Synthesis and Application of AgNPs-Chitosan Composite as a Self-Disinfecting Coating in Water-Based Polyurethane

: Infectious diseases still represent an important cause of mortality for humans. One of the main reasons is that various pathogenic bacteria can persist and survive on inanimate surfaces for many days. Therefore, self-disinfection coating technology has become of interest to deal with this problem. In this research, we propose to develop a self-disinfection coating containing AgNPs-chitosan composite in 50% water-based polyurethane (WPU), which has a strong short-and long-term antibacterial effect. The coating agent was synthesized by conventional composite approaches. The physical and chemical properties of AgNPs-chitosan nanocomposite are studied by TEM, SEM, and FTA 100 Drop Shape Instrument B Frame System. The results show that at a concentration of 39 µ g/mL, when reducing the size of AgNPs from 7.29 ± 1.65 to 4.66 ± 2.08 nm, the shape of a sphere turns into an asymmetrical circle and leads to increasing aggregation of AgNPs. Negative charges on the surface of AgNPs interact with amine (-NH 2 ) and hydroxyl (-OH) groups of chitosan through an electrostatic force. All formulations of the coating showed low hydrophobicity properties. Moreover, the short-and long-term antibacterial activity of the coating were investigated by application of the ISO 22196 standard protocol. The mean inhibition percentage of E. coli O157:H7 and S. aureus ATCC25722 of the formulation containing AgNPs at a concentration of 1280 µ g/mL and 50% v / v of WPU (Formula 4) and the formulation containing AgNPs at a concentration of 1280 µ g/mL, chitosan 39 µ g/mL and 50% v / v of WPU (Formula 8) from 1 day to 4 months after the coating completely dried was 81.72% ± 3.15% and 82.07% ± 3.01% on E. coli O157:H7, 84.64% ± 2.59% and 83.27% ± 3.12% on S. aureus ATCC25722, respectively. There was no signiﬁcant difference from statistical analysis at 95% conﬁdence interval ( p < 0.05). Furthermore, the quantify of silver ion from coating was measured by ICP-MS. The result reveal that the formulation containing AgNPs at a concentration of 1280 µ g/mL, chitosan 39 µ g/mL and 50% v / v of WPU (Formula 8) released an amount of silver ion lower than the formulation containing AgNPs at a concentration of 1280 µ g/mL and 50% v / v of WPU (Formula 4) by approximately 5.92 times, while the same concentration of AgNPs and inhibition efﬁcacy was not signiﬁcantly different. In addition, such a concentration was non-toxic on NHDF cells, which were investigated by MTT assay. Therefore, formulation containing AgNPs at a concentration of 1280 µ g/mL, chitosan 39 µ g/mL and 50% v / v of WPU coating (Formula 8) will be further developed into commercial self-disinfection coatings.


Introduction
Infectious diseases, also known as epidemic diseases, transmissible diseases or communicable diseases, are caused by pathogenic microorganisms that infect a host organism and can be extended, directly or indirectly, from one organism to another.Moreover, they still represent an important cause of morbidity and mortality in humans.The human behavior that causes emergence and re-emergence of infectious diseases is contact with a pathogenic cell-filled surface and then contact with other parts of the body and other people to spread the disease [1][2][3].In healthcare situations, microbes can contaminate an inanimate surface, which performs an important role in indirect transmission of infection.Specifically, surface areas are frequently exposed by the patient, who is full of germs, allowing transmission from animated sources to others by contaminated inanimate surfaces.Examples of pathogenic bacteria that can persist and survive on an inanimate surface, for example, Staphylococcus aureus, MRSA and Escherichia coli can survive on a plastic surface for 9, 12 and 27 days, respectively [4,5].Therefore, keeping the surface clean can reduce the spread of infectious diseases.Accordingly, preventive infection control strategies are performed by always cleaning the surface using liquid surface disinfectants, such as hydrogen peroxide, light-activated photosensitizers and no-touch (automated) approaches.However, the methods mentioned above also have a number of disadvantages or limitations, such as a smell similar to vinegar, irritation of the respiratory system and limited areas.
Nowadays, there is an interesting technology that can self-inhibit pathogens on surfaces called "Self-disinfecting coating" which is gathering attention in research and commercial fields worldwide [6,7].The evolution of self-disinfecting coating began via coating the inanimate surface with an antimicrobial compound such as metals (copper, titanium and silver) that have antimicrobial properties or utilizing surface equipment.Previous research that has shown antimicrobial properties caused by coating surfaces containing heavy metals for weeks or months has attracted some consideration as a new approach for disinfecting or preventing the growth of bacteria on surfaces [8].In particular silver nanoparticles have resulted in excellent properties; their distinctive optical, chemical, electrical and catalytic properties can be tuned to the nature of the surface, size, shapes, etc. AgNPs based disinfectants have drawn attention due to the practical applications in our daily lives [9].AgNPs have attracted much attention due to their potential as antimicrobial agents; they are widely applied in many applications in biological and medical fields, such as biosensors, wound healing, treating burns and cancer therapeutics [10,11].Additionally, at low concentrations, AgNPs are non-toxic to the human body and have high antibacterial properties [12].However, AgNPs have some limited properties for use in coating applications, which are a source of the development for silver nanocomposites with chitosan to improve their stability, distribution, aggregation, quantities, rate of Ag + release and cytotoxicity.Chitosan is a biopolymer and cationic heteropolymer obtained from chitin that is present in the exoskeleton of shrimp, crustaceans, fungi and yeasts.Chitosan has been broadly applied as a biological adaptation material, drug delivery vehicle, bone tissue scaffold and for wound healing biomaterials [13].Accordingly, the application of chitosan for stabilizing and controlled release of Ag ion agents can facilitate its application in biomedical industries [13].
Water-based polyurethanes (WPU) are one of most adaptable polymeric materials with high mechanical properties and a host of applications, such as coatings, adhesives, medical devices and packaging materials [14].WPUs are nontoxic, nonflammable, non-irritating, non-VOC and environmentally friendly.
However, the formulation for a self-inhibiting coating with long-term antimicrobial activity has not been developed.Using compounds of AgNPs-Chitosan composite in waterbased polyurethane is the novelty of this research.However, there has been similar research, for example, the invention of coatings containing AgNPs in PU (Oil based) and testing the effects in the short-term only [14][15][16].Namely, chitosan-starch silver nanoparticle (Cht-St-AgNPs) coated papers for antimicrobial packaging applications.However, it is just a test on paper and it is a short-term test.Some research has tested long-term effects [17].There is no research on AgNPs-Chitosan composites in WPU for coating applications [18,19].Therefore, in this study, we develop a novel self-disinfecting coating with long-term antibacterial activity by AgNPs-chitosan composites in WPU for antimicrobial inanimate surface applications.

