Antibiofilm Nitric Oxide-Releasing Polydopamine Coatings - ACS

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Applications of Polymer, Composite, and Coating Materials

Antibiofilm Nitric Oxide Releasing Polydopamine Coatings Zahra Sadrearhami, Farah Nabilah Shafiee, Kitty Ka Kit Ho, Naresh Kumar, Marta Krasowska, Anton Blencowe, Edgar H. H. Wong, and Cyrille Boyer ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b16853 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on February 4, 2019

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ACS Applied Materials & Interfaces

Antibiofilm Nitric Oxide Releasing Polydopamine Coatings Zahra Sadrearhami,a,b Farah Nabilah Shafiee,a,b Kitty K. K. Ho,b,c Naresh Kumar,b,c Marta Krasowska,d,e Anton Blencowe,d,f Edgar H. H. Wong*a,b and Cyrille Boyer*a,b

a

Centre for Advanced Macromolecular Design (CAMD), School of Chemical Engineering, UNSW

Australia, Sydney, NSW 2052, Australia. b

Australian Centre for NanoMedicine (ACN), School of Chemical Engineering, UNSW Australia, Sydney,

NSW 2052, Australia c

School of Chemistry, UNSW Australia, Sydney, NSW 2052, Australia

d Future e

Industries Institute, The University of South Australia, Mawson Lakes, SA 5095, Australia

School of Information Technology and Mathematical Sciences, University of South Australia, Mawson

Lakes Campus, Mawson Lakes, SA 5095, Australia. f

School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia

*Corresponding authors: [email protected]; [email protected]

Abstract The growing number of patient morbidity related to nosocomial infections has placed an importance on the development of new antibacterial coatings for medical devices. Here, we utilize the versatile adhesion property of polydopamine (pDA) to design an antibacterial coating that possesses low-fouling and nitric oxide (NO) releasing capabilities. To demonstrate this, glass substrates were functionalized with pDA via immersion in alkaline aqueous solution containing dopamine, followed by the grafting of low-fouling polymer (polyethylene glycol (PEG)) via Michael addition, and subsequent formation of N-diazeniumdiolate functionalities (NO precursors) by purging with NO gas. X-ray photoelectron spectroscopy (XPS) confirmed the successful grafting of PEG and formation of Ndiazeniumdiolate on polydopamine-coated substrates. NO release from the coating was observed over two days and the NO loading is tuneable by the pDA film thickness. The antibacterial efficiency of the coatings was assessed using Gram-negative Pseudomonas aeruginosa (i.e., wild type PAO1 and multidrug-resistant PA37) and Gram-positive Staphylococcus aureus (ATCC 29213). The NO releasing PEGylated pDA film inhibited biofilm attachment by 96% and 70% after exposure to bacterial culture solution for 24 and 36 h, respectively. In contrast, films that do not contain ACS Paragon Plus Environment

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NO failed to prevent biofilm formation on the surfaces at these time points. Furthermore, this coating also showed 99.9%, 97% and 99% killing efficiency against surface-attached PAO1, PA37 and S. aureus bacteria. Overall, the combination of low-fouling PEG and antibacterial activity of NO in pDA films makes this coating a potential therapeutic option to inhibit biofilm formation on medical devices.

Keywords: Polydopamine, Antimicrobial Coating, Nitric oxide, Biofilm, Multidrug-resistant Bacteria, Surface modification

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Introduction Infectious diseases caused by the formation of bacterial biofilms at the site of implanted medical devices such as catheters, artificial prosthetics, and joints pose significant problem in healthcare.1-2 Implantassociated infections cause human pain as well as a substantial economic burden on society.3 It has been found that significantly higher doses of antibiotics are required for biofilm treatment compared to bacterial suspensions.4 Multidrug-resistant (MDR) bacteria biofilms are even more difficult to treat, as there is a limited number of therapeutic options.5 The decline in effectiveness of available antibiotics on one hand, and the complexity of antibiotic resistance mechanisms on the other, necessitates the development of novel antibiofilm strategies. The initial attachment of bacteria to the surface of implanted medical devices is the first stage in biofilm formation. Thus, the inhibition of bacterial colonisation using antibacterial coatings, particularly during the most susceptible first 6 h period after implantation, is a fundamental strategy to prevent biofilm formation.3, 6-8

