Scalable Aqueous-Based Process for Coating ... - ACS Publications

Aug 25, 2016 - Scalable Aqueous-Based Process for Coating Polymer and Metal. Substrates with Stable Quaternized Chitosan Antibacterial Coatings. Debir...
2 downloads 8 Views 5MB Size
Download PDF
Article pubs.acs.org/IECR

Scalable Aqueous-Based Process for Coating Polymer and Metal Substrates with Stable Quaternized Chitosan Antibacterial Coatings Debirupa Mitra, Min Li, Rong Wang, Zhihao Tang, En-Tang Kang, and Koon Gee Neoh* Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, Singapore 117576 S Supporting Information *

ABSTRACT: Fabrication of antibacterial surfaces is an approach to reduce environmental bacterial burden and risk of infection transmission. Antibacterial coatings based on cationic polymers have been commonly used for this purpose, but coating methods to immobilize these polymers over large area substrates are limited. Herein, we report a facile aqueousbased method of immobilizing quaternized chitosan (QCS) on polymer and metal substrates via electrostatic interaction with substrate-anchored multivalent polyphosphate ions to form transparent antibacterial coatings of micron thickness. The QCS coatings are noncytotoxic and yet demonstrate a high killing efficacy against two clinically relevant bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, when challenged with bacterialoaded droplets or contacted with a bacteria-loaded dry surface. The coatings were stable when subjected to wiping, and the QCS-coated substrates retained their efficacy and could be reused for multiple cycles after wiping the contaminated surface with 70% ethanol. A distinct advantage of this coating method is the ease of scale-up using spray coating for preparation of uniform large area coatings.

■

wound dressings.17 However, CS has poor solubility in water, and one way to increase its solubility is by quaternization of the amino groups on its backbone, which results in permanent positively charged quaternary ammonium groups. Another advantage of quaternized chitosan (QCS) is that it has higher antimicrobial activity than CS, as shown by our earlier work on CS and QCS nanoparticles.18 Though the exact antibacterial mechanism of QCS is not fully established, the activity is mainly attributed to the electrostatic interaction between the positively charged quaternary ammonium groups and the negatively charged bacterial surface as well as hydrophobic interaction between the substituted alkyl chains and the lipophilic components of the bacterial cell wall.19,20 QCS derivatives, being water-soluble, have to be immobilized on the substrate surface in order to function as durable antimicrobial coatings. A few studies on immobilization of QCS on different types of substrates have been reported. In one study, QCS grafted poly(ethylene terephthalate) was prepared by first polymerizing acrylic acid (AA) on the surface followed by reaction of QCS with poly(AA).10 In another work, a polyelectrolyte complex between QCS and poly(AA) was immobilized on poly(ε-caprolactone) nanofibrous mats using UV-induced grafting.8 Despite a substantial amount of research on various antibacterial coatings, few coatings have been put to clinical

INTRODUCTION Bacterial attachment and colonization of surfaces often lead to undesired consequences such as early spoilage of food,1 medical device-associated infections,2 and nosocomial or hospitalacquired infections.3 Surface-attached bacteria such as those residing on curtains, floors, and various other high-touch areas in hospitals, clinics, and child-care centers play a vital role in the transmission of infectious diseases, and evidence of this was observed as early as 1960s.4 The fact that the inanimate environment acts as a potential reservoir of pathogens and aids in transmission of infection is now well established.5 A promising approach to overcome this ubiquitous problem is the implementation of antibacterial coatings on surfaces that are prone to contamination.3,5 Such coatings can inhibit the establishment of microbial reservoirs on the surface, either by being antifouling6 or bactericidal,7 thus leading to a cleaner and more hygienic environment.3 Surface functionalization of commonly used materials by immobilizing or grafting antibacterial polymers has emerged as one of the most frequently used approaches in the design of antibacterial coatings.6−12 Cationic compounds, and quaternary ammonium compounds specifically, are often a popular choice in the fabrication of antibacterial surfaces.13 Chitosan (CS), which is an inexpensive, nontoxic, and natural polysaccharide from chitin, is known to possess antimicrobial activity against a broad range of bacteria as well as fungi. The antibacterial activity of CS is mostly attributed to its cationic nature in acidic solutions when its amino groups are protonated.14 CS has been widely investigated for applications in food packaging and edible films,1 drug delivery,15 antimicrobial coatings,16 and © XXXX American Chemical Society

Received: June 7, 2016 Revised: August 22, 2016 Accepted: August 25, 2016

A

DOI: 10.1021/acs.iecr.6b02201 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research Table 1. QCS-Coated Substrates Prepared at Different Conditionsa

a

Sample

Substrate

Anion type

Anion concentration (wt %)

Cross-linking temperature (°C)

Cross-linking duration (h)

Water contact angle (±2°)

PVF-TPP-0.6 PVF-QCS-25-1.0 PVF-QCS-50-1.0 PVF-QCS-70-1.0 PVF-QCS-50-0.3 PVF-QCS-50-0.6 PVF-QCS-50-1.4 PVF-QCS-50-2.0 PVF-QCS-50-2.5 PVF-QCS-50-PP-0.5 PVF-QCS-50-PP-1.0 PVF-QCS-50-PP-1.5 PVF-QCS-50-HMP-1.0 PVF-QCS-50-HMP-1.5 PVF-QCS-50-HMP-2.0 SS-QCS-50-0.6

PVF PVF PVF PVF PVF PVF PVF PVF PVF PVF PVF PVF PVF PVF PVF SS

TPP TPP TPP TPP TPP TPP TPP TPP TPP PP PP PP HMP HMP HMP TPP

0.6 1.0 1.0 1.0 0.3 0.6 1.4 2.0 2.5 0.5 1.0 1.5 1.0 1.5 2.0 0.6

25 50 70 50 50 50 50 50 50 50 50 50 50 50 50

14 6 4 6 6 6 6 6 6 6 6 6 6 6 6

# 43 41 44 42 43 41 41 44 43 42 40 43 42 44 41

All samples (apart from PVF-TPP-0.6) were coated with 5 wt % QCS. # indicates no measurement was carried out.

or commercial practice,3 possibly due in part to the difficulty associated with scaling up of the process. Herein, we formulate a facile strategy of immobilizing QCS on polymer and metal substrates via electrostatic interactions with multivalent anionic agents such as sodium tripolyphosphate (TPP), sodium pyrophosphate (PP), and sodium hexametaphosphate (HMP) anchored on the surface. Electrostatic complexation of CS or CS derivatives with TPP, PP, and HMP for preparation of nanoparticles or hydrogels has been previously reported.15,21,22 However, to the best of our knowledge, this is the first report on electrostatic complexation of QCS with TPP, PP, and HMP for formulating micron scale coatings. In this study, these anionic agents, besides being surface anchors, also act as crosslinkers of the QCS polymer chains. The antibacterial efficacy of the QCS-coated substrates was tested against Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Pseudomonas aeruginosa (P. aeruginosa). Optimum coating conditions were established by evaluating antibacterial efficacy and stability of QCS-coated substrates prepared at different conditions. In addition, the reusability of the QCS-coated substrates and the scalability of the coating process were evaluated.

