waterborne polyurethanes

Progress in Organic Coatings 101 (2016) 253–261

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Progress in Organic Coatings journal homepage: www.elsevier.com/locate/porgcoat

Antibacterial sustained-release coatings from halloysite nanotubes/waterborne polyurethanes Saman Hendessi a , E. Billur Sevinis a , Serkan Unal b , Fevzi C. Cebeci a,b , Yusuf Z. Menceloglu a , Hayriye Unal b,∗ a b

Faculty of Engineering and Natural Sciences, Sabanci University, Tuzla 34956 Istanbul, Turkey Sabanci University Nanotechnology Research and Application Center (SUNUM), Tuzla 34956 Istanbul, Turkey

a r t i c l e

i n f o

Article history: Received 13 January 2016 Received in revised form 17 August 2016 Accepted 2 September 2016 Keywords: Halloysite nanotubes Carvacrol Antibacterial Anti-biofilm coatings Sustained release

a b s t r a c t Natural and safe antibacterial nanoparticles based on carvacrol loaded halloysite nanotubes and their waterborne polyurethane nanocomposite coatings with antibacterial and antibiofilm properties are presented. Halloysite nanotubes are natural clay nanoparticles with a hollow tubular structure that allows loading and sustained release of active agents. In this study, halloysite nanotubes were efficiently loaded with carvacrol, the active agent of essential thyme oil. Encapsulated carvacrol molecules were demonstrated to be released from halloysite nanotubes in a sustained manner over one week and effectively inhibit the growth of a pool of pathogenic microorganisms. Carvacrol loaded halloysite nanotubes were further investigated as antibacterial nanofillers in polymeric nanocomposites by incorporating them into waterborne polyurethane coatings. Polyurethane nanocomposite films containing 5 wt.% carvacrol loaded halloysite nanotubes showed uncompromised thermal and mechanical stability as compared to neat polyurethane films. Carvacrol/halloysite nanotubes/polyurethane films demonstrated sustained release of carvacrol and antibacterial activity on representative pathogens, Aeromonas hydrophila, as evidenced by growth inhibition in agar diffusion assays and reduction in bacterial count upon exposure to nanocomposite films. Furthermore, these nanocomposite films inhibited bacterial colonization on their surfaces at least for two days demonstrating their applicability as anti-biofilm surface coatings. Composed of safe and natural components, the sustained-release antibacterial coatings presented here have strong potential for being widely utilized to prevent and mitigate bacterial infections on materials surfaces without raising toxicity concerns. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Pathogenic microorganisms and infections caused by them are of great concern not only for the medical field but also for materials science. Adhesion of bacterial cells onto surfaces and interfaces initiates bacterial colonization and formation of resilient bacterial communities called biofilms, which turn these surfaces and interfaces susceptible to bacterial infections [1–3]. Materials that have the ability to kill pathogenic bacteria and prevent bacterial colonization are desired for utilization in several application areas such as food-contact materials, textiles, water purification systems, prosthetic devices and hospital equipment surfaces. While antibacterial materials benefit community health by controlling bacterial

∗ Corresponding author. E-mail address: [email protected] (H. Unal). http://dx.doi.org/10.1016/j.porgcoat.2016.09.005 0300-9440/© 2016 Elsevier B.V. All rights reserved.

infections, they also greatly contribute to the prevention of industrial economic losses caused by biofouling. Several approaches have been proposed to obtain antibacterial activity by the modification of surface pattern of materials to prevent bacterial adhesion or by the use of active agents to provide contact killing effect. These approaches include the manipulation of surfaces with steric barriers like polymer brushes [4–6], modification of the surface pattern [7–9], manipulation of hydrophobicity [10,11], and conjugation of materials’ surface with polycations [12–15], or antimicrobial peptides [16]. Another approach that is more versatile and effective is the utilization of nanoparticles and polymeric nanocomposites as antibacterial coatings or materials. Metal and metal oxide nanoparticles including silver, copper, gold, titanium and zinc along with carbon based nanoparticles have been demonstrated to have antibacterial properties which are reflected on their polymeric nanocomposites [17–22]. Among these, silver nanocomposites which demonstrated significant antibacterial effect based on the release of silver ions have attracted great

