Adhesive antibacterial coatings based on copolymers bearing thiazolium cationic groups and catechol moieties as robust anchors

Progress in Organic Coatings 136 (2019) 105272

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Adhesive antibacterial coatings based on copolymers bearing thiazolium cationic groups and catechol moieties as robust anchors

T

Alberto Chiloechesa,b, Coro Echeverríaa,b, , Rocío Cuervo-Rodríguezc, Daniela Plachàd, ⁎ Fátima López-Fabale, Marta Fernández-Garcíaa,b, Alexandra Muñoz-Bonillaa,b, ⁎

a

Instituto de Ciencia y Tecnología de Polímeros (ICTP-CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain Interdisciplinary Platform for Sustainable Plastics towards a Circular Economy-Spanish National Research Council (SusPlast-CSIC), Madrid, Spain c Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Avenida Complutense s/n, Ciudad Universitaria, 28040 Madrid, Spain d Nanotechnology Centre, VŠB–Technical University of Ostrava, 17. Listopadu 15/2172, Ostrava, Czech Republic e Hospital Universitario de Móstoles C/ Dr. Luis Montes, s/n, 28935 Móstoles, Madrid, Spain b

ARTICLE INFO

ABSTRACT

Keywords: Antibacterial coatings Cationic copolymers Dopamine RAFT polymerization

Herein, we describe a simple approach for the preparation of antibacterial polymeric coatings based on mussel inspired catechol chemistry. A series of statistical copolymers composed of 2-(4-methylthiazol-5-yl)ethyl methacrylate (MTA) and N-(3,4-dihydroxyphenethyl) methacrylamide (DOMA) were synthesized by conventional free radical and reversible addition fragmentation chain transfer (RAFT) polymerizations. Subsequently, the thiazole groups of MTA units were quaternized with methyl and butyl iodide as alkylating agents to provide cationic copolymers with also adhesive anchoring groups of catechol. The copolymers were systematically studied to investigate the effects of composition (MTA/DOMA ratio), molecular weight and alkylating agent on adhesive and antibacterial properties. It was proved that DOMA units play a major role in the adhesion, while the antibacterial activity only decreases slightly with content of DOMA up to 32%. Remarkable, for similar MTA molar equivalent, the copolymers with higher molecular weight exhibit better antimicrobial properties in solution, whereas when they are tethered onto a surface as a coating, the copolymers with lower molecular weight showed enhanced antibacterial performance even against Gram-negative bacteria. These findings confirm that antimicrobial polymers attached onto surfaces behave in a different manner than in solution, and can be more effective as the mobility and accessibility of the cationic groups increase.

1. Introduction The development of antimicrobial coatings is currently one of the main strategies of the global action plan to mitigate bacterial infections as preventive alternative. [1] In addition to the awareness of antimicrobial resistance, implantable device‐associated infections resulting from bacterial adhesion and subsequent biofilm formation are of equal concern. In the biofilm, bacteria are embedded in protective extracellular substances (EPSs), which provide a defensive against the action of antimicrobial agents [2]. Indeed, conventional antibiotics are typically ineffective against biofilms, which are up to 1000 times more resistant than their planktonic counterparts [3]. As the initial bacterial surface adhesion is the primary stage of biofilm formation process, the use of antibacterial coating to reduce the incidence of bacterial biofilms is a very promising approach. In addition to indwelling devices and implants, preventive strategies based on antibacterial surfaces are

⁎

required in surfaces near patient environments, which are also responsible of the spread of hospital-acquired infections. [4] Of all the main approaches to antibacterial surfaces, the contact-killing coatings stand out due to its unique properties and advantages over other strategies. Such surfaces present the ability to kill adhering bacteria upon contact rather than by releasing biocides. Thus, the drawbacks associated to biocide release surfaces such as side-effects and toxicity, generation of resistance, and the need of re-loading the surface with the biocide, are limited in contact-killing surfaces. These contact-killing coatings mainly comprise cationic polymers such as polymers bearing quaternary ammonium compounds, with efficient and broad-spectrum bactericidal properties [5–9]. Other important requirements for coatings are the facile and the rapid deposition as well as the durability. To this regards, mussel inspired surface chemistry [10,11] based on adhesive proteins containing catechol moieties has facilitated the design and the development of coatings in the last years. Catechol and

Corresponding authors. E-mail addresses: [email protected] (C. Echeverría), [email protected] (A. Muñoz-Bonilla).

https://doi.org/10.1016/j.porgcoat.2019.105272 Received 11 April 2019; Received in revised form 5 July 2019; Accepted 9 August 2019 Available online 18 September 2019 0300-9440/ © 2019 Elsevier B.V. All rights reserved.