Materials
AgNPs were produced chemically by the Prime Nanotechnology company (Bangkok, Thailand) and were spherical of 5-10 nm in size.In addition, 100% water based polyurethane (WPU) was obtained from the Sirocko company (Patumtani, Thailand).The AgNPs were diluted in deionized water (stock solution 1 mg/mL) and samples for the test were diluted with deionized water to achieve the final concentration of silver nanoparticles.The stock solutions were stored at room temperature to avoid salt solutions.Chitosan from Sigma, St. Louis, MO, USA was dissolved in distilled 3% v/v acetic acid as the stock solution for synthesis of the silver nanocomposite compound.

Bacterial Strain and Growth Condition
Escherichia coli O157:H7, Staphylococcus aureus ATCC25722, Vibrio cholera ATCC and Methicillin-resistant Staphylococcus aureus (MRSA) DMST20649 are well known as some common pathogenic bacteria used in laboratories for biological research and were stored at −70 • C in 20% glycerol before use.The bacterial cells were streaked on a Nutrient Agar (NA) plate and then incubated at 37 • C for 24 h.Single colonies were placed into 5 mL of Nutrient Broth (NB) and then incubated at 37 • C overnight (18-24 h) on a shaker at 160 rpm as starter bacterial cells.Then a subculture of starter bacterial cells at 37 • C were placed on a 160 rpm shaker and incubated for 2-3 h to yield a mid-logarithmic growth phase culture for studying antimicrobial activity and mechanism of action [20].

Antibacterial Activity Testing of AgNPs and Chitosan
Antibacterial efficiency is measured by the serial dilution and plate count method.The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values are used to determine susceptibilities of bacteria to drugs, and to evaluate the activity of new antimicrobial agents.This method is often used for preliminary screening of the antibacterial activity for a large number of agents.This technique is relatively insufficient to clearly determine live or dead cells and, therefore, results in much higher MBCs.
Four types of bacteria were investigated in this research; Escherichia coli O157:H7, Staphylococcus aureus ATCC25722, Vibrio cholera ATCC and Methicillin-resistant Staphylococcus aureus (MRSA) DMST20649, which are well known as common pathogenic bacteria in laboratories for biological research.All isolates were stored at −70 • C in 20% glycerol in micro-centrifuge tubes.The bacteria were streaked on nutrient agar (NA), cultured at 37 • C overnight and then kept in a cold room.
In the broth dilution method, all the bacteria were transferred to 5 mL of nutrient broth to produce concentrations of approximately 1 × 10 5 colony-forming units (CFU)/mL.This pre-culture was incubated for 24 h at 200 rpm and 37 • C. For preparation of the test inoculum, 0.1 mL of this pre-culture was transferred to 10 mL of nutrient broth, resulting in a suspension of 1 × 10 5 CFU/mL.We prepared AgNPs and AgNPs-Chitosan composites in many concentrations (following Table 1), diluted with deionized water.After that, each well of sterile 96-well plates for susceptibility testing were filled with 100 µL of suspension cells, and 100 µL of AgNPs and AgNPs-Chitosan composite added for test, incubated at 37 • C. The test results were evaluated 18-24 h later.The MIC value is estimated as a clear well, but this method cannot measure MBC values [21].In the serial dilution plate count method, preparation of all bacterial suspension cells for test was the same as for the broth dilution method; we prepared AgNPs in many concentrations (following Table 1), diluted with deionized water and CAZ.We added 100 µL of suspension cells with 100 µL of agents (AgNPs, and CAZ), incubated at 37 • C for 16-24 h.We extracted 50 µL of suspension to count cells by the serial dilution plate count method (10 −1 -10 −8 ).The MIC value corresponded to the lowest concentration that inhibited 99.99% of bacterial growth and the MBC value corresponded to the lowest concentration where 100% of the bacterial growth was inhibited, compared to the control (no treatment).Inhibition of bacterial cells was measured by the serial dilution plate count method assay.The percentage of inhibition was calculated using the Formula ((CFUcontrol − CFUtest)/CFUcontrol) × 100), in which CFUcontrol is the number in CFU of bacteria cells on a non-coated surface after 24 h.CFUtest is the number in CFU of bacteria cells on a surface coated with F1-F8 after 24 h [22].The average MIC and MBC values of AgNPs and chitosan were utilized to find the initial concentration for development of the formula for the self-disinfection coating.