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attention. Several nanocomposites containing silver nanoparticles alone and in combination with other nanofillers have been prepared with natural and biodegradable polymeric matrices [23–28]. While metal nanoparticles are effective antibacterial agents, concerns related to their safety and environmental effects limit their widespread use, commercialization and public acceptance especially in biomedical and food-contact applications. Therefore, there exists a significant need for natural antibacterial nanoparticles that are effective against pathogenic bacteria without raising any toxicity concerns and can be incorporated into nanocomposites with antibacterial effects. Essential oils that are volatile components of herbs and spices are widely studied due to their antibacterial properties [29,30] and attempts to incorporate them into structural materials and surfaces to act against pathogenic bacteria has been reported. One approach that has been studied widely is the direct incorporation of essential oils or their active components into polymeric materials [31–34]. While this method results in polymeric materials with some antibacterial activity, the fact that antibacterial agents are immediately released in an uncontrolled manner from the polymers diminishes the long term antibacterial activity of these materials. As another approach, active components of essential oils have been adsorbed on montmorillonite clay platelets which were then incorporated into polymeric materials as nanofillers. However, poor compatibility of montmorillonite clay with polymer matrices requires the use of compatibilizers or leads to poor mechanical properties in resulting nanocomposites [35,36]. While these examples demonstrate the utilization of essential oils in bulk polymeric materials such as thermoplastics, incorporation of essential oils into surface coating formulations with sustained release behavior has not been reported previously. In order to benefit from the antibacterial effects of essential oils to the best in the form of safe antibacterial nanocomposite coatings, they need to be efficiently encapsulated within natural and nontoxic nanocontainers that allow their sustained release and can be incorporated into a suitable polymeric coating system. Halloysite nanotubes (HNTs) are hollow tubular aluminum silicate nanoparticles with a high aspect ratio that can be utilized as nanocarriers and can be effectively dispersed in polymeric matrices resulting in active agent releasing nanocomposites [37–39]. Their nontoxic nature [40,41], effective encapsulation capacity and suitability for incorporation into polymers to prepare nanocomposite materials render HNTs ideal nanocontainers for antibacterial agents. An ideal polymer coating system for the effective utilization of such essential oil loaded halloysite nanotubes as sustainedrelease antibacterial nanofillers would be environmentally friendly waterborne polyurethanes which are widely employed in industrial applications due to their versatile chemistry to yield coatings with tunable thermo-mechanical properties [42]. Herein we studied the encapsulation of carvacrol, the active component of essential thyme oil, within HNTs to prepare natural and safe antibacterial nanoparticles, and their incorporation into waterborne polyurethane dispersions, to obtain sustained-release antibacterial and antibiofilm surface coatings.

from Medimark (France). Tryptic soy broth (TSB), Nutrient broth (NB), Brain-heart infusion (BHI) and Agar powder were purchased from Biolife (Italy). Anionic, aqueous polyurethane (PU) dispersion based on a polyester-polyol was kindly supplied by Punova R&D and Chemicals Inc. (Turkey) with a 35 wt.% solid content. 2.2. Loading of HNTs with carvacrol In order to load HNTs they were mixed with liquid carvacrol with a ratio of 0.1 g HNT per 1 mL carvacrol. Three different protocols were followed for the loading; (i) Ultrasonication: HNT-carvacrol mixture was subjected to ultrasonication with a microprobe (Qsonica, Q700) for 30 min with 2 s pulse on and 5 s pulse off time in an ice bath. (ii) Vacuum application: HNT-carvacrol mixture was transferred into a vacuum jar connected to a vacuum pump and 1 mbar pressure was applied for 30 min to remove air inside HNTs followed by application of atmospheric pressure for 10 min to allow carvacrol molecules enter evacuated HNTs. The cycle was repeated twice to increase loading efficiency. (iii) HNT-carvacrol mixture was subjected to ultrasonication and then treated with vacuum application as described in the first and second protocols above, respectively. For all three protocols, solid phase in the resulting suspension comprising carvacrol loaded HNTs was separated by centrifugation at 5000 rpm for 5 min, and the excess carvacrol was removed. Carvacrol loaded HNTs were washed with ethanol once or twice by centrifugation to remove surface adsorbed carvacrol molecules and were dried overnight at room temperature in an open container. Dry loaded HNTs were kept in a closed container at room temperature. 2.3. Determination of carvacrol loading efficiency Carvacrol loading efficiency was determined by thermogravimetric analysis (TGA) on a DTG-60H (Shimadzu, USA) instrument. Samples of carvacrol loaded HNTs and unloaded HNTs were heated in an alumina pan from 30 to 1000 ◦ C at a rate of 10 ◦ C/min under nitrogen flow. The resulting temperature dependent weight loss percentages were analyzed using TA-60WS Collection software. Loading efficiency was calculated as the difference in total weight loss of carvacrol loaded HNT sample and unloaded HNT sample. 2.4. Imaging of HNTs with transmission electron microscopy Transmission electron microscopic (TEM) analysis of loaded and unloaded HNTs was performed using JSM-2000FX (JEOL, Japan) at an operating voltage of 160 kV, using a 200 mesh Copper grid (Formvar film). 2.5. Measurement of carvacrol release from HNTs