substrates was carried out in two steps: an initial step for anchoring an anionic agent on the substrate surface and a subsequent step for cross-linking the QCS with the anchored anions. Preliminary experiments showed that the degrees of quaternization of coatings from QCS prepared at 60 and 80 °C were ∼38% and ∼53%, respectively, and the coating with the higher degree of quaternization resulted in higher antibacterial efficacy (Figure S1). Subsequently, QCS with the higher degree of quaternization was used for preparing coatings on different substrates with different anion type and concentration, and cross-linking temperature. The list of QCS-coated substrates prepared at different conditions is given in Table 1. For the preparation of QCS-coated PVF films, pristine PVF films measuring 2 × 2 cm2 were first cleaned by ultrasonication in ethanol and deionized (DI) water for 10 min each followed by drying in an oven maintained at 70 °C. To anchor the anionic agent, the dry and clean films were treated in a plasma chamber (Model ATTO 3xMFC, Diener electronic, Germany) filled with oxygen, operating at 150 W power for 10 min, to activate the surface of the PVF film. Fifty microliters of the anionic agent (TPP or PP or HMP solution of a specific concentration) was spread evenly on the film using a spatula and allowed to dry at 70 °C in a covered petridish for 4 h. In the second step, 50 μL of 5 wt % QCS was spread on the anionic agent-treated substrate using a spatula, and the modified substrate was then kept in a covered petridish maintained at a predetermined cross-linking temperature until the coating was thoroughly dry (∼4 to 14 h). QCS coated on SS foil with TPP as cross-linker was prepared in the same manner described above except that SS foils measuring 2 × 2 cm2 were cleaned by ultrasonication in DI water, acetone, and ethanol for 10 min each and dried with compressed air prior to the anchoring and cross-linking steps. All QCS-coated substrates were washed with DI water, airdried, and stored in a drybox before use in the experiments described below. Characterization of QCS-Coated Substrates. The crosssection of the QCS-coated PVF film was observed using scanning electron microscopy (SEM) (Model 5600 LV, JEOL, USA). A thin strip of the film measuring 2 cm × 6 mm was immersed in liquid nitrogen for 10 min, and then a sharp scalpel was used to cut across the cross-section while keeping

■

EXPERIMENTAL SECTION Materials. Low molecular weight chitosan (≥75% deacetylation, viscosity 0.02−0.3 kg/m·s for 1 wt % solution in 1 wt % acetic acid), hexyl bromide, TPP, PP, HMP, tryptic soy broth, lysogeny broth, and agar were bought from Sigma-Aldrich, USA. Potassium iodide and sodium hydroxide were purchased from Merck, Germany. 3-[4,5-Dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from Alfa Aesar, USA. Poly(vinyl fluoride) (PVF, 0.05 mm thickness) and stainless steel (SS, AISI-304, 0.05 mm thickness) foil were purchased from Goodfellow, UK. S. aureus (ATCC 25923) and 3T3 mouse fibroblast cells were bought from American Type Culture Collection (ATCC, USA). P. aeruginosa (PAO1) was bought from National Collection of Industrial Food and Marine Bacteria (NCIMB, UK). Preparation of QCS-Coated Substrates. QCS was synthesized using a method reported earlier18 with some minor modifications (details of the procedure can be found in the Supporting Information). QCS immobilization on the B

DOI: 10.1021/acs.iecr.6b02201 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research the film immersed in liquid nitrogen. The film was dried in an oven, fixed on metal stubs, and sputter-coated with platinum for SEM observations. QCS-coated substrates were characterized by X-ray photoelectron spectroscopy (XPS) using an AXIS UltraDLD spectrometer (Kratos Analytical, UK) with monochromatic Al Kα as the X-ray source (1468.6 eV photons). Peak fitting was carried out using XPSPEAK41 software, with the peaks referenced to the C 1s peak at 284.6 eV, and the fullwidth at half-maximum for all peaks within a core level was kept constant. The zeta potential of the TPP-coated and a selected QCS-coated substrate (PVF-QCS-50-0.6) was measured in DI water using an electrokinetic analyzer (Model SurPASS, Anton Paar, Austria). The water contact angle for pristine and QCScoated substrates was measured by the sessile drop method using a telescopic goniometer (Model 100-00-230, Rame-Hart, USA). Tests for Antibacterial Efficacy of QCS-Coated Substrates. Bacteria were cultured in their respective growth media (tryptic soy broth for S. aureus and lysogeny broth for P. aeruginosa) for 18−24 h in a shaker incubator (Model ES-20, Grant Instruments, UK). The suspension was centrifuged at 2700 rpm for 10 min, and the medium was decanted. The collected bacteria were then washed with phosphate-buffered saline (PBS) and resuspended in PBS at the desired concentrations. The bacterial concentration in PBS was established by measuring the optical density of the suspension using a microplate reader (Model Powerwave XS, Bio-Tek, USA). A reading of 0.1 at 540 nm was found to correspond to ∼108 cells/mL based on spread-plate counting. The antibacterial efficacy of QCS-coated substrates was determined primarily by two assays: “droplet” assay and “contact” assay. All pristine and QCS-coated substrates were cut into 1 × 1 cm2 pieces, placed in the wells of a tissue culture plate (24-well plate unless stated otherwise) and UV-sterilized for 20 min inside a BSL II biological safety cabinet fitted with a 30 W UV lamp (Model BH120, Gelman, Singapore). In the droplet assay, a 1 μL droplet of PBS containing ∼103 bacterial cells (S. aureus or P. aeruginosa) was placed on the surface of UV-sterilized pristine and QCS-coated substrates. The substrates were then incubated at 25 °C for 2 h, followed by quantitative counting of bacteria using spread-plate counting method. Briefly, each substrate with the droplet on its surface was placed in a tube containing 2 mL PBS and the bacteria were released into PBS by ultrasonication for 7 min followed by 10 s of vortexing. One hundred microliters of this bacterial suspension from each tube was spread onto agar plates and the number of colony forming units (CFU) was counted after overnight incubation at 37 °C. In the contact assay, a 1 μL droplet of PBS containing ∼104 bacterial cells (S. aureus or P. aeruginosa) was placed on a piece of sterile, hydrophilic 0.2 μm polyester track-etch membrane (Sterlitech Corporation, USA). After the membrane absorbed the water droplet (no visible droplet observed), the membrane was placed on pristine or QCS-coated PVF with the contaminated side of the membrane in contact with the film surface. A stainless steel rod (diameter of 0.7 cm) weighing 30 g was immediately placed on top of the membrane for 5 s. The rod and membrane were then removed and the film was allowed to incubate at 25 °C for 2 h. Following this, the number of viable bacteria on each film was quantified by the spread-plate counting method as described above. For both contact and droplet assays, the antibacterial efficacy was defined as the percentage reduction in CFU count from a QCScoated substrate as compared to the respective pristine surface.