Synthesis of AgNPs-Chitosan Composite
The synthesis approach used the conventional composite method to generate AgNPs-Chitosan composite.AgNPs (10 mL) were diluted and dissolved in deionized water (100 mL) for stock solution.Chitosan (0.2 g) was dissolved in 3% v/v acetic acid (40 mL).First, synthesized AgNPs-Chitosan composites were made by mixing AgNPs solution at various concentrations and chitosan solution at 156 µg/mL and stirring together at 600 rpm and room temperature for 30 min, which achieves AgNPs-Chitosan composite solution for synthesis of the self-disinfection coating.In this research, we divided formulas into two groups, with the first group containing especially AgNPs solution in 50% WPU (F1-F4) and the second group containing AgNPs-Chitosan composite (F5-F8) according to the information in Table 1.Coatings in F1-F4 were synthesized by mixing AgNPs solution in each concentration and 100% WPU by stirring machine at 600 rpm and room temperature for 15 min.Similarly, coatings in F5-F8 were synthesized by mixing AgNPs-Chitosan composite solution in each concentration and 100% WPU by stirring machine at 600 rpm and room temperature for 15 min [23,24].Then, the objectives were divided into two groups, which were to investigate whether chitosan had effects on stabilizing, controlling and releasing AgNPs, and enhancing its antibacterial activity.

Physical and Chemical Characterization of AgNPs and AgNPs-Chitosan Composite
The AgNPs and AgNPs-Chitosan composites were characterized for their shape, size and distribution by transmission electron microscope (FEI, TECNAI G2 20 version, Hillsboro, OR, USA) [25].For TEM measurement, 20 µL of nanocomposite solution was placed on carbon grids.The excess solution was removed by filter paper, and then the samples were evaporated slowly at 70 • C for 30 min when the preparation is ready for observation by TEM.The type of chemical reaction between AgNPs and chitosan was confirmed by Fourier transform infrared spectrophotometer (Bruker, TENSOR27 version, Billerica, MA, USA).In addition, the joints of the surfaces after coating in all formulas were investigated by scanning electron microscopy (LEO, Oberkochen, Germany).Finally, the hydrophobicity property of the coating on the surface was measured by FTA 100 Drop Shape Instrument B Frame System (First Ten Angstroms, Newark, CA, USA).

Preparation of Coating on Acrylic Piece as Sample Testing
The acrylic test pieces of 5-10 µm 0.3 cm thickness were cut by laser machine into the size of 5.0 × 5.0 cm 2 as a substrate.The solution of 50% WPU, AgNPs and AgNPs-Chitosan composite were coated on the acrylic pieces by electric high pressure airless sprayer in wind speed 854.95 kPa until its thickness was approximately 5-10 µm.After that, the test pieces were dried in a hot oven at 60 • C for 2 h until they were ready for characterization and antibacterial testing.

Antibacterial Activity Testing of Self-Disinfecting Coating
The antibacterial activity testing of the self-disinfecting coating was measured by ISO 22196 modified standard protocol.This is an internationally recognized test method for investigating the antibacterial activity of treated plastic materials (and other non-porous surfaces of products) to inhibit or kill the growth of test microorganisms [26][27][28].E. coli O157:H7 and S. aureus ATCC25722 were used as models of microorganisms.Both bacteria were in a nutrient broth and incubated at 37 • C for 18-24 h as starter cells.Then, starter cells were diluted for 2.5-10 × 10 5 CFU/mL with nutrient broth as bacterial stock.The test pieces were sterilized by UV light with laminar flow for 15 min on each side.The test pieces were placed on a sterile plate.Next, the 0.4 mL bacterial stock was applied to the sample surface and then covered by a clear polyethylene film (4.0 × 4.0 cm 2 ).Then, the sample plate was incubated at 37 • C for 24 h with a relative humidity higher than 90%.After 24 h, the living bacteria were extracted from the sample surface using 10 mL SCDLP broth.Next, ten-fold serial dilution (10 −1 -10 −7 ) of this solution by taking 1 mL of SCDLP solution mixed with 9 mL PBS buffer and then 0.1 mL of each serial dilution spread on the nutrient agar plate.The plate was incubated at 37 • C for another 24 h.For the evaluation, the results were determined by counting the CFU of the living bacteria on the agar plate.Finally, the percentage of growth reduction was calculated using the following Equation: % Reduction = [(CFUcontrol − CFUtest)/CFUcontrol] × 100, in which CFUcontrol is the number in CFU of bacteria cells on a non-coated surface after 24 h and CFUtest is the number in CFU of bacteria cells on a coated surface with F1-F8 after 24 h [29].The A schematic of antibacterial testing by the ISO 22196 modified standard is shown in Figure 1.

Quantification of Silver Ion Releasing from Self-Disinfection Coating
Quantification of silver ion release was measured by inductively coupled plasma mass spectroscopy (ICP-MS).The quantification and rates of silver ion (Ag + ) release were studied by means of inductively coupled plasma mass spectroscopy (ICP-MS) (Agilent, Santa Clara, CA, USA) at Khon Kaen branch, Central Laboratory (Thailand) Co., Ltd.(Bangkok, Thailand).The coated samples (F2-F4 and F6-F8) were immersed in flasks filled with 50 mL of deionized water at room temperature.The sample solution was collected after 1 day, 7 days, 30 days, 60 days, 120 days and 180 days [30].

Cytotoxicity Testing of AgNPs
The cytotoxicity of AgNPs was evaluated by the 3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT) assay, the cell proliferation rate and the reduction in cell viability when metabolic events led to apoptosis or necrosis.MTT, which is a yellow compound, is reduced by mitochondrial dehydrogenases into an insoluble purple formazan in living cells.A solubilizer, such as dimethyl sulfoxide, is added to dissolve the formazan crystals.The color can be measured in the range of 500-600 nm by a spectrophotometer.

Quantification of Silver Ion Releasing from Self-Disinfection Coating
Quantification of silver ion release was measured by inductively coupled plasma mass spectroscopy (ICP-MS).The quantification and rates of silver ion (Ag + ) release were studied by means of inductively coupled plasma mass spectroscopy (ICP-MS) (Agilent, Santa Clara, CA, USA) at Khon Kaen branch, Central Laboratory (Thailand) Co., Ltd.(Bangkok, Thailand).The coated samples (F2-F4 and F6-F8) were immersed in flasks filled with 50 mL of deionized water at room temperature.The sample solution was collected after 1 day, 7 days, 30 days, 60 days, 120 days and 180 days [30].

