Thiol Reactive Maleimido-Containing Tannic Acid for the Bioinspired

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Thiol Reactive Maleimido-Containing Tannic Acid for the Bioinspired Surface Anchoring and Post-Functionalization of Antifouling Coatings LiQun Xu, Dicky Pranantyo, Koon Gee Neoh, En-Tang Kang, and Guo-Dong Fu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00760 • Publication Date (Web): 19 Jun 2016 Downloaded from http://pubs.acs.org on June 25, 2016

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ACS Sustainable Chemistry & Engineering

Thiol Reactive Maleimido-Containing Tannic Acid for the Bioinspired Surface Anchoring and Post-Functionalization of Antifouling Coatings

Li Qun Xu, Dicky Pranantyo, Koon-Gee Neoh, En-Tang Kang* Department of Chemical & Biomolecular Engineering National University of Singapore 4, Engineering Drive 4 Kent Ridge, Singapore 117576

Guo Dong Fu School of Chemistry and Chemical Engineering Southeast University Jiangning District, Nanjing Jiangsu Province, P.R. China 211189

* To whom correspondence should be addressed: E-mail: [email protected]

1 ACS Paragon Plus Environment


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Abstract Inspired by tea stains, a new surface anchor, maleimido-containing tannic acid (TAMA), was developed to introduce the maleimido functionality onto the stainless steel (SS) surfaces. The feasibility of maleimido groups to serve as anchoring sites for surface functionalization via Michael

addition

was

explored

in

a

model

experiment

using

thiol-containing

1H,1H,2H,2H-perfluorodecanethiol. The surface conjugation efficiency of TAMA with the thiol-containing compounds via Michael addition was also compared to that of the surface with tannic acid (TA) only. Water-soluble thiolated carboxymethyl chitosan (CMCSSH) was then grafted on the SS surface pre-anchored with TAMA via solution immersion and spin coating. The deposition of CMCSSH was characterized by contact angle measurement, surface zeta potential, and X-ray photoelectron spectroscopy (XPS). The antifouling efficacy of the CMCSSH coatings was evaluated by protein adsorption and bacterial adhesion. The cytotoxic effect of the CMCSSH coatings on mammalian cells was evaluated using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with 3T3 fibroblasts.

Keywords: Bioinspired anchors; thiol-maleimide conjugation chemistry; antifouling; chitosan; tannic acid


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Introduction Tannic acid (TA), a naturally occurring plant polyphenol, has attracted considerable attention due to its natural antioxidant and antiviral activities.1,2 It consists of a center glucose molecule with all five hydroxyl moieties esterified with two gallic acid (3,4,5-trihydroxybenzoic acid) molecules. The trihydroxylphenyl groups play an important role in binding a variety of substrates through chemical and physical interactions,3-13 which are similar to the adhesion mechanism of 3,4-dihydroxyphenylalanine (DOPA)-enriched adhesive proteins of blue mussels.14 TA can be deposited onto various organic and inorganic substrates because of its inherent affinity towards surfaces.4,15-17 TA is generally recognized as safe and non-toxic to consume in small amounts by the US Food and Drug Administration.15 The commercial extraction of TA from plant part, such as Tara pods (Caesalpinia spinosa), gallnuts from Rhus semialata or Quercus infectoria, and Sicilian Sumac leaves (Rhus coriaria), is abundant and relatively cheap. Thus, the deposition of TA features an environmentally-friendly and economical approach to surface functionalization, in comparison to traditional silanzation, phosphonate chemistry, nitrene addition, diazonium radical chemistry, and ozone, plasma and electro-beam treatments.

