Tailoring of a Dual-active Antibacterial Coating for Polylactic Acid Fibres

The aim of this research was to develop a new, dual-active antibacterial coating for fi bres made from polylactic acid and, consequently, to increase the possibility of their use for a variety of technical textiles. The process of fi nishing was performed on a knitted fabric in three stages by applying silver chloride and 3-(trimethoxysilyl)propyldimethyltetradecyl ammonium chloride, which provided simultaneous dual antibacterial activity based on the mechanisms of controlled release and bio-barrier formation. The presence of the coating on the fi bres was confi rmed by scanning electron microscopy with energy–dispersive X–ray spectroscopy, inductively coupled plasma mass spectroscopy and a test with bromophenol blue. The results of microbiological tests confi rmed the excellent bactericidal activity of the coating, with a 99.99% reduction in the gram-positive bacteria Staphylococcus aureus and the gram-negative bacteria Escherichia coli. Application of the coating reduced the lightness and increased the yellowing of the fi bres from polylactic acid, which were disadvantages.


Izvleček
Namen raziskave je bil razviti novo protibakterijsko apreturo z dvojno aktivnostjo na vlaknih iz polimlečne kisline in s tem povečati možnost njihove uporabe za različne tehnične tekstilije.Apreturni postopek je bil izveden na pletivu v treh stopnjah z nanosom srebrovega klorida in 3-(trimetoksisilil)-propildimetiltetradecilamonijevega klorida (Si-QAC), ki sta zagotovila dvojno hkratno protibakterijsko aktivnost po mehanizmih nadzorovane sprostitve in tvorbe biobariere.Prisotnost apreture na vlaknih smo potrdili z vrstično elektronsko mikroskopijo z energijsko-disperzijsko spektroskopijo rentgenskih žarkov, masno spektroskopijo z induktivno sklopljeno plazmo ter testom z bromofenol modrim reagentom.Rezultati mikrobioloških testov so potrdili baktericidno delovanje apreture z 99,99-odstotno bakterijsko redukcijo za grampozitivno bakterijsko vrsto Staphylococcus aureus in gramnegativno bakterijsko vrsto Escherichia coli.Nanos apreture je zmanjšal belino in povečal porumenitev vlaken iz PLA, kar je njena pomanjkljivost.Ključne besede: vlakna iz polimlečne kisline, protibakterijska apretura, dvojna protimikrobna aktivnost, srebro, trialkoksisilan s kvarterno amonijevo skupino properties, PLA bres have become one of the most promising alternatives for polymer bres derived from petroleum [1−4].eir use has already been extended to the eld of technical textiles.ey are particularly suitable for single-use products, such as sanitary materials, speci c medical textiles and textile lters [5,6].For this type of textile products, a functional antimicrobial protection provides high added value and is therefore of great technological and economical importance.When preparing the antimicrobial protection for textiles, two groups of antimicrobial agents can generally be used, which vary according to the mechanism of antimicrobial activity [7−9].e rst group comprises antimicrobial agents that act by the mechanism of controlled release.Because most of these agents are bound to the bres with physical forces, they can be slowly released from the bres into the surrounding area in the presence of a sucient amount of moisture where they wholly destroy or inhibit the growth of microorganisms.An important weakness of physically bonded agents is the lowering of their concentration in the bres due to leaching, eventually falling below the limit of eciency.e second group includes agents that operate on the principle of bio-barrier formation.In this case, agents are chemically bonded to the textilebres where they create a biological obstacle for the microorganisms that come in contact with thebres.Because they do not leach from the bres, their concentration does not change with time.However, chemical bonding cannot ensure the permanent antimicrobial activity of agents because the settling of dead microorganisms on the bio-barrier can greatly reduce or even eliminate their e ectiveness.To eliminate the problems related to the mode of antimicrobial action and thereby to increase the e ectiveness of antimicrobial protection, the research work in recent years has been oriented towards nding new approaches for the preparation of antimicrobial coating preparations.One such approach is the tailoring of antimicrobial coatings to obtain dual activity.To this end, combinations have been used, consisting of antimicrobial agents that operate by the mechanism of controlled release and those that form a biological barrier [10−15].ese results have encouraged us to develop a novel, dual-active antimicrobial coating for the textile bres from PLA, which would also be appropriate for chemical modi cation of other hydrophobic and low-adhesive bres.e previous research on the PLA bres or PLA lms has been mostly directed towards the preparation of monocomponent antimicrobial coatings exhibiting either the controlled release of essential oils, antibiotics, silver nanoparticles, or zinc oxide, [16−20], or the formation of the chitosan bio-barrier [21,22].For the preparation of an antimicrobial coating with dual antimicrobial activity, we chose silver as a representative agent for the controlled-release mechanism of action, and an organic-inorganic hybrid sol-gel precursor with a quaternary ammonium functional group as a representative agent with the bio-barrier-forming antimicrobial mechanism.Assuming that because of their morphological and chemical structure, PLA bres have insu cient adhesive ability for silver, we decided to create a silica matrix on the bre surface to increase their adsorption capacity.In fact, it was found that the silica matrix signi cantly increases the concentration of the adsorbed silver, resulting in more uniform distribution as well as the reduced size of silver particles [23,24].us, we have developed a three-stage nishing procedure that includes the following stages: (i) the creation of a silica matrix, (ii) the in situ synthesis of AgCl and (iii) the creation of a bio-barrier.An important objective of our study was to determine the e ectiveness of the antibacterial coating as well as to determine the in uence of the coating on the colour of PLA bres, which is an important feature of the product from an aesthetic point of view.