Cytotoxicity Testing of AgNPs
The cytotoxicity of AgNPs was evaluated by the 3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT) assay, the cell proliferation rate and the reduction in cell viability when metabolic events led to apoptosis or necrosis.MTT, which is a yellow compound, is reduced by mitochondrial dehydrogenases into an insoluble purple formazan in living cells.A solubilizer, such as dimethyl sulfoxide, is added to dissolve the formazan crystals.The color can be measured in the range of 500-600 nm by a spectrophotometer.
Normal Human Dermal Fibroblasts (NHDF) were used to determine the cytotoxicity of AgNPs in different concentrations of mammalian cells.The NHDF cells were prepared in a 96-well plate at a concentration of 1 × 10 4 and 8 × 10 3 cells/well.After 24 h, AgNPs at different concentrations (2-512 μg/mL) were added to the plate.Cells without treatment agents served as the control.To measure their viability, the cells were incubated and investigated after 24 h.After this period of treatment, 10 μL of 5 μg/mL MTT stock solution was added to each well and incubated for 4 h at 37 °C.The medium was removed, and 150 μL of dimethyl sulfoxide (DMSO) was added into each well to dissolve the formazan crystals.The solution was measured at 570 nm by a Varioskan™ LUX multimode microplate reader (Thermo Scientific, USA.Finally, the fluorescence intensity of 570 nm was used in each concentration to calculate percentage of cell viability with intensity of control as 100% cell viability [31].Normal Human Dermal Fibroblasts (NHDF) were used to determine the cytotoxicity of AgNPs in different concentrations of mammalian cells.The NHDF cells were prepared in a 96-well plate at a concentration of 1 × 10 4 and 8 × 10 3 cells/well.After 24 h, AgNPs at different concentrations (2-512 µg/mL) were added to the plate.Cells without treatment agents served as the control.To measure their viability, the cells were incubated and investigated after 24 h.After this period of treatment, 10 µL of 5 µg/mL MTT stock solution was added to each well and incubated for 4 h at 37 • C. The medium was removed, and 150 µL of dimethyl sulfoxide (DMSO) was added into each well to dissolve the formazan crystals.The solution was measured at 570 nm by a Varioskan™ LUX multimode microplate reader (Thermo Scientific, Waltham, MA, USA).Finally, the fluorescence intensity of 570 nm was used in each concentration to calculate percentage of cell viability with intensity of control as 100% cell viability [31].

Statistical Analysis
All experiments had a sample size of n ≥ 3 and are representative of repeated trials.Sample error bars on plots represent ± SD.Tests for statistical significance of the difference of the means were performed using a two-tailed Student's t-test assuming unequal variances using the IBM SPSS Statistics program.p-values are indicated as follows on the figures: (* p < 0.05).

Antibacterial Activity Testing of AgNPs and Chitosan
The MIC value corresponds to the lowest concentration that inhibits ≥99% of bacterial growth.The MBC value corresponds to the lowest concentration that inhibits 100% of bacterial growth.Experiments were performed in triplicate with three independent experiments for each cell type.
MIC and MBC values were determined by the broth micro dilution method and the plate count method and the results are shown in Table 2.The purpose of this experiment was to determine the optimum concentration of each substance for the development of coating formulations.The results show that the MIC and MBC ranges of AgNPs against all four bacterial strains were 2.00-32.00µg/mL and 4.00-128.00µg/mL, respectively, while the MIC and MBC ranges of chitosan against all four bacterial strains were 78-208 µg/mL.The result concludes that both AgNPs and chitosan were able to inhibit all four species of bacteria.Indeed, MIC and MBC values of AgNPs were lower than chitosan, showing that antibacterial activity of AgNPs is better than chitosan.AgNPs were also found to have the ability to inhibit Gram-negative bacteria better than Gram-positive bacteria because Grampositive bacteria have a peptidoglycan layer that is thicker than that of Gram-negative bacteria.Therefore, the aforesaid concentration will be used to create and develop the selfdisinfection coating formulas.We used the MICmax, MBCmax, 10MICmax and 10MBCmax values of AgNPs mixed with chitosan at a concentration of 156 µg/mL, which is the median of the MIC and MBC of chitosan.The aforesaid concentrations will be used in the development of self-disinfection coating formulas.The researchers expected that AgNPs/Ag + would play the main role in bacteria inhibition and that the chitosan, as a biopolymer, would increase stability, reduce sedimentation and control the silver ion release of silver nanoparticles, resulting in a long-term inhibitory effect.If the silver ion release of silver nanoparticles is low, it will reduce the toxicity to both mammalian cells and the environment.The formula of self-disinfection coating was developed based on the antibacterial activity testing results shown in Table 1.There are eight coating formulas with short-and long-term inhibitory properties, as shown in Table 2, which is divided into two groups.The first group includes coatings containing only silver nanoparticles as an ingredient, consisting of Formulas 1-4 (F1-F4), which is a group containing silver nanoparticles at the concentration of 32 µg/mL (MICmax), 128 µg/mL (MBCmax), 320 µg/mL (10MICmax) and 1280 µg/mL (10MBCmax) mixed with 50% of the water-based polyurethane.These are low concentrations that are commonly used in the coating industry.The second group includes coatings containing silver nanoparticles and Chitosan known as 'silver nanocomposite particles' as an ingredient consisting of Formulas 5-8 (F5-F8), which group contains silver nanoparticles at concentrations of 32 µg/mL (MICmax), 128 µg/mL (MBCmax), 320 µg/mL (10MICmax) and 1280 µg/mL (10MBCmax) mixed with Chitosan at a concentration of 39 µg/mL.We used concentrations one-fourth times as much as the median of the MIC and MBC because silver nanocomposite solution could be synthesized with the least precipitation.However, before each coating was coated onto the test surface, the researchers homogenized it with the mixture at 100 rpm for 5 min before spraying, to reduce such an issue.

