoparticle array biosensor for clinical detection of serum p53 in head and neck squamous cell carcinoma.68 NSPs are also employed to produce dual-imaging/therapy-immunotargeted nanoshells to locate cancer cells and can absorb light and selectively destroy targeted cancer cells through photothermal therapy.69 In addition, NSPs can detect the interaction between amyloid β-derived diffusible ligands (ADDL) and the anti-ADDL antibody, which are related to the development of Alzheimer’s disease;70 however, silver is easily oxidized and forms plasmonically unattractive compounds such as halides in biological solutions, which deteriorates the plasmonic performance of NSPs.18
Other medical applications
NSPs have applications in the diagnosis and treatment of cancer,71 and are drug carriers that can deliver therapeutic agents,72 which are used in eye care for coating contact lenses.17 In addition, the use of nanosilver in combination with vanadium oxide in battery cell components is one example of advanced silver nanotechnology improving battery performance in next-generation active implantable medical devices.73
NSP TOXICITY
NSPs may have potential toxicities at some concentrations and can cause various health problems if used improperly. Thus, it is necessary to address the biosafety of NSPs in human health.
In vitro toxicity
NSPs have been reported to be cytotoxic to several types of cells, including human peripheral blood mononuclear cells,74 human alveolar epithelial cell line (A549)75, murine and human alveolar macrophage cell line,76 neuroendocrine cells,77 rat liver cell line,78 and mouse germline cells.79 Alt et al, however, found that bone cement containing 1.0% nanosilver did not lead to significant cytotoxicity in mouse fibroblasts (L929) and human osteoblast cell line.61 Although the details of the toxic mechanism are unclear, it suggests that NSPs are ionized in the cells, which leads to activate ion channels and changes the permeability of the cell membrane to both potassium and sodium,80 interaction with mitochondria,81and induction of the apoptosis pathway via the production of reactive oxygen species,82 which leads to cell death.
In vivo toxicity
Chen and Schluesener have reviewed biodistribution, organ accumulation, degradation, possible adverse effects, and toxicity associated with the medical use of nanosilver.13 Respiratory tract, gastrointestinal tract, skin, and female genital tract are the main entry portals of nanosilver into the human body through direct substance exchange with the environment. Additionally, systemic administration is also a potential route of entry, since colloidal silver nanoparticles have been exploited for diagnostic imaging or therapeutic purposes. Inhalation and instillation experiments in rats showed that low concentration, but detectable, ultrafine silver (14.6±1.0 nm) appeared in the lung and was subsequently distributed to the blood and other organs, such as heart, liver, kidney, and even brain.83 In a recent oral toxicity study of rats, Kim et al also found that silver nanoparticles accumulated in blood, liver, lungs, kidneys, stomach, testes, and brain, but NSPs showed no significant genotoxicity after oral administration of silver nanoparticles of 60 nm average size for 28 days at different doses.84 Lee et al showed that NSPs less than 12 nm in size affected early development of fish embryos, caused chromosomal aberrations and DNA damage, and induced proliferation arrest in cell lines of zebrafish;85 however, Lansdown found that silver was not a cause of neurotoxic damage, even though silver deposits have been identified in the region of cutaneous nerves,86 and Ji et al found that NSPs did not affect respiratory system in a 28-day in vivo study.87
Animal and human studies indicate that it is difficult to remove silver completely once it has been deposited in the body; however, nanosilver can be excreted through the hair, urine, and feces.88 There is no consensus on nanosilver’s toxicity to humans, and most toxicity investigations of silver nanoparticles are based on in vitro cellular experiments and relatively short-term animal experiments.
CONCLUSION
NSPs represent a prominent nanoproduct and are already widely used in medical applications, including wound dressing, diagnosis, and pharmacological treatment. Since the shape, size, and composition of NSPs can have significant effects on their function and possible risks to human health, extensive research is needed to fully understand their synthesis, characterization, and possible toxicity. In this review, we first gave an overview of NSP synthesis, then reviewed applications of NSPs in the field of biomedicine. Finally, possible toxicology was discussed.
There is a limited number of well-controlled studies on the potential toxicities of nanosilver, though these studies tend to suggest that NSPs can induce toxicity in living beings. It should be noted that in vitro conditions are drastically different from in vivo conditions; however, longer-term studies and assessment of NSP toxicity must be conducted so that NSP exposure does not exceed toxic levels.
ACKNOWLEDGMENTS
This work was supported by International Science and Technology Cooperation Program of China (S2013ZR0398), Chongqing Basic Scientific Research Grant (cstc2013jcyjC80001), Chongqing Agriculture Development Grant (14408, 12402), the NSERC Discovery Grant, Manitoba Health Research Council, Dr Moorehouse Fellowship, Manitoba Institute of Child Health and China 863 Project (Grant 2012AA020504).
REFERENCES
Medical textiles with silver/nanosilver and their potential application for the prevention and control of healthcare-associated infections
Silver nanoparticles as a medical device in healthcare settings
1. BACKGROUND
The production of auto-sanitizing products for healthcare is highly desirable given the increasing incidence of healthcare-associated infection (HCAIs) [1]. The use of nanoparticles as active components in composite materials in place of conventional chemical products such as ethanol or bleach can guarantee long lasting bactericidal effects while not being toxic to the human body. Silver nanoparticles (Ag NPs) are known for their antimicrobial applications in common/household items, and their use in commercial products is increasing. In fact, they are already widely found as antiseptic additives in packaging, fabric, and are also ideal candidates as additives for tile coatings [2]. The global Ag NP market is indeed expected to reach $2.45 billion by 2022, with increasing demand for antimicrobial materials in healthcare applications [3]. Healthcare is the largest sector of that market, accounting for over 30% of the global Ag NP market revenue in 2014. With the pressing need to prevent HCAIs, it is expected that the use of Ag NPs in medical devices, equipment and textiles will further expand. The increased usage of nanoparticle-based medical devices has raised the attention of the European and International regulators and occupational safety community [4–6], leading to new guidelines for safety assessment of nanotechnology-enabled medical devices [7]. Ag NPs are thought to exert their antimicrobial effect through the release of free metal Ag+ ions. Indeed, silver ions are powerful antimicrobials themselves, but they are easily sequestered by chloride, phosphate, proteins and other cellular components. However, Ag NPs are less susceptible to sequestration and are thus a more effective delivery method [8]. The biological effect of the nanoparticles is largely unproven, but recent results supported the theory that the cytotoxic effects of nanosilver are a combination of precipitated silver complexes and organic silver compounds rather than free silver ions [9]. It is suggested that the antibacterial activity is owing to the generation of silver ions in the aqueous solution binding with the proteins on the bacteria cell membrane and inhibiting cell respiration and reproduction [10–12]. Particles size, free surface area, shape and charge will affect the bioavailability of ions in terms of dissolution or transport and interaction with biological targets [13–15]. Toxicity refers to any deleterious effects on an organism upon exposure to silver. Obviously, if the practical intent is to disinfect or sterilize a specific type of organism, then toxicity may be interpreted as a positive outcome (e.g. antibacterial, antiviral, etc.). However, if the same material exerts unintended or undesired impacts to other organisms, then such toxicity may be interpreted as a potential hazard [1–16]. An ideal antimicrobial candidate, therefore, needs to be selectively toxic, i.e. it is antibacterial at a given concentration but not toxic to humans. For a realistic evaluation of risk/benefit ratio, the comparison between human toxicity and antimicrobial effect has to be considered in terms of exposure times which may differ considerably, [14] e.g. a treated inanimate surface (such as a wall) is unlikely to have a long human exposure time (contact) while the antiseptic effect can be evaluated over a longer period. Concerns have been raised currently regarding the potential toxicities of Ag NPs [17]. For example, the Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR committees) [18] highlighted the importance of considering the different forms of silver used in consumer and medical products, because Ag NPs undergo several transformations as aggregation, agglomeration, dissolution and subsequent speciation. The chemical species that are actually present determine the bioavailability and toxicity of silver in the environment. Focusing on medical devices, the ‘Guidance on the Determination of Potential Health Effects of Nanomaterials Used in Medical Devices’, where nanosilver is widely mentioned, addresses the specific aspects that need to be considered in the safety evaluation of nanomaterials. This guidance highlights the need for special considerations in relation to the safety evaluation of nanomaterials, in view of the possible distinct properties, interactions, and effects that may differ from conventional forms of the same materials [7]. It recommends a stepwise approach where the first step is the chemical identification and characterization of nanomaterials used in the production of a medical device [19]. Relevant methods for nanomaterial characterization may include size separation and extraction and chemical analysis/detection by spectroscopic or mass spectrometric techniques [7]. The European NanoSafety Cluster has also come forward by suggesting a multi-step approach based on the implementation of the three classes of characterization techniques: imaging-based, light scattering-based and separation-based. Such platform should be able to measure nanoparticle primary size, the size distribution in complex matrices, while providing information on different populations present and their surface properties [20]. To address all these issues and identify the purpose-specific applicability window of Ag NPs we propose a five-step approach based first on separation (through flow-field flow fractionation, FlFFF) and characterization of particles, obtaining information about the dimension, the shape and the effective coating of particles, and quantifying the initial free ion presence. FlFFF has been used widely to characterize, concentrate and quantify ion release of engineered nanoparticles, especially when combined with inductively coupled plasma-mass spectrometry (ICP-MS) [21] and has been also employed to evaluate protein corona onto Ag NPs [22,23]. By exploiting hollow fibre flow-field flow fractionation (HF5), the commercial miniaturized version of FlFFF, it has been also possible to collect fractions of purified particles, without destroying their colloidal properties as more traditional ultrafiltration systems do. Secondly, to rapidly and effectively study the antimicrobial effect of the particles over time, luminescent bacteria were used: luminescence is directly proportional to viability thanks to a plasmid modification thus reducing analysis time [24]. The third step involved in vitro toxicity tests on skin models. Further to that isolated particles have been tested for their antimicrobial activity and cellular toxicity to quantify particle-specific contribution. Finally, all the parameters were gathered together for each preparation of Ag NPs, in order to select the best antimicrobial candidate and the set of physicochemical properties required of silver nanomaterials to be used within medical devices in healthcare settings.