The distribution of surface-adhered dead and live bacteria on pristine PVF and QCS-coated PVF films after 2 h of incubation at 25 °C in the contact assay with S. aureus was evaluated using fluorescence microscopy. Fifty microliters of PI and SYTO 9 combination dye solution from the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, USA) was added to the surface of each film and the film was kept in the dark for 10 min. The excess dye solution was gently washed away with PBS and the stained films were observed under a fluorescence microscope (Model DM IL LED Fluo, Leica Microsystems, USA) fitted with red and green filters. Stability of QCS-Coated PVF Films. The stability of the QCS-coated PVF films was assessed by subjecting the coating to repeated wiping with a piece of cloth wetted with either DI water or 70% ethanol (v/v). The QCS-coated PVF films measuring 2 × 2 cm2 were wiped 30 times and between consecutive wipes, they were allowed to dry in air. The XPS [N+]:[C] ratio of the wiped films was then compared with that of the film before wiping and the antibacterial efficacy of DI water-wiped surfaces was assessed using the droplet assay as described above. Reusability of QCS-Coated PVF Films. The possibility of cleaning and reusing the QCS-coated PVF films was investigated by subjecting the films to five cycles of bacterial contamination followed by cleaning of the surface with 70% ethanol or DI water between consecutive cycles. Thirty pieces each of as-prepared QCS-coated PVF and pristine PVF films measuring 1 × 1 cm2 were placed in the wells of 12-well plates and UV sterilized for 20 min. The films were then subjected to contamination by, S. aureus using the contact assay described above. After the 2 h incubation period, all films were wiped with cloth wetted with 70% ethanol or DI water and allowed to dry completely at room temperature. Spread-plate counting was then carried out for six of the cleaned films (3 pristine PVF and 3 QCS-coated PVF). The remaining cleaned films were subjected to another round of contamination and spreadplate counting was performed on another six films (3 pristine PVF and 3 QCS-coated PVF) to obtain the antibacterial efficacy after one contamination-cleaning cycle. The remaining contaminated films were cleaned again and this cycle of contamination followed by cleaning was then repeated for four more cycles. Cytotoxicity of TPP and QCS-Coated Substrates. The possibility of cytotoxic effects arising from TPP and QCS was investigated using the standard MTT assay. 3T3 mouse fibroblast cells were cultured in growth medium comprising Dulbecco’s Modified Eagle Medium, 10% fetal bovine serum, 1 mM L-glutamine and 100 IU/mL penicillin. One milliliter of 3T3 cell suspension in the growth medium was seeded into each well of a 24-well plate at a density of 50,000 cells per well and incubated at 37 °C, 5% CO2 for 24 h. Pristine PVF, PVFTPP-0.6 (TPP coating only without QCS) and PVF-QCS-500.6 films measuring 1 × 1 cm2 were then gently placed in the wells with the coated surface touching the layer of cells and incubated for another 24 h at 37 °C, 5% CO2. Wells containing cells in growth medium without films served as the negative control. The films and media were then removed from each well and 200 μL of MTT reagent (5 mg/mL in sterile PBS) and 800 μL of fresh medium were added to each well. After incubation for 3 h, the solution containing media and MTT reagent was removed from the wells and 1 mL dimethyl sulfoxide was added to dissolve the formazan crystals. Absorbance was measured at 570 nm using a microplate reader C

DOI: 10.1021/acs.iecr.6b02201 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research

Scheme 1. Schematic Illustration of Preparation of QCS Coating on PVF via Oxygen Plasma Treatment (Step (1)) Followed by TPP Anchoring (Step (2)) and QCS Cross-Linking (Step (3))

Statistical Analysis. Measurements from all experiments were carried out in triplicate unless otherwise stated. All 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.

(Model Powerwave XS, Bio-Tek, USA) and the viability of the cells was expressed as a percentage relative to the negative control. Scale-up of Coating Process. Clean PVF film measuring 15 × 20 cm2 was plasma treated as described above and then placed on the movable stage of a semiautomatic spray coating system (Model SCS7400, Spraying Systems Co., Singapore). Fifteen milliliters of 0.6 wt % TPP solution was put in the solution reservoir and sprayed onto the plasma treated PVF film using an automatic spray nozzle (Model 38499-1/8JJAUSS, Spraying Systems Co., Singapore) at a nozzle moving speed of 50 mm/sec and air atomizing pressure of 0.25 bar. The distance between the spray nozzle and the film was kept at 45 mm. The excess TPP solution was removed from the surface using compressed air at 4 bar from a windjet air nozzle (Model AA727-1/4-15, Spraying Systems Co., Singapore) at a moving speed of 115 mm/sec and nozzle-substrate separation distance of 12 mm. The film was then placed inside a covered container and kept at 70 °C for 3 h. In the subsequent step for crosslinking with QCS, 15 mL of 5 wt % QCS solution (after filtering through a 0.2 μm syringe filter) was poured into another solution reservoir and sprayed on top of the TPP layer using the same automatic spray nozzle at a moving speed of 50 mm/sec and air atomizing pressure of 1 bar. Immediately after QCS deposition, excess QCS solution was removed from the surface using compressed air as described above. The spraycoated PVF-QCS-50-0.6 film was again placed inside the container and kept at 50 °C for 5 h. The dried coated film was then washed with DI water, dried in air and stored in a drybox for further experiments. The cross-section of spray-coated PVFQCS-50-0.6 film (at 3 random locations) was examined using SEM to confirm the success of the coating process. Water contact angle measurements were made on random areas of the spray-coated PVF-QCS-50-0.6 film and antibacterial efficacy was evaluated by droplet assay on six 1 × 1 cm2 samples cut out from random portions of the film.

■

RESULTS AND DISCUSSION Characterization of QCS-Coated Substrates. QCS was synthesized using a slight modification of a previously reported hexyl bromide alkylation process,18 and successful quaternization was confirmed by Fourier transform-infrared (FTIR) spectroscopy (Figure S2 in the Supporting Information). A new peak at 1520 cm−1 appeared in the FTIR spectrum of QCS due to stretching of the N+−C bond formed by quaternization of the amino groups at the C-2 position of CS, while the characteristic OH peaks of CS at 1032 and 1154 cm−1 were retained.18 The schematic illustration of the process for coating QCS on PVF using TPP as the anchor is given in Scheme 1. Oxygen plasma treatment of PVF is known to result in increased oxygen content23 due to the formation of oxygen and peroxyl radicals,10 which subsequently form terminal OH groups on the plasma-treated PVF surface when exposed to ambient air.10 When this OH-rich surface is coated with a polyphosphate salt (TPP), the phosphate anions are weakly physisorbed on the surface via hydrogen bonding. Upon heating, water molecules are driven off, which results in the covalent anchoring of the polyphosphate ions24,25 on the plasma-activated PVF as shown in Step 2 of Scheme 1. Positively charged QCS molecules can subsequently undergo electrostatic interaction with the negatively charged immobilized TPP molecules, resulting in a coating of QCS on the PVF films (Step 3 of Scheme 1). The same principle applies to SS foil as substrate or other multivalent polyphosphates such as HMP and PP as cross-linking agents. An advantage of this coating process is that both the anion and QCS solutions are D

