A novel non-releasing antibacterial poly(styrene-acrylate)/waterborne polyurethane composite containing gemini quaternary ammonium salt

Abstract

A series of novel non-releasing antibacterial polymer coatings, incorporating gemini quaternary ammonium salt modified waterborne polyurethane into a commercial poly(styrene-acrylate), were designed and prepared via a facile blending strategy. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and differential scanning calorimetry (DSC) results are used to prove the compatibility between the polyurethanes and poly(styrene-acrylate)s. X-ray photoelectron spectroscopy (XPS) and water contact angle (WCA) measurements are used to clarify the surface structure and properties of polymer coatings, indicating gemini quaternary ammonium salts (GQAS) attached onto waterborne polyurethane chains could migrate and aggregate onto surfaces of these polymer blending coatings. On the basis of the antibacterial characteristics of GQAS, these polymer blending coatings showed high efficiency in killing airborne bacteria, e.g. S. aureus and E. coli, in contact-killing tests. Thus, the antibacterial coatings and blending strategy are promising for the development of environmentally friendly materials.

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1. Introduction

Bacterial infections have become a serious public environmental issue, since the growing bacterial resistance and a diminishing useable antibiotic pipeline have made these infections hard to treat.^1–3 Attachment and the subsequent proliferation and colonization of bacteria on artificial surfaces always cause the formation of a biofilm, resulting in the spread of fatal community-acquired and nosocomial infections.^4–6 Therefore, the first step toward preventing community-acquired and nosocomial infections is the inhibition of bacterial colonization on wall surfaces,⁷ especially in those places where high hygiene standards are required, such as food processing environments,⁸ hospitals⁹ and schools. Generally, disinfectants are applied to prevent bacteria from infecting humans and deteriorating materials, these, of course, generate a considerable environmental problem and also support the development of resistant bacterial strains, e.g. methicillin resistant Staphylococcus aureus (MRSA).¹⁰ Thus various antibacterial surfaces have been designed to inhibit bacterial colonization and kill pathogenic microorganisms, which are loaded with antibacterial agents that are released into the surrounding environment to kill bacteria, such as silver,^11,12 antibiotics,¹³ triclosan,¹⁴ iodine and so on. Nevertheless, these surfaces work efficiently only in the short term,¹⁵ and constantly releasing these biocides into the environment could cause unpredictable long-term environmental problems¹⁶ and even the development of resistant bacteria.¹⁷ As a consequence, there is an urgent demand for novel antibacterial surfaces that possess non-releasing antibacterial activities but are less likely to cause the development of bacterial resistance.

Contact-active surfaces are well-known for their non-release and long-term activities.^10,18 In these surfaces, antimicrobial agents are generally covalently tethered to polymer backbones to avoid releasing. Various antibacterial agents from synthetic chemicals to natural biomolecules, such as quaternary ammonium compounds, polycations, antibiotics, chitosan, antimicrobial peptides, and antimicrobial enzymes, etc., are used in this field.^19–22 Among them, quaternary ammonium salts have become the most widely used antibacterial agent, owing to their excellent cell membrane penetration properties, low toxicity, good environmental stability, non-irritation, low corrosivity, extended residence time and excellent biological activity in comparison with the others.²³ Moreover, as developed in our group, gemini quaternary ammonium salts (GQAS) containing double quaternary ammonium groups have much stronger antibacterial activities than conventional quaternary ammonium salts, which show a great promise in antibacterial polymer materials.²⁴ However, such antibacterial surfaces are prepared through quite elaborate techniques that are useful in the laboratory but not in industry. Thus, developing a facile way to obtain antibacterial and contact-active materials is urgent. A promising method is directly blending the matrix with non-releasing antibacterial polymeric additives.