Several approaches have been developed recently to reduce microbial adhesion by surface modification

using polymers and can be broadly defined by two strategies.9-15 The first strategy entails the fabrication of low-fouling films using polymers such as polyethylene glycol (PEG) to minimize bacteria cells-substrate surface interactions, hence preventing bacteria adhesion. Another common strategy is to rely on the release of active biocidal agents (e.g., nitric oxide (NO),16-17 antibiotics18-19 and metals20-21) from polymer films to hinder biofilm formation, disperse and/or kill formed biofilms. Coatings that combined both strategies have also been reported as a promising option to combat biofilms.11, 22-23 NO is an endogenously produced molecule that plays key roles in the human body as well as in various bacterial systems.24-25 Nowadays, the use of NO has opened new opportunities in biofilm treatment26-27 as NO exhibits broad-spectrum activity against both Gram-positive and Gram-negative bacteria.28-29 However, the clinical applications of NO gas are hindered by the lack of localized delivery.24 To address this, polymeric nanoparticles,30-33 nanofibers34-35 and thin films36-38 have been used for NO delivery. For example, NO-functionalized polymeric substrates, where NO is released through the decomposition of NO precursors/donors such as N-diazeniumdiolates (NONOate) and S-nitrosothiols, have been shown to induce bacterial dispersal and killing, besides preventing bacterial attachment to surfaces.11,

36-37, 39-42

these advances, the optimization of the NO payload in delivery systems remains challenging. ACS Paragon Plus Environment

In spite of


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Recently, the immobilization of NONOate in polydopamine (pDA)-coated nanoparticles was reported to be an effective NO delivery strategy.43-45

PDA is synthesized via the oxidative self-polymerization of

dopamine and forms adherent thin films on a variety of substrates such as metals, polymers, glass, and ceramics by simple immersion.46 This polymer can be further functionalized with nucleophiles such as amines or thiols via Schiff base or Michael addition reactions, thus overall making pDA highly attractive to be employed as biomedical coatings.47 The formation of NONOate via the secondary amine of polycatecholamines was first reported by Lee’s group where a noticeable amount of NO release (1789 nmol cm-2) was achieved over 50 h with polystyrene particles coated with NONOate-modified polynorepinephrine and pDA.43 However, no biomedical application was investigated. Following that study, NONOatefunctionalized pDA hollow nanoparticles were prepared by template-mediated process and subsequent exposure to high pressure NO.44 The NONOate-functionalized hollow pDA particles released sufficient NO flux for bactericidal activity against planktonic Gram-negative bacteria and showed negligible toxicity due to the biocompatibility of catecholamines and low NO dose. In addition, we have also recently demonstrated the ability of NO releasing pDA-coated iron oxide nanoparticles to disperse (~80%) and kill Pseudomonas aeruginosa biofilms (up to 5-log10 reduction in bacterial cell viability).45 Despite the promising antibacterial activity of NO releasing pDA nanoparticles, only few studies have been reported, including a recent US patent published in 2017.48 In comparison with this recent patent,48 we describe the facile fabrication of new NO releasing pDA coatings for effective minimization of biofilm formation on substrate surfaces. Specifically, pDA films formed on glass or polymer substrates are further functionalized to incorporate low-fouling PEG and NONOate moieties. The introduction of both functionalities into the pDA film hinders the formation of P. aeruginosa biofilms, including an MDR strain, on glass substrate for up to 36 h. Furthermore, the coating not only hinders biofilm formation, but also kills 99.9%, 97% and 99% of any adhered P. aeruginosa PAO1, P. aeruginosa PA37 and Staphylococcus aureus ATCC 29213 bacteria biofilms, respectively. A tuneable NO loading is achieved by changing the pDA film thickness, and the technology described here represents potential utility in the clinical setting.


