Transparent Copper-based Antibacterial Coatings with Enhanced

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Biological and Medical Applications of Materials and Interfaces

Transparent Copper-based Antibacterial Coatings with Enhanced Efficacy against Pseudomonas aeruginosa Debirupa Mitra, Min Li, En-Tang Kang, and Koon-Gee Neoh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09640 • Publication Date (Web): 11 Dec 2018 Downloaded from http://pubs.acs.org on December 16, 2018

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ACS Applied Materials & Interfaces

Transparent

Copper-based

Antibacterial

Coatings

Enhanced Efficacy against Pseudomonas aeruginosa

Debirupa Mitra, Min Li, En-Tang Kang, Koon Gee Neoh*

Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, Singapore 117576

* Corresponding author: Tel: +65 65162176; Fax: +65 67791936; Email: [email protected]

KEYWORDS Antibacterial coating, EDTA, copper, P. aeruginosa, proteolytic activity

1 ACS Paragon Plus Environment

with

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Bacterial surface contamination is a major cause of hospital-associated infections. Antibacterial coatings can play an important role in reducing bacteria transmission via inanimate surfaces in healthcare settings. In this work, transparent copper-based antibacterial coatings were fabricated on commercial poly (vinyl fluoride) and stainless steel. Acrylated quaternized chitosan and ethylenediaminetetraacetic acid were covalently grafted on the substrate for complexation with copper ions. The number of viable Staphylococcus aureus (S. aureus) in a droplet (containing ~104 colony forming units (CFU)), deposited on the copper-containing coating decreased by ~96 % within 60 min at 25 °C. With Pseudomonas aeruginosa (P. aeruginosa), one of the most virulent and hardest to kill bacteria, no CFU could be observed within the same time span (killing efficacy >99.8% based on detection limit). An increase in copper release from the coating was observed in the presence of P. aeruginosa, which was postulated to be due to the proteolytic activity of P. aeruginosa. The higher efficacy of the coating against P. aeruginosa compared to S. aureus is thus attributed to this increased copper release from the coating, which resulted in extensive bacterial membrane damage and death. The copper-containing coating on poly (vinyl fluoride) retained its antibacterial efficacy after 100 wipes with a water-wetted cloth or isopropanol wipes, demonstrating its durability and long-term efficacy. The coating did not exhibit significant cytotoxicity towards mammalian fibroblasts, further demonstrating its potential for mitigating bacterial transmission in a clinical setting.


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INTRODUCTION Environmental surfaces contaminated with bacteria are microbe “reservoirs” that potentially transmit pathogens and consequently spread infection to healthy individuals.1-2 Inanimate surfaces may be contaminated through direct contact with a contaminated object, sedimentation of airborne bacteria or settling of infectious droplets generated by coughing, sneezing or even floor mopping.3 In a healthcare setting, surfaces like bedrails, door handles, computer accessories, instruments and privacy curtains are frequently contaminated with harmful bacteria contributing to increased occurrence of hospital-associated infections (HAIs), which are a leading cause of mortality as well as morbidity worldwide.4-5 Combating surface contamination is indeed a challenge since hand-hygiene compliance cannot be monitored or controlled, and commonly used surface cleaning and disinfection procedures lack efficacy.6-7 Thus, alternative interventions are needed, and antibacterial surfaces that either inhibit adhesion of bacteria or possess bactericidal properties may be one way to substantially lower the surface-associated microbial burden and thereby impeding the infection transmission cycle.4,8

Metals, specifically silver (Ag) and copper (Cu) are potent antibacterial agents with high efficacy against a wide range of microorganisms.9 Although Ag has been the preferred choice in many antibacterial studies,10-11 recent studies have demonstrated the superior efficacy of Cu at conditions (humidity and temperature) that are typical of the indoor environment.12-13 Cu has also been investigated for use in antifouling water-treatment membranes, antibacterial textiles and in heating ventilation and airconditioning systems.14-17 The use of Cu surfaces in a pediatric intensive care unit has been shown to significantly reduce the microbial burden,18 and in another study, the



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use of Cu oxide impregnated linens and personnel uniforms resulted in a significant decrease in the occurrence of HAIs.19 However, introduction of Cu surfaces in a clinical environment would require either bulk manufacturing of pure or alloyed Cu objects or Cu-coated objects to replace the existing ones. While that is feasible for objects like door handles and bed rails, other high-touch surfaces like instrument panels/screens and computer accessories cannot be made of or be coated with metallic Cu in order to preserve their functionality.

