Article pubs.acs.org/IECR
Synthesis of Novel N‑Halamine Epoxide Based on Cyanuric Acid and Its Application for Antimicrobial Finishing Kaikai Ma,† Ying Liu,† Zhiwei Xie,‡ Rong Li,† Zhiming Jiang,† Xuehong Ren,*,† and Tung-Shi Huang§ †
Key Laboratory of Eco-textiles of Ministry of Education, College of Textiles and Clothing, Jiangnan University, Wuxi, Jiangsu 214122, China ‡ Department of Bioengineering, University of Texas at Arlington, Arlington, Texas 76019, United States § Department of Poultry Science, Auburn University, Auburn, Alabama 36849, United States ABSTRACT: In this work, a novel N-halamine precursor, 1-glycidyl-s-triazine-2,4,6-trione (GTT), was synthesized through the reaction of cyanuric acid with epichlorohydrin in a facile condition. The pad−dry−cure technique was used to coat GTT onto cotton fabrics through the covalent surface modification of the cotton fibers. The GTT-coated cotton was characterized by FTIR spectroscopy and SEM. The N-halamine moieties attached to the cotton fibers could be rendered antimicrobial by treatment with a dilute sodium hypochlorite solution. The N-halamine-modified cotton fabrics demonstrated excellent antimicrobial efficacy against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli O157:H7) bacteria in brief contact times. Over 71% of the chlorine lost after the equivalent of 50 machine washes could be regained upon rechlorination. The chlorinated coated fabrics showed great rechargeability within one week under UVA light irradiation.
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INTRODUCTION Infections caused by microorganisms are among the greatest threats to humans, and textiles are suitable carriers and media for microorganisms such as bacteria, viruses, and fungi.1−3 Extensive research on producing antimicrobial materials has focused on biocidal agents, such as quaternary ammonium salts,4−6 metal ions,7 light-activated coatings,8 phosphonium salts,9 and N-halamines.10−17 N-Halamines are the most promising candidates for use in preparing antimicrobial textiles because of their broad-spectrum antibacterial activity, nontoxicity, and low environmental impacts. In addition, their antibacterial properties are durable and rechargeable. They have long shelf life once covalently attached to polymers, and their chlorine can be regenerated simply by exposure to household bleach in the washing process. Durable and renewable antibacterial cotton materials have been prepared using dimethylol-5,5-dimethylhydantoin (MDMH), monomethylol-5,5-dimethylhydantoin (DMDMH), and 3-methylol-2,2,5,5-tetramethyl-imidazolidin4-one (MTMIO).15−17 These compounds can be covalently bound to cellulose using a pad−dry−cure method. The monomeric precursor 3-allyl-5,5-dimethylhydantoin (ADMH) was developed and employed in grafting cotton fabrics.18 Series of N-halamine siloxane19−22 and N-halamine epoxide23,24 coatings have been prepared and coated on the surfaces of cotton and polyester swatches. N-Halamine derivatives containing two hydroxyl groups were synthesized and coated onto cotton fabrics using the cross-linking agent 1,2,3,4butanetetracarboxylic acid (BTCA) to impart antibacterial properties after chlorination with household bleach.12 These coatings exhibited excellent washing stability during laundering. The heterocyclic monomer 3-(4′-vinylbenzyl)-5,5-dimethylhydatoin was synthesized and used to form antibacterial thin films on the surface of cotton by admicellar polymerization.25 Most recently, the reactive melamine derivative 2-amino-4-chloro-6© XXXX American Chemical Society
hydroxy-s-triazine (ACHT) was synthesized and reacted with cellulosic materials, and the chemically bound ACHT moieties were transformed into chloromelamine derivatives after chlorination.26−28 Among the mentioned N-halamine precursors, especially hydantoin derivatives containing one amide group and one imide group, the amide nitrogen is retained for chlorination, whereas the imide nitrogen is employed as a linker to polymers and fibers.29 N-Halamines can contain one or more amine, amide, and imide halamine bonds. The inactivation efficacies of bacteria depend on the stabilities of the halamine bonds, which are directly related to the N-halamine structures. The rates of inactivating bacteria follow the order imide > amide > amine halamines, which is the reverse of the order of their stabilities.16 In this study, a noval cyclic N-halamine precursor, 1-glycidyl-striazine-2,4,6-trione (GTT), was synthesized through the reaction of cyanuric acid with epichlorohydrin (Scheme 1). The synthesis of GTT occurs at normal pressure and ambient temperature using water as the solvent, and it is easy to scale up in practical applications. In addition, the final reaction solution with GTT can be used directly as a treatment solution for Scheme 1. Synthesis of 1-Glycidyl-s-triazine-2,4,6-trione (GTT)
Received: January 13, 2013 Revised: April 24, 2013 Accepted: May 8, 2013