2. Experimental methods

The release rate of carvacrol from HNTs was measured by TGA under isothermal conditions on DTG-60H (Shimadzu, USA). Samples of carvacrol loaded HNTs and unloaded HNTs in an alumina pan were kept at 30 ◦ C for one week under air flow and the percent weight loss was monitored as a function of time.

2.1. Materials

2.6. Antimicrobial activity of carvacrol loaded HNTs

HNTs were provided by Eczacibasi Esan (Turkey). Carvacrol was supplied by Tokyo Chemical Industry Co. (Japan). The antibacterial activity of carvacrol loaded HNTs was determined by using different bacterial strains including Aeromonas hydrophila (A. hydrophila, ATCC 35654), Pseudomonas putida (P. putida, ATCC 49128), Listeria monocytogenes (L. monocytogenes, ATCC 7644) and Staphylococcus aureus (S. aureus, ATCC 25923). All bacterial strains were purchased

The agar diffusion method was used to evaluate the antimicrobial activity of carvacrol loaded HNTs against A. hydrophila, P. putida, L. monocytogenes and S. aureus. 3 mL overnight cultures of bacteria were grown in appropriate growth media. A. hydrophila were grown in TSB at 30 ◦ C, P. putida were grown in NB at 30 ◦ C, L. monocytogenes were grown in BHI at 37 ◦ C and S. aureus were grown in TSB at 37 ◦ C on a shaker incubator (200 rpm). 0.1 mL of overnight

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bacterial culture (109 CFU/ml) was spread on agar plates containing the appropriate growth medium. 0.01 g of HNTs was placed on the growth plates which were then incubated overnight in an incubator set at the respective optimal growth temperature. Images of plates were taken with a Bio-Rad Gel Imaging System. Minimum inhibitory concentration (MIC) values of carvacrol loaded HNTs for bacterial species were determined by broth dilution method. Serial two-fold dilutions of carvacrol loaded HNTs in growth medium were prepared in sterile culture tubes resulting in concentrations of 20–0.15 mg/mL. Tubes were then inoculated with diluted overnight broth culture at a final bacterial concentration of 5 × 105 CFU/mL and incubated overnight at the optimal growth temperature for each respective bacterial strain. MIC is determined as the lowest concentration without visible bacterial growth. Experiments were repeated three times. 2.7. Preparation of HNT/PU nanocomposites 0.2 g of carvacrol loaded HNTs (carvacrol loading ratio of 18.4%) were slowly added into 11.4 g of aqueous PU dispersion with a solid content of 35% under overhead agitation and allowed to mix for 1 h, resulting in 5 wt.% HNTs in solid PU. Following the mixing, the aqueous HNT/PU dispersion was cast onto glass plates which were kept at room temperature for 24 h followed by an overnight drying in an oven at 70◦ C to remove water and obtain a self-standing film. Theoretically, expected carvacrol content of final films was 0.9 wt.% as calculated by the HNT content in films (5 wt.%) and average carvacrol content in HNTs (18 wt.%). As a control experiment, carvacrol was directly incorporated into PU dispersion without HNTs (crvPU). For the preparation of this sample, 40 ␮L carvacrol was mixed with 12.7 g of PU dispersion that has 35 wt.% solid content (corresponding to 0.9 wt.% carvacrol in solid PU), using the same mixing, film casting and drying protocol to obtain films of them as described above. 2.8. Determination of mechanical properties of crv-HNT/PU nanocomposite films Mechanical properties of HNT/PU nanocomposite films were tested on a universal testing machine Zwick Roell Z100 UTM, with a load cell of 200 N and a crosshead speed of 25 mm/min according to the testing method determined by ASTM D1708-10, standard test method for tensile properties of plastics by use of micro-tensile specimens. The initial grip separation was 22 mm and average of at least five replicates of each sample was reported. 2.9. TGA analysis of crv-HNT/PU nanocomposite films TGA was performed on a DTG-60H (Shimadzu, USA) instrument. Crv-HNT/PU nanocomposite films were cut into small pieces and a total weight of approximately 20 mg were placed into an alumina pan. Samples were heated 30−1000◦ C at a heating rate of 10◦ C/min under nitrogen flow (flow rate 10 mL/min). The resulting temperature dependent weight loss percentages were analyzed using TA-60WS Collection software. PU films that do not contain HNTs were tested as controls. 2.10. Measurement of carvacrol release from HNT/PU nanocomposites The release rate of carvacrol from crv-HNT/PUs was measured by TGA under isothermal conditions on DTG-60H (Shimadzu, USA). Crv-HNT/PU, crv-PU and neat PU films were cut into small pieces and a total weight of approximately 20 mg were placed into an alu-