Herein, we report a strategy for fabricating Cu-based bactericidal coatings that takes into consideration the requirements for potential application in a healthcare environment (i.e. indoor ambient temperature and humidity, transparency for certain applications, durability and minimal cytotoxicity in addition to high efficacy). To achieve a transparent coating, Cu ions were used instead of metallic Cu, and suitable moieties were introduced on the substrate to complex with Cu ions and to modulate their

subsequent

release.

Acrylated

quaternized

chitosan

(AQCS)

and

ethylenediaminetetraacetic acid (EDTA) were selected as candidates for this purpose since EDTA is known to be a strong metal-ion chelating agent, and chitosan (CS) derivatives also possess affinity for Cu ions via complexation with their functional groups.20 The choice of these two agents was further motivated by their suitability for clinical applications since CS derivatives exhibit antibacterial activity with minimal toxicity,21 and EDTA is used clinically in chelation therapy and as an anticoagulant.22 Both AQCS and EDTA form clear aqueous solutions and hence, upon grafting on transparent substrates, the transparency of the substrate would not be adversely affected.


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To demonstrate the applicability of our strategy, poly (vinyl fluoride) (PVF) film was chosen as a model polymeric substrate suitable for placement over instrument panels. This strategy of using grafted AQCS and EDTA for incorporating Cu ions on metal substrates was demonstrated with stainless steel (SS) foil. SS was selected as a model metal substrate due to its extensive use in clinical settings such as door handles, bedrails, grab bars, etc.23 The antibacterial efficacy of the Cu-based coatings was evaluated against both Gram-positive Staphylococcus aureus (S. aureus) and Gramnegative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) since these bacteria are common HAI pathogens.24 P. aeruginosa is one of the hardest to kill bacterium because of its osmotic shock adaptability25 and multiple mechanisms of antimicrobial resistance.26 While S. aureus has been found to persist for 7 days to 7 months on dry inanimate surfaces, P. aeruginosa can persist from 6 hours to 16 months.24 As such, antibacterial surfaces with enhanced killing of P. aeruginosa would be highly beneficial for reduction of HAIs. We demonstrate herein that our coatings achieved a high kill rate against this bacterium. The potential applicability of these Cu-based coatings in the healthcare environment was further assessed by evaluating coating durability, transparency and cytotoxicity.

EXPERIMENTAL SECTION Materials Low molecular weight CS ( ≥75 % deacetylation, viscosity of 0.02−0.3 kg/(m·s) for 1 wt.% solution in 1 wt.% acetic acid, molecular weight of 50-190 kDa), hexyl bromide, acryloyl chloride, methane sulfonic acid, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC), copper (II) sulfate pentahydrate (CuSO4.5H2O), phosphate-buffered saline (PBS), mucin from porcine stomach Type



II, fluorescein sodium salt, tryptic soy broth, nutrient broth, Luria-Bertani (Miller) broth, agar, bacteriological agar and skim milk powder were purchased from SigmaAldrich, USA. EDTA-disodium salt was obtained from 1st Base, Singapore. Sodium hydroxide and potassium iodide were purchased from Merck, Germany. 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from Alfa Aesar, USA. Quant-iT Picogreen dsDNA assay kit was purchased from Invitrogen, USA. PVF film (thickness, 0.05 mm) and SS foil (AISI-304; thickness, 0.05 mm) were obtained from Goodfellow, U.K. S. aureus (ATCC 25923), P. aeruginosa (ATCC 15692), E. coli (ATCC 25922), 3T3 mouse fibroblast cells (ATCC CRL-1658) and adult human dermal fibroblasts (HDF, ATCC PCS-201-012) were procured from American Type Culture Collection (ATCC, USA). All other solvents used were of analytical reagent grade.