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15 min to produce a uniform solution of isocyanuric acid disodium salt. Then, 0.05 mol of epichlorohydrin was added, and the solution was stirred for 10 h at ambient temperature. After the reaction had completed, the mixture was neutralized (pH 6.8−7.0) with 10% sulfuric acid. Solvent water was removed by vacuum evaporation. The solid was dissolved in 100 mL of dimethylformamide (DMF), and the byproduct sodium salt was removed by filtration. After the DMF had been removed at reduced pressure, the desired white solid was produced in a yield of 79%. 1H NMR [400 MHz, deuterated dimethyl sulfoxide (DMSO-d6)]: δ 2.73−2.88 (2H), 3.30 (1H), 7.43−7.73 (2H), 11.05 (2H). Coating Procedures. A traditional pad−dry−cure process was used to coat GTT onto the cotton fibers. GTT was dissolved in water at concentrations ranging from 3% to 10%. Cotton swatches were soaked in the water bath for 15 min at a bath ratio of 30:1 (water to cotton by weight). Two dips and two nips were used to achieve a wet addition of 100 wt %. The swatches were dried at 95 °C for 5 min and then cured at 160− 170 °C for 10−20 min. The cured swatches were soaked in 0.5% detergent solution for 15 min, washed with water, and dried at ambient temperature. Chlorination and Titration. The coated swatches were soaked in a 10% commercial aqueous sodium hypochlorite solution (0.5% NaOCl) at pH 7 and room temperature for 1 h. The chlorinated cotton swatches were washed thoroughly with distilled water and dried at 45 °C for 1 h to remove any unreacted free chlorine from the surface of the cotton fabrics. The loaded chlorine concentration on the samples was determined by the iodometric/thiosulfate titration method. The chlorine weight percentage in each swatch was calculated as
coating cotton fabrics without any prior workup. Compared with hydantoin derivatives, cyanuric acid has three imide nitrogens. When one imide position of cyanuric acid is employed as a linker, there are still two imide positions that can be chlorinated. The two imide halamine bonds in GTTmodified materials might kill bacteria faster than the amide bonds in other materials. A regular wet finishing process, namely, the pad−dry−cure method, was employed to tether GTT to the surfaces of cellulosic fibers through covalent ether linkages. The biocidal activity of the coated cotton was rendered through bleach treatment (Scheme 2). The biocidal Scheme 2. Preparation of GTT-Coated Antimicrobial Cellulose
efficacies of chlorinated cotton swatches were evaluated against Escherichia coli O157:H7 and Staphylococcus aureus. The stabilities and rechargeabilities of the N−Cl bonds of the Nhalamine epoxide coatings were evaluated by standard washing tests and exposure to UV light. The biocompatibility of Nhalamine-coated fabrics was also studied by in vitro cytocompatibility tests.
Cl+ (wt %) =
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35.45NV × 100 2W
(1)
where N and V are the normality (equiv/L) and volume (L) of sodium thiosulfate, respectively, and W is the weight (g) of the swatch. Biocidal Efficacy Test. A modified version of AATCC Test Method 100-1999 was conducted to evaluate the biocidal efficacies of the coatings. In accordance with this testing method, both chlorinated and unchlorinated cotton swatches were challenged with S. aureus and E. coli O157:H7. A 25 μL aliquot of bacterial suspension (100 mM phosphate buffer, pH 7) was added to the center of two 1-in.2 cotton swatches, which were held in place by sterile weights to ensure good contact of the swatches with the inoculum. The amount of bacteria employed for the tests ranged from 106 to 107 colony forming units (CFU)/sample, and the actual bacterial numbers were determined by the count method. After contact times of 1, 5, 10, and 30 min, the samples were transferred to tubes containing 5 mL of sterile 0.02 N sodium thiosulfate solution and stirred to remove all oxidative chlorine and rinse off surviving bacteria, and the tubes were vortexed for 2 min. Serial dilutions of the quenched solution samples were made using 100 μM phosphate buffer (pH 7) and plated on Trypticase agar plates. After incubation at 37 °C for 24 h, the viable bacterial colonies were recorded for biocidal efficacy analysis. UV Light Stability Testing. An accelerated weathering tester (Q-Lab Corporation, Westlake, OH) was used to measure the UVA light stabilities of the chlorinated cotton fabrics coated with GTT. The chlorinated GTT-coated cotton swatches were placed in a UV chamber (0.89 W, 60 °C) for