In addition, polyurethanes have been extensively applied in organic coatings due to their extraordinary molecular tailorability, mechanical properties, biocompatibility and water dispersibility. Some antibacterial polyurethane coatings have been developed.^25,26 As recently reported by our group, a series of novel antibacterial and antifouling waterborne polyurethanes based on GQAS and non-toxic hydrophilic poly(ethylene glycol) (PEG) have also been designed and synthesized via a simple polymerization process without any organic solvent involved.²⁷ These waterborne polyurethanes possess high efficiency in killing airborne bacteria on contact without release, and could potentially be applied as antibacterial coatings or additives for preventing microbial contamination.

Herein, we report a new type of GQAS-based antibacterial polymer coating material prepared by incorporating antibacterial waterborne polyurethanes into a poly(styrene-acrylate) emulsion, since poly(styrene-acrylate) has been extensively used in latex paints. Polyurethane has good compatibility with poly(styrene-acrylate) by virtue of their carbonyl groups. The obtained antibacterial coating materials are characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, differential scanning calorimetry (DSC), X-ray photoelectron spectroscopy (XPS) and water contact angle (WCA) measurements. The antibacterial activities of the polymer surfaces are evaluated by contact killing tests.

2. Materials and methods

2.1 Materials

The antibacterial waterborne polyurethane (WPU, 16.78 wt%) was synthesized according to our previous report.²⁷ The structure of WPU is shown in Fig. 1, WPU was synthesized based on isophorone diisocyanate (IPDI), polyoxytetramethylene glycol (PTMG), poly(ethylene glycol) (PEG), L-lysine and a novel L-lysine-derivatized diamine containing gemini quaternary ammonium salt (EG12). IPDI was used as a hard segment, PTMG and PEG were used as soft segments, L-lysine and EG12 were used as chain extenders. Poly(styrene-acrylate) emulsion (PSA) (RS-998A, 53.62 wt%) was purchased from BATF Industry Co., Ltd, China. Agar was obtained from Biosharp, Japan. Tryptone was supplied by Oxoid Ltd, UK. Beef powder was purchased from Beijing Solarbio Science & Technology Co., Ltd, China. Sodium chloride (NaCl) was obtained from Chengdu Kelong Chemical Co., Ltd, China. 2,3,5-Triphenyltetrazolium chloride (TTC) was supplied by Sanland-chem International Inc. Staphylococcus aureus (S. aureus, Gram-positive, ATCC 6538) and Escherichia coli (E. coli, Gram-negative, ATCC 25922) were used for contact-killing tests.

Fig. 1 Schematic structure of waterborne polyurethane (WPU).

2.2 Measurements

Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was recorded on a Nicolet 6700 spectrometer (Thermo Electron Corporation, U.S.A.) between 4000 and 400 cm⁻¹, with a resolution of 4 cm⁻¹. Each sample spectrum was obtained by averaging 64 scans.

Differential scanning calorimetry (DSC) was performed on a Netzsch STA 449C Jupiter (Germany) at a heating rate of 10 °C min⁻¹ in the range of −120 to 100 °C under a steady flow of nitrogen. The DSC results were fit using the combination of Lorentzian and Gaussian equations by Origin-pro 9.0.

X-ray photoelectron spectroscopy (XPS) was carried out with a Kratos XSAM-800 Spectrometer with a Mg KR. The X-ray gun was operated at 20 kV and 10 mA current with a take-off angle of 30°. The relative atomic percentage of each element on the PSA/WPU surface was calculated by the peak areas using atomic sensitivity factors specified for the XSAM-800. C1s, O1s and N1s spectra bands were deconvoluted into sub-peaks by processing with the XPSPEAK4.0 spectrometer software.

Water contact angles were obtained on a Drop Shape Analysis System DSA 100 (Krüss, Hamburg, Germany) using 3 μL of distilled water at room temperature. The results were the mean values of three replicates.

2.3 Preparation of polymer coatings

Polymer composites were prepared by blending WPU and PSA emulsions in various proportions, as listed in Table 1. Polymer coatings were prepared by spreading the blending polymer emulsions on glass slides with a size of 15 mm × 15 mm layer by layer, and each layer contained 50 μL of emulsion. To obtain the best antibacterial activities, the second layer was spread after drying the first layer surface in air, so was the third one. At last, three-layer coating films were obtained. Then, the coatings were air dried for 24 h, and dried under vacuum at 40 °C for 48 h in sequence. The final coatings were placed in a desiccator before testing.