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mina pan. Samples were kept at 30◦ C for 72 h under air flow and the percent weight loss was monitored as a function of time. 2.11. Antibacterial activity of HNT/PU nanocomposites 2.11.1. Bacterial growth inhibition The ability of HNT/PU nanocomposites to inhibit bacterial growth was tested against A. hydrophila by the agar diffusion method. 100 ␮L of an A. hydrophila overnight culture (109 CFU/mL) was spread on an agar plate containing TSB medium. 1 cm × 1 cm samples of films were cut and placed onto the prepared growth plates containing A. hydrophila and incubated overnight at 30◦ C. Neat PU film was used as a control. The images of the plates were taken with Bio-Rad Gel Imaging System. 2.11.2. Bacterial killing A. hydrophila were grown in 3 mL TSB overnight at 30◦ C. Cells were harvested by centrifugation, washed twice in sterile Phosphate Buffered Saline (PBS) and resuspended in PBS at a concentration of 109 CFU/mL. 1 cm × 1 cm pieces of crv-HNT/PU films were incubated in A. hydrophila suspensions of 108 CFU/mL in PBS at 30◦ C in a shaker incubator. As controls, neat PU films of the same size were also incubated with cells along with a “cells only” sample without a film. At 48 h films were removed and aliquots of suspensions were serially diluted. 100 ␮L of each dilution was plated on TSB agar plates. Bacterial colonies were counted after overnight incubation at 30◦ C. Each sample was repeated 3 times. The viability values were calculated by comparing the number of colonies in crv-HNT/PU and PU incubated samples to the number of colonies in “cells only” sample and reported as the mean and standard deviation calculated from 3 separate tests. 2.12. Visualization of bacterial attachment on HNT/PU nanocomposites 1 cm x 1 cm pieces of crv-HNT/PU and PU film samples were incubated with A. hydrophila in wells of a 12-well plate. Each well contained 108 CFU/mL A. hydrophila in TSB. After incubation for 48 h at 30◦ C, cellular suspensions were removed from wells and films were rinsed twice with sterile PBS. Films were stained with Baclight Live/Dead stain (L-7012, Invitrogen) for 30 min in dark at room temperature followed by rinsing with PBS. Films were mounted onto coverslips and imaged with a Carl-Zeiss LSM 710 Laser Scanning Confocal Microscope equipped with a Plan-Apochromat 63x/1.40 oil objective. Reported images are 3-D renderings of Z-stacks created by using Zen 2010 software. 3. Results and discussion 3.1. Preparation of carvacrol loaded HNTs The natural antibacterial agent that was selected for encapsulation within HNTs was liquid carvacrol which is the active component of thyme oil, which is well-known for its antibacterial activity against pathogenic microorganisms [43]. For the preparation of carvacrol loaded HNTs, three different loading protocols were used in which mixtures of HNTs and excess carvacrol were subjected to (i) ultrasonication using a probe sonicator, (ii) vacuum treatment or (iii) ultrasonication and subsequent vacuum treatment in an attempt to replace the air inside the HNTs with carvacrol. Carvacrol molecules that are not encapsulated within HNTs and adsorbed on the surface were washed away with ethanol through ultracentrifugation after each loading protocol. The carvacrol loading efficiency of HNTs was determined by thermogravimetric analysis (TGA). Fig. 1 shows the weight loss that occurs as a function of temperature for blank HNTs in comparison with the weight loss