2. MATERIAL AND METHODS
2.1. Reagents
Foetal bovine serum (FBS) and culture media were purchased from Sigma-Aldrich (Dublin, Ireland). Calcein was purchased from Molecular Probes, Invitrogen (Dublin, Ireland). ThermoFisher (Dublin, Ireland) was the source of all the other chemicals, whenever not specified otherwise.
2.2. Silver nanoparticles
For this study, four different Ag NP suspensions have been employed. Ag Pristine (0.02 wt% silver concentration) was provided by Colorobbia SpA (Italy). Polyvinylpyrrolidone-coated sample (Ag PVP, 0.02 wt% silver concentration), citrate-coated sample (Ag CIT, 0.02 wt% silver concentration) and hydroxyethyl cellulose-coated sample (Ag HEC, 0.02 wt% silver concentration) were synthesized in ISTEC-CNR (Faenza, Italy). Pristine and Ag PVP were obtained within the same process on a different scale (industrial scale for the Pristine, laboratory scale for the PVP coated material) using the same reducing (glucose) and capping (polyvinylpyrrolidone) agents but an excess of polyvinylpyrrolidone for the industrial scale synthesis [25]. Ag CIT was obtained using sodium citrate (Sigma-Aldrich) both as reducing agent and as a stabilizer, starting from AgNO3 solution. The synthesis reaction occurred in basic environmental and at 70°C by microwave heating, which enables homogeneous heating and rapid achievement of the desired temperatures [26]. The Ag HEC was synthesized, at room temperature, reducing a solution of AgNO3 by hydroxyethyl cellulose, which was also used as the capping agent. The reduction synthesis was catalysed by NaOH (Sigma-Aldrich) [27].
2.3. Hollow-fibre flow-field flow fractionation (HF5)
HF5 analyses were performed using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) complete with degasser, autosampler, isocratic pump and an Agilent 1100 diode array detector (DAD) UV/Vis spectrophotometer combined with an Eclipse® DUALTEC separation system (Wyatt Technology Europe, WTE, Dernbach, Germany). The HF5 channel (Wyatt Technology Europe) consisted of the commercial PES fibre and cartridge provided by WTE. Detailed description of the system was reported in a previous work published by some of the co-authors [28]. The software ChemStation version B.04.02 (Agilent Technologies) and Wyatt Eclipse @ ChemStation version 3.5.02 (Wyatt Technology Europe) were used to handle the separation parameters. An 18-angle multi-angle light scattering (MALS) detector model DAWN HELEOS (Wyatt Technology Corporation, Santa Barbara, CA, USA) operating at a wavelength of 658 nm, was used to measure compute the Rg of particles in solution and was handled with ASTRA® software v. 5.3.2.14 (Wyatt Technology Corporation). An HF5 method is composed of four steps: focus, focus–injection, elution and elution–injection. During focus the mobile phase is split into two different streams entering from the fibre’s inlet and outlet; during focus–injection, the sample is introduced through the inlet and focalized in a narrow band. During the elution step, the inlet flow splits in two: the longitudinal flow (Vc, going to the detectors) and the crossflow (Vx, determining the applied field). Lastly, the crossflow is released and the stream of mobile phase passes through the injection line to clean it before the next injection. The flow conditions for the different HF5 analysis are shown in table 1. A volume of Ag NPs of 5 µl was injected for the characterization of the sample in order to avoid saturation of the scattering signal, while a volume of Ag NPs of 100 µl was injected to collect both the ionic fraction and the isolated nanoparticles. When particles travelling in a parabolic flow profile are subjected to a crossflow, they localize at different points of the flow profile, according to their diffusion coefficient D (proportional to their hydrodynamic radius Rh). Smaller particles experience a higher flow rate and faster elution (normal mode). The Rh can be obtained from direct calculation or through calibration with standards of known size. The FlFFF theory behind this work is described in the previous literature [7–29]. When the analysed particles are not spherical, Rh is an estimation of the equivalent radius of a sphere with the same coefficient of diffusion D. On the other hand, the Rg value provided by the MALS gives information about the compactness on the particles: two particles with same hydrodynamic radius (Rh), but with different Rgvalues, may have a different mass distribution, and thus, different shapes. Combining the two sizing techniques via Rg/Rh ratios, a shape factor is obtained, reflecting the compactness and shape of the particles. For example, this corresponds to a value of 0.77 for a compact sphere and increases to about 4 for needle-like conformation, or decreases to about 0.6/0.5 for particles presenting a hard core and a soft shell/coating.
Table 1.
Flow conditions for HF5 analyses. (Vc, longitudinal flow; Vx, cross/focus flow.)
| steps (versus) method | focus (ml min−1) | focus–injection (ml min−1) | elution (ml min−1) | elution–injection (ml min−1) |
|---|---|---|---|---|
| particle characterization | Vc = 0.35 | Vc = 0.35 | Vc = 0.35 | Vc = 0.35 |
| Vx = 0.80 | Vx = 0.80 | Vx = 0.1 | Vx = 0.0 | |
| time= 2 min | time = 3 min | time = 10 min | time = 3 min | |
| cationic Ag collection and fraction collection | Vc = 0.35 | Vc = 1.0 | Vc = 0.35 | Vc = 0.35 |
| Vx = 0.80 | Vx = 0.8 | Vx = 0.1 | Vx = 0.0 | |
| time = 0.5 min | time = 20 min | time = 10 min | time = 3 min |
2.4. Ag+/Ag determination
The Ag+/Ag ratio was determined in Milli-Q water, analysing samples through an ad hoc analytical method listed in table 2, able to retain Ag NPs and filtrate away Ag+ ions. The proof of concept of the efficacy of this method has already been demonstrated in a previous work [30] employing flame absorption atomic spectroscopy (FAAS). The subsequent quantification of ionic Ag with FAAS was used to estimate the Ag+/Ag ratio. This is a non-destructive method, allowing for the collection of filtered nanoparticles that can be, therefore, tested individually to investigate particle-specific activity. In this work, because the preliminary determination of Ag+ through FAAS showed a very low concentration of silver, graphite furnace was used instead. To quantify the ionic fraction a volume of 10 ml was collected, 1 ml of concentrated HNO3 was added and the concentration of Ag+ was determined with atomic absorption spectroscopy by interpolation on a standard calibration curve (LoD = 0.2 ppb) following opportune dilution. Pd and Mn(NO3)2 were used as modifiers to prevent analyte loss. Each quantitative analysis was repeated three times and the ionic silver amount, expressed as percentage on the total silver content, is listed in table 2. By subtraction, the amount of ‘nano’ silver present in the collected nanoparticles was obtained and by correlating it with the fraction volume the subsequent concentration was determined. The scheme of such a procedure divided in steps is further detailed in the electronic supplementary information.