Table 1 The formula of PSA/WPU polymer composites

Sample	Mass ratio of PSA/WPU^a	GQAS^b (wt%)	Tg (°C)
a Mass ratios were calculated by dry weight.b The weight fractions of GQAS were calculated by dry weight.
PSA	1 : 0	0	31.26
PSA/WPU-1	9.6 : 1	1.8	−56.18, 29.18
PSA/WPU-2	6.4 : 1	2.8	−72.72, 29.33
PSA/WPU-3	4.8 : 1	3.6	−76.43, 28.60
PSA/WPU-4	3.2 : 1	5.4	−77.21, 28.34
WPU	0 : 1	21.8	−77.65

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2.4 Antibacterial test

Antibacterial activities of the polymer coatings were evaluated by contact-killing tests.²⁸ Before each test, samples were sterilized by UV-irradiation for 30 min to avoid exogenous bacteria. The clinically relevant bacteria S. aureus and E. coli were grown for 12 h in suitable nutrient media at 37 °C. The 12 h grown bacteria were added to 30 mL PBS (pH = 7.4) and the final concentration of the bacterial solution was then adjusted to 10⁶ CFU mL⁻¹ for S. aureus and 10⁷ for CFU mL⁻¹ for E. coli, respectively. To mimic the scenario of natural deposition of bacteria onto a surface, aqueous suspensions of bacteria were sprayed onto both glass slides and films coated on glass slides, followed by air-drying. Afterward, the sprayed slides were transferred into Petri dishes and then the growth agar (0.8% agar in nutrient medium, autoclaved at 121 °C for 25 min, and cooled to 40 °C) was poured into Petri dishes slowly to make sure that the sprayed cells would not be washed off. The Petri dishes were closed and incubated at 37 °C for 12 h. This allows the initially sprayed cells to grow while being in contact with the surface. Finally, cells were stained with TTC (0.5 mg mL⁻¹) for observation.²⁹

3. Results and discussion

3.1 Bulk structure and compatibility of PSA/WPU composites

Poly(styrene-acrylate) emulsion, which is one of the most widely used coatings in various fields, has excellent weather resistance, durability, chemical corrosion and wear resistance, etc. As antibacterial waterborne polyurethanes are incorporated into this coating, the blending microstructure and compatibility of these two polymers are firstly investigated. To explore the compatibility and aggregation behavior of these two components in PSA/WPU composites, FTIR-ATR was used to characterize PSA/WPU coatings. Fig. 2 shows the FTIR-ATR infrared spectra of the two polymers and their blending coatings in the zone of 4000–800 cm⁻¹.

Fig. 2 FTIR-ATR spectra of PSA, WPU and blending coatings. (A) Full spectra and amplified regions. (B) Curve-fitting of PSA/WPU-4. (C) Fit peak 3s of free carbonyl group in WPU. (D) Fit peak 4s of hydrogen bonded carbonyl group in WPU.