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0

Fig. 1. Temperature dependent weight loss curves for unloaded HNTs (green), carvacrol loaded HNTs (black), and carvacrol (red) obtained by TGA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

for carvacrol loaded HNTs prepared by ultrasonication and subsequent vacuum treatment. Both unloaded and carvacrol loaded HNTs show an initial weight loss between 30 and 100◦ C due to the release of physically adsorbed water and another weight loss between 450 and 550◦ C primarily due to the dehydroxylation of structural AlOH groups. In the temperature range of 100–200◦ C, carvacrol loaded HNTs demonstrate an additional weight loss that is not observed in unloaded HNTs. The fact that this temperature coincides with the temperature of weight loss decay for free carvacrol clearly indicates the presence and release of carvacrol within HNTs, whereas the overall weight loss difference between both curves determines the carvacrol loading efficiency on HNTs. To elaborate on the effect of loading conditions on the encapsulation efficiency, various pre- and post-treatment methods were applied, of which the loading efficiency was determined (Table 1). While both ultrasonication and vacuum treatment on the HNT/carvacrol mixture resulted in significant amount of carvacrol loading potentially due to the replacement of the air inside the HNTs with carvacrol, the loading efficiency further increased when both treatments are applied consecutively. The finer particles sizes of HNTs obtained by ultrasonication of HNTs were also presumed to improve the loading efficiency. The post-loading treatment of HNT/carvacrol mixture was critical to obtain carvacrol loaded HNTs

Fig. 2. Representative TEM images of unloaded (A) and carvacrol loaded HNTs (B). Carvacrol loaded HNTs were obtained by ultrasonication and vacuum treatment on carvacrol-HNT mixture followed by 1× washing with ethanol.

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Fig. 3. (A) Carvacrol release curves represented by time dependent percent weight loss of carvacrol loaded HNTs (red) and unloaded HNTs (black) as calculated by isothermal TGA at 30◦ C. The Inset Table: Carvacrol release rates at different time periods. Rates were calculated by subtracting extrapolated values of unloaded HNT weight loss from carvacrol loaded HNT weight loss and taking the ratio of corrected weight loss over time. (B) Images demonstrating the growth inhibition around unloaded HNTs (top row) and the same amount of carvacrol loaded HNTs (bottom row) on plates seeded with the same concentration of P. putida, A. hydrophila, L. monocytogenes and S. aureus. (C) Minimum Inhibitory Concentration (MIC) values of carvacrol loaded HNTs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

B)

A)

Fig. 4. Characterization of crv-HNT/PU nanocomposites. (A) Temperature dependent percent weight loss curves for PU (black) and carvacrol loaded HNT/PU film (red). The Inset: Focused view of 100–300◦ C temperature range where carvacrol evaporates. (B) Representative Stress-Strain curves for PU (black) and carvacrol loaded HNT/PU film (red). The inset table: Statistical values for Young’s Modulus, elongation at break and tensile strength calculated from three different measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1 Carvacrol loading efficiencies obtained after various pre- and post-loading treatments. Treatment on Carvacrol/HNT mixture Ultrasonication

Post-loading treatment

excess carvacrol removed HNTs are washed (1×) HNTs are washed (2×) excess carvacrol removed Vacuum treatment HNTs are washed (1×) HNTs are washed (2×) Ultrasonication + vacuum treatment excess carvacrol removed HNTs are washed (1×) HNTs are washed (2×)

Loading Efficiency (%) 40.8 ± 1.5 15.7 ± 0.1 3.0 ± 0.3 39.3 ± 1.2 8.1 ± 0.2 0.4 ± 0.1 47.8 ± 1.8 18.4 ± 1.6 4.4 ± 1.0

in the form of dry, fine powder, in which surface adsorbed carvacrol was successfully removed and carvacrol was present only in the lumen of tubes. In all three samples, following the loading protocol,