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Materials and Methods Materials. Dopamine hydrochloride, Griess reagent (modified), methoxy-poly(ethylene glycol)-thiol (mPEG-SH; Mn = 2000 g mol-1), sodium methoxide and tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) were obtained from Sigma-Aldrich and used as received. Methanol, sodium phosphate dibasic (Na2HPO4), potassium phosphate monobasic (KH2PO4), sodium chloride (NaCl), ammonium chloride (NH4Cl), magnesium sulfate (MgSO4), glucose and calcium chloride (CaCl2) were obtained from ChemSupply and used as received. Glass and proprietary polyester Nunc™ Thermanox™ coverslips (13 mm diameter) were obtained from Thermo Fisher Scientific. Milli-Q water with a resistivity of > 18 MΩ⋅cm was obtained using an in-line Millipore RiOs/Origin water purification system. All other chemicals were used as received unless otherwise specified. Analytical Instruments. UV-Vis spectra were recorded using a CARY 300 Bruker spectrophotometer (Varian) equipped with a temperature controller. X-Ray Photoelectron Spectroscopy (XPS) measurements for pDA coatings were performed on a ESCALAB250Xi instrument (Thermo Scientific, UK) with a monochromated Al Kα source (energy 1486 eV) at 120 W over 500 μm at a 90o angle. Background vacuum pressure was 2 × 10–9 mbar. Static contact angle measurements were performed by a sessile drop method using a goniometer (model CAM 200, KSV Instruments LTD, Finland) equipped with a camera (Moticam 5.0). Contact angles were measured using 10 μL drops of Milli-Q water on the unmodified and coated glass coverslips. A minimum of four advancing water contact angle measurements were taken for each substrate. Angle analysis of captured droplets was performed with ImageJ software v1.51 using the tangent fitting. Atomic force microscopy (AFM) images of films on glass coverslips were acquired with a MultiMode 8 (Bruker, USA) instrument equipped with Nanoscope V controller and a vertical engagement scanner “E” (maximum scan range 10 μm in the x and y directions, and nominal 2.5 μm in the normal to the z direction) to assess morphological characteristics of coated substrates as well as the film thickness. 2 × 2 m2 and 5 × 5 m2 scans were collected in air and in phosphate-buffered saline (PBS) solution in PeakForce Tapping® mode. For the measurements in air SCANASYST-AIR (Bruker) SiN cantilevers (nominal resonance frequency of 70 kHz, nominal spring constant 0.4 N m-1, a tip of a nominal diameter of 2 nm) were used, while for the measurements in PBS SCANASYST-FLUID+ (nominal resonance frequency of 150 kHz, ACS Paragon Plus Environment


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nominal spring constant 0.7 N m-1, a tip of a nominal diameter of 2 nm) were utilized. Image processing and surface roughness analysis were performed using the NanoScope Analysis v1.7 and WSxM v5.049 software packages. Film thicknesses were estimated by film scratching (mechanical removal) and by imaging a profile along the film and the scratched zone. The height histograms of the remaining film were used to calculate the average film thickness. The layer structure and film thickness were also characterized using transmission electron microscopy (TEM). Samples were prepared by focused ion beam (FIB) lift-out technique using FEI xT Nova NanoLab 200. Layers of Au and Pt were deposited on the area of interest to protect the specimen during the FIB wedge milling. Samples were transferred onto a formvar-coated copper grid and the TEM analysis was performed with a FEI Tecnai G2 20 FEG transmission microscope operating at 200 kV. The high-resolution images were used for quantifying the thickness of coatings using ImageJ software v1.51. Preparation of pDA coatings. The glass and polyester coverslips were placed into a 6-well plate and 2step or 5-step pDA coatings were prepared as described in the literature.46 Briefly, for the first step coating, pDA was deposited on the surface by immersing the substrates in a 5 mL solution of 2 mg mL-1 dopamine (10 mM Tris buffer, pH 8.5) at 37 oC for 18 h. The substrates were sonicated 3 times, each time for 5 min in water and rinsed thoroughly to remove the weakly bonded dopamine. The second step of pDA coating was formed by repeating the incubation, sonication, and rinsing steps. 5-step pDA was obtained by repeating the abovementioned procedure. Grafting of PEG onto pDA coatings. 5-step pDA coatings were covered with 2 mL of 0.5 mM mPEGSH in 10 mM Tris buffer, pH 8.5 and incubated in a shaker at 50 oC for 6 h. The formed 5S-PEG surface was sonicated and washed with water before being dried overnight. Synthesis of NONOate-functionalized coatings. The pDA-based films were placed in a Parr apparatus containing 0.08 M sodium methoxide in methanol. The apparatus was purged and evacuated with N2 three times, followed by charging with excess NO gas (25 °C, 5 atm). After 48 h, the excess NO was vented by purging with N2 and the resultant NO releasing substrates were dried overnight and stored at -20 °C. Determination of in vitro NO release using Griess assay. NO released from the NO releasing substrates at specific time points were determined using standard Griess reagent kit.16,