Surface modification with AQCS, EDTA and Cu Synthesis of AQCS was carried out as described in the Supporting Information (SI). Surface modification of PVF was carried out as follows. In the first step, PVF was modified with AQCS as reported in our previous work.27 Briefly, PVF (measuring 14×8 cm2) was cleaned by ultrasonication in ethanol followed by de-ionized (DI) water, for 10 min each. The cleaned PVF film was dried and treated with oxygen plasma for 10 min at 150 W (Model ATTO, Diener Electronic, Germany). Then, 2.5 mL of 5 wt.% AQCS in DI water was spread evenly over the top surface of PVF, degassed with argon and placed inside a UV chamber (irradiation at 365 nm from 5×8 W lamps in a chamber of 30.5 cm (L)×12.7 cm (H)×25.4 cm (W); Model CL-1000L, UVP, USA) for 2 h. The film was then washed thoroughly with DI water to remove


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any unreacted AQCS, dried under nitrogen flow and stored in a dry box until further use (denoted as Film A).

Film A was further modified with EDTA in the following manner. A solution containing 100 mM each of di-sodium EDTA, EDC and NHS in PBS was prepared and kept at room temperature for 15 min. Film A (measuring 2×2 cm2) was placed in a 6-well plate, covered with 5 mL of the above solution and kept in a shaker (150 rpm) at 25 °C for 4 h. After 4 h, the EDTA-modified film (denoted as Film A-EDTA) was washed thoroughly with deionized (DI) water, dried in an oven at 60 °C for 2 h and stored in the dry box until further use. Cu loading was carried out by immersing Film A or Film A-EDTA (measuring 2×2 cm2) in 5 mL of 200 mM CuSO4.5H2O for 4 h in a shaker (150 rpm) at 25 °C. The Cu-loaded film (denoted as Film A-Cu or Film A-EDTA-Cu) was washed thoroughly with DI water, dried and stored in the manner mentioned above.

Modification of SS foil was carried out in a similar manner as described above, except the oxygen plasma treatment duration was shortened to 5 min. SS modified with AQCS, followed by reaction with EDTA and Cu loading is denoted as SS-EDTA-Cu.

Characterization Surface composition of pristine and modified substrates was analyzed by X-ray photoelectron spectroscopy (XPS; AXIS UltraDLD spectrometer, Kratos Analytical, U.K.) using Al Kα as the X-ray source (1468.6 eV photons). Peak fitting was carried out using XPSPEAK41 software, with the C-C peak (in the C 1s spectrum) at 284.6 eV as reference, and the full width at half-maximum of each peak within an elemental



core level was kept constant. Water contact angles were measured by a contact angle goniometer (Dataphysics, Germany). Energy dispersive X-ray spectroscopy (EDX; SEM Model 5600 LV, JEOL, USA fitted with EDX detector) was performed to obtain elemental maps of top, bottom and cross-section of Films A-EDTA and A-EDTA-Cu. To obtain cross-sections of the films for imaging, a sharp blade was used to cut through the film, with the cutting process initiated from the uncoated surface to prevent contamination of the cross-section by any coating that may adhere to the blade. The optical transmittance of Films A-Cu and A-EDTA-Cu in the visible region were measured using UV-Vis spectroscopy (UV 3600, Shimadzu, Japan) with pristine PVF as the reference. Cu content of Cu-containing PVF films or SS was measured using hot acid digestion (2 h in 65 % HNO3 at 70 °C) followed by inductively coupled plasma-optical emission spectrometry (ICP-OES; iCAP 6200 Duo, Thermo Scientific, USA) analysis.

Droplet assay for antibacterial efficacy S. aureus (in tryptic soy broth), P. aeruginosa (in Luria-Bertani (Miller) broth) and E. coli (in nutrient broth) were cultured for 18-24 h at 37 °C in a shaker-incubator. The bacterial suspension was centrifuged at 2700 rpm for 8 min, washed with PBS and redispersed in PBS at a concentration of ~107 cells/mL. The bacterial concentration in PBS was established by measuring the optical density of the suspension using a microplate reader (Multiskan GO, Thermo Scientific, USA). A reading of 0.1 at 540 nm was found to correspond to ~108 cells/mL based on spread-plate counting.