Table 2.
Physico-chemical properties of Ag NPs. (Rh, hydrodynamic radius determined by NTA; Rg, radius of gyration determined by MALS; ζ, zeta potential.)
| measured parameters (versus) Ag NPs | Rh(nm) | Rg (nm) | shape factor Rg/Rh | ζH2O(mV) | ζDMEM(mV) | % Ag+(w/w) |
|---|---|---|---|---|---|---|
| Pristine | 33.0 ± 2.5 | 18.5 ± 0.15 | 0.55 | −13.0 ± 9.0 | −7.61 ± 0.05 | 3.87 ± 0.08 |
| Ag PVP | 19.0 ± 3.6 | 13.5 ± 0.39 | 0.73 | −25.0 ± 9.0 | −6.99 ± 0.05 | 3.67 ± 0.09 |
| Ag CIT | 28.0 ± 2.9 | 13.6 ± 0.25 | 0.49 | −29.0 ± 12.4 | −22.00 ± 0.05 | 0.53 ± 0.03 |
| Ag HEC | 29.0 ± 4.0 | 25.0 ± 0.4 | 0.86 | +4.4 ± 4.3 | +0.06 ± 0.05 | 0.01 ± 0.02 |
2.5. Nanoparticle tracking analysis
The average hydrodynamic radius of Ag NPs in water was characterized using nanoparticle tracking analysis (NTA) developed by Malvern Instruments Limited (Wiltshire, UK). This technique uses the properties of light scattering and Brownian motion to obtain particle size distributions of samples in liquid suspension [31]. A NS500 instrument, equipped with a 405 nm laser in conjunction with software version NTA 3.1, was used for the purpose of this study. Ag NPs at the concentration of 200 µg ml−1 in Milli-Q water were vortexed for 5 s to disperse the particles and then diluted at 0.2 µg ml−1. The four different dispersions were then analysed via NTA for the measurement of hydrodynamic diameter at room temperature. All measurements were carried out three times in Milli-Q water to match the Rgdetermination. Results are reported as average mode ± standard deviation.
2.6. Zeta potential
Zeta potential of Ag NPs (200 µg ml−1), diluted 10-fold in Milli-Q water and Dulbecco’s Modified Eagle Medium (DMEM), were evaluated using a Zetasizer Nano Z (ZEN5600, Malvern Instruments, UK). Three zeta potential measurements were taken for each sample, each made of 20 accumulations. Measurements were carried out at 25°C, and elaborated using a Smoluchowski model.
2.7. Transmission electron microscopy
A Jeol 2100 transmission electron microscope (TEM; USA) was used to image the Ag NPs, with sizes of the Ag NPs being calculated using Image J software. A droplet of each preparation was deposited on a glass slide and left for 30 min. Then a grid was dragged onto the droplet surface to collect the nanoparticles, which accumulate on the droplet surface because of surface tension. Each grid was left to dry and then analysed.
2.8. Incubation with the silver nanoparticles
Ag NPs dispersed in a stock solution at a concentration of 200 µg ml−1 in Milli-Q water were diluted in the following way: for the cell treatments they were diluted in medium to reach the desired range of concentrations (2.5–100 µg ml−1), whereas for the antibacterial activity they were diluted in Milli-Q water to reach a range of concentrations from 0.625 to 100 µg ml−1.
2.9. Antibacterial activity testing
The antibacterial effect of the nanoparticles was tested against Escherichia coli. Escherichia coli strain TOP10 or CFT073 harbouring plasmid pGen-Lux [32] were incubated with various concentrations of nanoparticles over a time course of up to seven days. Plasmid pGen-Lux encodes the lux operon from Photorhabdus, the gene products of this operon imparts bioluminescence specifically on viable bacteria. One relative light unit (RLU) is approximately equal to 100 viable bacteria. Dead or non-viable bacteria are non-luminescent. All nanoparticles were tested up to a concentration of 100 µg ml−1 and were found to be non-luminescent, thus the effects of the particles on bacterial viability were amenable to measurement by luminometry. Bacteria were cultured at 37°C with shaking at 200 r.p.m. in L broth (Sigma, St Louis, MO, USA) to mid-logarithmic phase. Fifty microlitre aliquots of mid-logarithmic cultures (equivalent to approx. 106 cells) were incubated with an equal volume of the requisite nanoparticles in Lumitrac 200 96-well plates (Greiner). Milli-Q water was used as a negative control. Luminescence was read in a Thermofisher Luminoskan™ ascent microplate luminometer (Dublin, Ireland). Each concentration was tested in duplicate, and the experiment was repeated three times. The RLUs of untreated samples were normalized to 100% and treated samples were adjusted accordingly.
2.10. Cell culture and experimental treatments
The viability tests after exposure to Ag NP preparations were performed onto A431 (human epidermoid carcinoma) and HaCaT (human keratinocytes) cell lines representative of human skin models. A431 cells were obtained from ATCC (LGC Standard, UK) and cultured in Dulbecco’s modified Eagle’s medium (DMEM High Glucose) supplemented with 10% FBS, 2 mM L-glutamine, streptomycin (100 µg ml−1) and penicillin (100 U ml−1). HaCaT cells, obtained from ATCC (LGC Standard, UK), were cultured in DMEM (Dulbecco’s modified Eagle’s medium with Low Glucose) supplemented with 10% FBS, 2 mM L-glutamine, streptomycin (0.01 µg ml−1) and penicillin (0.01 U ml−1). Cells were routinely cultured in a humidified atmosphere of 5% CO2 in air in T75 cell culture flasks (Nunc, Fisher Scientific, Dublin, Ireland) For cytotoxicity experiments and ELISA assay, cells were seeded in complete growth medium on Nunc-96-well multiwell plates, at a density of 10 × 103cells well−1 and 20 × 103cells well−1, for A431 and HaCaT, respectively. For the recovery experiments cells were seeded in complete growth medium on Nunc-96-well multiwell plates, at a density of 2 × 103cells well−1 and 4 × 103cells well−1, for A431 and HaCaT, respectively. After 24 h, the growth medium of the cells was replaced with Ag NPs prepared as previously described. FBS was not employed for this stage to avoid the formation of artefact through protein corona effects. Doses of NPs were adjusted so as to obtain a silver concentration range from 4 µg cm−2 to 160 µg cm−2 (corresponding to a range from 2.5 to 100 µg ml−1). For the particle specific activity, the doses used were adjusted to obtain a final silver concentration of 5, 10, 20 µg ml−1 for Ag Pristine, 5, 10, 15 µg ml−1 for Ag PVP and 4, 6, 8 µg ml−1 for Ag HEC, corresponding to 3.12, 6.24, 12.48 µg cm−2, 3.12, 6.24, 9.36 µg cm−2 and 2.5, 3.75 and 5 µg cm−2 respectively. Since the fractionation led to dilution of the preparation, lower concentrations of total silver were used to assess toxicity. After 24 h exposure cell viability was assessed. In all the experiments, vehicle (1 : 1, Milli-Q water: DMEM) was added as negative control.