Progress in Organic Coatings 136 (2019) 105272

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1 H-NMR (700 MHz, DMSO-d6), δ (ppm): 8.81 (bs, 1H, CH 1,3thiazole), 8.79, 8.68 (bs, 2H, OH), 7.28–7.81 (bd, 1H, NH DOMA), 6.63 (bs, 1H, HAr), 6.58 (bs, 1H, HAr), 6.41 (bs, 1H, HAr), 4.02 (bs, 2H, OCH2), 3.06 (bs, 4H, CH2-1,3 thiazole, -NH-CH2-), 2.47 (bs, 2H, CH2Ph), 2.30 (s, 3H, -CH3 1,3-thiazole), 2.0-1.3 (m, 4H, 2 CH2), 1.07-0.4 (m, 6H, 2CH3). 13C-NMR (175 MHz, DMSO-d6), δ (ppm): 178.55174.88 (2 C = O),151.16 (-CH 1,3-thiazole), 149.74 (C-CH3 1,3-thiazole), 145.59 (CAr-OH), 143.95 (CAr-OH), 130.67 (CAr-CH2),127.32 (C-S 1,3-thiazole), 119.50 (CAr), 116.19 (CAr), 115.99 (CAr), 65.01 (OCH2), 54.94-50.26 (CH2 main chain) 45.28-44.47 (Cquat main chain), 42.05 (CH2-NH), 34.51 (CH2-Ph), 25.12 (CH2-1,3-thiazole), 20.61-16.74 (2 CH3 main chain), 15.11 (CH3-1,3-thiazole).

derivatives can adhere to various inorganic and organic materials, which opens a new route to the modification surfaces by a simple approach [12]. Combination of dopamine derivatives as adhesive anchoring groups with other functionalities has allowed the preparation of coatings with multiple functionalities [13] such as antimicrobial [11,14–16], antifouling [17], and biomedical adhesive [18]. Herein, we design a series of copolymers bearing thiazolium groups with antimicrobial activity and catechol moieties as anchoring groups, to obtain adhesive and efficient antibacterial coatings. Statistical copolymers of dopamine methacrylamide (DOMA) and 2-(4-methylthiazol-5-yl)ethyl methacrylate (MTA) were synthesized by two polymerization techniques, conventional radical polymerization and reversible addition fragmentation chain transfer (RAFT) polymerization. In this study, unprotected DOMA monomer was successfully employed, not only in conventional radical polymerization but also in RAFT polymerization, which is more susceptible to the presence of radical scavengers as catechol that is prone to oxidation. Subsequently N-alkylation of thiazole groups leading to cationic thiazolium moieties neither affects the catechol groups that remain intact. Thus, the adhesive properties as well as the antimicrobial activity of the copolymers were evaluated as a function of the copolymer composition, MTA/ DOMA ratio and of the molecular weight.

2.3. Synthesis of the P(DOMAx-co-MTAy) copolymers by RAFT polymerization Likewise, a set of copolymers were prepared by RAFT polymerization varying the monomer molar ratio MTA/DOMA = 90/10, 80/20, 70/30 and 60/40. In a typical procedure, e.g. for the sample MTA/ DOMA = 70/30, MTA (1.8487 g, 8.75 mmol) and DOMA (0.8287 g, 3.75 mmol) comonomers were placed in a Schlenk tube. Then, 0.125 mmol of AIBN, 0.25 mmol of CPDB and 12.5 mL of anhydrous DMF were added to the mixture. The reaction was deoxygenated by introducing argon gas for 30 min and the mixture was stirred at 70 °C for 24 h. After cooling to room temperature, the mixture was dialyzed in water (molecular weight cut-off of membranes was 1000 Da) and after lyophilization pure copolymers were obtained. The copolymers showed similar 1H and 13C-NMR spectra as those described in section 2.2.

2. Experimental section 2.1. Materials The monomers, N-(3,4-dihydroxyphenethyl) methacrylamide (DOMA) [19] and 2-(4-methylthiazol-5-yl)ethyl methacrylate (MTA) [20] were synthesized according to previous works. Iodomethane (MeI, 99%), 1-iodobutane (BuI, 99%) and 2-cyano-2-propyl benzodithioate (CPDB; > 97%) were purchased from Sigma-Aldrich. 2,2′-Azobisisobutyronitrile (AIBN, 98%) obtained from Across was recrystallized twice from methanol (MeOH, 99.9%; Sigma-Aldrich) prior to use. The solvents, anhydrous N,N-dimethylformamide (DMF, 99.8%), dimethylsulfoxide (DMSO, 99%), n-hexane (99%) and chloroform (99.8%), were purchased from Alfa-Aesar. Cellulose dialysis membranes (CelluSep T-series and H1) were obtained from Membrane Filtration Products, Inc. For microbiological studies: sodium chloride aqueous solution (NaCl, 0.9%, BioXtra, suitable for cell cultures) and phosphate buffered saline (PBS, pH 7.4) were purchased from Sigma-Aldrich. The 96 well microplates for the determination of the minimal inhibitory concentrations (MIC) were obtained from Thermo Scientific. Columbia agar plates with 5% sheep blood were purchased from BioMérieux. BBL™ Mueller–Hinton broth was supplied by Becton, Dickinson and Company and was used as a microbial growth medium. Strains of Grampositive Staphylococcus aureus (S. aureus, ATCC 29213) bacteria and Gram-negative Escherichia coli (E. coli, ATCC 25922) were obtained from Oxoid™.