Thiol-based conjugation reactions, involving the thiol-halo, thiol-ene and thiol-yne reactions exhibit ‘click’ reaction characteristics under appropriate conditions.18 Thiol-maleimide conjugation chemistry is another form of thiol-ene reaction performed by Michael addition of a thiolate anion to the electron-deficient double bond of the maleimide to form a succinimidyl thioether.19-21 This reaction can proceed rapidly under a physiological condition in quantitative yields even in the absence of a catalyst. It has been proved to be an efficient and versatile means



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for preparing cross-linking hydrogel precursors,22 synthesizing functional polymers,23-25 preparing protein bioconjugates,26 and labelling (macro)molecules with fluorophores and specific targets.27-29

Biofouling, the unwanted accumulation of biomolecules, microorganisms, plants, algae and animals on wetted surfaces is an economic and ecological problem.30-34 One of the key strategies for combating biofouling involves the functionalization of substrate surfaces with nontoxic polymer coatings.35,36 Deposition of antifouling polymer coatings onto the substrates requires reliable anchors or primers to activate the “inert” surfaces for further modification. The merge of TA-based coating technique with thiol-maleimide conjugation chemistry allows direct anchoring of the tailored antifouling functionality. Stainless steel (SS) has been widely utilized in food industry, as well as biomedical devices and implants, due to its durability and corrosion resistance properties. However, SS is susceptible to fouling by a variety of micro- and macro-organisms in aquatic environments.37,38 Thus, chemical modification of SS surfaces by TA-based coatings to confer the desired antifouling properties will be essential.

Herein, a new maleimido-containing TA (TAMA) was synthesized via etherification of TA with N-3-bromopropylmaleimide. The trihydroxyphenyl moieties in the bifunctional system can be used for the bio-inspired surface anchoring on SS surfaces, while the maleimido functionality is ready for the subsequent Michael addition with thiolated carboxymethyl chitosan (CMCSSH). Carboxymethyl chitosan (CMCS) was chosen as the polymer component due to its water solubility, biocompatibility, low toxicity, and antifouling and antibacterial characteristics.39-42 4 ACS Paragon Plus Environment

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The antifouling efficiency of the resulting CMCSSH coatings was assayed by protein adsorption and bacterial adhesion. The cytotoxic effect of the CMCSSH coatings was also evaluated with 3T3 fibroblasts.



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Experimental Section Materials SS foils (AISI type 304, Fe/Cr18/Ni10, 0.05 mm in thickness) were purchased from Goodfellow Ltd., Cambridge, UK. Maleic anhydride (99%), 3-bromopropylamine hydrobromide (98%), tannic acid (TA, ACS reagent, Product No. 403040), chitosan (1.9 - 3.1 × 105 g mol-1, 75-80% deacetylated,

Product

No.

448877),

2-iminothiolane

hydrochloride

(98%),

tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%), bovine serum albumin (BSA, Product No. A9771), BSA fluorescein isothiocyanate conjugate (BSA-FITC, Product No. A9771) and 1H,1H,2H,2H-perfluorodecanethiol (PFDSH, 97%) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Escherichia coli (E. coli, ATCC DH5α) was purchased from American Type Culture Collection (Manassas, VA, USA). All other regents and solvents were purchased from Sigma-Aldrich or Merck Chemical Co. (Hohenbrunn, Germany), and were used without further purification.

Synthesis of N-3-bromopropylmaleimide Maleic anhydride (19.6 g, 0.2 mol) and 3-bromopropylamine hydrobromide (43.8 g, 0.2 mol) were added into a 500-mL round-bottom flask containing 300 mL of dry dichloromethane (DCM). The solution was cooled in an ice bath and degassed with argon for 30 min. Then, triethylamine (27.9 mL, 0.2 mol) in 25 mL of DCM was added dropwise into the reaction mixture over 1 h. After additional 24 h of stirring at room temperature under bubbling argon, the solution was washed with 150 mL of 1 M HCl aqueous solution for three times. The organic


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layer was dried with anhydrous Na2SO4, filtered and evaporated to dryness. The obtained solid was used in the next step without further purification. N-3-Bromopropyl-substituted maleamic acid (12.5 g, 0.05 mol) was suspended in 100 mL of acetic anhydride in the presence of anhydrous sodium acetate (4.1 g, 0.05 mmol). The reaction mixture was stirred under reflux for 24 h. After cooling down to room temperature, the solution was poured into 500 mL of ice water and neutralized with NaHCO3 under vigorous stirring. The product was extracted three times with 100 mL of DCM. The combined organic layers were dried with anhydrous Na2SO4, filtered and concentrated in a rotary evaporator. The crude product was purified by column chromatography using ethyl acetate and hexane (1:1, v/v) as the eluent, to give a colorless and viscous liquid. The liquid was further crystalized in hexane, resulting in needle-like crystals. 1H NMR (600 MHz, CDCl3-d, δ, ppm): 6.71 (-CH=CH-, 2H), 3.67 (-CH2-Br, 2H), 3.36 (N-CH2-, 2H) and 2.16 (-CH2-CH2-CH2-, 2H).