50-53

The coated glass or

polyester coverslip was transferred into a 24-well plate (one coverslip per well) and 1 mL of PBS buffer ACS Paragon Plus Environment

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solution (pH 7.4) was added to each well and the plate was placed in an incubator at 37 °C. At various time points, 50 µL of the incubated PBS solution was taken for analysis and 50 µL of fresh PBS was added back into the well. The aliquot was mixed with 50 µL of Griess reagent and incubated at room temperature for 15 min. The sample was then diluted with Milli-Q water and the UV-Vis absorbance of the resulting solution was measured at 540 nm. The measurements were also repeated in the same manner in M9 minimal medium. The NO concentration in the sample solution was then calculated from a standard curve and converted to a cumulative NO release profile. Biofilm inhibition study. The Gram-negative bacteria strains P. aeruginosa PAO1 and PA37 were used to characterize the biofilm inhibition effects of the coatings. A single colony of PAO1 or PA37 was cultured overnight in 10 mL of Luria Bertani (LB) medium at 37 oC. The overnight cultures were then diluted 1:200 in freshly prepared M9 minimal medium (containing 48 mM Na2HPO4, 22 mM KH2PO4, 9 mM NaCl, 19 mM NH4Cl, 2 mM MgSO4, 20 mM glucose and 100 mM CaCl2, pH 7.0). The suspension was then aliquoted (1 mL per well) into 24-well plates (Costar, Corning®) in the presence of the (coated) glass coverslips (one sample per well). The plates were incubated at 37 oC in an orbital shaker (model OM11, Ratek, Boronia, Australia) with shaking at 180 rpm which does not stop agitation when the door is opened, and the glass coverslips were exposed to the bacteria culture for 6, 24 and 36 h.54 The medium was replaced with fresh M9 every 12 h to provide enough nutrient for biofilm growth. The well surface was washed twice with 1 mL of PBS to remove loosely attached biofilm cells. Crystal violet (CV) staining is typically used to evaluate biofilm attachment,30 however, this method was unsuitable for quantifying the bacterial attachment on coatings because of the absorption of CV by the pDA film. Therefore, the coverslips were mounted on glass microscope slides using PBS as the media and imaged with a tomographic microscope (3D Cell Explorer, NanoLive, Lausanne, Switzerland) equipped with a digital staining software to determine biofilm attachment on the coverslips. Images were taken with the best focus from different spots and all experiments were repeated in at least two independent experiments. Killing study. To characterize the bactericidal activity, PAO1 and PA37 biofilms were grown in the presence of coatings in the same way as abovementioned. For Staphylococcus aureus ATCC 29213 strain, a single colony of S. aureus was cultured overnight in 10 mL of tryptone soya broth (TSB) medium at 37 oC. The overnight culture was then diluted 1:1000 in TSB and the suspension was aliquoted (1 mL per well) into ACS Paragon Plus Environment