2.4. Synthesis of cationic copolymers by N-alkylation reaction Both series of copolymers, obtained by conventional free radical and RAFT polymerizations, were modified by N-alkylation reaction with either iodomethane or 1-iodobutane leading to the corresponding cationic copolymers. A typical quaternization reaction is described as follows for the copolymer with MTA/DOMA ratio of 90/10 as an example. The copolymer (222 mg, 0.90 meq of MTA) was dissolved in 5 mL of anhydrous DMF and then a large excess of methyl iodide was added (280 μL, 4.52 mmol, ratio MTA/alkyl iodide ≈ 1:5). The mixture was purged with argon during 15 min and then stirred at 70 °C for one week to ensure the complete reaction. Afterward, the cationic copolymer was purified by precipitation into n-hexane followed by dialysis against distilled water and finally was isolated by freeze-drying. The cationic copolymers quaternized with methyl iodide were denoted as P (DOMAx-co-MTAy)M whereas the copolymers quaternized with butyl iodide were labelled as P(DOMAx-co-MTAy)B. P(DOMAx-co-MTAy)M = 1H-NMR (700 MHz DMSO-d6), δ (ppm): 10.27 (bs, 1H, CH 1,3-thiazolium), 8.73 (bs, 2H, OH), 7.98 (bs, 1H, NH DOMA), 6.75-6.35 (m, 3H, 3 HAr), 4.15 (bs, 3H, +NCH3), 4.07 (bs, 2H, OCH2), 3.40 (bs, 2H, CH2-1,3-thiazolium), 2.86 (bs, 2H, CH2-N DOMA), 2.55 (s, 5H, CH3-1,3 thiazolium, CH2-Ph), 2.28-1.37 (m, 4H, 2 CH2), 1.37-0.20 (m, 6H, 2 CH3).13C-NMR (175 MHz, DMSO-d6), δ (ppm): 179.04-175.37 (2 C = O),157.53 (-CH 1,3-thiazolium), 144.00 (2 CArOH, C-CH3 1,3-thiazolium), 133.62 (Cquat-S), 64.73 (OCH2), 55.8849.80 (CH2 main chain), 47.10-43.25 (Cquat main chain), 41.42 (CH3N+), 30.23 (CH2-Ph), 25.86 (CH2-1,3-thiazolium), 20.84-15.45 (2 CH3 main chain), 12.65 (CH3-1,3-thiazolium). P(DOMAx-co-MTAy)B = 1H-NMR (700 MHz DMSO-d6), δ (ppm): 10.24 (bs, 1H, CH 1,3-thiazolium), 8.70 (bs, 2H, OH), 7.66 (bs, 1H, NH DOMA), 6.64 (bs, 1H, HAr), 6.55 (bs, 1H, HAr), 6.42 (bs, 1H, HAr), 4.53 (bs, 2H, +NCH2), 4.06 (bs, 2H, OCH2), 3.38 (bs, 2H, CH2-1,3-thiazolium), 3.09 (bs, 2H, CH2-N DOMA), 2.56 (s, 5H, CH3-1,3 thiazolium, CH2-Ph), 2.17-1.16 (m, 4H, 2 CH2), 1.83 (bs, 2H, CH2), 1.38 (bs, 2H, CH2), 0.93 (s, 3H, CH3) 1.16-0.25 (m, 6H, 2CH3).13C-NMR (175 MHz, DMSO-d6), δ (ppm): 177.35-176.31 (2 C = O),157.07 (-CH 1,3-

2.2. Synthesis of P(DOMAx-co-MTAy) copolymers by free radical polymerization Several copolymers with different chemical composition were prepared by conventional free radical polymerization (C-FRP) of MTA and DOMA comonomers using different feed ratio (monomer molar ratio MTA/DOMA = 90/10, 80/20, 70/30 and 60/40). Briefly (e.g. for the sample MTA/DOMA = 70/30), both monomers, MTA (3.6970 g, 17.5 mmol) and DOMA (1.6590 g, 7.5 mmol) at a total concentration of 1 M in anhydrous DMF (25 mL) were added into a Schlenk tube, together with the initiator, 40 mg of AIBN (0.01 M). The mixture was deoxygenated by purging argon during 15 min and then, the reaction was stirred at 70 °C for 24 h. The copolymers were obtained purified by precipitation in distilled water, followed by dialysis against water and freeze-drying. 2