For pure PSA, the typical five groups of peaks at 3300 cm⁻¹, 2924 cm⁻¹ to 2853 cm⁻¹, 1726 cm⁻¹, 1494 cm⁻¹ to 1450 cm⁻¹, and 1158 cm⁻¹ are due to the stretching vibrations of carboxyl group bands, alkyl group bands, the CO–O of acrylate, aromatic ring of styrene and C–O in acrylate, respectively.³⁰ On the other hand, distinguishing characteristic absorption peaks at 3306 cm⁻¹, 1716 cm⁻¹, 1644 cm⁻¹, 1557 cm⁻¹, 1365 cm⁻¹ and 1105 cm⁻¹ assigned to N–H stretching vibrations, urethane groups (NH–CO–O), urea groups (NH–CO–NH), N–H bending bands, C–N band, and C–O–C of ether groups, respectively, are present in WPU, which is consistent with our recent report.²⁷ For PSA/WPU blending coatings, characteristic absorption peaks of both components are present in the spectra. More importantly, with WPU as functional macromer additives, it can be clearly observed from Fig. 2A that the characteristic absorption peaks at 3306 cm⁻¹, 1644 cm⁻¹, 1557 cm⁻¹, 1365 cm⁻¹ and 1105 cm⁻¹ assigned to characteristic groups in WPU increase with increasing amounts of WPU in the composites. This indicates the WPU content on the surface of coatings increases when increasing the amount of WPU. The –NH stretching vibration of WPU is a very broad band centered at about 3310 cm⁻¹, characteristic of a wide distribution of hydrogen bonds of –NH with urethane C=O, urea C=O, and urethane alkoxy oxygen in different geometries. For the PSA/WPU samples (PSA/WPU-1, PSA/WPU-2, PSA/WPU-3 and PSA/WPU-4, Fig. 2A), as the WPU content increases, their –NH stretching bands (Fig. 2A) shift to higher wavenumbers and become narrow, suggesting that the hydrogen bond distribution between WPU has been changed when the PSA is present. In contrast to N–H stretching bands, carbonyl bands are much more complicated.³¹ In order to clarify the interaction between WPU and PSA, we analyze the stretching band region of carbonyl groups (around 1730 cm⁻¹) by curve-fitting analysis, as shown in Fig. 2B. These results from the curve-fitting suggest the presence of four Gaussian bands, indicating that some new interactions are formed between the two components to induce division. The representative C=O peak deconvolution of PSA/WPU-4 is shown in Fig. 2B and confirms these bands assignable to carbonyl groups in PSA are centered at approximately 1730 cm⁻¹ (peak 1, free C=O from acrylate and acrylic) and at 1724 cm⁻¹ (peak 2, hydrogen bonded C=O from acrylate and acrylic), whereas those assigned to carbonyl groups in WPU are centered at approximately 1710 cm⁻¹ (peak 3, free C=O from urethane and urea) and at 1698 cm⁻¹ (peak 4, hydrogen bonded C=O from urethane and urea).³² When we focus on the peaks of virgin C=O bands in WPU at 1710 cm⁻¹ (Fig. 2C) and at 1698 cm⁻¹ (Fig. 2D), the intensities of peaks decrease with an increasing amount of WPU in the composites, indicating that hydrogen bonds are formed between ester/carboxyl groups in PSA and urethane/urea groups in WPU. As a result, both free and hydrogen bonded carbonyl groups in WPU have been reduced. The existence of hydrogen bonds between PSA and WPU would benefit the compatibility of these two components. To further investigate the compatibility of PSA and WPU in these composites, DSC was carried out to determine the T_gs of PSA/WPU coatings and these data are listed in Table 1. The T_gs of pure PSA and WPU are at 31.26 °C and −77.65 °C, respectively. Two T_gs were obtained in blending coatings, in which the lower one is attributed to WPU, and the higher one should be from the chains of PSA. Apparently, the T_g of WPU in low WPU content blends (e.g. T_g of WPU in PSA/WPU-1: −56.78 °C) is drastically higher than that of WPU (−77.65 °C). Then, the T_g in high WPU content blends gets gradually closer to that of WPU (Table 1). This phenomenon can be explained by the formation of hydrogen bonds between ether groups of soft segments in WPU and carboxyl groups in PSA resulting in the soft segments’ mobility being restricted. In particular, most soft segments in WPU with a low content of blending have been involved in the formation of hydrogen bonds, resulting in sharp increases of their T_gs. On the other hand, with increasing WPU content in blends, the T_gs of chains in PSA affected by the soft segments of WPU decrease continuously. These results indicate that the two components show good compatibility and strong interaction with each other, in good agreement with the FTIR-ATR results.