Pristine and modified PVF films or SS foil, measuring 1×1 cm2 were placed in 24well plates and UV-sterilized (30 W lamp) for 20 min inside a biological safety


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cabinet. A 1 µL droplet of the bacterial suspension in PBS (containing ~104 cells) was placed on the surface of the substrates and incubated at 25 °C for 15, 30, 60 or 90 min. Another set of droplet assay was carried out with the bacteria re-suspended in PBS supplemented with 1 % mucin to simulate body fluids in cough/sneeze droplets. After the desired incubation time, each substrate was immersed in 2 mL PBS, ultrasonicated for 4 min and vortexed for 10 s. A 100 µL aliquot from the above suspension was then spread on medium-agar plate, incubated at 37 °C for 24 h and the number of colony forming units (CFU) was counted. Antibacterial efficacy was calculated as % reduction in the number of viable bacteria on the modified substrate as compared to the pristine substrate after a similar incubation time. If no CFU was observed from triplicate plates, the efficacy is expected to be >99.8 % based on the initial bacterial loading.

For evaluation of bacterial cell morphology using field emission scanning electron microscopy (FESEM; Model JSM 6700, JEOL, USA), a 1 µL droplet of the bacterial suspension in PBS (containing ~104 cells) was incubated on pristine or modified substrate for 30 min at 25 °C. Then, 1 mL 2.5 % glutaraldehyde (in PBS) was gently placed over the surfaces for overnight fixation at 4 °C. The surfaces were then washed very gently with DI water followed by serial dehydration with ethanol and drying, before observation under FESEM.

Durability assay To assess the durability of the coatings, Films A-Cu and A-EDTA-Cu (measuring 1×1 cm2) were either wiped 30, 60 or 100 times with cloth moistened with DI water or wiped 100 times with commercial 70 % isopropanol wipes, with air-drying between



each wipe. The antibacterial efficacy of the wiped films was analyzed using droplet assay with S. aureus, as described above. Additionally, the Cu content of Films A-Cu and A-EDTA-Cu after 100 wipes with DI water was analyzed by ICP-OES. The durability assay was also carried out on SS-EDTA-Cu wherein the antibacterial efficacy and Cu content were similarly analyzed after 100 wipes with DI water-wetted cloth.

Evaluation of membrane damage and Cu release The extent of bacterial membrane damage induced by the antibacterial surfaces was evaluated by measuring the amount of extracellular DNA released from the bacteria. The surfaces of pristine (control) and modified PVF films (measuring 1×1 cm2, placed in a 24-well plate) were covered with 300 µL suspension of S. aureus (or P. aeruginosa) in PBS, containing ~5×107 cells/mL and incubated at 25 °C for 60 min. Then, 250 µL suspension was removed and filtered through a 0.22 µm syringe filter. The amount of extracellular DNA in the filtered solution was measured quantitatively using the Quant-iT Picogreen dsDNA assay kit as per the manufacturer’s instructions. A 300 µL suspension of S. aureus (or P. aeruginosa), containing ~5×107 cells/mL incubated in the wells of a 24-well plate at 25 °C for 60 min without any film served as the reference. The DNA concentrations measured for the control and test samples were corrected for the amount measured for the reference.

To measure the amount of Cu released when the modified films were incubated with bacterial suspension, Films A-Cu and A-EDTA-Cu (measuring 1×1 cm2) were covered with either 300 µL suspension of bacteria (S. aureus or P. aeruginosa) in PBS containing ~5×107 cells/mL or just 300 µL PBS without bacteria. Corresponding


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experiments carried out with pristine PVF served as the control. After an incubation period of 60 min at 25 °C, 250 µL of the suspension was withdrawn and mixed with 2.75 mL of 5 % HNO3, and the Cu concentration was measured using inductively coupled plasma-mass spectrometry (ICP-MS; Model 7500, Agilent Technologies, USA).

Determination of proteolytic activity Proteolytic activity of the bacteria was assessed using skim milk agar plates (preparation described in SI).28 S. aureus, P. aeruginosa and E. coli were cultured in their respective medium for 24 h. The bacterial suspensions were centrifuged at 2700 rpm for 8 min, re-suspended in PBS at a concentration of ~107 cells/mL and incubated for 1 h at 25 °C. A 10 µL aliquot was then pipetted into holes punched in the skim milk agar plates and incubated at 37 °C. After 24 h incubation, the skim milk agar plates were inspected for any visible changes. PBS was used as the negative control and 0.1 and 0.01 mg/mL Proteinase K solutions in sterile PBS were used as the positive controls. In another set of experiment, 5 µL aliquots of the bacterial suspension (S. aureus or P. aeruginosa) were carefully pipetted into ~0.5 mm holes (t=0 h) punched in a thin layer of solidified skim milk-agar in the wells of a 96–well plate (preparation described in SI) and incubated at 37 °C for 1 to 12 h. PBS was used as the negative control. An optical microscope with 4× magnification lens (Model Eclipse Ti, Nikon, Japan) was used to image the holes at t=0, 1 and 12 h.

