Antimicrobial and mechanical properties of functionalized textile by nanoarchitectured photoinduced Ag@polymer coating

Photoinduced synthesis of the Ag@polymer coating

Specific monomers poly(ethylene glycol) 600 diacrylate (PEG600DA) and pentaerythritol triacrylate monomer (PETIA) used as comonomer (PEG600DA/PETIA with a 1:1 weight ratio) were mixed under magnetic stirring with diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (0.5 wt %) and the metal precursor AgNO3 (3 wt % and 5 wt %) for 1 h. After complete dissolution, this photosensitive formulation was applied to a cotton textile with an abyko-drive applicator (calibrated bar coater). The resulting smooth sample was then irradiated under UV light (600 mW/cm2) to obtain the final Ag@polymer textile material, with a coating thickness of 100 µm, measured with a Mitutoyo digital micrometer. Figure 1 shows images of the as-synthesized samples with and without AgNPs for the PEG600DA polymer and PEG600DA/PETIA copolymer matrixes coated onto cotton fabric.

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Figure 1: Cotton textile coated with Ag@PEG600DA (a), Ag@PEG600DA/PETIA (1:1) (b) and inserted images of PEG600DA and PEG600DA/PETIA polymers without AgNPs.

Optical and morphological properties

UV–vis spectroscopy was initially carried out on glass-coated Ag@PEG600DA or Ag@PEG600DA/PETIA samples, in order to follow the AgNP synthesis for different exposure times. The obtained spectra are presented in Figure 2, as well as images of the coatings taken for different exposure times. The characteristic surface plasmon resonance band of AgNPs for both coatings is observed around 430 nm and increases with exposure time, which indicates an increase in NP concentration. Since the absorption peak is 426 nm for the Ag@PEG600DA/PETIA coating, the particles are expected to be slightly smaller than those of the Ag@PEG600DA film. This can be explained by the fact that the PETIA comonomer is a triacrylate monomer, which increases the amount of cross-linked polymer chains compared to the diacrylate monomer (PEG600DA). Moreover, particle stabilization is ensured through interactions with the CO• groups resulting from photoinitiator decomposition [49]. In addition, AgNP interactions with the polymer’s non-bonding doublets of oxygen atoms create steric hindrance and avoid their aggregation. The coating, colorless before irradiation, becomes progressively pale yellow, orange, then finally brown. Due to the strong increase of AgNPs, the measurement system reaches the detection limit (OD > 3) after 15 seconds of exposure in the case of the Ag@PEG600DA coating (Figure 2a). As can be seen in Figure 2, AgNP synthesis is much faster in Ag@PEG600DA coatings than in the case of the PEG600DA/PETIA matrix. Indeed, as a diacrylate, PEG600DA offers less cross-linking sites than PETIA, which promotes nanoparticle formation and coalescence during UV exposure. When comparing the absorbance spectra for both coatings after 15 s irradiation, the full-widths at half maximum (FMWH) are calculated to be 134 and 131 nm, for Ag@PEG600DA and Ag@PEG600DA/PETIA, respectively (Figure 2c). Consequently, the nanoparticle size dispersion can be expected to be relatively similar for both coatings with a slightly smaller size dispersion for the Ag@PEG600DA/PETIA coating. Further exposure to UV light eventually turns the surface of the coating into a metallic silver layer with remarkable mirror-like properties (Figure 2).

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Figure 2: UV–vis spectroscopy monitoring of 100 µm-thick Ag@PEG600DA (a) and Ag@PEG600DA/PETIA (b) coatings, with images taken after increasing exposure times.

The optical properties exhibited by the Ag@polymer-functionalized textiles (Figure 1a and 1b) were then investigated via reflectance measurements. As shown in Figure 3, the Ag@PEG600DA coating is more reflective than the Ag@PEG600DA/PETIA sample (97% and 68% at 650 nm, respectively).

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Figure 3: Reflectance measurements and corresponding images of Ag@polymer (PEG600DA and PEG600DA/PETIA) functionalized textiles.

