Antimicrobial Performance of Thermoplastic Elastomers Containing Zinc Pyrithione and Silver Nanoparticles

The purpose of the present study was to evaluate the antimicrobial potential of styrene-ethylene/ butylene-styrene based thermoplastic elastomers (TPE) incorporated with zinc pyrithione (ZnPT) and silver nanoparticles (AgNano). Japan Industrial Standard was applied to evaluate the antimicrobial potential of incorporated TPE compounds against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Antifungal action was evaluated against Aspergillus niger, Candida albicans and Cladosporium cladosporioides. Samples prepared with ZnPT eliminated 99.9% of the E. coli and 99.7 % of the S. aureus population, and presented an inhibition zone in the fungal assay. Samples prepared with AgNano eliminated 99.7% of the E. coli and 95.5 % of the S. aureus population. There was no inhibition zone in samples containing AgNano; however, these samples did not present fungal growth on their surfaces. TPE samples containing ZnPT showed biocidal activity against the microorganisms tested and can be used to develop antimicrobial products.


Introduction
Styrene-ethylene/butylene-styrene (SEBS) based thermoplastic elastomers (TPE) are employed in a wide range of products with elastic properties, such as remote control keyboards, cell phone covers, tooth brush cables, toys and others.The World Health Organization has disclosed that the most common route for the transmission of diseases is by contact with surfaces contaminated with infectious droplets from coughs, sneezes or speech produced by contaminated persons; that can remain on surfaces for days 1 , and hands of health-care workers 2 .
With that in mind, assuming that a biofilm can build up within a few hours 3 and considering that these devices are not usually cleaned properly, the SEBS-based TPE materials can become a place for microbial growth, leading to material degradation and ultimately the spread of infections.In this sense, the production of SEBS-based TPE materials with antimicrobial properties is important in order to maintain microbial cells at low counts 4 .
An effective biocide should be able to migrate to the polymer surface, where it can act inhibiting susceptible surface-colonizing cells, and hence delay biofilm accumulation 5 .Antimicrobial additives can be categorized as organic and inorganic.Among organic additives, 5-chloro-2-(2,4dichlorophenoxy) phenol (Triclosan ® ), isothiazolone and zinc pyrithione (ZnPT) have shown efficacy and fast results toward a large range of microorganisms, such as Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Salmonella choleraesuis and Staphylococcus aureus 6 .The industrial use of ZnPT is more common as anti-dandruff in shampoo and soap, and in body wash due to its low water solubility, lack of color and odor when applied in cosmetics [7][8][9] .As antifungal and antibacterial, this substance has been used in paints 10 , textiles 11 and polymeric matrices as polyurethane foam 6 and polyvinyl chloride (PVC) 12 .The biocide action of ZnPT relies on the inhibition of membrane transport and efflux pumps of microorganisms taunting the accumulation of toxic substances inside the cells 13,14 .
The inorganic additive used in the present study was a nanoform of silver (AgNano).One of the mechanisms of silver ion release from polymeric matrix occurs through contact of metallic silver with dissolved oxygen in moisture 15,16 .Once released, AgNano will act on the microbial cells through several modes of action 17 , such as: (a) nanoparticles can bind to proteins of vital enzymes presents in the mycelial 18 and bacterial 19 cell wall, (b) also can damage the cellular structures and biomolecules 20 , (c) cause toxicity by the generation of reactive oxygen species 21 , besides (d) affecting in the molecular and cellular routes of bacteria.Recently, silver nanoparticles have been pointed out as the most innovative and efficient antibacterial form 16 , since they provide better dispersion in the polymeric matrix 22 .However, it is reported that in incorporated polymers, ZnPT leaches from the bulk toward to polymer surface in a faster way than silver.These differences in release rates could make silver additive less readily effective, but with bigger long-term effects than ZnPT Besides the antimicrobial efficacy, in order to be widely applied in consumer goods, antimicrobial TPEs needs to maintain their mechanical properties and color qualities.Toward to this end, this article evaluated the antimicrobial potential of TPE compounds incorporated with zinc pyrithione (ZnPT) and nanosilver (AgNano) in inhibiting the growth of microbial strains.This study describes: (i) the mechanical and color characteristics of metal-incorporated TPE compounds, (ii) the bactericidal performance of incorporated TPE compounds against Gram-positive and Gram-negative bacteria, (iii) the fungicide performance of incorporated TPE compounds against the yeast Candida albicans and filamentous fungi Aspergillus niger and Cladosporium cladosporioides.