In another experiment to confirm the ability of proteolytic enzymes (secreted by P. aeruginosa) to cleave amide bonds in a non-protein system, a CS film with fluorescein conjugated via amide bonds (CS-F) was incubated in a bacterial



suspension. The fluorescence emission spectrum of the incubation medium was measured after 60 min to detect the presence of cleaved fluorescein. Details of the experiment are given in the SI.

Cytotoxicity of modified films The potential cytotoxicity of the modified surfaces was evaluated using the standard MTT assay as described in the SI.

Statistical Analysis The above-mentioned experiments were carried out in triplicate unless otherwise stated. All experimental values were reported as mean±standard deviation, and statistical analysis was carried out using one way analysis of variance (ANOVA) with Tukey post hoc test. Statistical significance was accepted at p≤0.05.

RESULTS AND DISCUSSION Characterization of modified surfaces The preparation of the antibacterial surfaces was carried out via a four-step procedure as depicted in Scheme 1. PVF was chosen as a model polymeric substrate mainly for its transparency as well as its widespread use in different applications, low cost and availability.29. In the first step (Step 1 in Scheme 1), oxygen plasma was used to introduce reactive hydroxyl and peroxyl groups on the PVF substrate.30 The presence of these groups significantly lowered the hydrophobicity of pristine PVF, as confirmed by a decrease in water contact angle from 78 ° for pristine PVF to 30±3 ° (Table 1) for the plasma-treated surface. These functional groups facilitate UVinduced surface grafting of AQCS (chemical structure in Figure S1) and crosslinking


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via its acrylate groups in an oxygen-free environment (Step 2). The NH2 groups of AQCS-grafted PVF (Film A) were then reacted with EDTA (Step 3) using EDC/NHS chemistry and a depiction of the possible EDTA grafted moieties of Film A-EDTA is shown in Scheme 1. In the final step, Cu was loaded on Film A or Film A-EDTA by soaking in CuSO4.5H2O (Step 4) to obtain Films A-Cu or A-EDTA-Cu. The concentration of CuSO4.5H2O solution and the duration of soaking were fixed at 200 mM and 4 h respectively, since the Cu content of Film A-EDTA-Cu was found to be maximum using these parameters in a preliminary study (Figure S2). In this Scheme, the reactions are shown at the surface although for plasma-treated PVF, similar reactions can occur in the bulk, as discussed below. In the coating process, the volume of the AQCS solution that was spread on the top surface of the PVF film (in Step 2) was kept low (~20 µL per cm2 of surface) to avoid substantial spillage over the edges. Thus, only one side of PVF was coated and this was confirmed by EDX elemental maps that showed very little N and Cu on the bottom surface of Film A-EDTA-Cu compared to the top (coated) surface (Figure S3). To demonstrate that the surface modification procedure depicted in Scheme 1 can be applied to metal substrates, SS was modified via AQCS grafting followed by reaction with EDTA and Cu loading to obtain SS-EDTA-Cu.

The XPS wide-scan spectra of both Films A-Cu (Figure S4(b)) and A-EDTA-Cu (Figure S4(c)) showed the presence of N 1s (~400 eV) and Cu 2p (~930-960 eV) peaks that were absent in the spectrum of pristine PVF (Figure S4(a)). The F 1s peak (due to fluorine of PVF) was reduced as compared to the pristine PVF but it was still visible, indicating the surface coating thickness was less than the XPS probing depth of 6-8 nm.31 The N 1s core-spectrum of Film A-Cu (Figure 1(a)) was fitted with two