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24-well plates in the presence of the (coated) glass coverslips (one sample per well). The plates were incubated at 37 oC for 8 h. The biofilm viability analysis was determined by a drop plate method. The surface of the coverslip (surface area 1.33 cm2) was washed twice with sterile PBS to remove loosely attached bacteria, before being transferred to a clean well. The biofilm was then suspended and homogenized in 1 mL sterile PBS by ultrasonication (150 W, 40 kHz; Unisonics, Australia) for 20 min. Resuspended biofilm cells were serially diluted in sterile PBS and plated onto LB agar. Biofilm colonies were counted and biofilm colony forming unit (CFU) was calculated after overnight incubation at 37 oC. All experiments were repeated in at least three independent experiments Statistical Analysis. A one-way analysis of variance (ANOVA) function in Prism 7 (GraphPad Software) was used to statistically analyze and compare the treated samples to the control as well as comparing between treatments.


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Results and Discussion Preparation of Polydopamine Coatings Functionalized with PEG and NONOate. The versatility of pDA to form uniform films on a variety of substrate via simple dip coating or immersion techniques and the ability to further post-functionalize pDA films46,

55-56

provide the key motivations for us to utilize and

develop tailored pDA coatings for antibiofilm applications. The approach taken here to make such coatings is simple and efficient (Scheme 1). Firstly, pDA films were deposited onto glass coverslips by simply immersing in a 5 mL solution of 2 mg mL-1 dopamine (10 mM Tris buffer, pH 8.5) at 37 oC overnight. This deposition step was repeated once and four times more to produce uniform 2-step and 5-step coatings, respectively (hence the samples are referred to as 2S and 5S) (Figure S1, Supporting Information (SI)). Next, methoxy-poly(ethylene glycol)-thiol (mPEG-SH) was grafted onto 5S coating (to form 5S-PEG) to confer low-fouling ability. Static contact angle measurements were carried out on unmodified glass coverslip and the pDA-coated surfaces using water as the liquid phase. The unmodified glass coverslip and the 2S and 5S pDA-coated substrates showed similar advancing water contact angle values (ca. 45-52o) whereas the PEGylated surface, 5S-PEG, exhibited lower advancing water contact angle (29o), indicating an increase in surface hydrophilicity due to the presence of PEG (Figure S2, SI).

Scheme 1. Preparation of pDA, pDA-PEG, pDA-NO and pDA-PEG-NO coatings.

To characterize the surface morphology and film thickness, AFM was used. The representative topography images of all samples in air and in PBS are shown in Figure 1. According to the AFM measurements ACS Paragon Plus Environment


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performed in air (Figure S3, SI), the average film thicknesses of 2S, 5S and 5S-PEG were found to be 86, 191 and 229 nm, respectively (Table 1). Given that the NO release and antibiofilm studies were carried out in the wet state, we also measured the film thickness in PBS. In general, the film thickness in the wet state was ca. 4% higher compared to those measured in air (Table 1, Figures S4 and S5, SI). Additionally, it was found that the increase in pDA film thickness resulted in an increase in surface roughness, where the root mean square (RMS) roughness values for 2S and 5S in air were 19.4 and 35.5 nm, respectively (Table 1). Further functionalization with PEG did not result in any significant difference in surface roughness of the 5S-PEG coating (31.8 nm in air). Film thickness of coatings was also corroborated by TEM analysis (Figure S6, SI). TEM images of cross-sectional specimens of the pDA coatings revealed thicknesses of 50 ± 3, 152 ± 4 and 152 ± 8 nm, for 2S, 5S, and 5S-PEG respectively.

Figure 1. 2 μm × 2 μm AFM height images (left) in air and (right) in PBS for (a) 2S, (b) 5S and (c) 5SPEG.


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Table 1. Thickness and surface roughness parameters obtained from AFM height images. Samples

2S

5S

5S-PEG

Air

PBS

Air

PBS

Air

PBS

Thickness (nm)

86

89

191

200

229

239

RMS roughness (nm)

19.4

19.9

35.5

40.4

31.8

46.2

PTV roughness (nm)

166

188

204

207

220

236

Note: RMS stands for Root Mean Square (which corresponds to standard deviation for surface roughness).