3.2 Surface structures and properties of PSA/WPU coatings

As is well known, it is a critical principle that antibacterial components could migrate from bulks onto the surfaces of antibacterial coating materials. To investigate the surface structures and properties of these PSA/WPU coatings, XPS was used to determine the amount of C, N and O on the surfaces of these coatings. The XPS spectra of PSA, WPU and PSA/WPU blending coatings are shown in Fig. 3. The high-resolution C1s spectra as observed from the XPS analysis were resolved into three characteristic peaks. The deconvoluted peaks at 284.8, 285.9 and 288.6 eV are attributed to functional groups of C̲–C̲/C̲–H, C̲–O/C̲–N and C̲=O, respectively. For O1s spectra, the deconvoluted peaks at 531.8 and 533.1 eV are assigned to functional groups of C–O̲ and C=O̲, respectively. Moreover, for N1s spectra, deconvoluted peaks at 399.3, 400.5 and 402 eV are attributed to functional groups of N̲H–CO–N̲H, N̲H–CO–O and , respectively. These data are shown in Fig. 4 and listed in Table 2, the relative atomic percentage of C on the surfaces of PSA/WPU coatings decreases with increasing WPU content, while the relative atomic percentage of N and O increases. The theoretical atomic percentage of N⁺ in WPU bulk is 0.74%, while the atomic percentage of N⁺ on the WPU surface is 2.11%, indicating that GQAS have migrated to surface. In PSA/WPU-4 coatings, the atomic percentage of N⁺ on their surfaces (0.84%) is higher than that in pristine WPU bulk, even though WPU has been diluted to one fourth with PSA, which suggests that GQAS have been aggregated onto these surfaces. For the other blending coatings, their N⁺ atom percentages on surfaces are also higher than those in their bulks (calculated by mass ratio) indicating that GQAS have been aggregated onto the surfaces of these coatings too. This is due to the amphipathicity of GQAS in WPU, these GQAS chains tend to gradually move to the water–air interface during the drying process, leading to the enrichment of N⁺ and N1s on the surfaces of the coatings.

Fig. 3 XPS diagrams of PSA, WPU and PSA/WPU blends. (A) PSA; (B) PSA/WPU-1; (C) PSA/WPU-2; (D) PSA/WPU-3; (E) PSA/WPU-4; and (F) WPU.

Fig. 4 Graphical representation of the relative atomic percentage of C, O and N on the surface of PSA, WPU and PSA/WPU blend coatings.

Table 2 Atomic percentages on the surfaces of PSA, WPU and PSA/WPU films from XPS spectra at 30° take-off angle

	C (%)			O (%)		N (%)
	C–C/C–H	C–N/C–O	C=O	C–O	C=O	NH–CO–NH	NH–CO–O	C–N^+
a The theoretical atomic percentage in bulk. The formula of WPU can be found in ref. 27.
PSA	74.91	9.51	2.74	10.17	2.67	0	0	0
PSA/WPU-1	72.27	10.44	3.20	8.46	4.03	0.96	0.26	0.38
PSA/WPU-2	60.67	19.27	3.29	8.60	6.13	1.37	0.20	0.47
PSA/WPU-3	65.01	14.08	3.51	9.07	6.25	1.35	0.12	0.61
PSA/WPU-4	62.67	13.87	3.71	9.42	7.19	2.03	0.26	0.84
WPU	45.07	29.55	2.71	5.82	8.03	6.12	0.59	2.11 (0.74^a)

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To further clarify the surface properties of PSA/WPU coatings, water contact angle (WCA) measurement was applied to determine the hydrophilicity of these coatings. The data of static WCA over time on the coatings are shown in Fig. 5. Since the hydrophilic end groups of GQAS tend to aggregate onto the surfaces of pristine WPU coatings, their WCAs are merely 10.8° at beginning and then spreading (data are not shown in the figure). In contrast, the WCAs on PSA coatings remain nearly unchanged at around 82° within 30 s. These blends coatings are more hydrophilic as the amount of GQAS moieties on the surface increases.³³ With an increasing content of WPU in blending coatings, initial WCAs on these films decrease and their equilibrium WCAs sharply reduce within 5 s. Especially, the equilibrium WCAs of PSA/WPU-4 are merely 34.6°, indicating that GQAS moieties have migrated onto the surfaces of PSA/WPU blends spontaneously.²⁹ It is consistent with the XPS results that the amount of GQAS at the surface of coatings increases with increasing WPU. These results confirmed that the GQAS moieties could migrate and aggregate onto the surfaces of polymer blending coatings, this characteristic would enhance the contact-killing efficiency of these coatings. In addition, in spite of hydrophilicity being enhanced by the incorporation of WPU, no distinct swelling or shape change is observed in water resistance tests after immersing in water for 72 h. And the same phenomenon could be also observed in our contact-killing test (as shown in Fig. 6). No distinct swelling or shape change of the films was observed after incubating in gel at 37 °C for 12 h. Thus, these coatings can be used to kill airborne bacteria in humid environments.