Particle assembly and distribution, as well as the final thickness of the metallic layer, account for this difference in reflectivity. Scanning electron microscopy (SEM) carried out on the surface of functionalized textiles revealed the homogenous distribution of AgNPs, with average sizes of 62 ± 2 nm and 58 ± 1 nm for the Ag@PEG600DA (Figure 4a) and Ag@PEG600DA/PETIA (Figure 4b) coatings, respectively. The AgNP size dispersion is also slightly higher in the case of Ag@PEG600DA. Both results are coherent with the peak and FMWH values calculated from the absorbance spectra (Figure 2c).

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Figure 4: SEM of the 100 µm-thick Ag@PEG600DA (a) and Ag@PEG600DA/PETIA (b) coatings.

Transmission electron microscopy (TEM) cross sections of 100 µm-thick samples were then taken for both functionalized textiles (Figure 5). In both cases, a gradient distribution of AgNPs was observed after UV synthesis, with concentrations increasing from the depth to the surface of the sample. The top metal layer thickness of the Ag@PEG600DA film reached 250 nm (Figure 5a), compared to 90 nm for the Ag@PEG600DA/PETIA coating (Figure 5b). AgNPs trapped inside the polymers have sizes of 10–30 nm in the case of PEG600DA, while barely reaching 10 nm for PEG600DA/PETIA. Since the PETIA monomer has three reactive groups, the synthesized polymer network is denser than in the case of PEG600DA diacrylate monomer, which ultimately limits not only the increase in particle size but also the migration/diffusion of silver ions and NPs towards the surface. This affects the final thickness of the top metal layer, the depth of the concentration gradient and the final size of the AgNPs (see Figure 5). As a result, high reflectance is linked to the formation of a thick and compact top metal layer, which is indeed the case for the Ag@PEG600DA coating.

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Figure 5: TEM cross sections of the Ag@PEG600DA (a) and the Ag@PEG600DA/PETIA (b) with the selected area electron diffraction (SAED) patterns.

Moreover, the selected area electron diffraction (SAED) patterns obtained during TEM analysis proved the high crystallinity of the synthesized NPs and the diffraction rings which indicate the polycrystalline nature of the AgNPs have been indexed to the (111), (200), (220) and (311) planes and correspond only to the face-centered cubic (fcc) crystal structure of metallic silver (JCPDS, No. 04-0783) (Figure 5). This result is of great importance because it confirms the complete photoreduction of the precursor and the absence of any oxidized form of silver in the Ag@polymer coating.

Mechanical properties Flexibility and adhesion

Functionalization of the textiles with the Ag@polymer does not significantly affect the flexibility of the original cotton substrate (Figure 6). In the case of Ag@PEG600DA coatings, the samples do not show any cracking or loss of adhesion at the coating/textile interface following a bending-type deformation for example; the metal surface remains visually intact. However, the use of the PEG600DA/PETIA polymer matrix leads to an increase in rigidity, linked to the trifunctional monomer architecture, which favors polymer chain cross-linking. As such, the Ag@PEG600DA/PETIA coatings are rendered more sensitive to this type of deformation, when pressure is applied to the functionalized side of the fabric.

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Figure 6: Images of flexible Ag@PEG600DA (a) and Ag@PEG600DA/PETIA (b) samples; inserted post-scratch tests.

In order to assess the adhesion properties of the Ag@polymer coatings on the cotton fabric, scratch tests were carried out on the smooth surfaces of each type of coating, i.e., Ag@PEG600DA and Ag@PEG600DA/PETIA. In accordance with the NF EN ISO 2409 standard regarding paints and varnishes, a scratch test was performed by first creating a grid (1 mm evenly spaced incisions) in the surface of the coating. Then a standard-approved 8.75 N adhesive tape was applied and taken off. Depending on the amount of coating remaining after the scratch test, a value of 0 to 5 is attributed, 0 corresponding to perfect adhesion. Figure 6 shows the results for both the Ag@PEG600DA (Figure 6a) and Ag@PEG600DA/PETIA (Figure 6b) coatings.