Polydopamine coatings can serve as NO carrier since they possess primary and secondary amines, which can be converted to N-diazeniumdiolates.45 The ability of pDA to form uniform substrate-independent coatings opens up the possibility of immobilizing NO onto various substrate types via pDA films.43 To investigate the application of pDA coatings in NO delivery, 2S, 5S and 5S-PEG pDA based surfaces were functionalized with NONOate moieties in alkaline methanol solution purged with NO gas to form 2S-NO, 5S-NO and 5SPEG-NO films respectively. The immobilized NONOate moieties on the pDA coatings were characterized by XPS analysis. The analysis of N1s spectra of NO-functionalized pDA surfaces confirmed the successful formation of NONOate as judged by the presence of new peaks in N1s binding energy (around 407 eV) (Figure 2).37,

45, 57

Additionally, the appearance of S2p (163 eV) peaks in Figure 2 and an increase in

oxygen level (Figure S7 and Table S1, SI) attributed to ether group of PEG indicated that mPEG-SH brushes were successfully grafted to the pDA surfaces.13



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Figure 2. XPS analysis of 5S, 5S-PEG, and 5S-PEG-NO (top) and high resolution N1s XPS spectra (bottom) for 2S, 5S,5S-PEG, 2S-NO, 5S-NO and 5S-PEG-NO coatings.

Nitric Oxide Release from Polydopamine-Based Coatings. The NO release from the NO-modified coatings was estimated using Griess assay, which is a common indirect method to measure the NO concentration via detection of nitrite. The surfaces were rinsed with water before tested to remove the presence of ammonium nitrite salts formed during storage. The surfaces were immersed in 1 mL of PBS (pH 7.4) at 37 °C and cumulative NO release was quantified over 48 h by measuring the UV–vis absorbance of the sample at 540 nm.30, 58 Both 2S-NO and 5S-NO showed relatively high NO flux in the first 3 h (ca. 48 and 167 µM, respectively) followed by a steady release of NO over two days to reach the maximum of 105 and 233 µM, respectively (Figure 3). Interestingly, 5S-NO films released about twice as much NO than 2SNO, indicating that an increase in pDA film thickness can result in higher NO payload. Grafting of PEG onto the surface (5S-PEG-NO) did not change the rate of NO release (Figure 3). As the NO-release kinetics may be impacted by the media, NO release was also determined in M9 (medium used in biofilm inhibition experiments), where the NO release profile is similar to that in PBS (Figure S9, SI).As a control, bare glass coverslips treated with NO gas showed the absence of NO release compared to the pDA-coated substrates (Figure 3). Additionally, the absence of peaks corresponding to the NONOate groups in N1s spectra of ACS Paragon Plus Environment

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coatings after 48 h suggested that all stored NO is released (Figure S8, SI). The continuous release of NO over 48 h suggests these coatings as potential candidates for use in biomedical devices such as angiography catheters, etc.59 To demonstrate the versatility of this coating strategy on other substrates, polyester coverslips were similarly coated with 5S-NO pDA film as a polymeric substrate model and analyzed via Griess assay. The NO release profile of the polyester sample was comparable to that of glass substrate (Figure S10, SI).

Figure 3. Cumulative release of NO from pDA-coated substrates (surface area 1.33 cm2) in PBS (pH 7.4, 37 oC)

as measured by Griess assay.

Biofilm Inhibition of Nitric Oxide Releasing Polydopamine Coatings. Bacteria in the form of biofilms are protected by an extracellular polysaccharide matrix and are more difficult to eradicate than in their planktonic form.60 P. aeruginosa is one of the most common microorganisms that form highly resistant biofilms on moist surfaces in a hospital setting and is responsible for many catheter-associated urinary tract infection, infections related to mechanical heart valves, stents, and contact lens-associated corneal infections.61 Therefore, two strains of P. aeruginosa (wild type PAO1 and MDR PA37) were chosen to evaluate the antibiofilm activity of the NO releasing pDA films. For this, unmodified bare glass coverslip and the NO-modified pDA films were exposed to bacterial cultures and examined using tomographic microscopy where the bacteria biofilm density on the surface was quantified. Specifically, samples were incubated in bacterial culture for 6, 24 and 36 h and the surfaces were imaged with tomographic microscopy (3D Nanolive Cell Explorer) equipped with a digital staining software. Microscopy analysis of the ACS Paragon Plus Environment