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thiazolium), 145.56 (CAr-OH), 143.93 (CAr-OH), 143.35 (C-CH3 1,3thiazolium), 134.16 (Cquat-S), 130.63 (CAr-CH2), 119.48 (CAr), 116.19 (CAr), 115.93 (CAr), 64.51 (OCH2), 55.73-50.78 (CH2 main chain), 53.41 (CH2-N+), 47.10-43.25 (Cquat main chain), 41.82 (CH2-NH), 34.50 (CH2-Ph), 31.26 (CH2), 25.89 (CH2-1,3-thiazolium), 19.45 (CH2), 20.58-15.68 (2 CH3 main chain), 13.96 (CH3), 12.41 (CH3-1,3-thiazolium).

copolymers were dissolved in sterile water at a concentration of 1 mg/ mL. 96‐well microplates containing two-fold dilutions of copolymer solutions were prepared and inoculated with 50 μL of the final bacterial suspensions with an eight‐channel pipette. Positive and negative controls were also performed. The plates were sealed and incubated at 37 °C for 24 h. Minimal inhibitory concentration (MIC) values were determined as the lowest concentration of antimicrobial polymer where no visible growth was observed in the wells of the microtiter plates. Antimicrobial activities of the coatings were determined against Gram-negative E. coli bacteria. Each copolymer-coated glass was placed in a sterile falcon tube and then 2 mL of the bacterial suspension (106 CFU/mL) were added. Control experiments with glass substrates and with only inoculum were also performed. The tubes were shaken at 120 rpm during 24 h and then 200 μL of each solution were placed in a 96-well plate. Bacterial growth was determined by the absorption of optical density (OD) at 550 nm via a microplate reader (VirClia® Chemiluminescence). The measurements were made at least in triplicate and the antibacterial ratio was calculated as previously described accordingly to the following equation: [23]

2.5. Copolymer characterization All the copolymers were characterized before and after quaternization by 1H and 13C NMR measurements carried out at room temperature with a Bruker AVIII spectrometer (700 MHz for 1H and 175 MHz for 13C) using as solvent DMSO-d6 purchased from SigmaAldrich. Molecular weights and polydispersity indexes of P(DOMAx-coMTAy) copolymers were determined by gel permeation chromatography (GPC) on Waters Division Millipore system and a Waters 2414 refractive index detector, using as eluent 1 mL/min flow rate of GPC grade DMF (Scharlau) stabilized with 0.1 M LiBr (Sigma Aldrich, > 99.9%) at 50 °C. The calibration was made with poly(methyl methacrylate) standards (Polymer Laboratories LTD). Zeta potential measurements of the quaternized copolymers in distilled water were conducted in a Zetasizer Nano series ZS (Malvern Instruments Ltd) Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, England) using the Smoluchowski equation for electrophoretic mobility at 25 °C.

Antibacterial ratio =

ODcontrol

ODExpimental sample ODcontrol

× 100

(1)

3. Results and discussion 3.1. Preparation and characterization of the cationic copolymers P (DOMAx-co-MTAy)M and P(DOMAx-co-MTAy)B

2.6. Film formation and adhesive properties tests

The synthesis of the P(DOMAx-co-MTAy) copolymers was carried out by two different techniques, free radical polymerization and controlled radical polymerization via RAFT (Scheme 1). The first technique has many advantages over other polymerization processes as does not require rigorous conditions or special reagents, and permits polymerization of vinyl monomers bearing unprotected catechol groups, such as DOMA monomer. On the other hand, RAFT polymerization allows the preparation of well-defined polymers with controlled molecular weight, polydispersity and composition, but requires the use of chain transfer agents. Also catechol groups can act as radical scavengers, and in the case of RAFT procedure, the presence of DOMA can affect the radical polymerization [24]. In this work, two series of copolymers were prepared from similar feed molar composition MTA/DOMA = 90/10, 80/20, 70/30 and 60/ 40. Based on previous publications, the molar percentage of DOMA in the feed does not exceed 40% in order to obtain copolymers up to 30 mol% of DOMA, as it is reported to be an optimal composition with strong adhesive properties [18,25]. The copolymers were characterized by 1H and 13C NMR spectroscopy (see Fig. 1 and Figure S1 of Supporting Information), where it is clearly seen the successful incorporation of DOMA units with unprotected catechol groups. Remarkably, the hydroxyl groups of the catechol structure at 8.70 ppm are visible in the 1H NMR spectrum, and then we can confirm that the catechol groups incorporated in the copolymer as adhesive anchoring moieties were not affected by the radical polymerization conditions. The composition of the copolymers, thus the molar percentage of DOMA in the copolymers, was determined by 1H-NMR. The molecular characteristics of the synthesized copolymers are given in Table 1. As expected the polydispersity indices (Mw/Mn) and the molecular weights (Mn) determined by GPC measurements are found to be much lower in copolymers synthesized by RAFT polymerization. However, the polydispersity indices of all the copolymers obtained by RAFT ranged between 1.2 and 1.4, which are slightly higher in comparison with other systems probably due to the presence of unprotected catechol groups. On the other hand, the copolymer synthesized by conventional free radical polymerization presents higher Mw/Mn values, which are typical from this type of polymerization. The molecular weight values are in all cases above 20,000 g/mol. Remarkably, in both