Synthesis of TAMA TA (6.0 g, 3.5 mmol), N-3-bromopropylmaleimide (3.1 g, 14.1 mmol), potassium carbonate (5.8 g, 42.3 mmol) and dry N,N-dimethylformamide (DMF, 80 mL) were added into a 150-mL three-neck round-bottom flask. The reaction mixture was bubbled with argon for 30 min, and was mechanically stirred at 60 oC for 24 h under bubbling argon. After that, the solution was acidified to pH 4 using diluted HCl (2 M), and was dialyzed with doubly distilled water (molecular weight cut-off = 1000 daltons) for 4 days. During dialysis, the water was refreshed every 8 h. About 3.5 g of TAMA was obtained after lyophilization as a yellow-brown solid.



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Synthesis of CMCSSH CMCS was prepared according to the method reported in the literature with further purification.40 The obtained CMCS was dissolved in doubly distilled water. After neutralizing to pH 7 using NaOH aqueous solution (1 M), the solution was passed through a column with a cotton wool plug to remove the large aggregates. The solution was further passed through a 0.45 µm filter membrane, and dialyzed with doubly distilled water (molecular weight cut-off = 14000 daltons) for 3 days. During dialysis, the water was refreshed every 8 h. The lyophilized CMCS was a white solid.

CMCS (1 g) was dissolved in 100 mL of doubly distilled water with 1% (v/v) acetic acid. The solution was bubbled with argon for 30 min, followed by the addition of 2-iminothiolane hydrochloride (50 mg). The pH of the reaction mixture was adjusted to 7 using 5 M NaOH aqueous solution. In order to avoid an oxidation process, the argon was bubbled throughout the reaction. After stirring for 24 h, the solution was dialyzed with doubly distilled water (molecular weight cut-off = 14000 daltons) for 3 days. During dialysis, the water was refreshed every 8 h. The lyophilized CMCSSH was a white solid.

Preparation of TAMA- or TA-anchored SS surfaces The SS foils were cut into 1 cm × 1 cm pieces, and cleaned ultrasonically with doubly distilled water, acetone and ethanol for 15 min each. The SS substrates were dried by compressed air, and stored in a dry box for future usage.


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The SS substrates were immersed in ethanol solution containing 5 mg/mL of TAMA or TA at 37 oC for 48 h. The resulting TAMA- or TA-anchored SS substrates (denoted as SS-TAMA or SS-TA) were rinsed with ethanol, and dried with compressed air.

Preparation of PFDSH-functionalized SS surfaces The SS-TAMA or SS-TA substrates were immersed in PFDSH ethanol solution (2.5 mg/mL) at 37 oC for 24 h. The resulting PFDSH-functionalized SS-TAMA or SS-TA surfaces (denoted as SS-TAMA-PFDSH and SS-TA-PFDSH) were rinsed thoroughly with ethanol and dried with compressed air.

Preparation of CMCSSH-functionalized SS surfaces i)

via solution immersion coating

The SS-TAMA or SS-TA substrates were immersed in CMCSSH phosphate buffered saline (PBS, 10 mM, pH = 7.4) solution (2 mg/mL) at 37

o

C for 24 h. The resulting

CMCSSH-functionalized SS-TAMA or SS-TA surfaces (denoted as SS-TAMA-iCMCSSH and SS-TA-iCMCSSH) were rinsed thoroughly with PBS and doubly distilled water, and dried with compressed air.

ii)

via spin coating 9 ACS Paragon Plus Environment


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The CMCSSH PBS solution (2 mg/mL) was spin-coated onto the SS-TAMA substrates at a rotation rate of 1500 rpm (Spincoat G3P-B, Specialty Coating Systems Inc.). The CMCSSH-coated SS substrates (denoted as SS-TAMA-sCMCSSH) were cured at 37 oC in a humid environment for 24 h. After that, the SS-TAMA-sCMCSSH surfaces were rinsed thoroughly with PBS and doubly distilled water, and dried with compressed air.