Materials
We used a double we knitted fabric in 1 1 rib structure made from 100% PLA multi lament (10 capillaries) with linear density of 11.1 dtex, breaking force of 40.7 N and breaking elongation of 31.8%.e PLA multi lament was kindly supplied by Applied Polymer Innovations BV (Emmen, Netherlands).e thickness and weight of the fabrics were 5.2 mm and 428.3 g/m 2 , respectively.e commercial products 3-(trimethoxysilyl)-propyldimethyltetradecyl ammonium chloride (Si-QAC), namely, Sanitized T 99-19 (Sanitized, Switzerland) and silver chloride (AgCl), prepared from silver nitrate (AgNO 3 ; Sigma-Aldrich) and sodium chloride (NaCl; Carlo Erba) were selected as the antimicrobial agents.To create a silica matrix, we used the commercial for Polylactic Acid Fibres Tekstilec, 2016, 59(4), 289-297 product iSys MTX (CHT, Germany), which is a water-borne Si-and Zr-based sol-gel precursor (RV), in combination with Kollasol CDO (CHT, Germany), which is an anti-foaming and wetting agent.All solutions were prepared in bidistilled water.

Finishing
Chemical modi cation of the PLA samples was accomplished in a three-stage nishing procedure.In the rst stage (1S), the samples were immersed in 100.0 g/L RV and 10.0 g/L Kollasol CDO for 10 minutes at room temperature, followed by wringing via squeezing on a two-roll padder with a pick-up of 80 ± 5%, and drying in an oven at a temperature of 110 °C for 5 minutes.A er drying, the samples were le for 7 days at standard atmospheric conditions to allow complete crosslinking of iSys MTX.In the second stage (2S), the in situ synthesis of AgCl on the RV-treated samples was performed in the Gyrowash 815 (James Heal, UK) apparatus at room temperature, with occasional stirring, as follows: the specimens were immersed for 10 minutes in a 0.5 mM solution of AgNO 3 with a liquor ratio of 50:1 and then subsequently immersed for 10 minutes in a NaCl solution of the same concentration and liquor ratio.e procedure was repeated twice.en, the samples were washed in bidistilled water to remove the excess chemicals and dried at room temperature.In the third stage (3S), Si-QAC was applied to the samples by the pad-dry-cure method, with full immersion of the samples in a 100 g/L solution of Si-QAC, followed by squeezing on a two-roll padder with a pick-up of 80 ± 5% and drying in an oven at temperature of 110 °C for 5 minutes.A er drying, the samples were le for 7 days at standard atmospheric conditions to allow complete crosslinking of iSys MTX.
e three-stage nishing procedure is schematically presented in Figure 1.For comparison, the two-and one-step application procedures were also performed with the same antimicrobial agents under the same conditions as in the three-stage procedure.Accordingly, in the twostep procedure, RV was applied as in 1S, followed by the application of AgCl as in 2S.In the one-stage procedure, AgCl and Si-QAC were applied to the PLA samples as in 2S and 3S, respectively.e sample codes in relation to the application procedures are summarized in Table 1.