Films were formed onto glass substrates by drop casting from polymer solution in distilled water (1 mg/mL). The films were subsequently dried under vacuum at room temperature. Solvent erosion experiments were carried out to study the adhesion of the copolymers on glass substrate. For these experiments, distilled water was used as good solvent, while chloroform was employed as non-solvent. The films were immersed in the solvent (either water, chloroform or dimethyl sulfoxide) and washed during 24 h. 2.7. Film characterization The determination of static water contact angle was carried out on the surface of the prepared films using a KSV Theta goniometer (KSV Instruments Ltd.) from digital images of 3.0 μL water droplets on the film surface. The contact angle was measured at least three times on different sites of the surface. Each data reported is the average of triplicate measurements ± SD (standard deviation). The films were also characterized by Attenuated Total Reflectance Fourier Transform Infrared spectroscopy (ATR-FTIR) technique using a Perkin Elmer Spectrum Two instrument. The surface topography of the prepared films was analyzed by optical profilometry using a Zeta-20 optical profiler (Zeta Instruments) with a 100× optical objective and 13 nm of vertical resolution. 2.8. Antibacterial experiments Bacterial growth inhibition assays were carried out against the ATCC bacterial strains (S. aureus, ATCC 29213 and E. coli, ATCC 25922) according to the Clinical Laboratory Standards Institute (CLSI) microbroth dilution reference method [21,22]. All experiments were conducted in triplicate. The bacteria were previously incubated on 5% sheep blood and Columbia Agar plates for 24 h at 37 °C in a Jouan IQ050 incubator (Winchester, VA, USA). Subsequently, bacteria suspensions of about 108 colony-forming units (CFU) were prepared by adjusting concentration with saline solution to ca. 0.5 of the McFarland turbidity standard. Suspensions of 2 × 106 CFU mL−1 were finally obtained by further dilution with Mueller–Hinton broth. The 3

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Scheme 1. Synthesis of the P(DOMAx-co-MTAy) copolymers via conventional free radical (C-FRP) and RAFT polymerizations and quaternization reaction with alkylating agents to provide cationic P(DOMAx-co-MTAy)R copolymers.

systems the molecular weight of the copolymers decreases with the content of DOMA, and this behavior is more evident in the samples obtained by RAFT polymerization. This can be explained by the effect of the catechol moieties in the radical polymerization as radical scavengers. In addition, molar percentage for DOMA found in all isolated copolymers is lower than that in the feed, as previously described in other systems reported in literature [18,25]. Subsequently, the cationic copolymers were obtained from the P (DOMAx-co-MTAy) copolymers by simple quaternization reaction with either iodomethane (P(DOMAx-co-MTAy)M) and 1-iodobutane (P (DOMAx-co-MTAy)B). The reactions were performed at 70 °C during one week using a great excess of alkylation agent to assure the complete quaternization. NMR spectroscopy measurements demonstrated that practically complete quaternization was achieved in all the cases. A characteristic example of 1H-NMR of quaternized sample is given in Fig. 1b. The signal assigned to the aromatic protons of the 1,3-thiazole group at 8.79 ppm shifts to 10.09 ppm when 1,3-thiazolium moieties are formed. Similarly, the NMR spectrum reveals the presence of the hydroxyls of catechol groups after quaternization reaction, thus the non-oxidized adhesive form of DOMA units is preserved. Then, zeta potential measurements of quaternized copolymers were performed. As can be seen in Table 2, all the copolymers are positively charged as a result of quaternization reaction, with values ranging from +64 to +43 mV. As expected, the zeta potential values tend to become more positive as the content of MTA units increases in the copolymer. It

Table 1 Molecular characteristics of the P(DOMAx-co-MTAy) copolymers. Feed % mol DOMA

Feed % mol MTA

Copolymer % mol DOMA

Free radical polymerization 40 60 31 30 70 24 20 80 18 10 90 9 RAFT polymerization 40 60 32 30 70 22 20 80 13 10 90 6

Copolymer % mol MTA

Mn (g/ mol)

(Mw/Mn)

69 76 82 91

21600 22500 25200 24600

1.79 1.80 2.45 2.50

68 78 87 94

3100 3400 6100 9500

1.32 1.36 1.37 1.24

can be also appreciated in the series of copolymers prepared by free radical polymerization, that the zeta potential values are higher in the copolymers quaternized with methyl iodide in comparison with that in P(DOMAx-co-MTAy)B copolymers. The incorporation of a long length alkylating chain, i.e. butyl, in the copolymers with higher molecular weight reduces the zeta potential values and increases the hydrophobicity; even in the case of the copolymer with the lowest content of MTA units, provokes insolubility in aqueous media. It is worthy to remark that the hydrophobic-hydrophilic balance of antimicrobial