DOI: 10.1021/acs.iecr.6b02201 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research

does not contain nitrogen. On the other hand, the wide-scan spectrum of the PVF-QCS-50-0.6 film in Figure 1(c) shows the presence of the N 1s peak at ∼400 eV. The F 1s as well as F Auger peaks are no longer discernible, confirming the presence of a QCS coating of thickness that is greater than the probing depth of XPS (∼6−8 nm29). The N 1s core level spectrum of the PVF-QCS-50-0.6 film (inset of Figure 1(c)) can be fitted with a lower binding energy peak at 399.5 eV and a higher binding energy peak at 402 eV corresponding to N−H and N+ species, respectively,18 further confirming the presence of the QCS coating. The degree of quaternization of the QCS coating as calculated from the N+/total N peak area ratio was 53%. The O 1s peak in Figure 1(c) has higher intensity compared to Figure 1(b) due to the presence of oxygen atoms associated with QCS. XPS analysis was also performed for QCS-coated SS foil to ensure successful immobilization of QCS. The wide-scan spectra of pristine SS and SS-QCS-50-0.6 foil surfaces are shown in Figure S3. Since the Fe 2p peak of stainless steel overlaps strongly with the Ni LMM auger peak, the Cr 2p and Ni 2p peaks were monitored before and after the coating process. These two elements (Cr and Ni) are present in significant quantities in SS (type 304). Peaks at ∼576 eV and ∼853 eV corresponding to Cr 2p30 and Ni 2p,31 respectively, can be observed in the wide-scan spectrum of the pristine SS surface (Figure S3(a)). The wide-scan spectrum of the SSQCS-50-0.6 foil surface (Figure S3(b)) shows the appearance of the N 1s peak at ∼400 eV and a very substantial reduction in the intensity of the Cr 2p and Ni 2p peaks as compared to the pristine SS spectrum. The degree of quaternization as obtained from the N 1s spectrum (inset of Figure S3(b)) was calculated to be 52%, which is similar to that of the PVF-QCS-50-0.6 film surface. Thus, our coating method is applicable to both polymeric and metal substrates. In addition, XPS wide-scan and N 1s spectra of different QCS-coated PVF films cross-linked with PP and HMP were also obtained, and the spectra of the PVF-QCS-50-HMP-2.0 film surface are shown in Figure S4. The degree of quaternization for the PVF-QCS-50-HMP-2.0 film surface was calculated to be 54%, which is also similar to that of the PVF-QCS-50-0.6 film. The zeta potentials of PVF-TPP-0.6 and PVF-QCS-50-0.6 surfaces in DI water were measured to be −55 and 30 mV, respectively, consistent with a negatively charged TPP-coated surface and positively charged surface after QCS immobilization. The water contact angle of all QCS-coated substrates ranged from 40 to 44° (Table 1), while those of pristine PVF and pristine SS were 69° and 78°, respectively. The change in the zeta potential and water contact angle of the substrates before and after coating also provided evidence of successful immobilization of QCS. Effect of QCS Coating Conditions on Antibacterial Efficacy. In the preparation of QCS coatings, PVF was chosen as the model substrate for most of the experiments because of its optical transparency, excellent durability, and heat-resistance properties. PVF film is commonly used as a protective film for industrial products and architectural structures, making it a good candidate substrate for testing the antibacterial coatings. The other model substrate used was SS, as it is widely used in clinical, household, and industrial settings such as door handles, hand-wash faucets, machinery parts, and food-handling equipment, rendering it prone to bacterial contamination.32 In this work, the droplet assay was designed to simulate transmission of pathogenic microorganisms via infectious droplets on inanimate surfaces. These infectious droplets can

water-based, and by avoiding the use of organic solvents, the process is more environmentally friendly. Confirmation of the successful preparation of QCS-coated PVF films was carried out using SEM and XPS analyses. The SEM image of the cross-section of the PVF-QCS-50-0.6 film is shown in Figure 1(a). The presence of a ∼12 μm thick coating

Figure 1. (a) SEM image of the cross-section of the PVF-QCS-50-0.6 film showing a QCS coating over a PVF substrate, (b) XPS wide-scan spectrum of a pristine PVF film, and (c) XPS wide-scan spectrum of a PVF-QCS-50-0.6 film with an N 1s core level spectrum in the inset.

(indicated by the arrows in the figure) on the substrate indicates successful immobilization of QCS on PVF. The presence of QCS was further confirmed from the XPS spectra. The wide-scan spectrum of pristine PVF in Figure 1(b) shows an intense F 1s signal at ∼685 eV, which is associated with the fluorine atoms in the polymer. F Auger peaks can also be observed at 830 (KL1), 856 (KL2), and 875 (KL3) eV.26 The peak at 530 eV corresponds to O 1s, and although PVF does not contain oxygen atoms, the presence of the O 1s peak in the spectrum of clean PVF samples has also been reported in earlier studies.27,28 No N 1s peak was observed in Figure 1(b), as PVF E

DOI: 10.1021/acs.iecr.6b02201 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research

films showed antibacterial efficacy of 91%, 95%, and 92%, respectively. There was no significant difference (p > 0.05) in the antibacterial efficacy of these three films prepared at different cross-linking temperatures (25, 50, and 70 °C). XPS analyses of PVF-QCS-25-1.0, PVF-QCS-50-1.0, and PVF-QCS70-1.0 films also confirmed that the [N+]:[C] ratios of these films are similar (p > 0.05) (Table S1). As such, for subsequent QCS coatings, the cross-linking temperature was set at 50 °C. Figure 2(b) shows the droplet assay results of QCS-coated PVF films prepared with different concentrations of TPP crosslinker (0.3, 0.6, 1.0, 1.4, 2.0, and 2.5 wt %). As can be seen in Figure 2(b), QCS-coated PVF films cross-linked with 0.3, 0.6, 1.0, 1.4, and 2.0 wt % TPP, namely PVF-QCS-50-0.3, PVFQCS-50-0.6, PVF-QCS-50-1.0, PVF-QCS-50-1.4, and PVFQCS-50-2.0, respectively, exhibited antibacterial efficacy of 93− 95%. There was no significant difference (p > 0.05) in the antibacterial efficacy among these QCS-coated films. However, the PVF-QCS-50-2.5 film (prepared with 2.5 wt % TPP) could only achieve a 86% reduction in CFU count as compared to pristine PVF, which is significantly lower than those of other QCS-coated films. Since TPP acts as the anionic cross-linker for QCS, a high TPP concentration may increase cross-linking to the extent that the amount of free quaternary ammonium groups available on the QCS-coated surface is significantly decreased. As a result, the bactericidal activity of QCS may decrease under this condition. Besides TPP, QCS-coated substrates were also prepared with PP and HMP. The antibacterial efficacy of these films (shown in Figure S5) varied from 80% (PVF-QCS-50-PP-0.5) to 98% (PVF-QCS-50-HMP-2.0). This range of efficacy is quite similar to that obtained with TPP as cross-linker (Figure 2(b)). Thus, multivalent phosphate salts in general are suitable as anionic anchors to cross-link with QCS to form an antibacterial coating. However, as shown below, the coatings prepared at the same conditions with different anionic cross-linkers may not have the same degree of stability and for each anionic cross-linker; the preparation conditions have to be optimized to achieve high stability and antibacterial efficacy. To further illustrate the flexibility of this method of conferring surfaces with a QCS coating, SS foil was modified with QCS in a similar manner as PVF. The antibacterial efficacy of the QCS-coated SS foil (SSQCS-50-0.6) was 93% against S. aureus (Figure 3), which is not significantly different from that of the QCS-coated PVF film prepared under similar conditions (PVF-QCS-50-0.6, Figure 2(b)). This result is consistent with the XPS analyses, which