Fourier transform infrared spectroscopy (FT-IR)
FT-IR spectra were obtained on the Spectrum GX (Perkin -Elmer, UK) spectrophotometer equipped with a diamond cell.e spectra were recorded over the range of 4,000-600 cm -1 , with a resolution of 4 cm -1 and an average set of 32 spectra per sample.Before the measurements, the samples were dried to a constant weight.

Scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDS)
SEM was conducted on the Jeol JMS 6060LV and Jeol JSM 5610 microscopes, equipped with an Oxford-Link ISIS 300 EDS system with an ultra-thin window Si (Li) detector.Prior to performing the SEM and EDS analyses, we applied a 20-nm-thick carbon layer to each fabric sample to ensure su cient electrical conductivity and to avoid charging e ects.SEM micrographs were recorded using the secondary electron (SE) and backscattered electron (BSE) imaging modes.e BSE compositional contrast (Zcontrast) was applied to accentuate the di erences between the added particles and the bre matrix.Two parallel assessments were performed for each coated fabric sample, and the corresponding atomic concentration was reported as the mean value.

Inductively coupled plasma mass spectroscopy (ICP-MS)
ICP-MS was performed on the Perkin Elmer SCI-ED Elan DRC spectrophotometer.e fabric samples (0.5 g) were prepared in a Milestone microwave by acid decomposition, using 65% HNO 3 and 30% H 2 O 2 .ree measurements were taken for each sample, and the Ag concentrations were reported as the mean values.

Test with bromophenol blue (BPB) reagent
Qualitative determinations of Si-QAC on the coated samples were performed by using the BPB reagent, which is an alkaline dilution of the sodium salt 3'-3"-5'-5"-tetrabromophenolsulfonphtalein.e test was based on the formation of a complex between the BPB reagent anion and the quaternary ammonium group of Si-QAC on the surface of the fabric.Due to the formation of the complex, the samples were coloured blue.For the BPB test, 1 g of sample was immersed in 50 mL of 0.005% BPB reagent diluted in water and stirred vigorously for 20 min.e samples were subsequently removed from the BPB solution, thoroughly rinsed with cold tap water and dried at room temperature.e intensity of the blue coloration on the samples was assessed by the reectance, R, measurements of the samples on the Datacolor Spectra ash SF 600 spectrophotometer, using D 65/10° light.Before these measurements, the samples were conditioned at relative humidity of 65 ± 2% and temperature of 20 ± 1 °C for 24 hours.For each sample, ten measurements of the R value were obtained, and the corresponding K/S values were calculated according to the Kubelka-Munk equation: where K/S is the ratio of the coe cient of light absorption (K) to the coe cient of light scattering (S), and R the re ectance at the maximum absorption wavelength, determined at 610 nm.A erwards, the mean K/S value was determined.
Antibacterial activity e antibacterial activity was examined by a modied AATCC standard method 100-1999 for the bacteria Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 6538).In aseptic conditions, the sample was placed into a 250-mL container and inoculated with 400 µL of a nutrient broth culture containing 1−2 × 10 5 colony-forming units of bacteria.A er incubation at 37 °C for 24 hours, the bacteria were eluted from the swatches by shaking in 100 mL of neutralizing solution for 1 minute.After preparing serial dilutions in sterilised water, the suspensions were plated on nutrient agar and incubated at 37 °C for 24 hours.e number of bacteria was counted, and the reduction of bacteria, R, was calculated as follows: where A is the number of bacteria recovered from the inoculated swatch of the cotton sample in the jar incubated for the desired contact period (24 hours), and B is the number of bacteria recovered from the inoculated swatch of the cotton sample in the jar immediately a er inoculation (at "0" contact time).For each fabric sample, four parallel assessments were performed and the mean value was determined.for Polylactic Acid Fibres Tekstilec, 2016, 59(4), 289-297 Whiteness and yellowing index e whiteness of the samples was determined on the basis of the measurements of the CIE colour values using the Spectra ash 600 PLUS-CT spectrophotometer (Datacolor, Switzerland).e measurements were performed at the following conditions: 20 mm size of the measuring aperture, standard light D65 and T = 6500 K, using D65/10° with an excluded specular as an observer.
e whiteness, W 10 , was calculated from the following equation: where X, Y and Z represent the values in the CIE colour space.Before these measurements, the samples were conditioned at relative humidity of 65 ± 2% and temperature of 20 ± 1 °C for 24 hours.For each sample, ten measurements of Y 10 and YI were obtained and the mean values were calculated.