2.11. Calcein assay
Live cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually nonfluorescent cell-permeant calcein AM to the intensely fluorescent calcein. The polyanionic dye calcein is well retained within live cells, producing an intense uniform green fluorescence in live cells. After 24 h of incubation in the presence of Ag NPs, cell viability was tested replacing medium with a solution of calcein (1 mM) in serum-free medium. After 45 min of incubation at room temperature, protected from light, fluorescence was read at 635 nm with an Epoch microplate reader (Epoch, BioTek, UK). Since nanomaterials could interfere with this assay, a preliminary experiment was performed incubating both dyes with Ag NP preparations at the highest concentration used (100 µg ml−1). No fluorescence signal was detected above the background signal.
2.12. Resazurin assay
Resazurin is a substrate that changes colour in response to metabolic activity. It is a nonfluorescent molecule converted by intracellular enzymes in the fluorescent compound resorufin (λem = 590 nm). After 24 h of incubation in the presence of Ag NPs, cell viability was tested replacing medium with a solution of resazurin (44 mΜ) in serum-free medium. After 1 h of incubation, fluorescence was measured at 604 nm with Epoch microplate reader. Also in this case we performed a preliminary experiment to test the interference of Ag NPs with resazurin assay. No fluorescence signal was detected above the background.
2.13. Lactate dehydrogenase cytotoxicity assay
Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many different cell types. Plasma membrane damage releases LDH into the cell culture media. Extracellular LHD in the media can be quantified with Pierce LDH cytotoxicity assay kit (Thermo Scientific, UK). LDH catalyses the conversion of lactate to pyruvate via NAD+ reduction to NADH. Diaphorase then uses NADH to reduce the tetrazolium salt (INT) to a red formazan product that can be measured at 490 nm. The level of formazan formation is directly proportional to the amount of LDH released into the medium, which is indicative of cytotoxicity. In summary, after 24 h of incubation with Ag NPs, 50 µl of medium were transferred to a 96-well plate. Then, 50 µl of reaction mixture was added to each sample and, after 30 min of incubation at room temperature, 50 µl of stop solution was added. The absorbance was read at 490 and at 680 nm. To determinate the LDH activity, the value of absorbance at 680 nm (background) was subtracted from the 490 nm absorbance before calculation of per cent cytotoxicity. Total LDH activity (maximum LDH release control activity) was used as positive control and was performed by adding 10× lysis buffer (contained in the kit) to the cells.
2.14. Cytokine secretion
Details of the Materials, Methods and Results of the Cytokine secretion section are available in the electronic supplementary material.
2.15. Statistics
Statistic evaluation of effects has been performed with one-way ANOVA with a Bonferroni test. Statistics have been performed using GraphPad Prism™ software version 4.00 (GraphPad Software Inc., San Diego, CA). Differences have been considered significant for values of p < 0.05.
3. RESULTS
3.1. Physicochemical properties of nanoparticles: size, shape, surface charge and ionic content
Four different Ag NPs have been tested to correlate the particles antiseptic activity and toxicity to their physicochemical properties: Ag Pristine (commercial sample provided by Colorobbia SpA), Ag PVP, Ag CIT and Ag HEC, (CNR-ISTEC synthesized samples), respectively, coated by polyvynilpyrrolidone, citrate and hydroxyethilcellulose added during sol–gel synthesis as stabilizers. These preparations were characterized in terms of size, shape, charge and ionic content (table 2). By using a soft fractionation technique, the HF5 coupled with MALS, we exploited a hyphenated analytical platform able to in-flow size-separate analyte while calculating the gyration radius (Rg) of the particles, which—correlated to the hydrodynamic one (Rh) gives the shape factor. The absorption profile measured online for each preparation is reported in figure 1(a(i)), where the three dimensional (3D) absorption spectrum—collected during the separation is shown. With the exclusion of Ag Pristine, all the particles present a sharp absorption peak in a range between 395 and 425 nm, in accordance with the expected plasmon resonance of Ag NPs at such dimensions [33]. The broadening of the Ag Pristine absorption peak towards larger wavelengths can represent the influence of a small population of non-spherical particles. However, it is more likely that the lack of a stabilizing coating caused a partial, although minimal, aggregation process and the forming of aggregates are responsible for a red shift. In fact, we previously observed that PVP-stabilized nanoparticles tend to form chain-shaped aggregates when destabilized [30]. It is interesting to note that while TEM images (figure 1) are included as a comparison, the associated HF5-MALS fractograms (see the electronic supplementary material, figure S2) show a mono-modal size distribution, with only one band at each defined retention time. Furthermore, TEM images show a variety of species of different size and shape. Indeed, while drying Ag NPs could rearrange, agglomerate and nucleate into platelets, and cannot reflect the original state of the sample. Additionally, big agglomerates are usually omitted in TEM analysis. It is also difficult to assess aggregation by TEM owing to drying artefacts that can result in NP agglomeration during sample preparation [34]. Hence, all the calculations and the predictions have been made basing onto size/shape assessment performed in suspension. Hydrodynamic radius measurement, performed with NTA, was determined in Milli-Q water to match MALS measurements, in order to estimate particles shape [31]. Compared to dynamic light scattering (DLS), where the analysis is weighted towards larger particle size, and, therefore, tends to overestimate them [35], NTA has a lower concentration detection limit, and analyses NPs on a particle-by-particle basis. Among the four preparations, Ag PVP particles were the smallest whereas the other three were of similar dimensions (table 2). After the determination of the different radii, we considered the shape factor. This simple comparison of measurable dimensional parameters can in fact provide valuable information about the conformation/shape of particles in solution [36]. Observing the reported Rg/Rh ratios (table 2), ranging from 0.49 to 0.86, the particles appear to be spherical with a solid core and a less dense coating. In particular, even though the synthesis method is the same for Ag Pristine and PVP, both the difference in absorption spectra and Rg/Rh forecast a different behaviour between the two. Pristine nanoparticles have a smaller core compared to the coating, which is wider than that of Ag PVP particles, as confirmed by the low Rg/Rh ratio; indeed even commercial metal nanosols are stabilized by high amount of organic capping agents. Ag PVP, Ag CIT and Ag HEC have a spherical and coated shape as well, whereas Ag HEC shows a very compact nature and, therefore, a very thin coating. The zeta potential of the Ag PVP particles was found to be negative in pure water owing to the interaction of the surface with gluconate residue (generated during synthesis), but it was at least partially neutralized in medium (table 2). Ag Pristine particles undergo the same effect even though the decrease is less drastic. Ag CIT particles showed the expected negative charge, while Ag HEC had a neutral/weakly positive potential. The lower absolute values of zeta potential measured in medium, predict a low electrostatic repulsion even though from a visual observation of dispersed samples, the steric component of the coating seems to prevent the solid coagulation, segregation and sedimentation. In fact, a layer of ligands creates a repulsive potential to counteract the attractive van der Waals force [37]. This repulsion can be of steric nature (coating with polymers, such as PVP or uncharged molecules) or of electrostatic nature (coating with charged ligands, such as citrate). One direct consequence is that an increase in the ionic strength of the solution will shield the electrostatic repulsive potential, and lead to the aggregation of the nanoparticles, or their heterocoagulation onto living membranes [38]. The ionic content of Ag Pristine and Ag PVP is similar to predictable, because they were obtained through similar synthesis routes. Differences in activity and toxicity then can be dependent on the nature of the particles, in particular we expected Ag Pristine to be more active and toxic because the hard silver-based core is smaller. Citrate and HEC-coated particles have a considerably lower amount of free ions in the solution owing to the more precise stoichiometry applied to the synthesis (table 2). Being that the ionic percentage of Ag CIT particles is higher by an order of magnitude compared to Ag HEC, a consequent higher activity would have been expected if one considers the ionic bioavailability as the only mechanism of action. Nevertheless, the results we obtained showed an opposite trend, which makes necessary taking into account surface charge/particle specific activity. All the Ag NPs tested, are of a spherical shape; hence this parameter is kept constant. However, size, coating, charge and ion release vary for the four preparations and have been evaluated individually and as a combination. In this work, all the physical and chemical characterization of Ag NPs used has been made in suspension (water or cell medium), to allow us to predict the ‘real’ activity of nanoparticles in vitro for each preparation and to justify differences in antiseptic/toxic behaviour between similar preparations. In fact, the properties of Ag NPs can change during the life cycle of a nanomaterial and are partly depending on interactions with the surrounding environment, which may lead to a different behaviour of nanomaterials in different situations [39].