Fig. 1. 1H-NMR spectra of a) P(DOMA22-co-MTA78) and b) P(DOMA22-co-MTA78)B copolymers. 4

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in which the butylated polymers showed enhanced activity. Therefore, we can say that the hydrophobic DOMA units give enough hydrophobicity to methylated copolymers. In contrast, for the series of copolymers with lower molecular weight, those synthesized by RAFT, the copolymer quaternized with butyl iodide showed slightly better activity. It is also known that the molecular weight of polycations is also a crucial factor in the antimicrobial activity, and in these systems it is clear that the antibacterial properties are strongly influenced by the molecular weight. Remarkably, the copolymers synthesized by free radical polymerization exhibit enhanced antibacterial activity, with lower MIC values. The molecular weight of this series of copolymer ranges between 21000–25000 g/mol, values higher than that obtained by RAFT polymerization, 3100–9500 g/mol. As the concentration of antimicrobial polymers is given in μg/mL and the compositions (MTA/ DOMA ratio) are similar between copolymers of different series (Table 1), it can be said that for similar MTA molar equivalent, the copolymers with higher molecular weight exhibit better antimicrobial properties as previously observed. [30] In many investigations, it was revealed that antimicrobial activity of polymers increases with molecular weight but only up to certain range, over which the activity decreases due to impediments to diffuse through membrane [31,32].

Table 2 Zeta potential values and antimicrobial activities measured as MIC of the two series of synthesized cationic copolymers. Copolymer

Zeta potential (mV)

Free radical polymerization P(DOMA31-co-MTA69)M 62 ± 5 64 ± 3 P(DOMA24-co-MTA76)M P(DOMA18-co-MTA82)M 64 ± 5 62 ± 5 P(DOMA9-co-MTA91)M P(DOMA31-co-MTA69)B – P(DOMA24-co-MTA76)B 53 ± 5 43 ± 5 P(DOMA18-co-MTA82)B P(DOMA9-co-MTA91)B 49 ± 5 RAFT polymerization P(DOMA28-co-MTA72)M 48 ± 5 P(DOMA22-co-MTA78)M 54 ± 6 56 ± 4 P(DOMA13-co-MTA87)M P(DOMA6-co-MTA94)M 60 ± 6 48 ± 6 P(DOMA28-co-MTA72)B P(DOMA22-co-MTA78)B 45 ± 5 P(DOMA13-co-MTA87)B 61 ± 4 64 ± 5 P(DOMA6-co-MTA94)B

S. aureus MIC (μg/mL)

E. coli MIC (μg/mL)

64 32 32 32 – 32 32 32

64 32 32 32 – 125 125 125

> 500 125/64 32 32 32 32 32 32

> 500 500 250 250 250 250 125 125

polymers is closely associated with their effectivity, [26] as the cationic residues enhance their interactions with the anionic bacterial membranes, whereas the hydrophobic moieties interact with the inner hydrophobic part of the membrane, leading to a disruption and subsequent bacterial death.

3.3. Polymer coatings, adhesive properties Once, it was demonstrated that increasing amount of DOMA adhesive units, thus lower content of cationic MTA units, in the copolymers reduces the antibacterial performance; we evaluate the effect of DOMA/MTA ratio in the adhesive properties. Previous studies showed that increasing the catechol content involved enhanced adhesion but only up to ∼33% [25]. In this work, copolymers with variable DOMA content up to ∼32% were prepared and used to evaluate the influence of DOMA units on the surface adhesion. Films prepared by drop casting onto glass substrates were first tested by ATR-FTIR analysis. Figure S2 shows as an example the spectrum of the film obtained from the copolymer P(DOMA18-co-MTA82)M. The functional groups of both comonomer units were clearly identified in the spectrum, at 1654 cm−1 (C = O, DOMA), 1723 cm−1 (C꞊O, MTA), 1590 cm−1 (C꞊N+ thiazolium, MTA), 1141 cm−1 (C-O, MTA) [14,20]. Then, the films were evaluated by water contact angle measurements to determine the properties of coated surface. Subsequently, films were exposed to different treatments, such as water and chloroform washing under sonication. Then, the damage of the coatings was evaluated by the difference in the contact angle found after the solvent erosion. Table 3 summarized the results obtained for all the prepared films. In comparison with the contact angle values obtained in bare glass substrate, 14 ± 2°, the coatings increases the contact angle of the surfaces as a result of increasing hydrophobicity. On one hand, it is clearly seen that in general, the contact angle augments as the content of DOMA units increases in the copolymer. Secondly, it is also appreciated as a general insight, an increase in the hydrophobicity of the coatings when the copolymers are quaternized with butyl iodide. Also, when copolymers with similar composition and different molecular weight are compared, it is worthy to remark that copolymers with lower molecular weight (those synthesized by RAFT) provides coatings with lower contact angle, suggesting that the cationic groups are more exposed and available to the interface due to their enhanced mobility. After washing the surfaces either with chloroform (a bad solvent) or water (a good solvent), the contact angle of the coatings made from all copolymers P(DOMAx-co-MTAy)M and P(DOMAx-co-MTAy)B remained almost unchanged or even in most of the cases increases. Anyway, the