Physical and Chemical Characterization of AgNPs and AgNPs-Chitosan Composite
To determine the characteristics of AgNPs and AgNPs after adding chitosan, UV-Vis spectroscopy was used to investigate the formation of AgNPs by assessing the signature surface plasmon resonance (SPR) bands.These optical features can be used to assess the size, shape and distribution of the AgNPs in the solution.We also evaluated and confirmed the size and shape of AgNPs by TEM in Figure 2a,b.The TEM micrograph and size chart analyzed from the Image J program show that it was found that silver nanoparticles from Prim Nanotechnology consisted of a symmetric spherical shape with an average size of 7.29 ± 1.65 nm.In Figure 2b, when Chitosan at 39 ug/mL concentration was added, the size of the silver nanoparticles decreased by 2-3 nm with an average size of 4.66 ± 2.08 nm.It also affected the shape, making the silver nanoparticles more asymmetric.Moreover, scattered aggregation and distribution were noticeable when chitosan was added.This may be due to the fact that chitosan reduced chelating and acted as a stabilizing agent, thereby reducing the size of AgNPs and increasing precipitation [32,33].In Figure 2c, the interaction between silver nanoparticles and chitosan was found.There were negative ions on the surface of silver nanoparticles and amine and hydroxyl groups of Chitosan, because the IR spectrum of Chitosan showed, at 3359.87 cm −1 , 1651.00 cm −1 and 1597.07 cm −1 wavelengths, an area with reduced transmittance percentage when silver nanoparticles were added [1].The joints of the surface after coating with all formulas were investigated by scanning electron microscopy with the result shown in Figure 3, that show the characterization of the joint and the distribution of silver nanoparticles and silver nanocomposite particles of six coatings in SEM cross-section micrographs, which shows that all coatings were able to coat the tested acrylic surface and the texture was smooth without gaps or bubbles.However, there were some particles found on some areas of the surface, which may be dust or other foreign matter formed during the coating process, since the coating was conducted in an open system.It was also found that the coating thicknesses of Formulas F2, F3, F4, F6, F7 and F8 were 4.1 µm, 4.5 µm, 2.0 µm, 2.7 µm, 6.1 µm and 2.6 µm, respectively.FTA 100 Drop Shape Instrument B Frame System can measure the hydrophobic properties of coatings by means of the contact angle measurement principle.Surfaces with hydrophobic properties repel or prevent water droplets.Additionally, water traps air in the gaps of the surface and contact surface between liquid and solid surfaces and the contact surface between the liquid and the air decreases, resulting in the formation of spherical water droplets with less contact angle hysteresis that can be further reduced by increasing the surface roughness, resulting in a higher contact angle.In this regard, the surface contact angle and surface roughness create a hydrophobic structure of the surface which reduces contact areas in dirt.The presence of water on any surface can be explained by the Young's equation and the surface roughness results in hydrophobicity.A self-cleaning surface is a surface that can self-clean due to the liquid formation.When liquid particles or water drops onto the surface, they will flow away without sticking to it and sweep away dust and dirt particles on the surface.
Table 3 shows the mean contact angle (degrees) and Wetting Tension (dy/cm) of all coatings.It was found that the contact angle of uncoated acrylic test plate surface was 74.24 ± 3.25 degrees, while the acrylic surface coated with 50% WPU was 70.76 ± 3.18 degrees.When considering the range of difference, it can be seen that the coating with 50% WPU does not increase the hydrophobicity of the surface, which may be due to the coating thickness being only 5.20 ± 0.53 µm.Therefore, there was no difference in the contact angle for both conditions.Likewise, the contact angle of the acrylic test plate surface coated with the Formulas 1, 2, 3, 5, 6 and 7 was found to be 71.84 ± 3.50, 72.29 ± 4.77, 67.10 ± 3.14, 68.65 ± 1.74, 67.51 ± 4.23 and 66.93 ± 3.65 degrees, respectively, which did not differ from the surface of the two conditions mentioned above.However, the contact angle of the acrylic test sheet surface coated with Formulas 4 and 8 showed a slight increase in the values of 83.22 ± 2.33 and 81.73 ± 1.28 degrees.The high concentration of AgNPs is unlikely to contribute to the change of properties of other coatings, but it may be due to the coating thickness and surface characteristics of different test strips, which is difficult to control with this technique.However, the contact angle value of all formulas is in range 0 < θ < 90 • degree and this indicates that the surface is low in hydrophobicity properties.
Likewise, the results of wetting tension (dy/cm) of droplets on the test surface were consistent with the contact angle (degrees).It was found that the uncoated surface and the surface coated with 50% WPU had no difference in wetting tension, which was 19.75 ± 3.95 and 23.96 ± 3.97 dy/cm.The surface coated with silver nanoparticles and silver nanocomposite particles according to the Formulas 1-8 had no wetting tension from the uncoated surface and the surface coated with 50% PU-water based.However, Formulas 4 and 8 yielded reduced wetting tension, which was 10.11 ± 1.71 and 8.86 ± 1.62 dy/cm, respectively.It can be concluded that the acrylic surface coated with 50% PU-water based and the formulas containing silver nanoparticles and silver nanocomposite particles do not significantly increase hydrophobic properties of the acrylic surface.

The Suitable Method of Self-Disinfection Coating Coated on Acrylic Surface
The coatings were applied using a high-pressure pump for the spray dryer method as shown in Figure 4.It was found that this method was suitable due to the optimum thickness of the coating (approximately 5 nm) and the uniform color throughout the sheet, indicating good distribution of silver nanoparticles.The amount of the substance is lower than the previous method and it can dry faster as well.
Coatings 2022, 12, 1832 11 of and the formulas containing silver nanoparticles and silver nanocomposite particles d not significantly increase hydrophobic properties of the acrylic surface.