3D absorption spectra acquired in flow during HF5 characterization and NPs collection and representative TEM images of Ag Pristine (a), Ag PVP (b), AG CIT (c) and Ag HEC (d). Scale bar of TEM images represents 40 nm. For 3D spectra, horizontal axis represents wavelength (nm), depth axis represents time (min), height axis represent absorption intensity (mAU). Ag Pristine: min: 330 nm, max 450 nm. Ag PVP: min 325 nm, max 425 nm. Ag CIT: min 327 nm, max 420 nm. Ag HEC: min 320, max 410 nm.
3.2. Antimicrobial activity of silver nanoparticles over time and under re-contamination
We set different time points for bactericidal activity to simulate ageing of the medical device. Then, we designed a way to test the ability of Ag NPs to display a long lasting antiseptic effect. Lastly, the purified nanoparticles obtained through HF5 were tested to identify particle-specific effects. An important step is the choice of an appropriate model. Escherichia coli is a good model to test antibacterial activity of nanoparticles [40], however it is prudent to measure the activity of nanoparticles against pathogenic strains, because these are more likely encountered in a clinical situation. Bacterial strains TOP10 (a K-12 isolate) and CFT073 were chosen for analysis. Strain CFT073 is a uropathogenic strain that can form biofilms and cause urinary tract infections [41]. It belongs to sequence type 73 (ST73) of pathogenic E. coli and is one of the most frequent causes of E. coli extraintestinal infection. To maximize the information obtainable, the luminescence reads were performed until the negative control displayed a decrease in intensity when compared to the initial value. In this way, every experiment is balanced on the strain tested and the effect of Ag NPs can be evaluated over the longest period possible, accounting for strain-to-strain variability. Escherichia coli TOP10 was incubated with the nanoparticles over a time course of up to 72 h, while CFT073 allowed us to carry on the experiments up to 96 h without significant loss of luminescence on the untreated control (figure 2). At 24 h of exposure (red lines) the E. coli was not viable at concentrations ≥40 µg ml−1 for Ag Pristine and Ag PVP (figure 2a–c), while Ag CIT and Ag HEC displayed a smaller effect. Indeed, at the concentration of 100 µg ml−1 of Ag HEC viability dropped to 30% (figure 2g). After 72 h of exposure, a similar trend was registered for Ag Pristine and Ag PVP, with a decrease of viability to 75% and 65%, for Ag Pristine and Ag PVP respectively, even at the lowest concentration (figure 2a–c). However, Ag HEC induced a clear-cut killing effect for E. coli, with a complete loss of viability at concentrations ≥60 µg ml−1. On the contrary, Ag CIT was the least effective, with a decrease of viability to 50% for the highest concentration used (figure 2e). The particles were then tested against the clinical isolate CFT073 and all these agents displayed a bactericidal effect. Comparing these results to those obtained with E. coli TOP10 we observed a reduced sensitivity to all the preparations. CFT073 showed a higher resistance to Ag Pristine and Ag PVP, and the complete loss of viability was only observed at a concentration of ≥60 µg ml−1 (figure 2b–d), whereas Ag CIT displayed a mediocre effect (figure 2f). None of the three preparations showed a time-dependent antimicrobial effect. Instead, Ag HEC showed a time and dose dependent toxicity, with a complete loss of viability at doses ≥80 µg ml−1, thus maintaining its activity even towards a more resilient strain. This observation is in contrast with what could be expected when only applying the direct relationship between free ions and toxicant activity, which confirmed the presence of particle-specific interaction with living organisms. To test the ability of Ag NPs to maintain their antiseptic effect, an alternative experimental strategy was used. The used plates from the previous experiment with CFT073 were left to dry, the lack of remaining living bacteria was confirmed and another inoculation of viable luminescent bacteria was performed (figure 3). CFT073 viability was affected in a time-dependent manner when bacteria were treated with Ag NPs that had previously been used to test killing. The overall antiseptic effect was lower in comparison to that of freshly prepared nanoparticles, and one of the concurring factors is that particles were not uniformly distributed in the wells, because they were only left to dry after the previous experiment. After 24 h, particles are ineffective (Ag CIT) or only effective for the highest concentrations: this lack of acute toxicity (when compared to the previous experiment) finds explanation in the lack of starting free Ag+. This is also confirmed by the fact that Ag HEC displayed an almost unvaried dose–response pattern (figures 2h and and33d). All the preparations except for Ag CIT caused a time-dependent decrease in bacterial viability, supporting the hypothesis of the particles being interacting with bacteria.
Viability of bacterial strains E. coli Top10 and CFT073 after 24 h (red line) and 72 or 96 h (green or blue line). Bacteria were cultured at 37°C with shaking at 200 r.p.m. in L broth to mid-logarithmic phase. Of note, 50 µl aliquots of mid-logarithmic cultures (equivalent to approx. 106 cells) were incubated with an equal volume of Ag NPs (from 0.62 to 100 µg ml−1 final concentration) in 96-well plates. Milli-Q water was used as a negative control and ethanol as a positive control. After 24, 72 and 96 h luminescence was read and the RLUs of untreated samples were normalized to 100% (see Methods). (a,c,e,g) Escherichia coli TOP10; (b,d,f,h) E. coli CFT073. Data are means of three independent determinations ± s.d. Ag Pristine (a,b) Ag PVP (c,d) Ag CIT (e,f) and Ag HEC (g,h).
Viability of bacterial strain CFT073 after 24 h (red line) and 96 h (blue line) against reused Ag NPs. Bacteria were cultured at 37°C with shaking at 200 r.p.m. in L broth to mid-logarithmic phase. Of note, 50 µl aliquots of mid-logarithmic cultures (equivalent to approx. 106 cells) were incubated with an equal volume of water in the 96-well plates containing the previously employed nanoparticles at different concentrations (from 0.62 to 100 µg ml−1final concentration). The same well employed for the previous experiment was used as a negative control while ethanol was added instead of water as a positive control. After 24 and 96 h luminescence was read and the RLUs of untreated samples were normalized to 100% (see Material and methods). (a–d): CFT073. Data are means of three independent determinations ± s.d. Ag Pristine (a), Ag PVP (b), Ag CIT (c) and Ag HEC (d).