Preparation of the compounds
The samples were prepared using a co-rotating double screw extruder (L/D 40 and 16 mm screw diameter (AX Plásticos)), with a temperature profile ranging from 170 °C to 190°C, speed of 300 rpm, feed rate of 1.5 kg h -1 and melt discharge temperature of 200°C.The extrusion parameters were kept constant throughout the tests.Test samples in 2 mm thick plate form were prepared using injection molding machine (Haitian, PL860) at 190°C and an injection pressure of 17 bars.After molding, the test specimens were conditioned at 23 ± 2°C and 50 ± 5% relative humidity for 24 h before testing.A Standard sample with the same composition of the compounds without the inclusion of additives was also prepared.

Mechanical properties
The tensile at break, modulus at 100%, and elongation at break properties of the compounds were analyzed according to ASTM D 412 method.Dumbbell samples types C were tested in universal testing machine EMIC DL 2000 with a 0.1 kN load cell at room temperature.The cross-head speed and gauge length of the apparatus were 500 mm min -1 and 25 mm, respectively.The determination of the hardness Shore A of the compounds were performed according to ASTM D 2240, using 6 mm square samples and a Durometer Bareiss HPE-A, with a reading time of 3 seconds.For all the measurements mentioned above the result was the mean ± standard deviation of five test samples.The density measurement was performed in accordance to ASTM D 792, method A, namely hydrostatic method, with 2 mm square samples.For density the result was the mean ± standard deviation of three test samples.

Color analysis of treated samples
Color fastness was determined by exposing the samples to an Osram Ultra-Vitalux 300W UV lamp for 96 hours, color fastness delta values (∆E) were obtained using a portable colorimeter Delta Color Colorium 2 model.CIELAB color space was used to determine parameters L*, a*, and b*.L* value ranges from 0 (black) to 100 (white); a* value ranges from -80 (green) to 100 (red); and b* value ranges from -80 (blue) to 70 (yellow).∆E was calculated as a global parameter of color alteration according to equation 1.

Analysis of fourier transform infrared spectroscopy
Fourier transformed infrared spectroscopy attenuated total reflection (FTIR-ATR) of surface samples was recorded on a PerkinElmer spectroscope (Frontier).Each spectrum was recorded with a total of 10 scans at a resolution of 4 cm -1 at room temperature.Spectrum software was used for spectra analysis.were obtained from the Andre Tosello Fundation culture collection.The stock cultures were freeze-dried and stored at 20°C.Before use, bacterial cultures were resuscitated and grown in Brain Heart Infusion Broth (Oxoid) and in Potato Dextrose Agar (Oxoid) for fungus overnight at 37°C.Both media were prepared following the manufacturer's protocol.All media were sterilized by autoclaving at 121°C for 15 min.

Antibacterial test
Japan Industrial Standard (JIS) Z 2801 24 was applied to evaluate antibacterial efficiency of samples against S. aureus and E. coli strains.Prior the assay, the TPE specimen (square -50 mm x 50 mm) were disinfected with ethanol and then exposed to ultraviolet (UV) light with the wavelength between 300 and 400 nm for 2 h.The distance between the UV light and the specimen was kept at 10 cm.After that, the samples were placed in a sterile Petri dish followed by an inoculation of 7.6 x 10 6 CFU cm -2 of E. coli and 3.5 x 10 6 CFU cm -2 of S. aureus suspension on the specimen surface, and covered with polyethylene film (as shown in Figure 1).All of them were incubated for 24 h at 35 ± 1°C.The reduction in bacterial population (percentage, %), was calculated from the difference between the number of colony forming units (CFU) per square centimeter at zero hour (initial) and after 24 hours of incubation, equation (2): Where Ef is the reduction in bacterial population (percentage, %), Pi and Pf are, respectively, initial and final bacterial population (colony forming units per square centimeter, CFU cm -2 ).The result is the mean ± standard deviation of three test samples.
Antibacterial effectiveness -R, was validated in accordance with JIS Z 2801, with the equation ( 3): Where Ut is the average of logarithm numbers of viable bacteria after inoculation on control (additive free) sample after 24 h and At is the average logarithm numbers of viable bacteria after inoculation in antibacterial samples after 24 h.To be considered effective, R must be ≥ 2.0.

Fungal growth test
The Brazilian Association of Technical Standards (ABNT) NBR 15275 25 was used to evaluate the compounds' antifungal abilities toward the fungi A. niger, C. albicans and C. cladosporioides.Three test samples (square -25 mm x 25 mm) for each additive concentration were sterilized with ultraviolet (UV) light and then placed in a sterile Petri dish with agar and 100 µL of 1x10 5 -1x10 6 spores mL -1 of fungus suspension were inoculated on the specimen surface.All of them were incubated for seven days at 30 ± 2 °C.The presence of an inhibition zone (after 48 h incubation) and hyphal growth (after seven days incubation) were evaluated with a stereoscopic microscope.The results were expressed in millimeters of diameter of inhibition zone and the percentage of the specimen area covered by the fungus.