The Suitable Method of Self-Disinfection Coating Coated on Acrylic Surface
The coatings were applied using a high-pressure pump for the spray dryer metho as shown in Figure 4.It was found that this method was suitable due to the optimum thickness of the coating (approximately 5 nm) and the uniform color throughout the shee indicating good distribution of silver nanoparticles.The amount of the substance is lowe than the previous method and it can dry faster as well.

Short-and Long-Term Antibacterial Activity Testing of Self-Disinfection Coating
The antibacterial efficacy of self-disinfecting coating against two types of bacteri E.coli O157:H7 and S.aureus ATCC25722, from 1 day to 4 months after coating has com

Short-and Long-Term Antibacterial Activity Testing of Self-Disinfection Coating
The antibacterial efficacy of self-disinfecting coating against two types of bacteria, E. coli O157:H7 and S. aureus ATCC25722, from 1 day to 4 months after coating has completely dried is verified by the technique modified from the ISO 22196 standard protocol.The results are shown in Figure 5a,b.

Quantification of Silver Ion Release from Self-Disinfecting Coating
We measured the silver ion release of the coating by the inductively coupled plasma mass spectrometry (ICP-MS) technique.Figure 6 shows the amount of silver ions released from all 6 coatings at 1-180 days (1 day-6 months) after immersion in deionized water by the ICP-MS technique.According to Figure 6, the kinetics of the amount of silver ion released from all coatings gradually increased as time went by, and the increase was linear.However, the amount of release was found to depend on the concentration of AgNPs in each coating.Comparing the mean quantity of silver ions released in each coating (F2-F8) at 1-180 days, the sequence was found as follows: F4 (0.18759 ± 0.00352 μg/mL) > F3 (0.13798 ± 0.00409 μg/mL) > F2 (0.02330 ± 0.00067 μg/mL) > F8 (0.03168 ± 0.00622 μg/mL) > F5 (0.01585 ± 0.00494 μg/mL) > F6 (0.00582 ± 0.00019 μg/mL).The results show that the amount of silver element released from F8 was approximately 5.92 times lower than F4, while the effectiveness of inhibition is similar and did not differ significantly.
Therefore, the coatings of Formula 8 were better in terms of toxicity to mammalian cells and were more environmentally friendly than Formula 4. Figure 7 also shows that the coatings with chitosan had lower color intensity than the non-added coatings at the same AgNPs concentration for various reasons.Therefore, the coating of Formula 8 will be commercially developed into a self-disinfection coating.Moreover, Figure 7 reveals that coatings formulated with the same AgNPs were found to have significantly lower release of silver ions (95% confidence interval) at every measurement time.Therefore, it can be concluded that chitosan has the effect of reducing the amount of silver ions released but it does not affect the antibacterial efficacy of the self-disinfecting coating.The results show the kinetics of the inhibitory efficacy from 1 day to 16 weeks (4 months) of all coating formulas against E. coli O157:H7 (Figure 5a) and S. aureus ATCC25722 (Figure 5b).The pattern of both graphs is a sideway pattern, referring to the graph, without a clear trend, with the appearance of fluctuation like a zigzag.However, it was found that the F2 had the same tendency inhibition and that it was close to F6 and F3 had the same tendency inhibition and similar to F7 and F4 had the same tendency inhibition and was similar to F8.It is in accordance with the concentration of AgNPs present in the aforementioned coatings.Therefore, the graph looks like a sideways pattern, which may be due to the thickness of the coating in each piece not being the same, as the sample preparation method is quite difficult to control the thickness of the coating evenly for all pieces, unlike the use of industrial paint sprayers.Surprisingly, the results concluded that after 4 months coating drying, the inhibitory efficacies of all coating formulas were not significantly increased and decreased both against E. coli O157:H7 and S. aureus ATCC25722.

Quantification of Silver Ion Release from Self-Disinfecting Coating
We measured the silver ion release of the coating by the inductively coupled plasma mass spectrometry (ICP-MS) technique.Figure 6 shows the amount of silver ions released from all 6 coatings at 1-180 days (1 day-6 months) after immersion in deionized water by the ICP-MS technique.According to Figure 6, the kinetics of the amount of silver ion released from all coatings gradually increased as time went by, and the increase was linear.However, the amount of release was found to depend on the concentration of AgNPs in each coating.Comparing the mean quantity of silver ions released in each coating (F2-F8) at 1-180 days, the sequence was found as follows: F4 (0.18759 ± 0.00352 µg/mL) > F3 (0.13798 ± 0.00409 µg/mL) > F2 (0.02330 ± 0.00067 µg/mL) > F8 (0.03168 ± 0.00622 µg/mL) > F5 (0.01585 ± 0.00494 µg/mL) > F6 (0.00582 ± 0.00019 µg/mL).The results show that the amount of silver element released from F8 was approximately 5.92 times lower than F4, while the effectiveness of inhibition is similar and did not differ significantly.Therefore, the coatings of Formula 8 were better in terms of toxicity to mammalian cells and were more environmentally friendly than Formula 4. Figure 7 also shows that the coatings with chitosan had lower color intensity than the non-added coatings at the same AgNPs concentration for various reasons.Therefore, the coating of Formula 8 will be commercially developed into a self-disinfection coating.Moreover, Figure 7 reveals that coatings formulated with the same AgNPs were found to have significantly lower release of silver ions (95% confidence interval) at every measurement time.Therefore, it can be concluded that chitosan has the effect of reducing the amount of silver ions released but it does not affect the antibacterial efficacy of the self-disinfecting coating.