3.3. Cytotoxicity assessment of silver nanoparticles with two relevant cell models: A431 and HaCaT cells
To determine the safety of the Ag NP candidates for use as medical device we individuated the most relevant exposure scenario according to the SCENIHR guidelines [19]. Their use is meant to be as a surface coating, and can be then defined ‘surface contacting’, it can interact with consumers (workers, patients) through contact thus ‘facing/interacting with skin tissue’, and is not meant for topical use hence the scenario is of ‘limited contact’ (= or less than 24 h) [7]. Investigating the effect of Ag NPs on two human skin models is useful because they represent different skin layers. A431 are representative of the outer skin layer while HaCaT (keratinocytes), also part of the stratum granulosus of the skin, can simulate in vitro the first effects of penetration of the nanoparticles [29,42–45]. Different mechanisms of cytotoxicity, by carrying out three in vitro assays where the toxic effects of Ag NPs towards human skin cells were assessed (figures 4 and and5)5) in terms of presence of viable cells (calcein assay), damaging of cell membranes (LDH assay), or cellular metabolism (resazurin assay). Moreover, the cytotoxicity results obtained allowed us to calculate the IC50 for each preparation of Ag NPs and to compare the values of IC50 obtained between the different assays (table 3). A431 and HaCaT cells were exposed to the four preparations of Ag NPs and after 24 h the cell viability was assessed (figures 4 and and5,5, respectively). A431 and HaCaT cells reacted similarly to a 24 h exposure to Ag Pristine. Indeed this preparation induced an evident decrease in cell viability with calcein assay (figures 4a and and55a), whereas resazurin assay showed a dose-dependent decrease in viability (figures 4b and and55b). Moreover, LDH assay showed, for both cell lines, a cytotoxic effect starting from 20 µg ml−1, with an increase in LDH activity of 60–70% for the highest dose used (100 µg ml−1) (figures 4c and and55c). Ag PVP induced a similar cytotoxic effect onto A431 and HaCaT cells when compared to Ag Pristine. Both calcein and resazurin assay showed a dose-dependent decrease in cell viability (figures 4d,e and and55d,e). LDH assay confirmed the obtained results (figures 4f and and55f). On the contrary, Ag CIT, in both cell lines, did not show any cytotoxic effect (figure 4g–i and and55g–i) and the values of IC50 were greater than 100 µg ml−1 both for calcein and resazurin assays. Lastly, Ag HEC showed a moderate dose-dependent toxicity in both cell lines (figures 4j–l and and55j–l). However, no complete loss of viability was observed even at the highest concentrations used. In regard to the sensitivity of the assays, in our study we observed that calcein assay appears to be less sensitive compared with resazurin in detecting decrease of cell viability upon exposure to Ag NPs. The cytotoxic effect of Ag NPs on skin cell models was also investigated through the quantification of the cytokines TNF-α, IL-6, IL-8 and IL-1β secreted upon exposure (electronic supplementary material, figure S3). Ag HEC displayed a lack of acute inflammatory response, which can be a promising feature for surface treating applications. Even though skin contact is a possible pathway for contamination, a long-term effect for human health is avoided.
Cytotoxicity of Ag NPs towards A431 cell line. Cells, grown for 24 h in complete growth medium, were treated with different concentrations of Ag NPs or with ethanol (80%), used as a positive control. After 24 h of exposure cell viability was assessed using calcein assay, resazurin assay or LDH assay (see Material and methods). (a,d,g,j) Calcein assay; (b,e,h,k) resazurin assay; (c,f,i,l) LDH assay. Data are means of three independent determinations ± s.d. Ag Pristine (a–c), Ag PVP (d–f), Ag CIT (g–i) and Ag HEC (j–l).
Cytotoxicity of Ag NPs towards HaCaT cells. Cells, grown for 24 h in complete growth medium, were treated with different concentrations of Ag NPs or with ethanol (80%), used as a positive control. After 24 h of exposure cell viability was assessed using calcein assay, or resazurin assay, or LDH assay (see Material and methods). (a,d,g,j) Calcein assay; (b,e,h,k) resazurin assay; (c,f,i,l) LDH assay. Data are means of three independent determinations ± s.d. Ag Pristine (a–c), Ag PVP (d–f), Ag CIT (g–i) and Ag HEC (j–l).
Table 3.
IC50 values (µg ml−1) of A431 and HaCaT cells exposed for 24 h to Ag NPs.
| A431 | HaCaT | |||
|---|---|---|---|---|
| IC50 values (µg ml−1) | calcein | resazurin | calcein | resazurin |
| Ag Pristine | 21.87 | 9.33 | 10.00 | 7.58 |
| Ag PVP | 18.62 | 8.31 | 12.02 | 10.96 |
| Ag CIT | >100 | >100 | >100 | >100 |
| Ag HEC | 45.7 | 21.87 | 28.84 | 29.51 |
To investigate the effect of long-lasting toxicity of Ag NPs, A431 and HaCaT cells were exposed for 24 h to the four preparations of Ag NPs. Then, the cells were rinsed and allowed to recover in a complete growth medium for additional 6 days (figure 6). After the recovery period of 6 days, an increase in viability (assessed with calcein assay) of both cell lines treated with Ag NPs was observed, especially for Ag Pristine, Ag PVP and Ag HEC at the higher concentrations used (60–80–100 µg ml−1), as shown in figure 6. Moreover, the recovery experiment confirmed the lower cytotoxicity of Ag CIT (figure 6e,f). In summary, the cellular toxicity followed the same pattern we observed for bacterial strains, with a higher toxicity for Ag Pristine and Ag PVP, and a lower and moderate toxicity for Ag CIT and Ag HEC, respectively.
Recovery A431 and HaCaT cells after exposure to Ag NPs. Cells, grown for 24 h in complete growth medium, were treated with different concentrations of Ag NPs or with ethanol (80%), used as a positive control. After 24 h of exposure cell medium was replaced with full growth medium and cells were cultured for six additional days. On the seventh day viability was assessed using calcein assay. (a,c,e,g) A431; (b,d,f,h) HaCaT. Data are means of three independent determinations ± s.d. *p < 0.05, **p < 0.01 and ***p < 0.001 versus untreated, control cells. Ag Pristine (a,b), Ag PVP (c,d), Ag CIT (e,f) and Ag HEC (g,h).
3.4. Specific toxicity and antimicrobial activity of flow-field flow fractionated silver nanoparticles
In order to assess the toxic and antiseptic effects ascribable to nanoparticles themselves, isolated from the suspension of each Ag NP sample and from the starting concentration of Ag+, we treated both bacteria and cell lines with the FlFFF fractionated nanoparticles, according to the FlFFF process described in the Material and methods section. The concentrations used for the specific exposure are calculated, keeping in consideration the dilution factor occurred during fractionation and the measured initial Ag+ amount. These are 5, 10, 20 µg ml−1 for Ag Pristine, 5, 10, 15 µg ml−1 for Ag PVP and 4, 6, 8 µg ml−1 for Ag HEC. On these, viability of bacterial strain CFT073 after 24 and 96 h and cytotoxicity has been measured for the three relevant AgNPs and results are shown in figure 7. Of note, citrate-coated particles have not been screened in this further study because their antimicrobial effect was found to be negligible (as shown in figures 2 and and3).3). Interestingly, Pristine and PVP-coated nanoparticles exerted a similar effect on the pathogenic strain CFT073, at both time points. The antimicrobial activity is preserved and increased with respect to unfractionated samples (e.g. for fractionated Ag PVP we observed a loss of viability of 80% at 15 µg ml−1 as opposed to 60% for the unfractionated sample). The acute effect of these two preparations could reflect the fact that nanoparticles establish a new equilibrium by releasing ions in the new medium, and, therefore, a certain (and equal) amount of Ag+ is present both for Ag Pristine and Ag PVP. Ag HEC nanoparticles did not show a strong antimicrobial effect after 24 h, which is linked to their initial lesser amount of free Ag+ ions; we however observed a dose-dependent response. Nevertheless, after 96 h, viability of CFT073 was reduced to 27% even at the very low concentrations used (4, 6, 8 µg ml−1) (figure 7e). Fractionated Ag Pristine and Ag PVP determined a higher decrease in viability of A431 and HaCaT cells than the unfractionated samples (figure 7b–d). Ag HEC did not produce any significant toxic effect in A431 cells, and HaCaT viability remained well over 50% when compared to the untreated cells (figure 7f).