Statistical analysis
Statistical analysis of variance (ANOVA) and t-test was applied in tensile strength at break, modulus at 100%, elongation at break, hardness, density and antibacterial results using MYSTAT, student version 12 (Systat Software, Inc., CA, USA).The level of significance was set at 0.05.

Results and Discussion
In the materials industry, the control of standard characteristics such as mechanical properties is important to ensure the product quality.Therefore, the incorporation of an additive in polymeric materials should allow for an improvement in performance without prejudice to the original characteristics.
Table 1 shows the variations in mechanical properties after the incorporation of 1.5% (w/w) of ZnPT and AgNano.There were no significant changes in tensile and elongation at break values of metal-incorporated samples when compared to the Standard compounds (tensile -p=0.47;elongation at break p=0.09).However, the modulus, density and hardness values in ZnPT incorporated compounds presented a significant difference (p<0.05) when compared with Standard and AgNano compounds, which could be due to the polypropylene fraction in masterbatch, present in ZnPT additive, that have higher modulus, density and hardness than the SEBS (Table 1).
In order to verify if the addition of ZnPT and AgNano may cause any color modification or molecular organization difference in TPE incorporated materials, a color fastness and a FTIR-ATR analysis were performed.
As seen in Table 2, with the incorporation of ZnPT a loss of transparency (L*) was noted in TPE samples, which may be related to the high amount of polypropylene present in the masterbatch.After 96 hours of UV light, the sample incorporated with ZnPT turned completely yellow.This color modification is visually perceptible in Figure 2 and proven by improving the yellowness value (b*), that leapt from 1.9 to 22.This modification in color of ZnPT-loaded material can be related to its extreme instability 12 accelerated by UV light resulting in photodegradation 26 .In SEBS compounds, color variation is normal and could be ascribed to degradation of styrene and conjugated bond sequences in the polymer backbone, hence becoming similar to stilbene (which is yellow) 28,29 .In the Standard sample, the global color (∆E) change after 96 h under UV light was of 2.4, results up to 2.0 are visually imperceptible.
At infrared assay, there were no difference between the spectra from Standard and AgNano (Figure 3a and Figure   3b).Differences were only noticed in the ZnPT spectra, this fact may be due to the easy leaching of zinc pyrithione to the surface becoming detectable.In Figure 3c, an increment can be observed in the band intensity between 1300 cm -1 and 800 cm -1 , which could be assigned to an increase in isotactic PP amount from the masterbach 30 .An increase in band intensity at 763 cm -1 was attributed to aromatic =C-H out-of-plane deformation vibrations from the pirithione structure.The weak band at 1536 cm -1 depicts that the coordination of oxygen atoms to Zn is in chelated form, if they were in ionic form, a peak at 1576 cm -1 would be observed 31 The reduction in E. coli population did not differ significantly between ZnPT and AgNano incorporated TPE samples (E. coli -t = 2.60; df = 2.01; p = 0.12).The S. aureus counts were significantly different between ZnPT and AgNano incorporated compounds (S. aureus -t =4.43; df =2.01; p = 0.05), with better effectiveness of ZnPT (Figure 4).A good inhibitory effect towards Gram-negative and Gram-positive bacteria was obtained in both additives tested, even after undergoing typical polymer industrial processing.Moreover, a better bactericide effect of ZnPT compared to AgNano was observed.Dagostin and coworkers 6 also found a better antimicrobial result of ZnPT incorporated into polyurethane foam matrices than Triclosan® and isothiazolone.Previous studies report that pyrithione can penetrate bacterial cytosol besides acting in cell membrane 32 .Once inside the cell, the chelating mechanism of ZnPT promotes a disorder in the cell envelope, owing to the leakage of intracellular components as well as the inhibition of nutrient uptake 7 .
In the present investigation, metal uptake by bacteria was not explored.However, Gram-negative bacteria were most susceptible to the action of both additives while S. aureus was more susceptible to ZnPT.The differential effectiveness observed between the additives tested herein may be related to the way in which these metals operate.Nanosilver is positively charged and bacterial cell wall is negative, which leads to an electrical disturbance; pyrithione is a chelating agent that can sequestrate metal ions which are important in bacterial cell conformation 7 .This property also causes an electrical disorder in bacteria membranes, mainly Gram-negative ones, due to the action on lipopolysaccharide groups, present in the outer membrane of these microorganisms 33 .It was shown that zinc pyrithione leads to decrease in intracellular adenosine triphosphate amount in Gram-negative bacteria species Escherichia coli and Pseudomonas aeruginosa 7 .Also, the treatment with ZnPT induces the production of reactive oxygen species (ROS) 34,35 , which are related to the antibacterial action 36 .Thus, the ROS from zinc ions, such as hydrogen peroxide, would be associated to the toxic effects of ZnPT 37 .
According to Standard JIS Z 2801 24 , in order to accept the material as being antibacterial, the effectiveness value must be equal to or higher than 2. In the present study, the antimicrobial incorporated compounds did not reach the R value of 2.0 in the measurement against the S. aureus population.However, against the E. coli population, the ZnPT compounds presented better antibacterial effectiveness (R = 3.1) than AgNano (R = 2.0) compounds Table 3.
Results of antifungal activity are presented in Figure 5.The ZnPT antifungal activity against A. niger (Figure 5a), C. albicans (Figure 5b) and C. cladosporioides (Figure 5c) are indicated by the appearance of inhibition zones of 7 mm, 2 mm and 6 mm, respectively.There was no inhibition zone in samples containing AgNano; however, these samples did not present fungal growth on their surfaces (Figure 5).
Inhibition zone in sample with ZnPT and other organic antifungal agents have been ascribed to the fact that this component leaches from the polymer to the ambient 5,38,39 .According to Coulthwaite et al. 5 a zone of inhibition means that free molecules of the biocide are on the surface of the polymer and are released in sufficient quantities into the surrounding culture medium where the sensitive organisms  are present.The characteristics of the polymeric matrix related to its water diffusion ability 40,41 , and the structure of the polymer system 42 can influence biocidal action, since it may facilitate the release of the antifungal agent.For example, Silaparson et al. 43 show that it is more difficult for Triclosan® to be diffused through amorphous and rigid thermoplastics (such as polystyrene and polyvinyl chloride) than through soft and crystalline thermoplastics (such as polypropylene and polyethylene).
The antifungal capability of ZnPT is widely known 44 .Chandler and Segel 13 have demonstrated that inhibition of fungi growth by pyrithione is the result of the reduction in membrane transport systems.In addition, the source of ZnPT antifungal efficacy is related to the balance between the acquisition, storage and usage of metals by the fungus 45 .In that case, the metal starvation 34 or high intracellular availability 8,45 will affect the activity of iron-sulfur proteins involved in diverse metabolic functions required for microbial growth 46 .Ion exchange is a slow process and this can enlarge the biocidal effect of metal-based antimicrobials 47 .Silver nanoparticles have been indicated to prevent the growth of C. albicans through modifications in cell dynamics 18,42,48, .
On the basis of the results, we infer that better biocide effect of ZnPT than the AgNano can be related to the different mechanisms that these substances use to reach the polymer surface.Although few studies report the specific mechanisms of ZnPT in polymers, it is known that organic additives as ZnPT, n-octylisothiazolin-one (OIT) and Triclosan® are known as migratory and easily leached from polymer matrices, forming an inhibition zone in fungal assays 23 .
Even with no inhibition zone, the sample incorporated with AgNano presented no mycelia growth on its surface.For silver nanoparticles is reported that to achieve biocide properties in polymer, silver ions must migrate to the polymer surface to get in contact with fungi and bacteria cells, for this, water must permeate the polymer chain to oxidize the silver nanoparticles (as shown in equation 4) 16,40,49,50 .