Cytotoxicity Testing of AgNPs Solution
The toxicity testing of silver nanoparticles solution against Normal Human Dermal Fibroblasts (NHDF) was measured by the MTT assay technique.The researchers wanted to find out the concentration of silver nanoparticles toxic to animal cells.The researchers chose to use animal dermal fibroblast cells as an example.Figure 8 shows that the silver nanoparticles can cause toxicity to dermal fibroblasts (NHDF) in a dose-dependent manner.That is, when the concentration is increased, it will lead to increased toxicity.The safe concentration of silver particles is 0-16 µg/mL, since this concentration yields the percentage of cell viability of more than 50%, which represents non-toxicity to mammalian cells.According to the previous experiments, Figure 8 shows the quantity of highest silver ion release from self-disinfection coating being 0.18759 ± 0.00352 µg/mL.This represents 0.01466% of the initial concentration, which is considered to be a very low concentration and is not toxic to such animal cells.However, further studies on the toxicity to mammalian cells and the environment are still needed.

Cytotoxicity Testing of AgNPs Solution
The toxicity testing of silver nanoparticles solution against Normal Human Dermal Fibroblasts (NHDF) was measured by the MTT assay technique.The researchers wanted to find out the concentration of silver nanoparticles toxic to animal cells.The researchers chose to use animal dermal fibroblast cells as an example.Figure 8 shows that the silver nanoparticles can cause toxicity to dermal fibroblasts (NHDF) in a dose-dependent manner.That is, when the concentration is increased, it will lead to increased toxicity.The safe concentration of silver particles is 0-16 μg/mL, since this concentration yields the percentage of cell viability of more than 50%, which represents non-toxicity to mammalian cells.According to the previous experiments, Figure 8 shows the quantity of highest silver ion release from self-disinfection coating being 0.18759 ± 0.00352 μg/mL.This represents 0.01466% of the initial concentration, which is considered to be a very low concentration and is not toxic to such animal cells.However, further studies on the toxicity to mammalian cells and the environment are still needed.

Discussion
Currently, a primary way that infectious diseases are spread is by contact with the environment, such as toys, door handles, bench tops, bedding and toilets, and then to another person who comes in contact with the contaminated environmental source.Many research studies have reported that many types of bacteria can persist and survive on in-

Discussion
Currently, a primary way that infectious diseases are spread is by contact with the environment, such as toys, door handles, bench tops, bedding and toilets, and then to another person who comes in contact with the contaminated environmental source.Many research studies have reported that many types of bacteria can persist and survive on inanimate surfaces for many days or months [34,35].Staphylococcus aureus including MRSA and MSSA can persist and survive on plastic surfaces for 9-12 days, stainless steel for 72 h and on a dry mop for ≤28 days [36].E. coli O157:H7 can survive on spinach leaves for 27 days, in soil for 179 days and in water for 98 days [37,38].Pseudomonas aeruginosa can survive on a dry floor for 6 h to 16 months [39].Antimicrobial impregnated surfaces are increasingly being discussed and used to eliminate microorganisms that can persist and survive for long periods on inanimate surfaces [40].However, because of the long contact period needed for microorganisms on antimicrobial surfaces [41][42][43], such technologies may be useful for surfaces with a low frequency of hand contacts.Therefore, this is the reason that the self-disinfecting coating has been of interest recently.
Many previous research studies tried to develop self-disinfecting coatings containing heavy metals effective for weeks or months and they have been accepted for consideration as a new approach to disinfecting or preventing the growth of bacteria on surfaces.Silver nanoparticles (AgNPs) are more interesting due to an increased catalytic efficiency and causing more antibacterial activity than the bulk counterpart.Additionally, at low concentrations, AgNPs are non-toxic to the human body and have high antibacterial properties.In 2017, Khwanmuang et al. reported that Ag-TiO 2 /polyurethane nanocomposites had high potential for self-disinfecting coating applications with short-term antibacterial activity [44].In addition, WPU/Ag nanocomposite has the potential to be used as a coating for antimicrobial products with enhanced mechanical properties [45].
In this research, we aimed to develop self-disinfection coating with short-and longterm antibacterial activity by AgNPs-chitosan composite in WPU.First, we investigated the efficiency of AgNPs which were derived from Prime Nanotechnology Co., Ltd.(Bangkok, Thailand) and chitosan by the broth microdilution method and plate count method.The minimum and maximum MIC values of AgNPs against all four species of bacteria were 2 and 32 µg/mL, respectively.MBC values were 4 and 128 µg/mL, respectively, and the median of MIC and MBC for chitosan was 128 µg/mL (in Table 2).The aforesaid concentration was used to develop self-disinfection coating formulas.Our formulas for self-disinfection coating are divided into two groups, with the first group (F1-F4) receiving a coating agent containing AgNPs in various concentrations and the second group receiving coating agents containing AgNPs-Chitosan composite (F5-F8) with the details shown in Table 1.The self-disinfection coating was synthesized by conventional composite approaches at room temperature.The physical and chemical properties of the AgNPs-chitosan nanocomposite were studied by transmission electron microscopy (TEM), scanning electron microscope (SEM), atomic force microscopes (AFM) and FTA 100 Drop Shape Instrument B Frame System.The results in Figure 2 demonstrate that at a concentration of 39 µg/mL in acetic acid, the size of AgNPs was reduced from 7.29 ± 1.65 to 4.66 ± 2.08 nm due to chitosan acting as a reducing agent [46], the shape changes from a sphere into an asymmetrical circle and increasing aggregation of AgNPs due to acidic pH and physiological electrolyte content universally induce micron-scale aggregation [47].Negative charges on the surface of AgNPs interact with amine (-NH 2 ) and hydroxyl (-OH) groups of chitosan by an electrostatic force.All formulations of the coating were shown to have low hydrophobicity properties (in Figure 2 and Table 3).The suitable method for fabricating self-disinfecting coatings is a high-pressure pump for a spray dryer machine.This method derived optimum thickness of the coating (approximately 5 nm) and the uniform color throughout the sheet indicating good distribution of silver nanoparticles with the intensity of color being varied by the concentration of NPS (in Figure 4).Furthermore, antibacterial activity of coating on the surface was measured by the ISO 22196 standard protocol.The inhibitory efficacy of F4 is 81.72% ± 3.15% on E. coli O157:H7 and 84.64% ± 2.59% on S. aureus ATCC25722 and F8 is 82.07%± 3.01% on E. coli O157:H7 and 83.27% ± 3.12% on S. aureus ATCC25722.Furthermore, the quantity of silver ion from the coating was measured by inductively coupled plasma mass spectrometry (ICP-MS).The results revealed that F8 released an amount of silver ions approximately 5.92 times lower than F4, while for the same concentration of AgNPs and the inhibition efficacy were not significantly different (in Figures 6 and 7).The safe concentration of silver particles is 0-16 µg/mL, since this concentration yields a percentage of cell viability of more than 50%, which represents non-toxicity to mammalian cells (in Figure 8).
In summary, we were successfully able to synthesize a water-based polyurethane coating with both short-and long-term antimicrobial effects.Thus, after the F8 coating was applied to the test surface for 4 months, it was found to be effective in inhibiting E. coli O157:H7 (82.07%± 3.01%) and S. aureus ATCC25722 (83.27% ± 3.12%).Moreover, this coating released silver ions just 0.18759 ± 0.00352 µg/mL (accounting for 0.01466% of the initial concentration).This coating formulation has low toxicity on NHDF cells (representing mammalian cells) and is an environmentally friendly product.Therefore, the Formula 8 coating will be further developed into commercial self-disinfecting coatings.