main peaks at 399 eV and 401.4 eV, corresponding to N-H (or N-C) and N+, respectively, confirming the successful grafting of AQCS.27 A small peak at 400.4 eV corresponding to NHC=O32 (NHC=O/total N=0.04) was also observed, attributed to the acetyl groups of the CS used (degree of deacetylation ≥75 %). On the other hand, the NHC=O peak (400.3 eV) in the N 1s core spectrum of Film A-EDTA-Cu (Figure 1(b)) was prominent and accounted for ~29 % of the total N 1s peak area (NHC=O/total N=0.29), implying successful amide bond formation between EDTA and AQCS as shown in Scheme 1. The Cu 2p3/2 peaks in the Cu 2p spectra of both Films A-Cu (Figure 1(a’)) and A-EDTA-Cu (Figure 1(b’)) were deconvoluted into two component peaks. The more prominent higher binding energy peak, attributed to Cu (II) species,33 was observed at 934.9 eV for Film A-Cu (Figure 1(a’)) and 934.3 eV for Film A-EDTA-Cu (Figure 1(b’)). The lower binding energy peak in the Cu 2p spectrum observed at 932.9 eV for Film A-Cu and at 932.7 eV for Film A-EDTA-Cu may be either Cu (0) or Cu (I) since the binding energies of these species are very close and indistinguishable in the Cu 2p spectrum.34 Thus, Cu LMM Auger spectrum was used for differentiating between these two Cu species. The Auger kinetic energy peak was observed at 916.2 and 916.7 eV for Film A-Cu (Figure 1(a”)) and AEDTA-Cu (Figure 1(b”)) respectively, which corresponds to Cu (I) species.35 The existence of Cu (I) is attributed to the partial reduction of Cu (II) by X-rays during XPS analysis, a common phenomenon reported in the literature.34,36

The XPS wide-scan spectrum of SS-EDTA-Cu foil (Figure S4(e)) also showed the presence of N 1s and Cu 2p peaks at ~400 eV and 930-960 eV respectively, which were absent in the corresponding spectra of pristine Type 304 SS foil (Figure S4(d)). While the intensities of the Fe 2p and Cr 2p peaks of SS-EDTA-Cu at 710-716 eV and


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574-581 eV respectively37 were reduced compared to pristine SS, their presence indicated that the coating was 0.05) from the as-prepared Film A-EDTA-Cu. Thus, Film A-EDTA-Cu exhibits a high degree of durability when subjected to a combination of friction and aqueous medium from the wiping process, demonstrating its promise as an antibacterial coating with long-term efficacy. However, SS-EDTA-Cu does not possess the same degree of durability, and after 100 wipes with DI water, its antibacterial efficacy against S. aureus after 60 min incubation decreased to ~44 % (from ~95 % for the as-prepared substrate). XPS analysis of the wiped SS-EDTA-Cu showed that most of the Cu has been lost. Thus, the reservoir of Cu ions in the bulk of the PVF substrate, but which was absent in the SS substrate, endowed the former with a much higher degree of durability.

For application of antibacterial films on high-touch surfaces such as ventilator and syringe pump driver panels, computer monitors and keyboards in hospitals, a high level of transparency of the overlying antibacterial film is essential to preserve the functionality of the device. Thus, elemental Cu that has been used as bedrails,



intravenous stand poles and faucet handles18,68 in a hospital environment in earlier investigations would not be suitable. On the other hand, the transparent Film AEDTA-Cu would be advantageous for such an application. The transmittance of Film A-EDTA-Cu in the visible region (350-750 nm) was measured to be 73-75 % of that of pristine PVF (Figure 9(a)). Figure 9(b) shows that with the placement of Film AEDTA-Cu (measuring 12×8 cm2) over a syringe pump touch-screen panel, the panel remained clearly visible and its touch functionality was preserved.

Lastly, the possible cytotoxicity of the antibacterial films was evaluated using mammalian cells (Figure S13). The viability of mouse fibroblasts (3T3) after 24 h incubation on films without Cu (Film A and Film A-EDTA) was similar to that on pristine PVF (Figure S13(a)). CS derivatives as well as EDTA have been widely investigated for healthcare and biomedical applications69-70 and thus, coatings based on these components can be expected to have minimal cytotoxicity. The Cucontaining Film A-Cu and Film A-EDTA-Cu resulted in a slight decrease in 3T3 cell viability as compared to their non-Cu counterparts, but there was no significant difference (p>0.05) in cell viability when compared to the control (cells grown in the absence of any film). The viability of 3T3 cells incubated with Film A-Cu or Film AEDTA-Cu for 24 h was still >94 %. The potential cytotoxicity of Cu-containing films was further evaluated using HDF, and the cell viability of HDF when incubated with Films A-Cu or A-EDTA-Cu for 24 h was >90 % (Figure S13(b)) indicating minimal cytotoxicity.