Fig. 5 Water contact angle (WCA) results of PSA and PSA/WPU blending coatings. (1) PSA; (2) PSA/WPU-1; (3) PSA/WPU-2; (4) PSA/WPU-3; (5) PSA/WPU-4. Photographs on the right are the corresponding images of the equilibrium water contact angles on coatings.

Fig. 6 Antibacterial activity of polymer coatings on glass surface. (a) for S. aureus, (b) for E. coli. Each black dot corresponds to a bacterial colony grown from a single surviving bacterial cell.

3.3 Antibacterial property of PSA/WPU coatings

A contact-killing test was carried out to measure the antibacterial activity of these blending coatings.^28,29 Fig. 6 shows typical samples of antibacterial PSA/WPU blending coatings (two square-shape coatings for each blend). Bacteria were sprayed onto both glass slides and coatings coated on glass slides. Generally, bacteria in contact with the antibacterial coatings will be killed while those inoculated on glass slides will form colonies. As shown in Fig. 6, numerous S. aureus and E. coli colonies grown on surfaces of these coatings are well distinguishable from the glass control. The number of bacterial colonies on PSA/WPU coatings is markedly less than that on glass control. No inhibition zone is observed in all cases, which indicates that these bacteria are killed on contact and no biocides are released from PSA/WPU coatings.^12,34 Additionally, it is found that the higher content of WPU in blends, the fewer S. aureus survive. A similar phenomenon is observed in E. coli colonies. The results further confirm that the amount of antibacterial GQAS on these coating surfaces increase with increasing content of WPU in blends. According to these pictures, these coatings show better antibacterial efficiency to S. aureus colonies compared with E. coli colonies. Few S. aureus colonies are observed as the WPU is above 13.5%, e.g. PSA/WPU-2, and the bactericidal rate of PSA/WPU-4 is almost 100%. However, it should be noted that it is hard to kill Gram-positive and Gram-negative bacteria at the same time with a mono-component antibacterial agent.^35,36 More often, Gram-negative bacteria are much more difficult to kill due to their additional outer lipid membranes.⁷ Herein, the number of E. coli colonies decreased gradually with increasing WPU content. When the WPU content is up to 24% in PSA/WPU-4, merely several E. coli colonies could be observed and the bacteriostasis rate is up to 90%, indicating that these coatings possess good antibacterial efficiency. The results confirm that the blending PSA/WPU coatings have excellent antibacterial ability in killing airborne bacteria, both S. aureus and E. coli colonies.

4. Conclusions

In summary, a novel coating material with good antibacterial activity was successfully prepared by incorporating gemini quaternary ammonium salt (GQAS) modified waterborne polyurethane into poly(styrene-acrylate) via a facile blending strategy. The non-releasing GQAS attached onto waterborne polyurethane chains could migrate to the surfaces of these coatings and endow antibacterial activities, as demonstrated by XPS and WCA measurements. The surfaces of coatings can kill both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria on contact with high efficiency. This work develops a facile and effective strategy to prepare non-releasing antibacterial coatings. The antibacterial coatings and blending strategy could potentially be widely used for the development of environmentally friendly materials.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (51173118 and 51273124), the National Science Fund for Distinguished Young Scholars of China (51425305), and the Opening Project of State Key Laboratory of Polymer Materials Engineering (Sichuan University) (sklpme 2014-2-03).

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