be generated from coughing and sneezing or other activities, such as wet cleaning of contaminated indoor surfaces or atomization of contaminated tap/shower water.33 For the droplet assay, 1 × 1 cm2 surfaces (pristine and QCS-coated) were contaminated with ∼103 bacterial cells. This bacterial loading was chosen, as earlier studies on bacterial contamination in hospitals and clinics reported typical bacterial counts ranging from 1 to 103 CFU/cm2.33,34 The antibacterial efficacy also depends on the contact time between the bacteria and the antibacterial surfaces, and our preliminary experiments showed that 2 h of contact was sufficient to result in high antibacterial efficacy (data not shown). S. aureus was used as the model bacteria in most of the experiments, as it is a virulent pathogen that is very commonly found in clinical settings.34,35 Optimization of the conditions (cross-linking temperature and duration, and concentration of TPP cross-linker) for preparation of the QCS-coated PVF films was carried out by evaluating the antibacterial efficacy of coated films using the droplet assay. The CFU count in a 1 μL droplet of a bacterial suspension in PBS at 25 °C was taken as the control for the droplet assays. Figure 2(a) shows the results obtained with the

Figure 2. Quantitative count of viable S. aureus from droplet assay on (a) QCS-coated PVF films cross-linked at different temperatures and (b) QCS-coated PVF films cross-linked with different TPP concentrations before (as-prepared) and after being subjected to 30 wipes with DI water. Control is the number of CFU in a 1 μL droplet of suspension. The wiping experiments were not carried out for Film H. * Significant difference (p = 0.05 for (a) and p < 0.05 for (b)) between pristine PVF and control. # Significant difference (p < 0.05) as compared to pristine PVF. $ Significant difference (p < 0.05) between as-prepared PVF-QCS-50-2.5 and PVF-QCS-50-2.0 films in (b). @ Significant difference (p < 0.05) between films before and after wiping with DI water in (b).

pristine and QCS-coated PVF films prepared at different crosslinking temperatures. It can be seen that the CFU count on the pristine PVF surface was lower as compared to the control (p = 0.05), which can be attributed to the natural death of S. aureus in the absence of nutrients over the course of the 2 h incubation period.36 For the QCS-coated PVF films, it was observed that the PVF-QCS-25-1.0, PVF-QCS-50-1.0, and PVF-QCS-70-1.0

Figure 3. Quantitative count of viable S. aureus and P. aeruginosa from droplet assay on SS-QCS-50-0.6 and pristine SS. Control is the number of CFU in a 1 μL droplet of suspension. * Significant difference (p < 0.05) between pristine SS and control. # Significant difference (p < 0.05) between QCS-coated and pristine SS. F

DOI: 10.1021/acs.iecr.6b02201 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research

Figure 4. Fluorescence microscopy images of S. aureus stained by SYTO 9 (a, c) and PI (b, d) on pristine PVF (a, b) and PVF-QCS-50-0.6 film (c, d) after contact assay.

show that both PVF-QCS-50-0.6 film and SS-QCS-50-0.6 foil surfaces have similar [N+]:[C] ratios (Table S1). Stability of QCS Coatings. The ability of antibacterial coatings to withstand cleaning and wiping is crucial because most surfaces within bacteria-burdened environments such as hospitals and clinics are subjected to a daily cleaning routine.37 To assess the stability of the prepared coatings, QCS-coated PVF films prepared with 0.3−2.0 wt % TPP were wiped 30 times with a DI water-wetted cloth and then subjected to the droplet assay. The films were allowed to dry completely at room temperature between consecutive wipes. This study was carried out to simulate the effect of a 30-day daily cleaning routine on the antibacterial efficacy of the QCS-coated PVF films. As shown in Figure 2(b), most QCS-coated PVF films maintained their antibacterial efficacy after the wiping routine. The exception to the above was the PVF-QCS-50-0.3 film, which showed a slight but significant increase in CFU count after wiping. The [N+]:[C] ratios of the PVF-QCS-50-0.6 film before and after the wiping routine with DI water were compared and found to be similar (Table S1). These results indicated that with sufficient TPP on the substrate surface to cross-link with the QCS, a stable QCS coating can be achieved. The wiping routine was also carried out 30 times with 70% ethanol instead of DI water and the surface composition of the wiped films remained unaltered (Table S1), indicating the stability of the QCS-coated films to ethanol as well. However, the same degree of stability was not observed with PVF-QCS50-HMP-2.0 and PVF-QCS-50-PP-1.0 films. The water contact angle of these films after the wiping routine with water increased to ∼61° (from 44° and 42°, respectively before wiping), indicating some loss of the QCS coating. The lower stability of QCS-coated films cross-linked with PP is consistent

with earlier findings that association of CS with PP is weaker than with TPP.38,39 Although there are no reports comparing the electrostatic complexation of HMP and TPP with CS or QCS, the lower stability of QCS-coated films cross-linked with HMP may be due to a weaker association of HMP with QCS because of the cyclic structure of HMP.40 It is possible that the stability of the QCS-coated films cross-linked with HMP and PP can be improved by changing the coating conditions. Nonetheless, the remaining tests were carried out with PVFQCS-50-0.6 surfaces. Efficacy of QCS-Coated PVF Films against Bacterial Transmission via “Contact”. The transmission of bacteria from contaminated inanimate surfaces to healthy humans via direct physical contact is another major route, especially in a bacteria-burdened environment such as health-care units and hospitals.33 Health-care workers’ hands/gloves have been shown to become contaminated after touching bacteria-loaded inanimate surfaces inside a patient’s room.41 To test whether the QCS-coated surface can inhibit this mode of transmission, a contact assay was carried out by pressing a S. aureus-loaded dry surface against pristine PVF and QCS-coated PVF films. The contact force in this experiment was fixed at 0.3 N, which is within the range of normal touch force of adults42 to simulate bacterial transmission upon touch by an average adult human finger, and the duration of contact was fixed at 5 s. After a 2 h incubation period at 25 °C, the proportion of live and dead bacteria on the film surface was qualitatively assessed by staining with a combination dye. Figure 4 shows the fluorescence microscopy images of these films where viable bacteria and dead or membrane-compromised bacteria appear green and red, respectively. On a pristine PVF film, most bacteria were observed to be green (Figure 4(a)) and a few G

DOI: 10.1021/acs.iecr.6b02201 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research were stained red (Figure 4(b)), indicating that most of the surface-adhered bacteria were viable even after 2 h of incubation under dry conditions. On the other hand, only a few bacteria on the PVF-QCS-50-0.6 film were observed to be green (Figure 4(c)) and most of the bacteria appeared red (Figure 4(d)), indicating that most bacteria on this film were no longer viable. This result is consistent with our previous work, which showed that adherent bacteria on surfaces of bone cement containing QCS nanoparticles were mostly dead or membrane-compromised due to the bactericidal nature of QCS.18 The quantitative spread-plate results from QCS-coated and pristine PVF films are shown in Figure 5. In this