Results and discussion
e ATR spectra of PLA-N and PLA-RV-Ag-SiQAC samples (Figure 2) show that the application of the antimicrobial coating caused chemical changes in the PLA bres, resulting in the increase of the intensity of the absorption peaks at wavenumbers 2927 and 2856 cm -1 , which are characteristic of asymmetric and symmetric stretching of the C-H bond in aliphatic alkyl groups [25]. is can be attributed to the tetradecyl groups in the structure of the Si-QAC lm.Furthermore, the application of the coating caused a reduction in the intensity of the absorption peak at the wavenumber of 1759 cm -1 , which is characteristic of the CϭO stretch of ester groups in the macromolecules of PLA.
is result indicates that the antimicrobial polymeric lm coated the PLA bres, which resulted in partial shading of the peaks characteristic of the bre structure.Furthermore, a broad absorption peak appeared at the wavelength of 1565 cm -1 in the spectrum of the nished PLA bres, which is characteristic of amide II, which shows a strong absorption in the spectral region between 1570 and 1515 cm -1 [25].In this spectral region, high intensity absorption peaks can be detected in the spectrum of RV, which suggests the presence of this group in the structure of RV.However, because RV is a commercial product, its exact structure is not evaluable by the producer.In the spectrum of the PLA-RV-Ag-SiQAC sample, the absorption peaks at the wavelengths 1129, 1083 and 1043 cm -1 , which are characteristic of the asymmetric stretching vibration of the Si-O-Si group in the polysiloxane network [25,26], are overlapped by the peaks characteristic of the ngerprint of PLA.Silver could also not be detected in this spectrum.erefore, to prove the presence of Si-QAC in the coating, the BPB test was used, and the results are presented in Figure 3. Blue colour in the samples indicated the binding of the BPB anions to the cationic nitrogen atoms of the quaternary ammonium groups of Si-QAC via electrostatic attractive interactions.Accordingly, the K/S values, determined for the samples a er shaking in the solution of BPB, highly increased from 0.2 for the PLA-N sample without Si-QAC to 10.2 and 11.4 for the PLA-SiQAC and PLA-RV-Ag-SiQAC samples, respectively.e presence of the antimicrobial coating on the PLA bres was also con rmed by the SEM and EDS analysis.e SEM/BSE images of the PLA-N, PLA-Ag, PLA-RV-Ag and PLA-RV-Ag-SiQAC samples (Figure 4) revealed that spherical silver particles, visible as bright spots, were formed over the entire surface of the bres in the in situ synthesis of AgCl (Figure 4b).e presence of AgCl was also con rmed by the EDS analysis, from the peaks of Ag-Lα and Cl-Kα (Table 2).e application of RV and Si-QAC greatly increased the roughness of the bres, con rming the forma-tion of the polymer coating from the sol-gel precursors (Figures 4 c and d).In these images, the presence of AgCl is not clearly perceptible.In addition, the Si-Kα peaks and Zr-Lα belong to the RV and Si-QAC silica matrix, while the C-Kα and   O-Kα peaks belong to the silicon matrix as well as to the PLA bres.e peak for nitrogen in the structure of Si-QAC could not be determined on a PLA-RV-Ag-SiQAC sample because it was overshadowed by the much more intense peaks of carbon and oxygen.Table 3 shows that di erent samples absorbed different amounts of silver, namely: PLA-Ag <PLA-RV-Ag-SiQAC <PLA-RV-Ag, from solutions with the same concentrations of AgNO 3 and NaCl.ese results con rm that the silica matrix created by RV highly increased the adsorptive capacity of bres, resulting in the incorporation of a een times higher concentration of silver into the PLA-RV-Ag sample in comparison to the PLA-Ag sample.e decrease of the concentration of silver on the bres a er the application of Si-QAC from 140 mg/kg (PLA-RV-Ag sample) to 53 mg/kg (PLA-RV-Ag-SiQAC sample) was expected because we assumed that silver particles were physically bonded to the silica matrix, enabling them to partially leach out of the sample when it was immersed in the solution of Si-QAC.
e results of the antimicrobial test, presented in Table 3, clearly demonstrate that the PLA bres do not show any bacterial reduction.e concentration of silver on the PLA-Ag sample was too low to provide a bacterial reduction higher than 60%, which represents a threshold value for the biostatic action of the antimicrobial agent.Even the antibacterial activity of the one-component coating prepared with Si-QAC was only biostatic with the R values of 67.5% and 78.1% for E. coli and S. aureus, respectively.e biocidal activity with the R values equal to 100% for both studied bacteria was achieved on the PLA-RV-Ag and PLA-RV-Ag-SiQAC samples, which proves that the creation of a silica matrix is absolutely necessary for the PLA bres with very low adhesion ability to AgCl, to achieve e ective antimicrobial activity in the coating.e results also show that the presence of the Si-QAC polymer lm on the PLA bres did not hinder the leaching of silver particles from the coating, resulting in the synergistic action of the bio-barrier and controlled-release antimicrobial mechanisms.
e results of the colorimetric measurements presented in Figures 5 and 6 reveal that the presence of coatings decreased the lightness and increased the yellowing of the PLA bres, especially in the case of the application containing RV and Si-QAC.At the same solution concentration (100 g/L), the application of Si-QAC decreased the whiteness of the bres by 25%, but the application of RV decreased the whiteness by more than 50%.Accordingly, the whiteness of PLA-RV and PLA-RV-Ag-SiQAC fell below the value of 40 (Figure 5), which represents the threshold for which the equation ( 3) is valid.
is represents a signi cant weakness of the studied antimicrobial coating.In line with the decrease of the whiteness, the highest yellowing was caused by the application of RV; the yellowing did not signicantly increase with further application of silver and Si-QAC (Figure 6).