In both cases, excellent adhesion was observed (value of 0), which can be explained by the remarkable textile wetting properties of the photosensitive formulation. The choice of polymer matrix effectively dictates the observed wettability and adhesion to the chosen substrate. The adhesion properties can also be explained by the innovative structure of the depth-wise gradient distribution of AgNPs in the metallic coatings. Indeed, as the AgNPs and the polymer matrix are synthesized simultaneously, the polymer chains are able to insinuate themselves between the particles, effectively anchoring them to the rest of the coating.

Abrasion

The longevity of the functionalized textiles was next assessed by testing the abrasion resistance of the coatings, with a particular emphasis on the optical properties of the top metal layer. Circular samples (3.8 cm in diameter) underwent the Martindale test (ISO 12947 standard), which imitates the natural deterioration of a material with time. Throughout the test, the samples sustain a fixed number of abrasion cycles, during which an abrasive material (wool here) is frictioned linearly on the surface with a force of 12 N, following a Lissajous curves pattern. Figure 7a shows the Ag@PEG600DA-functionalized textile before and after 500 and 1000 abrasion cycles. The total reflectance spectra and its diffuse component are also presented (Figure 7b). Before abrasion, the Ag@PEG600DA coating is highly reflective, with a total reflectance value of 97% at 700 nm, of which 52% comes from the diffuse component. The applied friction seems to polish the surface, increasing its mirror-like aspect. After 500 cycles, the total reflectance increased significantly between 400 and 580 nm, while dropping slightly (10%) in the 580–1000 nm range. The diffuse reflectance effectively decreased from 52 to 20% at 700 nm.

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Figure 7: Influence of 500 and 1000 abrasion cycles on the surface of an Ag@PEG600DA coating (a) and on the total and diffuse reflectance (b). Associated SEM surface images (c).

Indeed, friction causes a localized rise in temperature between particles, which promotes a reduction in surface roughness, as can be seen in the SEM images (Figure 7c). Consequently, the diffuse reflectivity drops in favor of the specular reflectance. The particles are no longer simply juxtaposed but form a continuous silver layer, especially after 1000 friction cycles. The characteristic silver plasmon band, identifiable between 380 and 500 nm on the pre-abrasion reflectance spectra, has effectively disappeared and the resulting curve resembles that of bulk silver.

Regarding the Ag@PEG600DA/PETIA coating, surface images and reflectance spectra of the samples pre- and post-abrasion are shown in Figure 8a and 8b. Interestingly, the total reflectance is lower than that of the Ag@PEG600DA coating (68% compared to 95% at 600 nm). After 500 abrasion cycles, the metallic layer was damaged, even more so after 1000 cycles, as can be seen in Figure 8a. This deterioration causes a significant drop in reflectance, mainly diffuse. The underlying metallic layer appears brown and exhibits an absorption band at 430 nm, linked to the plasmon resonance of AgNPs (Figure 8c), trapped inside the polymer matrix.

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Figure 8: Influence of 500 and 1000 abrasion cycles on the surface of an Ag@PEG600DA/PETIA coating (1:1) (a) and on the total and diffuse reflectance (b). Associated SEM surface images (c).

To better understand this sudden decrease in reflectance, the surface was characterized by SEM (Figure 8c). Before the abrasion test, the surface was completely covered with homogenously distributed and almost monodispersed nanoparticles (25–50 nm). Post-abrasion, only a few nanoparticles remain visible, which corroborates the resurgence of the AgNP plasmon band.

Unsurprisingly, the nature of the polymer matrix affects the resistance of the final material to abrasion. The rigid nature of the PETIA monomer could account for the low abrasion resistance and decrease in reflectance of the Ag@PEG600DA/PETIA coating, compared to that of Ag@PEG600DA. As previously explained, the PETIA monomer prevents the easy diffusion of silver ions towards the surface, resulting in the observed thin top metal layer (90 nm) of the Ag@PEG600DA/PETIA film, as confirmed in the TEM images (Figure 5). The metal layer is not thick enough to withstand the damage inflicted by the abrasion process.