Figure 6. Quantitative count of viable P. aeruginosa on PVF-QCS-500.6 film from droplet and contact assays. Control for the droplet assay is the number of CFU in a 1 μL droplet of suspension, and that for the contact assay is the number of CFU on the membrane surface prior to contact with the films. * Significant difference (p < 0.05) between pristine PVF and control. # Significant difference (p < 0.05) between QCS-coated and pristine PVF.

permeabilizing effect.45 Thus, the QCS coatings can be used effectively against both Gram-positive and Gram-negative bacteria. Reusability of QCS-Coated PVF Films. While the contact-killing characteristic of QCS endows it with high antibacterial efficacy, the accumulation of dead bacteria on the QCS-coated surface (as shown by Figure 4(d)) may eventually lead to loss of efficacy due to cloaking of the quaternary ammonium groups. Since the QCS coating is stable when subjected to wiping as shown in Figure 2(b), it was hypothesized that the accumulated bacteria could be removed by cleaning and the antibacterial efficacy of the QCS coating would be restored. To test this hypothesis, cycles of bacterial contamination by contact assay followed by cleaning with either DI water- or 70% ethanol-wetted cloth were investigated to assess the reusability of the QCS-coated PVF films. Figure 7

Figure 5. Quantitative count of viable S. aureus on PVF-QCS-50-0.6 film from contact assay. Control is the number of CFU on the membrane surface prior to contact with the films. * Significant difference (p < 0.05) between pristine PVF and control. # Significant difference (p < 0.05) between QCS-coated and pristine PVF.

experiment, the CFU count on the dry bacteria-loaded membrane surface (prior to placing it on the pristine or QCS-coated films) was used as the control. It was observed that the CFU count from the pristine PVF film after 2 h of incubation is significantly lower (p < 0.05) compared to the control (Figure 5). This is probably because not all bacteria present on the membrane surface were transferred to the films since dry surfaces have been shown to transfer substantially fewer bacteria as compared to their wet counterparts.43 Furthermore, some bacteria transferred to the surfaces may die in the absence of nutrients, as mentioned above. Figure 5 shows that the antibacterial efficacy of the PVF-QCS-50-0.6 film was 90%, consistent with the qualitative results in Figure 4. The results confirmed that QCS-coated films are able to substantially reduce the transmission of bacteria under dry conditions as well as via droplets. The antibacterial efficacy of QCS-coated PVF films was further evaluated using Gram-negative P. aeruginosa. P. aeruginosa is frequently found on inanimate surfaces within hospitals and health-care centers, and the survival period of P. aeruginosa on dry inanimate surfaces can be up to 16 months compared to 7 months for S. aureus, indicating longer persistence of P. aeruginosa.44 The results for antibacterial efficacy of PVF-QCS-50-0.6 film against P. aeruginosa are shown in Figure 6. In both the droplet and contact assays, the antibacterial efficacy was ∼90%, which is similar to the efficacy observed against Gram-positive S. aureus (Figure 2(b) and Figure 5). QCS-coated SS also exhibited 91% efficacy against P. aeruginosa, as shown in Figure 3. Although the outer membrane of Gram-negative bacteria acts as a barrier to macromolecules, polycationic polymers such as CS have been previously shown to disrupt the outer membrane of Gram-negative bacteria via a

Figure 7. Antibacterial efficacy of PVF-QCS-50-0.6 films after repeated cycles of contamination via contact and cleaning. The PVF-QCS-500.6 film was cleaned with 70% ethanol-wetted cloth after each contact assay.

shows that, over five cycles of contamination followed by cleaning with 70% ethanol, the antibacterial efficacy of the PVFQCS-50-0.6 film was maintained at ∼90%. A comparison of the fluorescence microscopy images of the contaminated PVFQCS-50-0.6 film before and after cleaning with 70% ethanol showed that almost all adherent bacteria (live as well as dead) were removed after the cleaning process (Figure S6). Spreadplate counting confirmed that, after the contaminated surfaces were cleaned with 70% ethanol in each cycle, no CFU was observed. On the other hand, when DI water was used instead of 70% ethanol in the cleaning process, the antibacterial efficacy was reduced to 87% after the first cycle (Figure S7) and to 54% after the fifth cycle, which is significantly (p < 0.05) lower than H

DOI: 10.1021/acs.iecr.6b02201 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research that of the as-prepared film (90%). More viable as well as dead bacteria remained on the DI water-cleaned surfaces (Figure S6(e) and S6(f)) as compared to the 70% ethanol-cleaned ones (Figure S6(c) and S6(d)). Thus, DI water may not be an effective medium for cleaning the QCS-coated surface, whereas, with 70% ethanol, the accumulated bacterial cells can be readily removed to restore the antibacterial efficacy of the QCS coating. Cytotoxicity of QCS-Coated and TPP-Coated PVF Films. Cytotoxicity of TPP-coated PVF films was first evaluated using the MTT assay, and no cytotoxic effects were observed from the PVF-TPP-0.6 film (Figure 8). This result is

surface with high antibacterial efficacy without significant cytotoxic effects. Scale-up of QCS-Coated PVF Film. The results discussed above were obtained with films of 2 × 2 cm2. Since many applications of antibacterial coatings call for large area surfaces, a preparative procedure that is facile and easily scalable would be highly desirable. A spray coating technique was employed herein to demonstrate the scalability of the coating process. QCS was coated on PVF films measuring 15 × 20 cm2 using a semiautomatic spray coating robot equipped with an airatomizing spray nozzle (Figure 9(a)). The spray coating parameters, air-atomizing pressure, nozzle-substrate separation distance, nozzle speed (coating speed), and flow rate, were adjusted to obtain a spray-coated PVF-QCS-50-0.6 film with a similar water contact angle as that of the manually coated PVFQCS-50-0.6 films. A comparison of the visual appearance of the spray-coated PVF-QCS-50-0.6 film and the pristine PVF film is shown in Figure 9(b). The transparency of the PVF film was retained after spray coating and, hence, such spray-coated films can be used as antibacterial coatings on computer or touch screens and equipment panels where transparency is critical. The thickness of the coating on a spray-coated PVF-QCS-500.6 film was determined to be ∼8 μm from the SEM image in Figure 10(a), which is thinner than that of the manually coated PVF-QCS-50-0.6 film (∼12 μm, Figure 1(a)). The SEM image also shows that the coating adhered well to the PVF substrate. Since uniformity of coatings is an important aspect upon scaleup, the uniformity of the spray-coated PVF-QCS-50-0.6 film was tested by analyzing the antibacterial efficacy and surface hydrophilicity at different locations on the film surface. The antibacterial efficacy of the spray-coated PVF-QCS-50-0.6 film was determined by performing the droplet assay against S. aureus on six 1 × 1 cm2 samples, cut out from random areas of the 15 × 20 cm2 film. The results obtained (Figure 10(b)) show that the mean reduction in CFU count on the spraycoated PVF-QCS-50-0.6 film was 92% (with standard deviation of 2%) compared to pristine PVF, similar to that of the manually coated films. The water contact angle measured at 12 random locations was found to be similar at 43 ± 3°. These results indicate that QCS was uniformly coated on the spraycoated PVF-QCS-50-0.6 film. Hence, spray coating would be an efficient, fast, and reliable method for the preparation of QCS coatings via electrostatic interactions with surface-immobilized TPP on larger substrates.