Conclusions
Overall data obtained in our study showed a good antimicrobial activity of ZnPT-incorporated TPE materials against all the fungus and bacterial species tested.However, the precise mechanisms require further investigation.Samples prepared with AgNano also presented bactericidal action, however with no fungistatic effect.No relevant modification in mechanical properties was observed, showing that there was a small interaction between the additive and polymer.Owing to the color modification in TPE materials containing both additives, the industrial utilization of these incorporated materials will be restricted to applications in which pigmentation would not represent a market problem.

Figure 1 .
Figure 1.Representative scheme of antibacterial assay composed by TPE specimen, bacterial suspension, and the polyethylene film.

Table 1 .Figure 2 .
Figure 2. Color variation comparing Standard TPE samples and TPE incorporated with 1.5% of ZnPT and 1.5% AgNano, before and after 96 hours of UV light exposure.

Figure 4 .
Figure 4. Variation in the number of colony forming units (CFU) of: (a) Escherichia coli and (b) Staphylococcus aureus population between the initial and final time in ZnPT and AgNano TPE incorporated samples.

Table 2 .
Color analysis of Standard samples and TPE incorporated with 1.5% of ZnPT and 1.5% of AgNano before and after 96 h of UV light exposure.

Table 3 .
Percentage of bacterial population reduction after 24 h of incubation and (R) antibacterial effectiveness values.