removal of excess carvacrol by centrifugation resulted in relatively very high loading efficiencies; however, resulting HNTs were oily and clumpy, suggesting the presence of carvacrol molecules not only in the lumen but also on the outer surface of HNTs. Upon washing carvacrol loaded HNTs with ethanol once, oily and clumpy HNTs turned into dry and fine powder form with a significant average loading efficiency of 18.4% as demonstrated by TGA. Further washing of carvacrol loaded HNTs (2× washing) significantly decreased the loading efficiency, indicating potential extraction of the encapsulated carvacrol. Carvacrol loaded HNTs that were quantified in terms of carvacrol loading efficiency by TGA were visualized with Transmission Electron Microscopy (TEM) in order to define the mode of interaction of carvacrol with HNTs. Fig. 2A shows representative TEM images of unloaded HNTs where their hollow tubular structures are visible. When carvacrol loaded HNTs obtained by consecutive ultrasonication and vacuum treatment followed by a washing step

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Carvacrol release (% Weight Loss of Films)

0,8

crv-PU crv-HNT/PU PU

0,6

0,4

0,2

0,0 0

10

20

30

40

50

60

70

80

90

100

Time (h) Fig. 5. Carvacrol release curves represented by time dependent percent weight loss of carvacrol/PU (black), carvacrol loaded HNT/PU (red) and neat PU (green) films as calculated by isothermal TGA at 30◦ C. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

carvacrol loaded HNTs that could lead to release of antibacterial agents lasting up to one week. The release of carvacrol from HNTs was further studied in terms of the resulting antibacterial activity of loaded HNTs. Equal amounts of unloaded HNTs and carvacrol loaded HNTs were placed on growth plates that were seeded with equal amounts of bacteria and the presence of any growth inhibition zone was monitored (Fig. 3B). Pictures obtained from growth plates demonstrate that carvacrol loaded HNTs result in growth inhibition in a variety of bacteria to different degrees while unloaded HNTs themselves do not have any growth inhibition effect. Minimum inhibitory concentrations of carvacrol loaded HNTs range from 1.25 to 2.5 mg/mL for tested bacterial strains (Fig. 3C). Carvacrol molecules released from HNTs inhibit the exponential growth of bacteria leading to a killing zone around the nanoparticles. Pseudomonas putida, Aeromonas hydrophila, Listeria monocytogenes and Staphylococcus aureus tested in this experiment represent gram negative and gram positive bacteria that are mainly responsible for foodborne infections, thus demonstrate the potential of carvacrol loaded HNTs as nanoparticles that can be utilized for the preparation of materials for food safety applications. 3.2. Nanocomposites of carvacrol loaded HNTs

are analyzed, significantly higher electron densities are observed in the lumen of HNTs indicating the presence of carvacrol molecules (Fig. 2B). TEM images demonstrate the fact that as opposed to only being adsorbed on the outer surface, carvacrol molecules were actually encapsulated by HNTs, which is expected to allow their sustained release. The release of carvacrol from HNTs over time was studied by using isothermal TGA. The weight loss of carvacrol loaded HNTs due to released carvacrol was monitored over one week at 30 ◦ C, slightly over room temperature for the proper stabilization of the TGA instrument. Fig. 3A shows the weight loss curve of carvacrol loaded HNTs in comparison with the weight loss curve of unloaded HNTs. While unloaded HNTs sustain a constant weight over one week except a minimal initial weight loss due to moisture, carvacrol loaded HNTs present a gradual weight loss of approximately 17% which is due to the release of volatile carvacrol from HNTs. A relatively faster release occurs within the first hour potentially due to the release of surface adsorbed carvacrol whereas the release rate decreases over time resulting in a sustained release over one week. This result clearly demonstrates the sustained release property of

In order to demonstrate potential applications of carvacrol loaded HNTs, their incorporation into nanocomposites and the antibacterial activity of resulting materials were investigated. For this purpose, waterborne polyurethane (PU) dispersions were used, which is commonly utilized for a variety of industrial applications as surface coatings. PU dispersions composed of 35 wt.% polyesterbased PU and 65 wt.% water were mixed under strong agitation with 5 wt.% carvacrol loaded HNTs containing 18.4% carvacrol from which self-standing films are cast through evaporation of the water content. The TGA analysis of cast films demonstrates that carvacrol loaded HNTs do not negatively affect the thermal stability, as the decomposition of the PU occurs at the same temperature for both carvacrol loaded HNT containing PU (crv-HNT/PU) and neat PU films (Fig. 4A). A closer look at the temperature range between 50 and 300◦ C demonstrates that the crv-HNT/PU nanocomposite presents an additional weight loss that is not present in neat PU which is due to the release of carvacrol from the nanocomposite film. This demonstrates the successful incorporation of carvacrol into the nanocomposites through HNTs.