Conclusions
The self-disinfecting coating of the AgNPs-Chitosan composite in WPU was successfully prepared using conventional composite approaches and coating on an acrylic surface by electric high pressure airless sprayer was accomplished with thickness of coating about 5-10 µm.From the results, we conclude that the F8 coating will be further developed into commercial self-disinfecting coatings.F8 (the formulation containing AgNPs at a concentration of 1280 µg/mL and 50% v/v of WPU) showed the highest mean percentage of long-term inhibition effect from 1 day to 4 months after the coating completely dried being 82.07%± 3.01% on E. coli O157:H7 and 83.27% ± 3.12% on S. aureus ATCC25722.The amount of silver element released from F8 is approximately 5.92 times lower than F4, although the effectiveness of inhibition is similar and did not differ significantly.In this study, we have shown that when adding chitosan at 39 µg/mL in acetic acid solution the effect is to decrease size, which results in better antimicrobial efficiency [46,47].However, this did not increase the inhibitory efficiency of the coating status.Additionally, it increases aggregation due to acidic pH and physiological electrolyte content universally induced micron scale aggregation [45].Finally, chitosan has the effect of reducing the release of silver ions, the antibacterial effect was prolonged and environmental toxicity was reduced.In addition, from the NHDF cell toxicity test results, it was found that the concentration and amount of AgNPs or Ag ions released from the F8 coating were non-toxic on mammalian cells.However, the yellow color of the F8 coating after drying was still visible, which was quite clearly the characteristic of AgNPs and makes it difficult to apply to white or bright surfaces.Therefore, in future research, it may be necessary to improve this property in order to be able to bring real commercial benefits.

Figure 2 .
Figure 2. TEM images and average size of silver nanoparticles (a) and silver nanoparticle-Chitosan (b) corresponding to the photograph sample by Image J program.(c) The comparison of FTIR spectra for Chitosan and Chitosan combined with silver nanoparticles.

Figure 2 .
Figure 2. TEM images and average size of silver nanoparticles (a) and silver nanoparticle-Chitosan (b) corresponding to the photograph sample by Image J program.(c) The comparison of FTIR spectra for Chitosan and Chitosan combined with silver nanoparticles.

Figure 2 .
Figure 2. TEM images and average size of silver nanoparticles (a) and silver nanoparticle-Chitosan (b) corresponding to the photograph sample by Image J program.(c) The comparison of FTIR spectra for Chitosan and Chitosan combined with silver nanoparticles.

Figure 4 .
Figure 4. Tested acrylic sheets coated with all eight coating formulas (size 5 × 5 cm 2 .)with EURO MAX S-710 high pressure spray gun MAX S-710.

Figure 5 .
Figure 5.The kinetics of the inhibitory efficacy from 1 day to 16 weeks (4 months) of all coating formulas: (a) tested against E.coli O157:H7 and (b) tested against S. aureus ATCC25722.

Figure 5 .
Figure 5.The kinetics of the inhibitory efficacy from 1 day to 16 weeks (4 months) of all coating formulas: (a) tested against E. coli O157:H7 and (b) tested against S. aureus ATCC25722.

Figure 6 .
Figure 6.The kinetics of silver nanoparticles and silver ion release of the six coatings from a period of 1 day to 180 days.

Figure 6 .
Figure 6.The kinetics of silver nanoparticles and silver ion release of the six coatings from a period of 1 day to 180 days.

Figure 6 .
Figure 6.The kinetics of silver nanoparticles and silver ion release of the six coatings from a period of 1 day to 180 days.

Figure 8 .
Figure 8.The percentage of dermal fibroblast (NHDF) survival after incubation with the silver nanoparticles solution at a concentration of 2-512 μg/mL for a period of 24 h.

Figure 8 .
Figure 8.The percentage of dermal fibroblast (NHDF) survival after incubation with the silver nanoparticles solution at a concentration of 2-512 µg/mL for a period of 24 h.

Table 1 .
Formulas of self-disinfection coating developed based on antibacterial activity testing result.

Table 2 .
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of AgNPs and Chitosan against bacteria by serial dilution plate count assay.

Table 3 .
Average values of contact angle (degrees) and wetting tension (dy/cm) of coating agents by FTA 100 Drop Shape Instrument B Frame System.