3.2. Antibacterial properties The antibacterial properties of the synthesized copolymers were quantitatively tested by determining the minimum inhibition concentration (MIC), which is the lowest concentration of the polymer that inhibits bacterial growth. For this study, S. aureus (Gram-positive) and E. coli (Gram-negative) were selected as representative bacteria. MIC values obtained for both series of copolymers are also listed in Table 2. It can be clearly seen that most of the synthesized copolymers showed antibacterial activities against both bacterial strains S. aureus and E. coli. In general, and as it could be expected, the higher the content of cationic thiazolium groups (MTA units) results the lower MIC values against both tested bacteria. Compared to the MIC obtained for the homopolymers, PMTAM (32 and 64 μg/mL against S. aureus and E. coli, respectively) and PMTAB (8 and 32 μg/mL against S. aureus and E. coli, respectively), [20] the incorporation of DOMA units reduces the activity, but only to certain extend. Notice that the evaluation of P (DOMA31-co-MTA69)B copolymer was not possible due to its water insolubility. The results are particularly good for S. aureus with MIC values up to 32 μg/mL, while the copolymers exhibit moderate activity against Gram-negative E. coli bacteria. These results agree well with other published data of cationic antimicrobial polymers, [20,27] in which the different susceptibility may arise from the differences in the structure of the bacterial membrane. As mentioned, hydrophobic-hydrophilic balance has a strong influence on the activity, and it is required hydrophobic groups to promote insertion and disruption of the inner/outer membranes [28,29]. In this case, the DOMA units as well as the alkylating agent provided the hydrophobic segments to the copolymers. From the results summarized in Table 2 for the copolymers obtained by free radical polymerization, it is appreciated that the copolymers quaternized with methyl iodide exhibit lower MIC values than the corresponding copolymers with a longer alkylating chain, butyl group. This behavior differs from that obtained in the homopolymers,

5

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3.4. Antibacterial properties of the polymeric coatings

Table 3 Water contact angle values of the polymeric coatings before rinse, and after chloroform and water rinse treatments. Copolymer

The antibacterial activity of the coatings based on P(DOMAx-coMTAy)M and P(DOMAx-co-MTAy)B copolymers was investigated against Gram-negative E.coli bacteria by optical density method. Many nosocomial infections are ascribable to Gram-negative bacteria, which are, in general, difficult to treat due to the dwindling supply of new antibiotics to which they are susceptible [33]. Before the test, films were immersed in distilled water during 24 h, to remove the polymers non-attached onto the surface. Fig. 3 shows the antibacterial ratio determined for all the prepared films. Remarkably, coatings made from the homopolymers PMTAM and PMTAB, with 100% of MTA units, did not show any activity and were not able to inhibit bacterial growth. This can be explained by the fact that these coatings without catechol units were not strongly attached onto the surface, which points out the relevance of the DOMA units in the coating performance. Conversely, the rest of films bearing adhesive anchoring groups of dopamine show antibacterial action meaning the presence of films after the water rinse due to strong attachment of the polymers to the substrate. Remarkably, the family of copolymers of low molecular weight (synthesized by RAFT) exhibits excellent killing efficiency, as shown in Fig. 3b, in which higher antibacterial ratio values are observed. On the other hand, the series of copolymers obtained by conventional radical polymerization, only the copolymers quaternized with methyl iodide present outstanding antibacterial action, whereas in the butylated copolymers, the activity decreases as the content of DOMA augments. This capacity to inhibit bacterial growth can be related to the hydrophilicity of the surface previously studied by contact angle measurements (see Table 3). The coatings with lower values of contact angle are more effective, meaning better accessibility of cationic groups to contact the bacterial membrane. In this case, films made from copolymers with lower molecular weight showed better antibacterial action, contrary to the MIC results obtained in solution. Therefore, the activity of the copolymers is different in aqueous solution than that exerts when are tethered to the surface. [7,15] In fact, in some cases cationic coatings are able to kill bacteria, that in contrast are resistant in solution [34]. In solution, longer polymers increase chain entanglement and augment positive charge density, which is more concentrated. On the other hand, when attached onto a surface, the mobility is impeded and the active cationic groups in higher molecular weight polymers can be less accessible for a good contact with the bacteria cell membrane.