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CONCLUSION The above results show that transparent highly efficacious and durable antibacterial coatings on PVF can be fabricated by incorporating Cu ions in EDTA conjugated to AQCS. The grafted EDTA enables Cu ions to be readily loaded, and the subsequent release of these ions endows these coatings with high killing efficacy against both Gram-positive and Gram-negative bacteria. The release of Cu from this coating was higher in the presence of P. aeruginosa than S. aureus, which resulted in a higher killing efficacy of the former. The increase in Cu release is postulated to be due to the cleavage of amide bonds between EDTA and AQCS by proteolytic enzymes secreted by P. aeruginosa. Despite the coating’s high bactericidal efficacy, there is minimal cytotoxicity towards mammalian cells. As such, this type of coating shows promise for inhibiting pathogen transmission from high-touch devices in a hospital environment, particularly those requiring transparency of the overlying antibacterial film.

ASSOCIATED CONTENT Supporting Information. Detailed methods for AQCS synthesis, AQCS grafting, droplet assay, preparation of skim milk agar plates and MTT assay; Figures: chemical structure of AQCS (Figure S1), Cu content of Film A-EDTA-Cu under different conditions (Figure S2), EDX maps of Film A-EDTA-Cu (Figure S3 and S6), XPS spectra of Film A-Cu, Film A-EDTA-Cu and SS-EDTA-Cu (Figures S4 and S5), droplet assays: (i) SS-EDTA-Cu against S. aureus and P. aeruginosa (Figure S7), (ii) Films A-Cu and A-EDTA-Cu against S. aureus and P. aeruginosa at different incubation times (Figure S8), (iii) Film A-EDTA-Cu against S. aureus and P. aeruginosa in the presence of mucin (Figure S9), (iv) Films A-Cu and A-EDTA-Cu



against E. coli (Figure S11) and (v) Film A-EDTA-Cu after 100× wiping with isopropanol (Figure S12), observation of proteolytic activity after 1 and 12 h of incubation with S. aureus and P. aeruginosa (Figure S10) and viability of 3T3 fibroblasts and HDF when incubated with modified PVF films (Figure S13).

ACKNOWLEDGEMENT This work was financially supported by the National University of Singapore (Grant No. R279000416112).

REFERENCES (1) Boyce, J. M. Environmental contamination makes an important contribution to hospital infection. J. Hosp. Infect. 2007, 65, 50-54. (2) Kassem, I. I. Chinks in the armor: The role of the nonclinical environment in the transmission of Staphylococcus bacteria. Am. J. Infect. Control 2011, 39, 539-541. (3) Morawska, L. Droplet fate in indoor environments, or can we prevent the spread of infection? Indoor air 2006, 16, 335-347. (4) Page, K.; Wilson, M.; Parkin, I. P. Antimicrobial surfaces and their potential in reducing the role of the inanimate environment in the incidence of hospital-acquired infections. J. Mater. Chem. 2009, 19, 3819-3831. (5) Shek, K.; Patidar, R.; Kohja, Z.; Liu, S.; Gawaziuk, J. P.; Gawthrop, M.; Kumar, A.; Logsetty, S. Rate of contamination of hospital privacy curtains in a burns/plastic ward: A longitudinal study. Am. J. Infect. Control 2018. (6) Bogusz, A.; Stewart, M.; Hunter, J.; Yip, B.; Reid, D.; Robertson, C.; Dancer, S. J. How quickly do hospital surfaces become contaminated after detergent cleaning? Healthcare infection 2013, 18, 3-9.


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SCHEME

Scheme 1 Schematic showing the preparation of Film A, Film A-EDTA and the corresponding Cu-loaded films. In this Scheme, the reactions are shown at the surface although for plasma-treated PVF, similar reactions can occur in the bulk.



FIGURES

Figure 1 XPS (a,b) N 1s, (a’,b’) Cu 2p and (a”,b”) Cu LMM Auger spectra of (a,a’,a”) Film A-Cu and (b,b’,b”) of Film A-EDTA-Cu.

Figure 2 Quantitative count of viable (a) S. aureus and (b) P. aeruginosa after incubation of a droplet containing ~ 104 cells in PBS on pristine and modified PVF films for 60 min at 25 °C. * Significant difference (p

Transparent Copper-based Antibacterial Coatings with Enhanced

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