Rheological properties

Potential significant changes in the viscoelastic properties of the functionalized material were suspected of being induced by the addition of AgNPs in the polymer matrix, which was assayed by rheometry measurements. This analysis confirms that coatings with and without Ag exhibit a viscoelastic behavior (Figure 9a–d). The storage modulus G’ is not significantly affected by the polymer matrix, but changes with the presence of Ag in the coating. Both storage moduli of uncoated and PEG600DA/PETIA-coated textiles are about 1 MPa (1.1 ± 0.1 MPa and 1.0 ± 0.1 MPa, respectively), whereas they are 0.3 ± 0.1 MPa and 0.6 ± 0.1 MPa for Ag@PEG600DA/PETIA 3 and 5 wt % coatings, respectively. This decrease in the elastic component may be due to the presence of AgNPs in the polymer matrix. In addition, their general viscoelastic behaviors differ: PEG600DA/PETIA-coated textile has a predominant elastic behavior in the tested frequency range (G’/G” > 1), while Ag@PEG600DA (3 and 5 wt %) coatings display similar levels in viscous and elastic behaviors in the same frequency range (Figure 9b). This appears to result from the decrease in the elastic component rather than from a viscosity modification, which may be due to the textile. These changes are not expected to negatively impact the use of the functionalized textile; rather, the improvement of the elastic properties induced by addition of Ag may be beneficial for elongation properties without any permanent, undesirable deformation.

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Figure 9: Rheological characteristics of uncoated textile (a), PEG600DA/PETIA (b) and Ag@PEG600DA/PETIA (3 and 5 wt %) (c and d, respectively)-coated textiles in the 0.5–5 Hz frequency range.

Antimicrobial properties

The antimicrobial activity of the functionalized textiles was quantified via a liquid diffusion assay, while a plate diffusion assay was used to visualize the microbial growth inhibition. Preliminary tests showed that the Ag@PEG600DA-coated textile does not stay flat during immersion into microbial suspensions, despite being glued to the glass dishes. For this reason, antimicrobial characterizations were only carried out on Ag@PEG600DA/PETIA loaded with 3 wt % and 5 wt % of AgNO3 in order to observe the impact of the silver concentration on the E. coli bacteria and the C. albicans fungus.

Liquid diffusion assay

Attenuance measurements (OD600, i.e., OD at 600 nm), performed on microbial suspensions in contact with the functionalized textiles after 1, 3, 6, and 24 h of immersion, are plotted in Figure 10. For both E. coli and C. albicans microorganisms, control cultures (without any textile or with silver-free, functionalized textile) show an increase of CFU/mL with sample immersion time, in agreement with growth rates expected in the M63G medium [50]. Contrarily, Ag@PEG600DA/PETIA samples indicate a decrease of CFU/mL values, thus reflecting a lower cell concentration in this suspension than in those cultivated without the sample. This effect can be attributed to the presence of silver, since microbial growth is also observed in the case of the control material (cotton coated with the Ag-free PEG600DA/PETIA polymer matrix). Nevertheless, this growth is lesser than that of the control culture for the C. albicans microorganism, suggesting that the polymer matrix releases one or more compounds capable of inhibiting fungal growth. In a previous study [51], we demonstrated that the polymer matrix is capable of releasing several organic compounds due to photodegradation, including formic acid. This weak acid has been shown to exhibit antifungal properties. Lastauskiené et al. [52] showed that formic acid can induce programmed cell death in several Candida species, including C. albicans pathogens. In vitro testing revealed MIC values of 1.22 mg/mL (26.5 mM) on C. albicans thus suggesting a mild but significant fungicidal effect. This is similar to the efficacy of acetic acid and can be classified as slightly fungicidal [53,54].

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Figure 10: Colony forming units (CFU) per mL of suspension calculated from OD600nm measurements of E. coli (a) or C. albicans (b) cultures after 0, 1, 3, 6, or 24 hours contact with the samples; inserted image of immersed functionalized textile. For both microorganisms, 3 wt % and 5 wt % conditions are significantly different (p-value < 0.05) from the controls whatever the time (except at time = 0, i.e., inoculation suspension, and at 1 h for C. albicans). The 3 wt % and 5 wt % conditions are not significantly different from each other. Controls are not significantly different from each other, except at 24 h of culture for C. albicans.