Conclusion
In this study, we have successfully developed a new three-stage procedure to tailor a dual-active antimicrobial nishing for the PLA bres, using AgCl and Si-QAC.e procedure included the following steps: the application of RV with the aim to create a sili-ca matrix on the surface of the bres, which was important for increasing the adhesive ability of the bres; the in situ synthesis of silver in a silica matrix with two sequential immersions of the PLA samples in the solutions of AgNO 3 and NaCl to create an antimicrobial coating with physically incorporated silver particles, which can be released into the environment in a controlled manner, acting as a poison for microorganisms; the application of Si-QAC with quaternary ammo-nium groups with the aim to create a polymer lm on the bre surface, which could act as a biological barrier and destroy microorganisms that come in contact with the bres.e mode of preparation of the coating allows its application to other hydrophobic bres, such as polyethylene terephthalate, polypropylene, and polyamide bres.

Figure 1 :
Figure 1: Schematic presentation of the three-stage procedure of antimicrobial nishing

W 10 =
Y 10 + 800(0.3138x 10 ) + 1700(0.3310y 10 ) (3), where Y 10 is the standardized colour value of the sample, and x 10 and y 10 are the standardized colour portions of the sample.e yellowing index, YI, was from the following equation:

Figure 3 :
Figure 3: Photos of PLA samples a er shaking in the solution of the BPB reagent

Table 2 :
Elemental composition of the coated samples obtained by EDS analysis

Table 3 :
Concentration of silver, c Ag , on the nished samples and the bacterial reduction, R, against bacteria E. coli in S. aureus