Figure 8. Viability of 3T3 fibroblast cells incubated with pristine PVF, PVF-TPP-0.6, or PVF-QCS-50-0.6 films expressed as percentage relative to the negative control (cells incubated in medium without any film).

expected since the US Food and Drug Administration lists TPP as a “generally regarded as safe” compound46 and TPP is also an ingredient in commercially available detergents and food preservatives. The cytotoxicity of QCS-coated PVF films was then evaluated, and the results are shown in Figure 8. The viability of fibroblast cells on the QCS-coated PVF film (PVFQCS-50-0.6) was not significantly different from that in the negative control, where cells were incubated in medium without any film. The lack of cytotoxicity of PVF-QCS-50-0.6 film is also expected since CS, a naturally derived polymer, is known for its biocompatibility, and it has gained FDA approval for use in bandages and wound dressings.14,17 Derivatives of CS containing quaternary ammonium groups have also been previously demonstrated to be biocompatible and noncytotoxic.18,19,47 Thus, the QCS coating prepared via electrostatic interactions with surface-immobilized TPP endows the

Figure 9. Photograph of (a) the semiautomatic spray-coater used for preparing a spray-coated PVF-QCS-50-0.6 film, and (b) spray-coated PVFQCS-50-0.6 and pristine PVF films placed over a white sheet with text for comparison of transparency. I

DOI: 10.1021/acs.iecr.6b02201 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research

Figure 10. (a) SEM image of the cross-section of a spray-coated PVF-QCS-50-0.6 film and (b) quantitative count of viable S. aureus after droplet assay on spray-coated PVF-QCS-50-0.6 and pristine PVF films. Control is the number of CFU in a 1 μL droplet of suspension. * Significant difference (p < 0.05) between pristine PVF and control. # Significant difference (p < 0.05) between QCS-coated and pristine PVF.

■

■

CONCLUSION This work demonstrates a facile and environmentally friendly strategy to immobilize QCS on PVF and SS substrates via electrostatic interaction with multivalent polyphosphate ions anchored on the substrate. QCS coatings of micron thickness prepared with 5 wt % QCS, 0.6 wt % TPP at 50 °C were found to have optimal coating performance in terms of antibacterial efficacy and stability. The QCS coating reduced the CFU count on surfaces in contact with bacteria-laden droplets or bacteriacontaminated objects by ∼90% as compared to the unmodified surfaces for both Gram-positive S. aureus and Gram-negative P. aeruginosa. The coating is highly stable to wiping, and by using 70% ethanol to wipe the contaminated surfaces, the antibacterial efficacy can be restored. The coating process can be easily scaled up using a spray coating technique, and QCScoated films of 15 × 20 cm2 prepared using this method exhibited transparency, uniformity, and favorable antibacterial efficacy. Thus, the coating principle employed in this work shows good promise for preparing large-scale, stable, antibacterial coatings for reducing surface bacterial burden and inhibiting transmission of pathogens.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.6b02201. Synthesis and characterization of QCS, additional XPS spectra of QCS coated substrates, antibacterial efficacy of QCS-coated SS and QCS-coated PVF cross-linked with PP, HMP, reusability of QCS-coated PVF when cleaned with water, and [N+]:[C] ratios of different coated substrates (PDF)

■

REFERENCES

(1) Dutta, P. K.; Tripathi, S.; Mehrotra, G. K.; Dutta, J. Perspectives for chitosan based antimicrobial films in food applications. Food Chem. 2009, 114, 1173−1182. (2) Weinstein, R. A.; Darouiche, R. O. Device-Associated Infections: a macroproblem that starts with microadherence. Clin. Infect. Dis. 2001, 33, 1567−1572. (3) Cloutier, M.; Mantovani, D.; Rosei, F. Antibacterial coatings: challenges, perspectives, and opportunities. Trends Biotechnol. 2015, 33, 637−652. (4) Sanborn, L. W. R. The relation of surface contamination to the transmission of disease. Am. J. Public Health Nations Health 1963, 53, 1278−1283. (5) 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. (6) Li, M.; Neoh, K.-G.; Kang, E.-T.; Lau, T.; Chiong, E. Surface modification of silicone with covalently immobilized and crosslinked agarose for potential application in the inhibition of infection and omental wrapping. Adv. Funct. Mater. 2014, 24, 1631−1643. (7) Tiller, J. C.; Liao, C. J.; Lewis, K.; Klibanov, A. M. Designing surfaces that kill bacteria on contact. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 5981−5985. (8) Kalinov, K.; Ignatova, M.; Maximova, V.; Rashkov, I.; Manolova, N. Modification of electrospun poly(ε-caprolactone) mats by formation of a polyelectrolyte complex between poly(acrylic acid) and quaternized chitosan for tuning of their antibacterial properties. Eur. Polym. J. 2014, 50, 18−29. (9) Campoccia, D.; Montanaro, L.; Arciola, C. R. A review of the biomaterials technologies for infection-resistant surfaces. Biomaterials 2013, 34, 8533−8554. (10) Huh, M. W.; Kang, I.-K.; Lee, D. H.; Kim, W. S.; Lee, D. H.; Park, L. S.; Min, K. E.; Seo, K. H. Surface characterization and antibacterial activity of chitosan-grafted poly(ethylene terephthalate) prepared by plasma glow discharge. J. Appl. Polym. Sci. 2001, 81, 2769−2778. (11) Hasan, J.; Crawford, R. J.; Ivanova, E. P. Antibacterial surfaces: the quest for a new generation of biomaterials. Trends Biotechnol. 2013, 31, 295−304. (12) Kugel, A.; Stafslien, S.; Chisholm, B. J. Antimicrobial coatings produced by “tethering” biocides to the coating matrix: a comprehensive review. Prog. Org. Coat. 2011, 72, 222−252. (13) Madkour, A. E.; Tew, G. N. Towards self-sterilizing medical devices: Controlling infection. Polym. Int. 2008, 57, 6−10. (14) Rabea, E. I.; Badawy, M. E.-T.; Stevens, C. V.; Smagghe, G.; Steurbaut, W. Chitosan as antimicrobial agent: applications and mode of action. Biomacromolecules 2003, 4, 1457−1465. (15) Liu, H.; Gao, C. Preparation and properties of ionically crosslinked chitosan nanoparticles. Polym. Adv. Technol. 2009, 20, 613−619.