Water contact angle (º) Before rinse

Free radical polymerization P(DOMA31-co-MTA69)M 52 ± 4 69 ± 6 P(DOMA24-co-MTA76)M P(DOMA18-co-MTA82)M 60 ± 4 P(DOMA9-co-MTA91)M 55 ± 4 PMTAM 38 ± 4 P(DOMA31-co-MTA69)B – 77 ± 6 P(DOMA24-co-MTA76)B P(DOMA18-co-MTA82)B 76 ± 4 75 ± 4 P(DOMA9-co-MTA91)B PMTAB 65 ± 7 RAFT polymerization P(DOMA28-co-MTA72)M 46 ± 2 P(DOMA22-co-MTA78)M 39 ± 1 31 ± 3 P(DOMA13-co-MTA87)M P(DOMA6-co-MTA94)M 29 ± 4 46 ± 3 P(DOMA28-co-MTA72)B P(DOMA22-co-MTA78)B 40 ± 1 P(DOMA13-co-MTA87)B 39 ± 1 41 ± 1 P(DOMA6-co-MTA94)B

Chloroform rinse

Water rinse

45 69 63 59 71 – 77 76 50 75

± ± ± ± ±

2 1 3 5 5

± ± ± ± ±

4 2 1 3 3

± ± ± ±

1 3 2 1

55 55 76 60 22 – 72 69 75 31

± ± ± ±

3 1 3 2

51 46 32 28 65 61 42 36

± ± ± ± ± ± ± ±

1 4 2 3 1 1 2 3

51 52 50 29 81 72 68 44

± ± ± ± ± ± ± ±

1 5 4 2 2 4 1 2

contact angle values did not decrease to reach the values of bare glass substrate, which means that the coatings on the substrate are maintained after solvent rinse. The increase in the contact angle values observed in the films after solvent washing can be explained due to the surface roughness, which was induced by swelling process (Fig. 2). In contrast, the water contact angles of the PMTAM and PMTAB coatings decreased drastically after water rinse, indicating the films were practically washed off from the substrates. However, the values did not achieve the contact angle of bare glass, and then probably rest of coatings remains at the surface. This could be explained by the presence of electrostatic interactions between the positive charged polymers and the negatively charged glass surfaces. Thus, another good solvent it was then employed to washed off the coatings from the surface, DMSO. The washing step with DMSO over films of PMTAM and PMTAB conducts to recovering the contact angle of bare glass surfaces, meaning their complete removal from the surface. In contrast, copolymeric coatings with DOMA units remained unaltered after DMSO washing, for instance, with contact angle values of 66 ± 3° and 72 ± 2°, for P(DOMA18-co-MTA82)M and P(DOMA18co-MTA82)B, respectively. These results demonstrated a robust adhesion of copolymers to glass substrates.

4. Conclusions In conclusion, novel antibacterial and adhesive coatings have been successfully developed by combining a mussel inspired adhesion

Fig. 2. Optical profilometry image of a surface obtained from the P(DOMA31-co-MTA69)M copolymer partially washed with water.

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Progress in Organic Coatings 136 (2019) 105272

A. Chiloeches, et al.

Fig. 3. Antibacterial ratio of films obtained from methylated P(DOMAx-co-MTAy)M and butylated P(DOMAx-co-MTAy)B copolymers synthesized by a) free radical polymerization and b) RAFT polymerization. The copolymers are labeled as the percentage of MTA.

component DOMA, with a cationic thiazolium group MTA, which exert antibacterial properties. Two series of copolymers were prepared by either RAFT or conventional radical polymerization, in which DOMA/ MTA ratio was varied as well as the molecular weight. The resulting copolymers demonstrated high antibacterial activities against both E. coli and S. aureus in aqueous solution. It was found that the activity only decreases slightly as the content of DOMA increases in the copolymer, with acceptable MIC values for content of DOMA up to 32%. The molecular weight also exerts a crucial influence on the antibacterial action, the higher the molecular weight, the higher the antibacterial activity. In contrast, when the copolymers are attached onto surface the antibacterial activities of copolymers with lower molecular weight are higher, probably as a result of their better mobility and accessibility of the cationic MTA units. The resulting coatings also demonstrated a strong attachment to the substrate and washing permanence as well as excellent antibacterial action.

[8] [9] [10] [11] [12] [13] [14] [15]

Acknowledgements

[16]

This work was funded by the MINECO (Project MAT2016-78437-R), the Agencia Estatal de Investigación (AEI, Spain) and Fondo Europeo de Desarrollo Regional (FEDER, EU) and by CSIC (Project I-LINK1191). C. Echeverria also acknowledges MINECO for her IJCI-2015-26432 contract. D. Plachá also thanks to MŠMT CR (Project SP 2019/23).

[17] [18]

Appendix A. Supplementary data

[19]

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.porgcoat.2019. 105272.

[20] [21]

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