The inhibition rate of microorganism growth was determined from the OD600nm measurements, for suspensions in contact with 3 wt % and 5 wt % AgNO3-loaded samples. The results are presented in Figure 11a for E. coli and remarkably a total inhibition of growth was reached within 24 hours. The silver concentration has no significant impact on antibacterial activity, which indicates that a mere 3 wt % Ag-loaded sample is sufficient to obtain complete elimination of bacteria. The released silver quantity was determined by ICP analysis and has been correlated with the bacteria growth inhibition rate (Figure 11b). For equal immersion times in microbial suspensions, the amount of silver released is more important for the 5 wt %-loaded samples, but in both cases (3 wt % and 5 wt %) a linear evolution is observed between the growth inhibition and the released silver content. A complete inhibition was achieved for a silver quantity of 15 µg/g released in liquid media. As demonstrated by Hsueh et al. [55] AgNPs can be considered as containers continuously releasing silver ions, thus indirectly avoiding planktonic bacteria proliferation by the direct action of silver ions. The capacity of 3 wt % and 5 wt %-loaded textiles to inhibit the growth of E. coli is thought to be related to this mechanism.

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Figure 11: Growth inhibition of bacteria (E. coli) for different sample immersion times (a), and for the corresponding amount of released silver (b).

Concerning the fungus, similarly to bacteria, the amount of loaded silver does not have a significant impact on the antifungal activity (Figure 12a). Nevertheless, the antifungal activity of the Ag@PEG600DA/PETIA coating (3 and 5 wt %) results in total inhibition of fungal growth within 6 hours. The silver amount, released immediately after sample immersion, is sufficient in blocking C. albicans growth (Figure 12b). It should be noted that the required silver quantity to effectively inhibit microorganism growth is lower for C. albicans fungus (≈10 µg/g) than for E. coli bacteria (≈15 µg/g), which enables a faster growth inhibition of the fungi. In addition, the response of C. albicans to doses lower than the maximum effective dose is not linear, unlike that of E. coli bacteria, confirming a higher sensitivity to silver, even in the presence of low amounts of released silver (90% inhibition rate for approximately 2.5 µg/g released silver).

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Figure 12: Growth inhibition of fungus (C. albicans) for different sample immersion times (a), and for the corresponding amount of released silver (b).

This effect, more pronounced for C. albicans than E. coli has also been observed by Akinsiku et al. [56], who determined that the sensitivity to silver varies by a factor of 5 between clinical isolates of E. coli and C. albicans. The mechanisms leading to this difference do not seem to have been elucidated so far. However, some hypotheses can be put forward. In the present study, the antimicrobial activity of E. coli and C. albicans is only due to the action of silver ions released by AgNPs [20]. Ionic silver first perforates the cell wall, enters the microorganism and eventually induces cell apoptosis. Indeed, once inside the cell, silver interacts with compounds generating reactive oxygen species (ROS) or blocks DNA replication and protein action (enzymes), thereby leading to cell death. The increased growth inhibition observed for C. albicans compared to E. coli could be explained in part by the difference in kinetics of silver penetration into microorganisms, possibly due to the significant difference in the external cell wall composition. Peptidoglycan is a component of bacterial cell wall that was suggested as the main target of silver [57]. In E. coli and the other Gram-negative bacteria, the peptidoglycan layer is protected by an outer membrane, which may limit the interactions with silver in comparison to Gram-positive bacteria, that exhibit a peptidoglycan layer directly in contact with the surrounding medium. Similar to Gram-positive bacteria, C. albicans exhibits an external cell wall, in direct contact with the surrounding medium, which also contains N-acetylglucosamine, one of the two main components of peptidoglycan, and may therefore be a target for silver [58]. In addition, silver has been shown to target several fatty acids of the membrane of C. albicans, which is thought to affect the hyphal morphogenesis of yeast, a vital process of the cell [59]. Moreover, C. albicans has been reported as particularly sensitive to ROS, which may be one of the reasons for its higher sensitivity to silver than other microorganisms [60].