Viability of bacterial strain CFT073 after 24 and 96 h (a,c,e) and skin cells (b,d,f) after 24 h when treated with fractionated Ag NPs. (a–e) Bacteria were cultured at 37°C with shaking at 200 r.p.m. in L broth to mid-logarithmic phase. Of note, 50 µl aliquots of mid-logarithmic cultures (equivalent to approx. 106 cells) were incubated with an equal volume of Ag NPs (5, 10, 20 µg ml−1 for Ag Pristine, 5, 10, 15 µg ml−1 for Ag PVP and 4, 6, 8 µg ml−1 for Ag HEC) in 96-well plates. Milli-Q water was used as a negative control and ethanol as a positive control. After 24 and 96 h luminescence was read and the RLUs of untreated samples were normalized to 100% (see Material and methods). (a,c,e): CFT073. Data are means of three independent determinations ± s.d. *p < 0.05, **p < 0.01 and ***p < 0.001 versus untreated, control bacterial cells. # p < 0.05 and ## p < 0.01 versus 5 µg ml−1 Ag Pristine; ; $ p < 0.05 versus 10 µg ml−1 Ag Pristine;: ## p < 0.01 versus 5 µg ml−1 Ag PVP; : # p < 0.05 and ### p < 0.001 versus 4 µg ml−1 Ag HEC; versus 4 µg ml−1. (b,d,f) Cells, A431 and HaCaT, grown in complete growth medium for 24 h, were treated with different concentrations of Ag NPs (5, 10, 20 µg ml−1 for Ag Pristine, 5, 10, 15 µg ml−1 for Ag PVP and 4, 6, 8 µg ml−1 for Ag HEC) or with ethanol (80%) used as a positive control. After 24 h of exposure cell viability was assessed using calcein assay. Data are means of three independent determinations ± s.d. *p < 0.05, **p < 0.01 and ***p < 0.001 versus untreated, control bacterial cells. # p < 0.05 and ### p < 0.001 versus 5 µg ml−1 Ag Pristine; $ p < 0.05 and $$ p < 0.01 versus 10 µg ml−1 Ag Pristine; ## p < 0.01 versus 5 µg ml−1 Ag PVP; $ p < 0.05 versus 10 µg ml−1 Ag PVP. Ag Pristine (a,b), Ag PVP (c,d) and Ag HEC (e,f).
4. DISCUSSION
Our work intends to address the lack of existing platforms to obtain coherent information pertaining the Ag NP samples, and to provide more than simple characterization. In fact, we choose to characterize Ag NPs based on a separation step (achieved with FlFFF) followed by characterization through light scattering techniques and fraction collection, to assess together the antibacterial activity and the toxic response of human cells to different Ag NPs. HF5 is a miniaturized and disposable field-flow fractionation device, with the advantage of a lower channel volume, flow rates and, therefore, lower dilution factors. Both narrow peaks and low dilution contribute to increase detectability and sensitivity [46,47]. Short analysis time and high throughput, ease of use and minimum downtime are determining factors for a productive analytical tool; moreover the sample is analysed in suspension thus not creating artefacts through handling (e.g. drying). The advantage of using characterization techniques that can process samples in suspension, e.g. NTA together with FlFFF, UV–vis detector and MALS, is that realistic size and shape can be determined while the orthogonal data provides the information on the particles’ activity [48]. By using a DAD as a concentration detector we were also able to acquire the absorption spectra of the nanoparticles, thus providing a contemporary surface evaluation in terms of different coatings. In fact, when acquired during a separation, an absorption spectrum consists of a 3D plot, where one dimension is time, one is wavelength and the third is intensity of absorption. Last, by coupling graphite FAAS to quantify the collected free ions, a simultaneous quantification of free Ag+ and nanoparticles characterization and collection allowed for a direct calculation of ‘nano’ silver, to understand the amount of nano-dimensioned metal with respect to the total [49]. Indeed another advantage of using a miniaturized device such as HF5, is that the collected cross-flow resulted in being concentrated enough to allow for low-cost techniques such as graphite FAAS to be employed, as opposed to ICP-MS. The nature of the separation, and the simultaneous quantification of free ions, allowed for the recollection of separated Ag NPs that could be selectively tested for toxicity and activity. This is a new feature that overcomes the a priori calculation of the contributions of ions, medium, and other contaminants and provides a direct quantification of particle-specific effect. In this setting, our approach for the evaluation of Ag NPs was based on a five-step procedure (figure 8), able to accomplish: (i) characterization of the particles in suspension to match in vitro tests, (ii) testing of the nanoparticles to quantify their antibacterial response (acute and in a life cycle scenario), (iii) in vitro test to assess toxic response upon contact (skin model), (iv) testing of collected, purified nanoparticles to assess particle-specific activity, and (v) correlation of relevant properties and nanoparticles activity (antiseptic/toxic). The ranking and evaluation of the most suitable Ag NPs across the four under investigation to be used as an antimicrobial agent with reduced acute toxicity was performed by taking into account all the parameters involved from the physicochemical properties (PCP) to the in vitro and antiseptic response. Our results reported a highest toxicity (assessed with viability assays and the determination of cytokines secretion) towards human skin cells and antiseptic activity on bacteria, respectively, for both Ag Pristine and Ag PVP, suggesting that the higher content of Ag+ plays a crucial role in determining the toxicity of Ag NPs (figures 2–5) [50]. However, the effect on the viability of CFT073, A431 and HaCaT cells was evaluated also using the fractionated and collected nanoparticles where the only available silver was the solid phase contained in the nanostructure and the ionic phase adsorbed on it. The results showed a higher toxic/antiseptic effect compared to that of the entire sol (figure 7). These results seem to correlate with the hypothesis of a particle size-dependent activity/toxicity. Particle size plays an important role in toxicity and antimicrobial activity; many studies suggest that smaller particles have a higher chance to interact with the cell membrane and are, therefore, more toxic [11,51,52]. A direct prediction and comparison in activity (versus bacteria) and toxicity (versus human cell lines) is possible between Ag Pristine and Ag PVP, where size was the main distinguishing factor, as reported in table 2. Ag Pristine and Ag PVP derived from similar sol–gel synthesis route [25] were obtained with different reagents concentration and this led to both different dimension and a different absorption spectrum. We predicted that the different Rg/Rh ratio—hence the different coating thickness—together with the bigger size, could lead to a lesser activity of Ag Pristine, because a lower ratio is indicative of a thicker layer of polymer onto the surface of the particles, making the active surface less available for interactions. Nevertheless, the silver ion content, which has been quantified through flow field flow fractionation and atomic absorption, is almost the same between the two preparations (table 2). Fractionated Ag PVP, as showed in figure 7a–c, is more active/toxic than fractionated Ag Pristine, and this is coherent with the lower size of Ag PVP particles. In fact, free silver ions can be sequestrated by the medium and made less available, while the particles—being stable—maintain their potential. Moreover, the increase observed for fractionated samples of Ag Pristine and Ag PVP can also be partially owing to a destabilizing effect caused by dilution and subsequent different equilibrium achieved between Ag NPs and free ions, compared to the initial preparations. However, when diluted to obtain stock concentration for the previous experiments the nanoparticles did not show this deviation from linearity, and the dilution effect can be taken out from the concurring parameters to cause destabilization and hence increased toxicity. Purification of nanoparticles through FlFFF is also able to remove the impurities without affecting the characteristics of the nanoparticles. Those impurities present in Ag NPs suspensions do not only include Ag+ but also the residual reducing and stabilizing agents from the synthesis process that could hinder or modify the overall activity. Ag CIT and Ag HEC were expected to be both less toxic and less antiseptic, because the acute effect mediated by ion release is minor. Indeed, they are both less toxic than Ag Pristine and Ag PVP, either to bacteria strains or to the cell lines. For Ag HEC the difference between the activity of unfractionated and fractionated samples was very low (figures 2g,h, ,33d, ,44j–l, ,55j–l, ,77e,f), and this can be explained by the fact that the free ion concentration in the starting material was low enough not to interfere particularly with the particle-specific activity. Moreover, our work reported that Ag HEC is more effective in terms of activity and toxicity (viability assays) than Ag CIT, suggesting that other parameters than ionic concentration become relevant in this case. By simply following the theory of silver ions being the active element, Ag CIT should have been toxic (even though less than Ag Pristine and Ag PVP) and Ag HEC should have had a negligible activity, because the difference in ionic content of the two sols is more than one order of magnitude (table 2). However, these two preparations have the same size but we reported opposite surface charge (Ag CIT is negatively charged and Ag HEC is neutral/weakly positive). When considering surface charge of the nanoparticles, one should also consider the counterpart’s one, and both cells and bacteria have a negatively charged membrane. Similarly charged particles tend to repulse each other in proportion to the magnitude of the (negative) potential. Therefore, by taking in account