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unmodified glass coverslip showed high density of bacterial cells at 6, 24 and 36 h (Figure 4a and Figure S11, SI). In contrast, 2S–NO, 5S-NO and 5S-PEG-NO surfaces showed significantly reduced adhesion of P. aeruginosa (PAO1) after 6 h (Figure 4), with 77, 97, and 98% inhibition, respectively, relative to the unmodified glass coverslip. It is worthwhile noting that the prevention of bacterial adhesion for the first 6 h in post-implantation is critical for the long-term success of an implant.3 Increasing the bacterial exposure time to 24 h led to 35, 78 and 96% inhibition for 2S-NO, 5S-NO, and 5S-PEG-NO, respectively. On the other hand, pDA surfaces alone (2S and 5S) generally exhibited non-significant difference or a modest increase in bacterial attachment in all exposure times when compared to the control unmodified glass coverslip (Figure 4b). This is in agreement with previous studies which showed that pDA films support cell attachment,13, 46 with the exception of 5S coating which prevented bacteria attachment at the 6 h mark. This may be due to the difference in surface roughness between the 5S and 2S coatings as it is been reported that the surface roughness decreases bacteria attachment.62-64 At 36 h, 5S-PEG-NO demonstrated 70% inhibition of PAO1 biofilm formation compared to 5S-NO (32% inhibition) (Figure 4b). The surface which contains PEG and no NO (5S-PEG) failed to prevent bacterial attachment during longer incubation times (24 and 36 h). Generally, low-fouling coatings without antimicrobial agents are insufficient for long-term applications due to the contamination and deterioration of the coating in physiological media.59 Encouraged by these results, we examined the attachment of a MDR strain of P. aeruginosa (PA37) on 5SNO and 5S-PEG-NO for 36 h. Images of the unmodified glass coverslip showed extensive colonisation of bacterial cells on the surface whereas 5S-NO and 5S-PEG-NO inhibited bacterial attachment by 48 and 69%, respectively (Figure 5). This showed that NO releasing pDA coatings are capable of inhibiting MDR biofilm formation on surfaces.


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Figure 4. (a) 2D Tomographic microscopy images and (b) normalized attachment of P.aeruginosa (PAO1) on polydopamine-modified substrates. 1 × 108 CFU mL-1 of P.aeruginosa (PAO1) was grown on the surfaces for 6, 24 and 36 h. Images were taken and analyzed using 3D Nanolive Cell Explorer Steve digital staining software for total % area coverage of both live and dead cells. Scale bar = 20 μm. All values were normalized to % bacteria coverage on unmodified glass coverslip. Statistical significance compared to control was evaluated via one-way ANOVA test. (* p < 0.1, ** p< 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant (p > 0.05)). Data are the average of at least two biological replicates and images were taken from no fewer than five spots. Note: Green colour shows both live and dead bacteria.



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Figure 5. 2D (top) and 3D (bottom) tomographic microscopy images of P.aeruginosa (PA37) attachment on polydopamine-modified substrates. 1 × 108 CFU mL-1 of P.aeruginosa (PA37) was grown on the surfaces for 36 h. Images were taken and analyzed using 3D Nanolive Cell Explorer Steve digital staining software. Scale bar = 20 μm. Note: Green colour shows both live and dead bacteria.