EXPERIMENTAL METHODS Materials. Fabrics of 100% bleached cotton were provided by Zhejiang Guangdong Printing & Dyeing Company, Zhejiang, China. Cyanuric acid was purchased from J&K Chemicals, Shanghai, China. Other chemicals used in this research were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. Staphylococcus aureus ATCC 6538 and Escherichia coli O157:H7 ATCC 43895 (American Type Culture Collection, Rockville, MD) were used in this study, and the bacteria were grown in Trypticase soy broth (TSB, Becton, Dickinson and Company, Detroit, MI). NIH mouse 3T3 fibroblasts were purchased from ATCC (Manassas, VA). Instruments. Nuclear magnetic resonance (NMR) spectra of the synthesized compound were recorded on an AVANCE III 400 MHz Digital NMR spectrometer (Bruker AXS GmbH, Karlsruhe, Germany). Fourier transform infrared (FTIR) spectra of cotton, coated cotton, and chlorinated coated cotton were obtained with a Nicolet NEXUS 470 spectrometer (Nicolet Instrument Corporation, Madison, WI). An SU-1510 field-emission scanning electron microscope (Hitachi, Tokyo, Japan) was used to characterize the surface morphology of control and coated cotton fibers. The UV light stabilities of chlorinated cotton fabrics were measured using an accelerated weathering tester (Q-Lab Corporation, Westlake, OH). Synthesis of 1-Glycidyl-s-triazine-2,4,6-trione (GTT). Cyanuric acid (CA, 0.05 mol) and NaOH (0.1 mol ) were stirred in 125 mL of water in a 500 mL round-bottom flask for B
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(BTCA) as a cross-linking agent to link 3-(2,3-dihydroxypropyl)-5,5-dimethylimidazolidine-2,4-dione to cotton cellulose as in our previous report.12 The coated cotton swatches contained imide functional groups that could be rendered antimicrobial by exposure to household bleach solution (Scheme 2). Characterization of Cotton Coated with GTT. The FTIR spectra of cotton, cotton coated with GTT, and chlorinated cotton coated with GTT are shown in Figure 1.
contact times of up to 168 h. The cotton swatches were removed from the UVA chamber and titrated or rechlorinated and titrated. Standard Washing Testing. AATCC Test Method 611996 (Test 2A Procedure) was used to evaluate the durability of the coatings and the stability of chlorine on the samples after repeated standard washings. Stainless steel canisters (1 in. × 2 in.) containing 150 mL of 0.15% AATCC detergent water solution and 50 stainless steel balls were fixed in a LaunderOmeter (Darong Textile Instrument Co., Ltd., Zhejiang, China) and rotated at 42 rpm and 49 °C for 45 min. Each test sample was rinsed three times with distilled water and then dried at ambient temperature. Each cycle of washing in this method is equivalent to five machine washings. The cotton swatches were washed for the equivalents of 5, 10, 25, and 50 washing cycles. The chlorine loadings on the cotton swatches before and after washing were determined by titration. In some cases, chlorinated and unchlorinated samples were rechlorinated to assess the chlorine loadings after variable numbers of washing cycles. Cell Culture and Cytocompatibility Testing. NIH mouse 3T3 fibroblasts were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) with supplements of 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified atmosphere of 95% air and 5% CO2. For in vitro cytocompatibility tests, all samples were cut into round meshes that fit into 96-well tissue culture plate then UV-sterilized for 1 h. The cell seeding density was 8000 cells/well. A tissue culture plate was used as the control. MTS assays were performed at time points of 4 and 24 h for this study following the manufacturer’s instructions. The cell viability was characterized as the percentage with respect to the control. Statistical analysis of all data was performed by one-way ANOVA (StatView), and p values of 0.05), indicating that the N-halamine-modified cotton fabrics are nontoxic to mouse 3T3 fibroblast cells in vitro. Thus, these bacteria-killing fabrics are cytocompatible.
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CONCLUSIONS A novel N-halamine precursor, 1-glycidyl-s-triazine-2,4,6-trione (GTT), has been synthesized and coated onto cotton fabrics through covalent bonding. Structural and surface characterization of the cotton fabrics treated with GTT were accomplished using FTIR spectroscopy and SEM, respectively. The imide groups of GTT attached onto the cotton samples could be converted to halamines upon exposure to aqueous solutions of household bleach, and the coated cotton became biocidal. The N-halamine-modified cotton demonstrated a logarithmic reduction of 3.67 or 99.98% reduction against S. aureus and a logarithmic reduction of 4.73 or 99.99% reduction against E. coli within a contact time of 1 min. S. aureus and E. coli could be completely inactivated within 5 min of contact with logarithmic reductions of 6.99 and 7.40, respectively. The imide halamines of the cotton fabrics were found to be stable after repeated laundry washing, and about 71% of the chlorine lost after 50 washing cycles could be regenerated after rechlorination. Washing tests showed that the ether bond between GTT and cotton exhibited excellent stability during repeated washes and could endure 50 washing cycles with low loss (30%). The UVA light stability of the chlorine on the samples coated with GTT was excellent compared with that of E
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