surface charge then the lowest activity found in Ag CIT finds explanation and suggests that for a low ionic content (less than 1%), the main role is played by attractive forces [14,50]. As found in previous studies [14], on differently charged particles, as the absolute value of the negative potential decreases, the electrostatic barrier between membranes is reduced and the chance of cell-particle interaction increases, determining a higher toxicity. Repulsion turns to attraction when cells and bacteria are exposed to more positively charged particles like Ag HEC. Hence, when screening for the best Ag NP candidate, particle activity is a relevant aspect to be considered: differently charged materials exert a relevant influence on the overall activity, especially when longer exposure is considered. Therefore, particles cannot be considered only as Ag ion-release devices. When focusing on a real-life scenario, a further step is necessary and it involves accounting for the time of action of the candidate compounds. Indeed, if the 24 h exposure is sufficient to describe skin contact, the antimicrobial potential needs to be monitored over a longer amount of time, and a specific experimental design has to be set up for the scope. In fact, nanoparticles can have a long-term effect and measuring their activity over too short a span can lead to a biased evaluation. This long-term activity is also linked to the fact that bacteria can be selected through the previous use of silver ion-based antiseptics, widely used, that can increase resistance and cancel the acute toxicity determined by free ions [12]. On the other hand, nanoparticles need time to exert their particle-specific interaction and could have a delayed bacterial toxicity. By re-infusing living bacteria on dried plates containing used nanoparticles, the long-lasting antiseptic potential of Ag Pristine, Ag PVP, Ag CIT and Ag HEC was confirmed and so was the particle-specific activity: the dose-dependent effect is similar to that of newly diluted nanoparticles and ion-dependent effect is to be considered negligible (figure 4). The applicability of these nanoparticles, or of similar engineered ones, is desirable as they have shown to be good candidates for surface treatment when the correct time points were considered [53]. In fact the lack of individuation of long-term antiseptic effect would have disqualified Ag HEC, which instead seems to be the most interesting candidate because we did not report an inflammatory effect on human skin cells and, moreover, after the recovery experiment cell viability is above 50% for all the concentration used (figure 6). To summarize the results in a compact view we rationalized the property-effect relationships between the evaluated parameters, establishing a basis to categorize the key parameters needed to predict nanoparticle activity. Figure 9 shows how the different PCP of the Ag NPs can impact toxicity on skin cells and antiseptic activity. By exploiting this multi-step approach, based on characterization, toxicity assessment and activity evaluation, it is possible to extrapolate which combination of PCP is more effective. This result can be expanded over the four candidates screened and represents a process for the selection of the required PCP set needed for a successful antiseptic medical device. This selection is visualized in figure 10. As shown in figures 9 and and10,10, small particles with a negative surface charge and a higher ionic content like Ag PVP are acutely toxic and, even though antiseptic, have a small window of concentrations that can be exploited for surface treatment because the two trends go accordingly. Even bigger particles, such as Ag Pristine, that possess the same charge and ion content parameters, display a similar behaviour. Strongly negatively charged particles like Ag CIT do not show remarkable effects and are not good candidates. Instead, the combination of a more positive surface charge, a very low amount of free silver ions, and a size above 20 nm leads to the best candidate, represented in this work by Ag HEC. In fact, this preparation showed a low acute toxicity, a good cellular recovery after a 24 h exposure, and a remarkable long-term antiseptic activity. This is especially true for purified particles, where the decrease in bacterial viability was 80% (at 96 h of exposure) even for very low concentrations of nanoparticles.
Schematic representation of the multi-step approach used. (i) Characterization of the particles in suspension to match in vitro tests, (ii) testing of the nanoparticles to quantify their antibacterial response (acute and in a life-cycle scenario), (iii) in vitro test to assess toxic response upon contact (skin model), (iv) testing of collected, purified nanoparticle to assess particle-specific activity, and (v) correlation of relevant properties and nanoparticles activity (antiseptic/toxic and particle-specific).
Summary of physico-chemical properties of the four Ag NPs preparations and their impact on skin toxicity and antiseptic activity.
Qualitative representation of positive outcomes and correlated physico-chemical properties of the four candidates. Colours refer to toxicity towards human skin cells/bacteria. Red, high toxicity; orange, low toxicity; yellow, very low toxicity and green, non-toxic.
5. CONCLUSION
In our work, we developed and performed a five-step approach to assess and identify the purpose-specific applicability window of candidate Ag NPs, as antimicrobials in healthcare settings while also protecting consumer safety upon occasional or unintentional exposure.
FlFFF, coupled online with UV and MALS detectors, and offline with atomic absorption, provided together with NTA all the needed information to evaluate each nanoparticle descriptor in a realistic medium, allowing for the determination of the key parameters for the safe development of antiseptic nanoparticles. Moreover, the separation step provided purified particles for individual testing. We evaluated efficacy aspects by monitoring the long-term effect of nanoparticles onto luminescent strains of E. coli and of CFT073, a pathogenic strain present in hospitals and responsible for urinary tract infections. We addressed safety aspects by studying toxicity, inflammatory response and cellular recovery upon exposure of skin models to Ag NPs. Lastly, the design of experiments to verify preservation of antiseptic activity and particle-specific effects, led us to a realistic evaluation of the best candidate materials as coating agents, in correlation with their physicochemical requirements.
ACKNOWLEDGEMENTS
G. Clarke of the Department of Clinical Medicine, Trinity College Dublin is acknowledged for technical assistance in generating the TEM data. S. Casolari of the Department of Chemistry, University of Bologna, is acknowledged for the technical support regarding atomic absorption measurements.
DATA ACCESSIBILITY
The datasets supporting the results presented in this article are uploaded as part of the electronic supplementary material in this submission and are available online under the Data Dryad archive (http://dx.doi.org/10.5061/dryad.36j5d) [54] and under the Trinity College Access to Research Archive system (http://hdl.handle.net/2262/81721) under A. P.-M. research publication. This work was also included as part of V.Ms PhD thesis, as a result of her two internships and collaborative work with the co-authors of this manuscript carried out at Trinity College Dublin, Ireland in 2015 and 2016 (supervised by A.P.-M.). (http://dx.doi.org/10.6092/unibo/amsdottorato/7995).
AUTHORS’ CONTRIBUTIONS
A.P.-M., S.G. J.S. and L.D.C. conceived this study and designed the experiments, with V.M., and structured the paper, V.M. and L.D.C. performed the experiments, analysed the data, and carried out the statistical analysis. A.P-M., L.D.C. and V.M., drafted the paper. A.L.C, M.B. and S.O. synthesized the Ag NPs. V.M., L.D.C., S.G.J.S., A.L.C., P.R., Y.V. and A.P.-M. revised the paper. L.D.C. and A.P.-M. finalized the paper.
FUNDING
The research leading to these results has received partial funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) through the projects SANOWORK (EC-GA no. 280716) (A.L.C., M.B., S.O.), MARINA (EC-GA no. 263215) (L.D.C., A.L.C., M.B., S.O.), NANoREG (EC-GA no. 310584) (A.P.-M., L.D.C.) and the Erasmus +/UNIPHARMA project (V.M.).
REFERENCES
Conductive, antibacterial, and electromagnetic shielding silver‐plated cotton fabrics activated by dopamine.
ABSTRACT
A two‐step method was developed in this article to fabricate conductive silver‐plated cotton fabrics, imparting them with antibacterial and electromagnetic shielding properties. Cotton fabric was firstly functionalized by a spontaneous polymerization of dopamine, which acted as an activation and adsorption layer to initiate the following silver plating through the catechol and indole functional groups. The chemical composition of the functional cotton fabric was investigated by X‐ray photoelectron spectroscopy and the surface morphology of the fabric was observed by scanning electron microscopy. The crystalline structure of the silver‐coated cotton fibers was characterized by power X‐ray diffraction, and thermogravimetric analysis of the fabric was also studied to show thermal stability. The homogeneous silver plating was highly conductive with surface resistance about 23.55 mΩ sq.−1 and shielding effectiveness was about 55~95 dB. It also demonstrated excellent and durable antibacterial property against Staphylococcus aureus and Escherichia coli both with reduction percent of bacteria over 99.99%. All of above features made this silver‐plated cotton fabrics a promising candidate as multifunctional textiles. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 46766.
Reference:
https://onlinelibrary.wiley.com/doi/full/10.1002/app.46766