The culture medium could also impact microorganism susceptibility to silver ions. For comparison purposes, the same medium was chosen for both bacteria and fungus. M63G medium has been preferred since it does not reduce silver antimicrobial activity contrary to media containing components such as Cl− or other biomolecules, which interact with ionic silver [20]. In addition, it provides nutrient conditions more likely to mimic real conditions of use of the functionalized textiles developed in this study than rich nutritive media that supply microorganisms with an unusual, easy access to large amounts of nutrients. M63G is known to offer suitable conditions for E. coli growth [41] and it was shown to also allow C. albicans growth in a preliminary study (data not shown). Of course, E. coli and C. albicans growths are limited in M63G compared to that in the LB and Sabouraud nutritive media that are perfectly adapted to these microorganisms, respectively. In addition, physiological changes induced by a different access to nutrient and other compounts are expected to impact the microbial susceptibility to antimicrobials including silver [61]. They may lead to reduced, increased, or even unaltered minimal inhibitory concentrations, but the exact changes are unknown. In this study, finally, even if a direct impact of the M63G environment on microorganism susceptibility is plausible, it is not currently possible to confirm that the culture medium played a significant role in the observed difference of inhibition kinetics.

Plate diffusion assay

Functionalized and uncoated textiles were placed on a microbial film and the resulting microbial growth around the samples was examined to identify the potential presence of a growth inhibition zone. This study provides visual information from which quantitative trends could be extracted. The results obtained after 24 h incubation, at 30 °C for E. coli and 37 °C for C. albicans, are presented in Figure 13 and Table 1. For E. coli, a growth inhibition zone is clearly noticeable around the silver-loaded samples and increases with silver concentration. The control sample (cotton coated with the PEG600DA/PETIA polymer matrix) exhibits a slight inhibition zone. Concerning fungus, the control sample shows little change but an inhibition zone was clearly observed around the silver-loaded samples. The antimicrobial activities on E. coli and C. albicans, confirmed by quantitative data reported in Table 1, can both be attributed to silver ions released from the functionalized materials, as explained above for assays in liquid. As experiment replications show important variation, no conclusive information could be extracted from the differences noticed between E. coli and C. albicans or between 3 wt % and 5 wt % Ag-loaded samples. Finally, the plate diffusion assay demonstrates lower antimicrobial activity than the liquid diffusion tests. This can be first explained by silver’s limited diffusion through agar, compared to liquid media. In addition, the sample surface contribution is limited to its immediate vicinity in the case of the plate diffusion test, whereas the whole surface contributes in liquid tests. In other words, regarding the surrounding medium, the functionalized textile can protect its surface and immediate vicinity without unwished release in the environment, or provide a protective inhibitory effect in the whole liquid surrounding.

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Figure 13: Plate diffusion test for the observation of growth inhibition zones of E. coli and C. albicans after 24 h incubation.

Table 1: Growth inhibition zone sizes for E. coli and C. albicans. Both total (i.e., sample and inhibition zones together) and sample areas are measured by image analysis with the diameter of the Petri dish as the distance reference). Neat inhibition area was defined as the difference between total and sample areas. The 3 wt % and 5 wt % conditions are significantly different from the controls (p-value < 0.05) for both microorganisms and for E. coli only, respectively. The 3 wt % and 5 wt % conditions are not significantly different from each other. Samples are not significantly different from each other.

  Surface (mm2) Polymer 3 wt % 5 wt % microorganism mean sample surface 112 ± 7 E .coli sample + inhibition zone 140 ± 19 218 ± 40 185 ± 25 neat inhibition zone 31 ± 15 109 ± 40 75 ± 25 C. albicans total inhibition zone 115 ± 17 133 ± 10 179 ± 26 neat inhibition zone 8 ± 19 16 ± 14 68 ± 0

The antimicrobial activity of the Ag@PEG600DA/PETIA coating was thus confirmed for both E. coli and C. albicans. As observed during the abrasion tests, this sample exhibits a thinner silver top layer (90 nm) at the sample surface than the Ag@PEG600DA coating (250 nm), suggesting the latter would present superior antimicrobial activity due to the increased amount of available silver.

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