Fig. 6. Antibacterial activity of crv-HNT/PU nanocomposite films. (A) Agar diffusion assay on a plate seeded with A. hydrophila cells demonstrating the growth inhibition around neat PU (a) and crv-HNT/PU film (b). (B) Viability assay performed by incubation of neat PU film (red) and crv-HNT/PU film (green) with a suspension of A. hydrophila for 48 h and determining the viability of cells by plate spreading and colony counting. Viability values were calculated in comparison to a control A. hydrophila suspension that was not incubated with any films. Error bars represent standard deviation calculated from 3 separate samples. Red columns represent fresh films; green columns represent 45-days old films kept in a closed container at room temperature. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 7. Representative Laser Scanning Confocal Microscopy images of films incubated with a suspension of A. hydrophila for 48 h followed by Live/Dead staining. (A) crv-HNT/PU nanocomposite films; (B) unloaded-HNT/PU nanocomposite films. Top images are rendered confocal Z-stacks. Lower panel shows cross sections of imaged films.

The crv-HNT/PU films were further characterized in terms of their mechanical properties. As demonstrated in Fig. 4B, films that contain 5 wt.% carvacrol loaded HNTs showed similar Young’s Modulus, slightly lower tensile strength at break and increased elongation at break values compared to neat PU films. It is important to note that nanocomposite films were mechanically strong, presenting 22.4 ± 1.2 MPa tensile strength and 1558% elongation at break, which favors their utilization in high performance coating and elastomer applications requiring physical durability. Crv-HNT/PU nanocomposite films were also investigated in terms of their ability to release carvacrol and the corresponding antibacterial activity. Isothermal TGA at 30◦ C was utilized to demonstrate the release of carvacrol from polyurethane matrices. The weight loss that occurs in polyurethane films containing carvacrol loaded HNTs due to the evaporation of carvacrol was monitored over four days. Fig. 5 shows the sustained release of carvacrol from the nanocomposite film. Over 100 h 0.52% of the initial weight of crv-HNT/PU film was gradually lost and the release curve did not reach saturation indicating that not all carvacrol content was released yet. Considering the theoretical amount of 0.9 wt.% carvacrol within the films as calculated by the product of HNT content (5 wt.%) and carvacrol loading of these HNTs (18.4 wt.%) it can be stated that the nanocomposite still contains carvacrol to release even after four days and the sustained antibacterial effect can be expected for longer time. On the other hand, when the same amount of carvacrol was directly incorporated into PU matrix without the HNTs (crv-PU), the release rate for the resulting cast films was significantly faster where half of the total carvacrol release occurred within the first 10 h. This result demonstrates that loaded HNTs embedded into the PU matrix act as nanocontainers that allow the sustained release of carvacrol that would result in a longer-lasting antibacterial effect. The resulting antibacterial activity of carvacrol-HNT/PU nanocomposites against A. hydrophila was demonstrated with

growth inhibition tests on solid growth medium. A. hydrophila are gram negative aquatic pathogens responsible for gastroenteritis and are known to form biofilms on surfaces [44,45]. In this work they were chosen as a representative species on which the antibacterial activity of crv-HNT/PU nanocomposites is tested. Pieces of PU films were placed onto growth plates that were seeded with bacteria. While the neat PU film itself did not have any effect on the growth of A. hydrophila, carvacrol-HNT/PU composite film that can simply be prepared by mixing PU emulsions with carvacrol loaded HNTs for 1 h inhibited the growth of cells as seen by the killing zone around the film (Fig. 6A). The amount of carvacrol that was released from the HNTs and the polyurethane matrix was enough to stop the growth of bacteria, proving the antibacterial activity of the carvacrol-HNT/PU nanocomposite films. Besides the ability of nanocomposite films to inhibit the growth of bacteria, their efficiency to kill bacteria in an environment lacking the growth conditions was also demonstrated. Equal size films of crv-HNT/PU nanocomposites and neat PU were incubated in aqueous suspensions that contain the same number of A. hydrophila. At the end of 48 h, dilutions from both incubations were plated on growth plates to compare the amounts of remaining live cells in comparison to a “cells only” control. As seen on Fig. 6B, crvHNT/PU film lead to a significant reduction in the number of alive cells whereas the neat PU film itself did not demonstrate any killing effect on cells. Nanocomposites films containing the carvacrol loaded HNTs were able to kill bacteria due to the sustained release of carvacrol over two days demonstrating the strong antibacterial properties of these nanocomposites. The decrease in number of cells after being incubated with crv-HNT/PU films corresponds to 54–97% (0.3–1.1 log reduction) which is significant considering that the films contain less than 1 wt.% carvacrol. When the experiment was repeated with crv-HNT/PU films that were prepared 45 days ago and kept in an airtight container at room temperature, bacterial reduction values within the same range were