Biofilm Killing Study of Nitric Oxide Releasing Polydopamine Coatings. The bactericidal efficiency of the coatings against P. aeruginosa PAO1, PA37 and S. aureus biofilms were assessed using colony-forming unit (CFU) assay under static conditions. P. aeruginosa biofilms were grown in the same manner as in the biofilm inhibition study for 6.5 h. For S. aureus, TSB media was used where the biofilms were grown for 8 h. As shown in Figure 6, 5S-NO and 5S-PEG-NO reduced the number of viable adhered P. aeruginosa PAO1 cells by 99% and 99.9% relative to the unmodified glass coverslip control. On the other hand, 2S-NO displayed less than 1-log10 reduction in CFU. This result is not unexpected, as it has been previously shown that NO has a dose-dependent antimicrobial activity where bactericidal efficiency increases at higher doses.58, 65-67 5S-NO and 5S-PEG-NO also reduced the cell viability of MDR P. aeruginosa (PA37) by 95% and 97%, respectively, relative to the unmodified glass coverslip control. Additionally, these two coatings showed bactericidal activity against Gram-positive S. aureus reducing the number of viable biofilm cells by 98% and 99%, respectively. As expected, coatings without NO (2S, 5S, and 5S-PEG) did not exhibit any noticeable reductions in biofilm CFU of these three microorganisms due to the lack of active bactericidal component. Considering the biofilm inhibition and killing experiments, dual anti-adhesion and bacterial eradication properties at longer treatment time (36 h) were only observed in 5S-PEG-NO films. Concomitantly, the number of planktonic cells incubated with all the types of coatings here did not show any ACS Paragon Plus Environment

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significant decrease in the culture wells, which suggests the nontoxic effect of NO releasing coatings on planktonic cells (Figure S12, SI).

Figure 6. Cell viability of P. aeruginosa PAO1, PA37 and S. aureus biofilms bacteria. Coatings were incubated in 1 × 108 CFU mL-1 of bacteria. Statistical significance was evaluated via one-way ANOVA test. (** p< 0.01, *** p < 0.001, ns = not significant (p > 0.05)). Data are the average of at least three biological replicates.

According to previous reports, pDA films form strong covalent and noncovalent interactions with substrates and show good stability in various conditions, except in strongly alkaline solution (pH >13), and do not exhibit cytotoxicity on cells.46, 68-70 We also did not observe any microscopic change in the pDA films after NO purging, several washing steps and biological experiments. Given the fact that NONOate compounds may result in the formation of potentially toxic by-products (nitrosamines) after the release of NO,71 a comprehensive cytotoxicity and film stability study is needed to determine the applicability of these coatings in human patients.

Conclusion In this study, we developed an efficient strategy for antibiofilm applications via the generation of multifunctional polydopamine films endowed with low-fouling and nitric oxide releasing capabilities. Films that contain both NONOate and PEG functionalities released NO over 48 h and inhibited > 96 and 70% of P. aeruginosa bacterial attachment after 24 and 36 h of exposure time, respectively. These films are also effective in inhibiting the attachment of a multidrug-resistant strain P. aeruginosa. In addition, this coating efficiently killed biofilm cells of Gram-negative P. aeruginosa and Gram-positive S. aureus opportunistic ACS Paragon Plus Environment


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pathogens. Overall, the presented strategy may find potential applications in biomedical devices fabrication as this strategy can be applied for a broad range of substrates, including glass and polymer films. However, factors such as time of coating process and NO Parr chamber size need to be considered for practical applications. Additionally, further cytotoxicity experiments including the potential formation of nitrosamines by the reaction between released NO and amine groups from pDA are needed to ensure in-vivo feasibility of these coatings.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Figures S1-S12 and Table S1 include digital photos of samples, contact angle results, AFM and TEM images, XPS and NO release data and microscopy images.

Author Contributions The manuscript was written through contribution of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

Acknowledgments This work was supported by the Australian Research Council via the Future Fellowship (FT120100096, C.B.) and 2016 UNSW Vice-Chancellor’s Research Fellowship (E.H.H.W.) schemes, respectively. Z.S. acknowledges the receipt of an Australian Government Research Training Program (RTP) Scholarship. AFM analysis was performed at the South Australian node of the Australian National Fabrication Facility under the National Collaborative Research Infrastructure Strategy.


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Antibiofilm Nitric Oxide-Releasing Polydopamine Coatings - ACS

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