Protein adsorption assay Protein adsorption assay was carried out by immersing the pristine and modified SS substrates in a BSA protein solution (1 mg/mL in PBS (10 mM, pH 7.4)) for 12 h, followed by rinsing with PBS and doubly distilled water thoroughly. After drying under reduced pressure at room temperature for 24 h, the compositions of protein-adsorbed SS surfaces were analyzed by X-ray photoelectron spectroscopy (XPS).43

The pristine and modified SS substrates were also incubated in a BSA-FITC solution (1 mg/mL in PBS (10 mM, pH 7.4)) for 12 h. After rinsing with PBS and doubly distilled water, the SS substrates were viewed under a Nikon Eclipse Ti microscope equipped with a 470 nm excitation filter and a 540 nm emission filter for green fluorescence.

Bacterial adhesion assay E. coli were used to evaluate the anti-adhesion characteristics of the pristine and modified SS surfaces to bacteria. E. coli was cultured following the methods reported in the literature.39 After 10 ACS Paragon Plus Environment

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overnight culture, the culture broth was centrifuged at 2700 rpm for 10 min to remove the supernatant. The bacteria cake was washed with sterile PBS twice, and dispersed in PBS to give rise to a final concentration of 5 × 107 cells/mL. The pristine and surface-modified SS coupons of 1 × 1 cm2 in size were placed in a 24-well plate and covered with 1 mL bacterial suspension for 4 h at 37 °C. After that, the bacterial suspension in each well was removed, and the SS substrates were washed with PBS thrice to remove any non-adhered or loosely adhered bacteria. For the scanning electron microscope (SEM) observation, these substrates were fixed in glutaraldehyde solution, dehydrated with ethanol water mixtures and dried under reduced pressure.39 Quantification of bacteria adhesion on the pristine and modified SS substrates was carried out using the spread plate method.44

Cytotoxicity tests The cytotoxicity assays with 3T3 fibroblasts were carried out according to procedures described in the literature.45

Characterization The chemical structures of obtained compounds and polymers were characterized by 1H NMR spectroscopy on a Varian 600 MHz spectrometer. XPS measurements were carried out on a Kratos AXIS Ultra HSA spectrometer equipped with a monochromatized AlKα X-ray source (1468.71 eV photons). SEM (JEOL JSM 5600 LV) was used for the observation of the adhered bacteria on the SS substrates. An electrokinetic analyzer (SurPass, Anton Paar) comprising an



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adjustable-gap cell was used to determine the ζ potential of the SS surfaces. The static water contact angle measurements were performed using an optical video contact angle system (OCA-15-plus, Dataphysics). A 3-µL droplet of doubly distilled water was dispensed on the SS surfaces using the electronic syringe unit of the instrument. The static water contact angle was measured using the sessile drop method with the dedicated software (SCA20).


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Results and Discussion The synthesis of TAMA used for the surface functionalization of SS surfaces is shown in Scheme 1. TA was etherified with N-3-bromopropylmaleimide, with potassium carbonate as a base, in DMF. The structure of TAMA was characterized by 1H NMR spectroscopy. Figure 1 shows the 1H NMR spectra of TA and TAMA in DMSO-d6 at 25 oC. The 1H NMR spectrum of TA shows the glucoside proton signals at 6.44, 6.03, 5.48, 4.63 and 4.33 ppm, the aromatic proton signals in the range of 6.74 - 7.55 ppm, and hydroxyl proton signals at 8.74 - 10.33 ppm. After etherifying, the peaks of methylene protons in N-3-bromopropylmaleimide at 3.98, 3.60 and 1.92 ppm are present in the 1H NMR spectrum of TAMA. The integrated area ratios of methylene protons adjacent to maleimido group (9) and proton of C-1 (1) in the glucoside were utilized to determine the efficiency of etherifying step. The average substitutions of maleimido groups in each TA molecule are 2.36.

The trihydroxyphenyl moieties of TA can readily anchor on metal substrates via metal chelation interactions.4 SS substrates were then functionalized with TAMA to introduce maleimido functionality on the surfaces for subsequent conjugation with thiol-containing compounds via thiol-maleimide conjugation chemistry (Scheme 2). The immobilization of TAMA onto the SS surfaces was performed by immersion of SS substrates in the ethanol solution of TAMA. The successful anchoring of TAMA onto the SS surfaces was ascertained by XPS measurements. Parts a-b of Figure 2 show the XPS wide-scan spectra of the pristine SS and SS-TAMA surfaces. In comparison to the XPS wide-scan spectrum of pristine SS surface, the increase in C 1s signal intensities coupled with the presence of N 1s signals and the decrease in



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relative intensity of the Fe 2p, Cr 2p and Ni 2p signals in the XPS wide-scan spectrum of the SS-TAMA surface, are consistent with the surface anchoring process. When the SS-TAMA substrates were immersed in ethanol (a good solvent for TAMA) for two weeks, no apparent change in the XPS C 1s core-level spectral line shape of the SS-TAMA surface was observed. Thus, the TAMA anchor on the SS surface is relatively stable.

To illustrate that the bifunctional TAMA can be used for attachment of molecules of interest via thiol-maleimide conjugation chemistry, a model thiol-containing compound, PFDSH, was used to functionalize the SS-TAMA surfaces. Since the trihydroxyphenyl moieties can react with thiol-containing compounds via Michael addition,17,46 TA was also anchored onto the SS surfaces to compare the conjugation efficiency of TA and TAMA with thiol groups. The XPS core-level spectral area ratios were employed to determine the change in surface compositions during modification of the SS-TA and SS-TAMA surfaces with PFDSH. As shown in Table 1, the [S]/[Fe] and [F]/[Fe] molar ratios of the SS-TA-PFDSH surfaces are about 0.156 and 2.345, respectively. Upon reaction with PFDSH, higher [S]/[Fe] and [F]/[Fe] molar ratios are observed for the SS-TAMA-PFDSH surface. These results suggest that the bifunctional TAMA with both trihydroxyphenyl and maleimido groups exhibit a higher conjugation efficiency with the thiol-containing compound than TA.

The precursor necessary for the subsequent surface antifouling coatings, CMCSSH was prepared via carboxymethylation of chitosan powder with monochloroacetic acid and thiolation of CMCS in the presence of Traut’s reagent (2-iminothiolane hydrochloride). Thiolated chitosan 14 ACS Paragon Plus Environment

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can be prepared via conjugation of 2-iminothiolane on the primary amino groups of chitosan backbone.47 The optimal condition for the preparation of thiolated chitosan is at pH 7.47 However, as the pH of the reaction mixture increases, the solubility of chitosan becomes poor in aqueous media due to deprotonation of the amino groups. To overcome the drawback of poor solubility of chitosan at neutral pH, the water-soluble CMCS was chosen to synthesize the CMCSSH. After the reaction, the solution was dialyzed and freeze-dried. The lyophilized CMCSSH is a white solid with a mild skunk odor. It is readily soluble in doubly distilled water and PBS (10 mM, pH 7.4). The presence of thiol functionality in CMCSSH was confirmed by 1H NMR spectroscopy (Figure S2, Supporting Information). The chemical shifts at 3.31, 2.43-2.55 and 2.14-2.19 ppm are associated with the protons on 4-mercaptobutyramidine,48 suggesting that the ring-opening reaction of 2-iminothiolane with CMCS has taken place. The amount of free thiol groups immobilized on CMCS was determined by Ellman’s reagent assay. The amount of free thiol groups in CMCSSH prepared from CMCS to 2-iminothiolane hydrochloride weight ratio of 20:1 was determined to be 63 µmol per gram of CMCSSH. The thiol-containing CMCSSH was then utilized to functionalize the SS-TAMA surface via Michael addition. The conjugation efficiency of SS-TAMA surface with CMCSSH was also compared to that of the SS-TA surfaces via solution immersion coating. The conjugation efficiency of SS-TAMA and SS-TA surfaces with CMCSSH was evaluated from the XPS core-level

spectral

area

ratios.

The

[S]/[Fe]

and

[N]/[Fe]

molar

ratios

of

the

CMCSSH-conjugated SS-TAMA (SS-TAMA-iCMCSSH) surface from solution immersion coating are about 0.260 and 1.425, respectively, which are significantly higher than those (0.125 and 0.720) of the SS-TA-CMCSSH surface (Table 1). Together with the results of conjugation with PFDSH, the SS-TAMA surface exhibits higher conjugation efficiency with thiol-containing 15 ACS Paragon Plus Environment


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PFDSH and CMCSSH. Thus, the coatings on the SS-TAMA surfaces were used in the subsequent work.

Since the half-reaction RS-SR + 2e- + 2H+ → 2RSH has a standard reduction potential of about -0.25 eV,49 the oxidation of thiols to disulfides are achieved under mild conditions.50 Upon prolonged exposure to air, thiol groups can be oxidized to disulfides.51 To prove the easy cross-linking of CMCSSH, the CMCSSH aqueous solution was bubbled with air at 37 oC and the jelly-like white precipitates were found to form after 1 h. Upon addition of TCEP to the above solution, most of the precipitates had disappeared. These results suggest that CMCSSH is susceptible to oxidation in air with the formation of disulfide linkages. The self-crosslinking property of CMCSSH was utilized to fabricate a thicker coating on SS-TAMA surfaces via the spin coating method and air-mediated oxidative coupling. After thorough rinsing, the presence of thicker

CMCSSH

coating

on

the

resulting

CMCSSH-conjugated

SS-TAMA

(SS-TAMA-sCMCSSH) surface from spin coating was ascertained by XPS measurements. In comparison to the XPS wide-scan spectrum of the SS-TAMA-iCMCSSH surface (Figure 2c), the metallic elements from the underlying SS substrates (Fe 2p, Ni 2p and Cr 2p) are barely discernible in the XPS wide-scan spectrum of SS-TAMA-sCMCSSH surface (Figure 2d). Three replicates were measured by XPS to confirm the coating thickness of the SS-TAMA-sCMCSSH surface is higher than that of the SS-TAMA-iCMCSSH surface.

The successful functionalization of SS surfaces by TAMA and CMCSSH was confirmed by the static water contact angle measurements (Figure 3). The pristine SS surface is hydrophobic 16 ACS Paragon Plus Environment

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with a static water contact angle of 72o. After anchoring of TAMA, the contact angle of the surface decreases to 55o. Deposition of CMCSSH via solution immersion coating and spin coating increases the surface hydrophilicity significantly, with the contact angles of 20o and 17o, respectively, for the SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces. The highly hydrophilic nature of the CMCS-coated SS-TAMA surface is consistent with that observed for the CMCS-coated polydopamine surfaces.39 The deposition of CMCSSH onto the SS-TAMA surfaces was also evidenced by the surface zeta (ζ) potential measurements. Figure 4 shows the zeta potential measurements of SS-TAMA, SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces as a function of pH. For all of these surfaces, the zeta potential increases with the decrease in pH. The SS-TAMA surface is negatively charged from pH 3 to 9. The SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces are negatively charged at high pH and positively charged at low pH with isoelectric points between pH 4-5. The presence of cationic amino/amidine groups and anionic carboxyl moieties in CMCSSH results in the isoelectric characteristics of the SS surfaces.52 The SS-TAMA-sCMCSSH surface has a thicker CMCSSH layer and more anionic carboxyl moieties than the SS-TAMA-iCMCSSH surface. Thus, the SS-TAMA-sCMCSSH surface has more negative surface charges at high pH than that of the SS-TAMA-iCMCSSH surface.

Protein adsorption plays a significant role in the subsequent adhesion of fouling organisms,35 as the adsorbed proteins may adversely affect the surface functionality.30,31 The adsorption of BSA on the modified SS substrates was investigated. The extent of BSA adsorption on the SS surfaces was determined from the XPS N 1s and C 1s core-level spectral peak area ([N]/[C]) ratio.43

Figure

5

compares

the

[N]/[C]

ratios

of

the

pristine

SS,

SS-TAMA, 17

ACS Paragon Plus Environment


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SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces before and after BSA exposure. The [N]/[C] ratios of the pristine SS, SS-TAMA, SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces increase from 0.03 to 0.19, from 0.05 to 0.19, from 0.08 to 0.12, and from 0.09 to 0.11, respectively, after BSA exposure. The significant increase in [N]/[C] ratios of the pristine SS and SS-TAMA surfaces indicates that these surfaces can adsorb BSA readily. The minor changes in [N]/[C] ratios of the SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces before and after BSA exposure suggest that the non-specific adsorption of BSA can be largely suppressed. In comparison to the SS-TAMA-iCMCSSH surface, the SS-TAMA-sCMCSSH surface exhibits slightly better anti-nonspecific protein adsorption performance. To visualize the protein adsorption, these surfaces were further incubated with fluorescent BSA-FITC and imaged by a fluorescence microscope. Insets of Figure 5 show the respective fluorescence images of the pristine SS, SS-TAMA, SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces. The contrast in the extent of fluorescence from the surfaces provided by the pristine SS and SS-TAMA surfaces and that of SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces confirms that the hydrophilic CMCSSH coatings exhibit much lower protein adsorption. The fluorescence image assays are congruent with the results obtained from the XPS [N]/[C] ratios.

To further examine the antifouling properties of modified SS surfaces, the resistance to bacterial

adhesion

on

the

pristine

SS,

SS-TAMA,

SS-TAMA-iCMCSSH

and

SS-TAMA-sCMCSSH surfaces was assayed. Figure 6a-d shows the representative SEM images of these surfaces after incubation in E. coli suspension for 4 h. The relatively hydrophobic pristine SS and SS-TAMA surfaces favor the adhesion of E. coli due to the hydrophobic interactions (Figures 6a-b). After surface functionalization with hydrophilic CMCSSH coating, 18 ACS Paragon Plus Environment

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there is a large reduction in the number of adhered E. coli (Figures 6c-d). The bacterial-resistant properties of the CMCSSH coatings can be attributed to chain dynamics and steric repulsion of the hydrated CMCSSH layer, as well as the bactericidal effect provided by the positively charged amino groups.31,39,40,53 The number of adherent bacteria on the pristine and modified SS surfaces was also quantified by the spread plate method as shown in Figure 6e. The number of adhered E. coli on the pristine SS surface is 4.0 × 105 cells/cm2. The anchoring of TAMA reduces the number of adhered cells to slightly 2.2 × 105 cells/cm2. However, there is a clear reduction in the number of adhered cells on the SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces, as compared to the pristine SS and SS-TAMA surfaces. The number of adhered E. coli on the SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces further decreases to 5.8 × 104 and 1.8 × 104 cells/cm2, respectively. The SS-TAMA-sCMCSSH surface is also less susceptible to bacterial adhesion than the SS-TAMA-iCMCSSH surface. These results are consistent with the observation in the SEM images of Figure 6a-d.

Possible cytotoxic effect of the CMCSSH coatings on mammalian cells was evaluated using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with 3T3 fibroblasts. The pristine SS, SS-TAMA, SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces were immersed in the Dulbecco’s Modified Eagle Medium (DMEM) at 4 oC for 1, 4 and 7 days. Subsequently, the media were utilized to culture the 3T3 fibroblasts for 24 h. As shown in the quantitative MTT assay (Figure 7), all the SS surfaces exhibit more than 97% cell viabilities, relative to the cell viability in the control experiment. This result indicates that the CMCSSH coatings pose no significant cytotoxicity to the mammalian cells.



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Conclusions The SS surfaces were conferred with maleimido moieties via a simple method of pre-treatment with TAMA. The maleimido groups can be utilized to conjugate with thiol-containing compounds via Michael addition under mild conditions. Water-soluble CMCSSH was successfully grafted on the TAMA-anchored SS surfaces via solution immersion and spin coatings. The CMCSSH coatings inhibit the non-specific adsorption of bovine serum albumin. The CMCSSH coatings fabricated by solution immersion and spin coatings can also significantly reduce Escherichia coli adhesion by > 85% and 95%, respectively. Furthermore, the CMCSSH coating exhibits minimal cytotoxicity toward 3T3 fibroblasts. Spray coating is much faster than immersion and spin coatings, and is better suited for coating large and nonplanar substrates at the industrial scale.54 In future work, spray coating will be utilized to deposit TAMA anchor and CMCSSH coating on SS surfaces to facilitate the development of chitosan-based coatings for practical industrial applications.

Supporting Information The 1H NMR spectra of TAMA, CS, CMCS and CMCSSH; the XPS S 2p and N 1s core-level spectra of SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces. The Supporting Information is available free of charge on the ACS Publications website at DOI: **.****/*.*.*.

Acknowledgments The authors would like to acknowledge the financial support of this study from the Singapore Millennium Foundation under Grant No. 1123004048 (NUS WBS no. R279-000-428-592).


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Legends for Schemes and Figures Scheme 1. Synthesis of TAMA and CMCSSH.

Scheme 2. Schematic illustration of the bioinspired approach to prepare the TA- or TAMA-anchored SS surfaces and the subsequent surface functionalization with thiol-containing compounds via Michael addition.

Figure 1. 1H NMR

spectra of (a) TA and (b) TAMA in DMSO-d6.

Figure 2. XPS wide-scan spectra of the (a) pristine SS-TAMA-iCMCSSH and (d) SS-TAMA-sCMCSSH surfaces.

SS,

(b)

SS-TAMA,

(c)

Figure 3. Static water contact angles of the pristine SS, SS-TAMA, SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces.

Figure 4. Zeta (ζ) potential of the SS-TAMA, SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces as a function of pH.

Figure 5. Evaluation of protein adsorption, expressed as XPS-derived surface [N]/[C] ratios on the pristine SS, SS-TAMA, SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces after incubation in PBS (10 mM, pH = 7.4) containing 1 mg/mL of BSA for 12 h. Inset: fluorescence microscopy images of the pristine SS, SS-TAMA, SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces after exposure to BSA-FITC PBS (10 mM, pH = 7.4) solution (1 mg/mL) for 12 h.



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Figure 6. Representative SEM images of of the (a) pristine SS, (b) SS-TAMA, (c) SS-TAMA-iCMCSSH and (d) SS-TAMA-sCMCSSH surfaces, and (e) the number of adhered E. coli cells per cm2, after exposure to E. coli suspension in PBS (5 × 107 cells/mL) for 4 h.

Figure 7. Relative cell viability of 3T3 fibroblasts after 24 h culturing in DMEM pretreated with the pristine SS, SS-TAMA, SS-TAMA-iCMCSSH and SS-TAMA-sCMCSSH surfaces for 1, 4 and 7 days.


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Table 1: Surface compositions (molar ratios) of SS-TA and SS-TAMA surfaces after conjugation with thiol-containing compounds [S]/[Fe] ratio

[F]/[Fe] ratio

[N]/[Fe] ratio

SS-TA-PFDSH

0.15 ± 0.02

2.35 ± 0.18

–

SS-TAMA-PFDSH

0.23 ± 0.02

4.12 ± 0.20

–

SS-TA-iCMCSSH

0.12 ± 0.02

–

0.72 ± 0.04

SS-TAMA-iCMCSSH

0.26 ± 0.03

–

1.43 ± 0.06



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Scheme 1 28 ACS Paragon Plus Environment

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Scheme 2 29 ACS Paragon Plus Environment


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Figure 1


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Figure 2



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Figure 3


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Figure 4



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Figure 5


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Figure 6



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Figure 7


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TOC Title: Thiol reactive maleimido-containing tannic acid for the bioinspired surface anchoring and post-functionalization of antifouling coatings

Author: Xu L. Q., Pranantyo D., Neoh K. G., Kang E. T., Fu G. D.

Synopsis: Inspired by tea stains, maleimido-containing tannic acid (TAMA) was developed to serve as anchoring sites for surface antifouling coatings.


Thiol Reactive Maleimido-Containing Tannic Acid for the Bioinspired

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