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obtained indicating the long term antibacterial activity of prepared nanocomposites. Anti-biofilm properties of carvacrol-HNT/PU nanocomposites were also investigated. crv-HNT/PU nanocomposite films along with films made by incorporating unloaded HNTs into PU matrix (HNT/PU) as a control were incubated with equal amounts of A. hydrophila in static growth conditions for 48 h to allow adhesion of bacteria onto film surfaces. The incubation was followed by washing of both surfaces to remove unadhered cells and staining with fluorescent dyes that allow visualization of bacteria on the surface. Representative Confocal Laser Scanning Microscopy (CLSM) images obtained are demonstrated in Fig. 7. UnloadedHNT/PU surfaces were highly colonized with bacteria as seen by the green (live cells) and red (dead cells) stained cells on the surface. Crv-HNT/PU surfaces, on the other hand, did demonstrate only background fluorescence due to absorption of fluorescent dyes by the film, but did not demonstrate any bacterial attachment. The slow release of carvacrol molecules over two days of incubation was presumed to prevent the attachment of bacteria on the surface of the nanocomposite film. These results suggest that carvacrol–HNT/PU nanocomposites have strong potential as antifouling coatings and can be utilized on surfaces that are prone to biofilm formation. 4. Conclusions A novel and feasible method for the preparation of antibacterial agent releasing nanoparticles that are entirely natural and their incorporation into waterborne polyurethane nanocomposites was demonstrated. HNTs provide a natural and safe environment for the encapsulation and sustained release of antibacterial essential oil leading to strong antibacterial effect. Compared to other antibacterial nanoparticles reported in literature, carvacrol loaded HNTs are advantageous as they do not pose any risk to human health being composed of only natural components. As a demonstration for the utilization of carvacrol loaded HNTs they were incorporated into waterborne polymer dispersion as antibacterial nanofillers resulting in nanocomposite films that show sustained release of natural antibacterial agents. While nanocomposites prepared with carvacrol loaded HNTs demonstrated growth inhibition and killing activity on pathogenic bacteria they also provided surfaces that prevent bacterial colonization. As natural and safe antibacterial nanoparticles, essential oil loaded HNTs and their waterborne polyurethane nanocomposites have strong potential for the prevention and mitigation of bacterial infections on materials surfaces. Acknowledgments The authors thank Eczacıbası ESAN (Turkey) for providing HNTs and Mr. Turgay Gonul for assistance with TEM analysis. This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK), Grant No: 113O872. References [1] J.W. Costerton, P.S. Stewart, E.P. Greenberg, Bacterial biofilms:. a common cause of persistent infections, Science 284 (5418) (1999) 1318–1322. [2] T.R. Garrett, M. Bhakoo, Z. Zhang, Bacterial adhesion and biofilms on surfaces, Prog. Nat. Sci. 18 (9) (2008) 1049–1056. [3] P. Gupta, S. Sarkar, B. Das, S. Bhattacharjee, P. Tribedi, Biofilm, pathogenesis and prevention—a journey to break the wall: a review, Arch. Microbiol. 198 (1) (2016) 1–15. [4] M. Krishnamoorthy, S. Hakobyan, M. Ramstedt, J.E. Gautrot, Surface-initiated polymer brushes in the biomedical field: applications in membrane science, biosensing, cell culture, regenerative medicine and antibacterial coatings, Chem. Rev. 114 (21) (2014) 10976–11026. [5] W.J. Yang, K.-G. Neoh, E.-T. Kang, S.L.-M. Teo, D. Rittschof, Polymer brush coatings for combating marine biofouling, Prog. Polym. Sci. 39 (5) (2014) 1017–1042.

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