AUTHOR INFORMATION

Corresponding Author

*Tel: +65 65162176; Fax: +65 67791936; Email: chenkg@nus. edu.sg. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was financially supported by the National University of Singapore (grant number R279000416112). J

DOI: 10.1021/acs.iecr.6b02201 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research (16) Chua, P. H.; Neoh, K. G.; Shi, Z.; Kang, E. T. Structural stability and bioapplicability assessment of hyaluronic acid-chitosan polyelectrolyte multilayers on titanium substrates. J. Biomed. Mater. Res., Part A 2008, 87, 1061−1074. (17) Dai, T.; Tanaka, M.; Huang, Y. Y.; Hamblin, M. R. Chitosan preparations for wounds and burns: antimicrobial and wound-healing effects. Expert Rev. Anti-Infect. Ther. 2011, 9, 857−879. (18) Shi, Z.; Neoh, K. G.; Kang, E. T.; Wang, W. Antibacterial and mechanical properties of bone cement impregnated with chitosan nanoparticles. Biomaterials 2006, 27, 2440−2449. (19) Tan, H.; Ma, R.; Lin, C.; Liu, Z.; Tang, T. Quaternized chitosan as an antimicrobial agent: antimicrobial activity, mechanism of action and biomedical applications in orthopedics. Int. J. Mol. Sci. 2013, 14, 1854−1869. (20) Kong, M.; Chen, X. G.; Xing, K.; Park, H. J. Antimicrobial properties of chitosan and mode of action: a state of the art review. Int. J. Food Microbiol. 2010, 144, 51−63. (21) Cai, Y.; Lapitsky, Y. Formation and dissolution of chitosan/ pyrophosphate nanoparticles: is the ionic crosslinking of chitosan reversible? Colloids Surf., B 2014, 115, 100−108. (22) Angelova, N.; Hunkeler, D. Effect of preparation conditions on properties and permeability of chitosan−sodium hexametaphosphate capsules. J. Biomater. Sci., Polym. Ed. 2001, 12, 1317−1337. (23) Vandencasteele, N.; Merche, D.; Reniers, F. XPS and contact angle study of N2 and O2 plasma-modified PTFE, PVDF and PVF surfaces. Surf. Interface Anal. 2006, 38, 526−530. (24) Adden, N.; Gamble, L. J.; Castner, D. G.; Hoffmann, A.; Gross, G.; Menzel, H. Phosphonic acid monolayers for binding of bioactive molecules to titanium surfaces. Langmuir 2006, 22, 8197−8204. (25) Lim, M. S.; Smiley, K. J.; Gawalt, E. S. Thermally driven stability of octadecylphosphonic acid thin films grown on SS316L. Scanning 2010, 32, 304−311. (26) Li, H. Y.; Su, L.; Kuang, S. Y.; Pan, C. F.; Zhu, G.; Wang, Z. L. Significant enhancement of triboelectric charge density by fluorinated surface modification in nanoscale for converting mechanical energy. Adv. Funct. Mater. 2015, 25, 5691−5697. (27) Tanaka, K.; Inomata, T.; Kogoma, M. Improvement in adhesive strength of fluorinated polymer films by atmospheric pressure glow plasma. Thin Solid Films 2001, 386, 217−221. (28) Paynter, R.; Ratner, B. D.; Horbett, T.; Thomas, H. XPS studies on the organization of adsorbed protein films on fluoropolymers. J. Colloid Interface Sci. 1984, 101, 233−245. (29) Xu, Z.; Chen, L.-M.; Yang, G.; Huang, C.-H.; Hou, J.; Wu, Y.; Li, G.; Hsu, C.-S.; Yang, Y. Vertical Phase Separation in poly(3hexylthiophene): fullerene derivative blends and its advantage for inverted structure solar cells. Adv. Funct. Mater. 2009, 19, 1227−1234. (30) Yuan, S. J.; Pehkonen, S. O. Microbiologically influenced corrosion of 304 stainless steel by aerobic Pseudomonas NCIMB 2021 bacteria: AFM and XPS study. Colloids Surf., B 2007, 59, 87−99. (31) Lorang, G.; Da Cunha Belo, M.; Simões, A. M. P.; Ferreira, M. G. S. Chemical composition of passive films on AISI 304 stainless steel. J. Electrochem. Soc. 1994, 141, 3347−3356. (32) Weaver, L.; Michels, H. T.; Keevil, C. W. Survival of Clostridium dif f icile on copper and steel: futuristic options for hospital hygiene. J. Hosp. Infect. 2008, 68, 145−151. (33) Morawska, L. Droplet fate in indoor environments, or can we prevent the spread of infection? Indoor Air 2006, 16, 335−347. (34) Archer, G. L. Staphylococcus aureus: a well-armed pathogen. Clin. Infect. Dis. 1998, 26, 1179−1181. (35) Hedin, G.; Blomkvist, A.; Janson, M.; Lindblom, A. Occurrence of potentially pathogenic bacteria on the hands of hospital patients before and after the introduction of patient hand disinfection. APMIS 2012, 120, 802−807. (36) Liao, C. H.; Shollenberger, L. Survivability and long-term preservation of bacteria in water and in phosphate-buffered saline. Lett. Appl. Microbiol. 2003, 37, 45−50. (37) Dharan, S.; Mourouga, P.; Copin, P.; Bessmer, G.; Tschanz, B.; Pittet, D. Routine disinfection of patients’ environmental surfaces. Myth or reality? J. Hosp. Infect. 1999, 42, 113−117.

(38) Shu, X.; Zhu, K. The influence of multivalent phosphate structure on the properties of ionically cross-linked chitosan films for controlled drug release. Eur. J. Pharm. Biopharm. 2002, 54, 235−243. (39) Huang, Y.; Lapitsky, Y. Determining the colloidal behavior of ionically cross-linked polyelectrolytes with isothermal titration calorimetry. J. Phys. Chem. B 2013, 117, 9548−9557. (40) Wilson, H. R.; Williams, R. J. Electrostatic interactions between organic ions. Part 2.Phosphates with amines. J. Chem. Soc., Faraday Trans. 1 1987, 83, 1885−1892. (41) Boyce, J. M. Environmental contamination makes an important contribution to hospital infection. J. Hosp. Infect. 2007, 65, 50−54. (42) Mascaro, S. A.; Asada, H. H. Measurement of finger posture and three-axis fingertip touch force using fingernail sensors. IEEE Trans. Robot. Autom. 2004, 20, 26−35. (43) Patrick, D. R.; Findon, G.; Miller, T. E. Residual moisture determines the level of touch-contact-associated bacterial transfer following hand washing. Epidemiol. Infect. 1997, 119, 319−325. (44) Kramer, A.; Schwebke, I.; Kampf, G. How long do nosocomial pathogens persist on inanimate surfaces? A systematic review. BMC Infect. Dis. 2006, 6, 130−137. (45) Helander, I. M.; Nurmiaho-Lassila, E. L.; Ahvenainen, R.; Rhoades, J. Roller, S. Chitosan disrupts the barrier properties of the outer membrane of gram-negative bacteria. Int. J. Food Microbiol. 2001, 71, 235−244. (46) Weiner, M. L.; Salminen, W. F.; Larson, P. R.; Barter, R. A.; Kranetz, J. L.; Simon, G. S. Toxicological review of inorganic phosphates. Food Chem. Toxicol. 2001, 39, 759−786. (47) Li, P.; Poon, Y. F.; Li, W.; Zhu, H. Y.; Yeap, S. H.; Cao, Y.; Qi, X.; Zhou, C.; Lamrani, M.; Beuerman, R. W.; Kang, E. T.; Mu, Y.; Li, C. M.; Chang, M. W.; Leong, S. S.; Chan-Park, M. B. A polycationic antimicrobial and biocompatible hydrogel with microbe membrane suctioning ability. Nat. Mater. 2011, 10, 149−156.

K

DOI: 10.1021/acs.iecr.6b02201 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX