Improved Antimicrobial Siloxane - Industrial & Engineering Chemistry

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Ind. Eng. Chem. Res. 2007, 46, 1861-1866

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APPLIED CHEMISTRY Improved Antimicrobial Siloxane Jie Liang,† Kevin Barnes,† Akin Akdag,† S. Davis Worley,*,† Jae Lee,‡ Royall M. Broughton,‡ and Tung-Shi Huang§ Departments of Chemistry and Biochemistry, Polymer and Fiber Engineering, and Nutrition and Food Science, Auburn UniVersity, Auburn, Alabama 36849

The monomer and polymer of the compound 3-(3-triethoxysilylpropyl)-7,7,9,9-tetramethyl-1,3,8-triaza spiro[4.5]decane-2,4-dione have been prepared and bonded to the surfaces of silica gel particles and cellulose, and the monomer has been copolymerized with a polyurethane formulation. Treatment of these polymers with dilute sodium hypochlorite solutions renders them antimicrobial. The oxidative chlorine immobilized on the materials is stable for extended periods and can be regenerated upon eventual loss by further exposure to dilute chlorine bleaching solutions. Data are presented showing that the treated materials are biocidal against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli O157:H7. The new siloxane is superior to previous N-halamine siloxane technology in that the N-halamine moiety contains two nitrogen binding sites for chlorine, one of which is a very stable hindered amine site. Introduction For many years work has been proceeding designed to produce the ideal antimicrobial surface coating which could be used to minimize the spread of infections in medical facilities, in other public venues, and in the home environment. Biocidal materials have been blended into paints for this pupose, but unfortunately such materials tend to leach out over time such that the coating becomes ineffective and cannot be refurbished. The best choice for an antimicrobial surface coating is one in which the biocidal component is chemically bonded to surface molecules comprising the coating. A variety of functional groups have been employed for this purpose over the years, such as quaternary ammoniums,1-5 quaternary phosphoniums,5-7 sulfoniums,8 and N-halamines.5,9-16 The N-halamine derivatized coatings do have several advantages over the others mentioned. They are broad-spectrum biocidal materials which are effective against Gram-positive and Gram-negative bacteria equally, fungi, protozoa, and viruses. Their mechanism of action is thought to involve direct transfer of oxidative halogen to cells at which point cell inactivation is induced by oxidation. Since the active oxidant is halogen, there is no possibility of organisms developing resistance to them. They act rapidly in their disinfection function, within seconds to less than 30 min contact, dependent upon the surface concentration of oxidative halogen and the strength of the nitrogen-halogen chemical bonds. Possibly of greatest significance, they can be regenerated once the chlorine or bromine is lost by simply re-exposure to free chlorine (e.g., from household bleach) or free bromine, an advantage not possessed by the other functional groups mentioned above. Much work has been accomplished in these laboratories over the past 2 decades concerning the preparation and use of * Corresponding author. Tel.: 334-844-6980. Fax: 334-844-6959. E-mail: [email protected]. † Department of Chemistry and Biochemistry. ‡ Department of Polymer and Fiber Engineering. § Department of Nutrition and Food Science.

N-halamine biocidal materials. Recently, an N-chloraminederivatized siloxane (5,5-dimethyl-3-(3′-triethoxysilylpropyl)hydantoin, I, in Figure 1), was reported.9,17 Siloxane derivatives are excellent species for tethering active functional groups to surfaces and have been employed extensively for this purpose utilizing biocidal quats.18,19 The siloxane labeled I in Figure 1 and its polymeric form have been demonstrated to provide biocidal activity to a variety of surfaces including cellulose,9 sand particles,20 silica gel,21 ceramics,17 poly(vinyl chloride),17 and paint.9 Although this material certainly possesses commercial potential, it was surmised that a material capable of loading higher amounts of chlorine which would also bind the chlorine more tightly, and thus exist for an extended time without the need for recharge, would be desirable for a wide variety of applications. This thinking led to the preparation of 3-(3-triethoxysilylpropyl)-7,7,9,9-tetramethyl-1,3,8-triazaspiro[4.5]decane-2,4-dione (TS in Figure 1) and its polymerized derivative (PTS in Figure 1). The precursor to these siloxane molecules is 7,7,9,9-tetramethyl-1,3,8-triazaspiro[4.5]decane2,4-dione, the structure of which is labeled as T in Figure 1. This molecule is very intriguing because it contains three different types of nitrogen functionalities: amine, amide, and imide. Thus, upon chlorination, the resulting N-chloramine contains three different types of nitrogen-chlorine bonds generally having bond strengths in the order amine N-Cl > amide N-Cl > imide N-Cl, which should provide stability toward transfer to receptors on biological cells in that same order, although ab initio computations indicate that, for this particular molecule, the amide N-Cl bond is slightly more labile than is the imide N-Cl bond due to steric interactions with protons on the six-membered ring.22 Nevertheless, T contains no protons on carbon atoms alpha to the nitrogen atoms, so there is no possibility of dehydrohalogenation for the chlorinated molecule which would cause loss of oxidative chlorine, and hence loss of biocidal activity. The imide proton in T is the most acidic one in the molecule; hence, the sodium or potassium salts of T are produced at the imide nitrogen, and nucleophilic

10.1021/ie061583+ CCC: $37.00 © 2007 American Chemical Society Published on Web 03/06/2007

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Ind. Eng. Chem. Res., Vol. 46, No. 7, 2007 Scheme 1. Production of Antimicrobial Silica Gel

Figure 1. Structures of compounds considered in this study.

substitution at this position by 3-chloropropyltriethoxysilane then yields TS. The new compound TS would be expected to have two distinct advantages for antimicrobial use as compared to siloxane I: two nitrogen atoms are available for oxidative chlorine atom substitution as compared to only one for I and a very stable hindered amine N-Cl functionality is available upon bonding to chlorine. The polymerized derivative PTS is easily formed from reaction of the sodium or potassium salts of T with poly(3-chloropropylsiloxane). This paper will discuss the syntheses and testing of TS and PTS and their chlorinated derivatives and their uses in coatings for several important applications. Experimental Section Preparation and Characterization of Precursor Biocidal Materials. The compound 7,7,9,9-tetramethyl-1,3,8-triazaspiro[4.5]decane-2,4-dione (T in Figure 1) and other starting chemicals could be purchased from the Aldrich Chemical Co. (Milwaukee, WI) and employed as received. Compound T can also be prepared in greater than 97% yield by reaction of 2,2,6,6tetramethyl-4-piperidone, potassium cyanide, and ammonium carbonate in a molar ratio of 1:2:4, respectively, in ethanol/ water (1:1 v/v) in a high-pressure reactor.23 Purification can be effected by precipitation and filtration from aqueous solution. Purified T has a sharp melting point at 371 °C as determined from DSC (differential scanning calorimetry); the literature melting point was 360-365 °C.23 The potassium salt of T was prepared by adding 11.25 g (0.05 mol) of T and 3.16 g (0.05 mol) of 88.7% potassium hydroxide to 100 mL of ethanol and refluxing for 10 min. Following removal of the ethanol and water produced during salt formation, the salt was dried under vacuum at 45 °C and obtained in near quantitative yield. Then 0.05 mol of the potassium salt of T was added to 150 mL of anhydrous N,N-dimethylformamide (DMF) and stirred at 60 °C until maximum dissolution occurred. Then 12.67 g (0.05 mol) of 95% 3-chloropropyltriethoxysilane was added to the mixture dropwise over 30 min, and the reaction mixture was stirred at 100 °C for 8 h. The potassium chloride produced was removed by filtration, and most of the DMF was

removed by vacuum distillation. Hexanes were used to extract the TS from the residue. After drying, 19.01 g of solid product TS was obtained (yield, 88%). IR (KBr, cm-1): 3371, 2972, 2929, 1770, 1707, 1454, 1425, 1365, 1269, 1105, 1082, 956, 792, 758. 1H NMR (CDCl3): δ 6.01 (s, 1H), 3.80 (q, 6H), 3.49 (t, 2H), 1.69 (m, 7H), 1.22 (m, 21H), 0.62 (m, 2H). 13C NMR (CDCl3): δ 177.19, 156.79, 61.71, 58.44, 49.02, 43.07, 41.27, 35.41, 30.72, 21.61, 18.30, 7.64. m/e 429. Poly(3-chloropropyltriethoxysilane) was prepared by stirring 63.37 g (0.25 mol) of 95% 3-chloropropyltriethoxysilane in 200 mL of 0.1 M HCl in a 500 mL flask at ambient temperature for 2 h. After removal of water and formed ethanol, the crude product was washed with water and dried, producing 34.45 g of viscous white gel (yield, 99%). Then 5.54 g (0.04 mol) of poly(3-chloropropylsiloxane) and 10.52 g (0.04 mol) of the potassium salt of T were added to 80 mL of anhydrous DMF, and the temperature was increased to 100 °C for 8 h. After cooling to ambient temperature, the potassium chloride produced in the reaction was removed by filtration, and most of the DMF was removed by evaporation. Hexanes were used to extract the PTS from the residue. After drying under vacuum overnight at 50 °C, 12.01 g of white solid product was obtained (yield, 92%). IR (KBr, cm-1): 3450, 3371, 2956, 2935, 1768, 1707, 1452, 1423, 1369, 1271, 1190, 1122, 763, 704. NMR and IR data were obtained using Bruker 400 MHz and Shimadzu Prestige-21 FTIR spectrometers, respectively. Coating Procedures. To demonstate the efficacies of chlorinated TS and PTS as antimicrobial compounds in coatings, the unchlorinated precursor compounds were reacted with silica gel, a polyurethane paint formulation, and cellulose. In the case of silica gel, typically 5.0 g of 30-60 mesh silica gel (SigmaAldrich Chemical Co., St. Louis, MO) were placed in a 100 mL flask with 20.0 g of ethanol/water (1:1 w/w) and 0.5-1.5 g of TS. The mixture was refluxed for time periods in the range of 1-6 h. The coated silica gel (silica gel-TS in Scheme 1) was removed by filtration, rinsed three times with 100 g portions of ethanol/water (1:1 w/w), and dried in air at ambient temperature. For PTS a similar procedure was employed except that the reaction time was in the range of 2-20 h. For example, a mixture of 0.75 g of PTS, 2.5 g of silica gel, and 10.0 g of ethanol/water (1:1 w/w) were refluxed for 2-20 h to produce silica gel-PTS. Chlorination of the coated silica gel samples to produce silica gel-TS-Cl and silica gel-PTS-Cl was effected by soaking them in dilute bleach solution. Typically, 25.0 g of coated silica gel were soaked in 300 mL of a 50% aqueous solution of sodium hypochlorite bleach buffered to pH 7.0 for 1 h at ambient temperature. The chlorinated silica gel samples (silica gel-TS-Cl and silica gel-PTS-Cl) were removed by filtration, rinsed with three 100 mL portions of distilled, deionized water, and dried at 45 °C for 2 h to remove

Ind. Eng. Chem. Res., Vol. 46, No. 7, 2007 1863 Scheme 2. Production of Antimicrobial Cellulose (Cotton)

any occluded free chlorine. The chlorine loadings on the silica gel particles (% by weight) were determined by standard iodometric/thiosulfate titration. Antimicrobial paint was produced by adding 2.00 g of commercial water-borne acrylic polyol formulation to 0.050.25 g of TS, stirring for 2 min, and then mixing in 0.50 g of commercial isocyanate formulation, followed by the addition and mixing of 1.50 g of distilled water. The resulting formulation was immediately spread onto the surfaces of transparency slides, which were dried in air at an ambient temperature for 16 h. The painted slides were then chlorinated with commercial bleach (6.0% sodium hypochlorite) at different concentrations for 0.52.5 h. After rinsing thoroughly with distilled water, the painted slides were dried at first in air at ambient temperature for 20 h and then at 45 °C for 5 h. The bound oxidative chlorine was determined by using a modified iodometric/thiosulfate titration method.10 The surface concentration of the bound oxidative chlorine was calculated according to following equation:

Cl+ (atom/cm2) ) 6.02 × 1023 × N × V/(2 × A)

(1)

where N and V are the normality (equiv/L) and volume (L), respectively, of the Na2S2O3 consumed in the titration and A is the area in cm2 of the painted slide. For antimicrobial cellulose (Scheme 2), 1 square in swatches of Style 400 Bleached 100% Cotton Print Cloth (Testfabrics, Inc., West Pittston, PA) were soaked in a bath containing typically 5.0% by weight TS in ethanol/water (3:1 v/v) solution for 15 min and then cured at 95 °C for 1 h. After the curing process, the swatches were soaked in a 0.5% detergent solution for 15 min, washed with water, and dried in air. Then the swatches were soaked in a 10% solution of household bleach (pH buffered to 7) at ambient temperature for 45 min, rinsed with water, and dried at 45 °C for 1 h. An iodometric/thiosulfate titration method was again used to determine Cl+ %. For PTS, a similar procedure was employed except that the initial coating bath contained 5.0% weight of PTS in 0.1 N acetic acid solution, and the soaked swatches were cured at 95 °C for 1 h and then at 145 °C for 20 min. In some cases coated swatches were chlorinated in a commercial washing machine using a modified version of the AATCC Test method 61 (Test 2A). In those cases the concentration of free chlorine (expressed as Cl+) added was about 200 mg/L. For the cotton swatches the chlorine loadings were calculated from eq 2 below,

%Cl+ ) [N × V × 35.45/(2 × W)] × 100%

(2)

where N and V are the normality (eqv/L) and volume (L) of sodium thiosulfate, respectively, and W is the weight (g) of the cotton swatch.

Antimicrobial Efficacy Testing. In all of the testing two species of bacteria, Gram-positive Staphylococcus aureus ATCC 6538 and Gram-negative Escherichia coli O157:H7 ATCC 43895, were employed. In all cases both chlorinated and unchlorinated control samples were evaluated for antimicrobial efficacy. In most cases uncoated control samples were also analyzed. For the silica gel samples, glass columns of length 25.0 cm and inside diameter 1.0 cm were packed to about 18.0 cm with the various forms of treated or untreated silica gel. The emptybed volumes of the columns were measured, so as to provide a measure of contact time, to be in the range 3.00-5.00 mL. A peristaltic pump (MasterFlex L/S, Cole Palmer Inc., Vernon Hills, IL) was used to control the flow rate of 50 mL portions of inoculum (containing about 107 CFU of bacteria and buffered to pH 7.0) through the columns. By repeated recirculation of the inoculum through the columns, contact times could be varied. Measured portions of effluent (0.05 mL) were collected at specific time intervals in sterile tubes; they were immediately quenched with 0.5 mL of 0.1 N sodium thiosulfate to prevent any subsequent inactivation of the bacteria by any free chlorine which might have leached out of the biocidal silica gel. Then serial dilutions of the quenched effluents were plated onto Trypticase soy agar, and colony counts were performed after incubation at 37 °C for 24 h. Colony counts were made to determine the viability of the bacteria. For antimicrobial paint samples, the solution was spread onto commercial transparency slides which were cut into 1 in. square pieces, and a “sandwich test” was employed to evaluate the efficacies of the samples. In this test, 25 µL of bacterial suspension were placed in the center of a slide lying in a sterile Petri dish, and a second identical slide was laid upon it which was held in place by a sterile weight to ensure good contact of the slides with the inoculum. The bacterial suspensions employed for the tests contained from 103 to 105 colony forming units (CFU), the actual number determined by counting after spread-plating on Trypticase soy agar plates. After contact times of 0.5, 1, and 2 h, the various slides were placed in sterile conical centrifuge tubes, each containing 5.0 mL of sterile 0.1 N sodium thiosulfate to quench any oxidative free chlorine which might have been present, and vortexed for 120 s to remove bacteria. Then the slides were removed, and serial dilutions of the quenched solutions were plated on Trypticase soy agar. The plates were incubated at 37 °C for 24 h and then counted for viable CFU of bacteria. For the antimicrobial cellulose tests, 1 in. square cotton swatches were subjected to the same type of “sandwich test” as noted above for the painted slides. Excellent contact between two swatches containing the inoculum (in this case about 2 × 107 CFU) was ensured by laying a sterile weight onto a sterile piece of aluminum foil which covered the two swatches in the Petri dish. In this case contact times of 1, 5, and 10 min were employed in the antimicrobial testing. Stability and Regeneration of Bound Chlorine Testing. Stability and recharge capability of chlorine on the coated silica gel samples (silica gel-TS-Cl and silica gel-PTS-Cl) were evaluated by analytically determining their weight % Cl+ contents before and after flowing distilled water through a column containing them at 5 mL/min for 168 h. The % Cl+ contents were again measured after a recharge with household bleach as discussed in a previous section. Stability and recharge capability measurements for the antmicrobial paint have not been completed; however, extensive testing of these parameters has been performed for coated

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cellulose. Washing tests were performed on the cotton swatches coated with TS, TS-Cl, PTS, and PTS-Cl using AATCC Test Method 61 (Test 2A Procedure). In this standard method each washing cycle is equivalent to five household machine washings (Standard Laboratory Washing Machine of the American Association of Textile Chemists and Colorists, Atlas Electric Devices, Inc., Type LHD-EF, Model B5). Evaluation of the retention of the coatings was monitored by analytical determination of % Cl+ loading following chlorination either before or after the washing cycles. Results and Discussion Materials. Each TS monomer possesses three ethoxy groups bonded to the silicon atom which can easily be hydrolyzed in aqueous solution. The resulting hydroxyl groups then react with those on silica gel or cellulose, or they copolymerize with polyol/ isocyanate formulations, to bind the functional group to the surfaces covalently. Binding can also occur to surfaces through hydrogen-bonding and physical interactions. In this way, the TS can be grafted onto a variety of different surfaces such as PVC and metal oxides in addition to silica gel, cellulose, and polyurethane paints. Since TS is not soluble in water alone, ethanol and water mixed solvents (3:1 v/v) were used to dissolve TS to prepare coating solutions. The hydrolyzed TS will homopolymerize upon standing, so it should be employed for coating soon after exposure to water. On the other hand, we have observed that PTS, which presumably contains one hydroxyl group for covalent grafting onto surfaces, is inert to homopolymerization in a moist environment and is not soluble in water alone. However, it is soluble in dilute acidic solution because of its weak basicity at the amine nitrogen functionality; thus, 0.1 N acetic acid can be used to dissolve PTS to prepare its coating solution. This could be an advantage in that organic solvents can be avoided during coating and curing. Considerable effort was made to optimize the coating conditions for the compounds TS and PTS on the substrates. In the case of silica gel, it was found that optimum chlorine loading (2.71 wt % Cl+) was obtained for a weight ratio of 1.5 g of TS to 5.0 g of silica gel in 20.0 g of ethanol/water (1:1 w/w) with a 4 h reaction time. Significant improvement in loading was not observed when the amount of TS or reaction time was increased. For PTS the corresponding data which provided a maximum 2.60 wt % Cl+ were 0.75 g of PTS, 2.5 g of silica gel, and 10.0 g of ethanol/water (1:1 w/w) with a 16 h reaction time. Thus, from the standpoint of reaction time, TS is the better choice for coating silica gel if it can be employed before it homopolymerizes (if moisture is present) to produce a uniform coating. It should be noted that uncoated silica gel (control) did not load any chlorine upon treatment with bleach solution. For the antimicrobial paint, it was found that the chlorine loading increased with concentration of TS in the formulation, strength of the bleach solution used for chlorination, and time of exposure to the bleach solution. The highest loading (1.30 × 1017 atoms Cl+/cm2) was obtained for the polyurethane formulation containing 0.25 g of TS, 2.00 g of commercial polyol, 0.50 g of commercial isocyanate, and 1.50 g of water when chlorinated with 10% aqueous bleach for 2 h. Even when a 5% solution of aqueous bleach was employed for a period of 2.5 h, the loading was 1.01 × 1017 atoms Cl+/cm2. However, the polyurethane formulation, without TS added, initially under the same conditions also loads chlorine to the level of 3.18 × 1016 atoms Cl+/ cm2, but this latter “background chlorine” declines to sub-biocidal concentration levels (below 1.0 × 1016

Table 1. Biocidal Efficacies of Coated Silica Gel Particles against S. aureus and E. coli O157:H7 coating material/ chlorine loading

contact time (s)

log reduction of S. aureusa

log reduction of E. coli O157:H7b

silica gel control 0 wt % Cl+

0 5 10 30

0.319 0.666 0.880

0.125 0.171 0.187

silica gel control coated with TS 0 wt % Cl

0 5 10 30

0.837 2.342 2.851

0.215 0.251 0.534

silica gel coated with TS-Cl 2.72 wt % Cl+

0 5 10 30

7.204 7.204 7.204

3.865 7.467 7.467

silica gel coated with PTS 0 wt % Cl+

0 5 10 30

2.942 3.176 4.021

0.171 0.186 0.429

silica gel coated with PTS-Cl 2.59 wt % Cl+

0 5 10 30

7.166 7.166 7.166

7.437 7.437 7.437

a The inoculum concentration varied from 1.40 × 107 to 1.67 × 107 CFU (colony forming units). b The inoculum concentration varied from 2.67 × 107 to 2.93 × 107 CFU (colony forming units).

atoms Cl+/cm2) within 24 h. The N-Cl bonds in the polyurethane copolymer are much weaker than those in the spirocyclic moiety on TS. For antimicrobial cellulose, the optimum conditions for coating are those discussed in the Experimental Section. Higher curing temperatures lead to deterioration of the fiber strengths. Antimicrobial Efficacies. The results of the antimicrobial testing versus S. aureus and E. coli O157:H7 for silica gel coated with TS and PTS and chlorinated are presented in Table 1. It is evident from the data for unchlorinated coatings that both TS and PTS caused significant reductions in the bacteria. However, this was due to adhesion to the coated particles, not inactivation, for live bacteria could be isolated in the control columns. The effect was less pronounced for E. coli O157:H7 than for S. aureus probably due to the shapes of the colonies (spheres for E. coli O157:H7 and elongated rods for S. aureus). Nevertheless, there was clearly a biocidal effect for the chlorinated coated silica gel with complete inactivation of S. aureus within 5 s and for E. coli O157:H7 within 10 s. In contrast, the silica gel particles coated with the hydrolyzed chlorinated monomer of 5,5-dimethyl-3-(3′-triethoxysilylpropyl)hydantoin (I in Figure 1) required 30 s for complete inactivation of both organisms.21 The polymeric form of I on silica gel did effect a complete inactivation within 10 s for the two bacteria.21 Thus, the new coatings with TS and PTS do give an improved performance over those with I, probably because of higher chlorine loadings due to two chlorination sites on the molecule. The antimicrobial efficacies against the two bacteria for TS copolymerized into the commercial polyurethane paint formulation and spread on transparency slides are given in Table 2. It is evident that the surface was antimicrobial as the inactivations clearly exceeded the control losses. The results were less gratifying than those for the silica gel tests; however, there are many fewer surface sites for binding chlorine in the paint, and sites beneath the surface are not available for chlorination. Nevertheless, the surface should resist the colonization of bacteria and the establishment of biofilm as long as active chlorine is present. Results for the efficacies against S. aureus and E. coli O157: H7 for chlorinated cellulose coated with TS and PTS are shown

Ind. Eng. Chem. Res., Vol. 46, No. 7, 2007 1865 Table 2. Biocidal Efficacies of TS in a Polyurethane Paint Formulation against S. aureus and E. coli O157:H7 coating material/ chlorine loading

Table 4. Stability and Recharge of Chlorinated Silica Gel Particles Coated with TS and PTS and Exposed to Flowing Water

contact time (h)

log reduction of S. aureusa

log reduction of E. coli O157:H7b

coating materiala

time of flow (h)

wt % Cl+ b

unchlorinated TS control 0 atoms/cm2 Cl+

0 0.5 1 2

0.21 0.78 2.31

0.17 0.38 0.41

TS-Cl TS-Cl PTS-Cl PTS-Cl

0 168 0 168

2.72 2.57 2.59 2.47

TS-Cl formulation 9.61 × 1016 atoms/cm2 Cl+

0 0.5 1 2

0.27 2.98 5.88

0.47 3.38 3.38

a The inoculum concentration was 7.67 × 105 CFU (colony forming units). b The inoculum concentration was 2.40 × 103 CFU (colony forming units).

Table 3. Biocidal Efficacies of Coated Cellulose against S. aureus and E. coli O157:H7 coating material/ chlorine loading

contact log reduction log reduction of time (min) of S. aureusa E. coli O157:H7b

cotton control 0 wt % Cl+

0 1 5 10

NDc 0.142 0.179

0.044 0.185 0.201

cotton control coated with TS 0 wt % Cl

0 1 5 10d

NDc 0.761 4.585

0.068 0.168 0.392

cotton coated with TS-Cl 0.82 wt % Cl+

0 1 5 10d

NDc 3.142 7.367

4.697 5.299 7.426

cotton coated with PTS 0 wt % Cl+

0 1 5 10

1.193 1.379 1.518

2.174 3.019 3.349

cotton coated with PTS-Cl 0.91 wt % Cl+

0 1 5 10

7.068 7.068 7.068

6.623 6.623 6.623

a The inoculum concentration varied from 1.17 × 107 to 2.33 × 107 CFU (colony forming units). b The inoculum concentration varied from 4.20 × 106 to 2.67 × 107 CFU (colony forming units). c No determination. d For E. coli O157:H7 this contact time was 15 min.

in Table 3. In this case the chlorinated PTS functioned significantly better in inactivating both species of bacteria than did the chlorinated TS. The explanation for this observation is not apparent, as the chlorine loadings for the two types of coatings were similar (nearly 0.9% by weight Cl+). Both coatings were more effective than was chlorinated I, which required a contact time of 30-60 min to provide a 7.6 log reduction of S. aureus, although in that case only a 0.42% by weight Cl+ loading could be obtained.9 It should be noted that when cotton coated with TS was chlorinated under the conditions experienced during a machine-washing procedure, the chlorine loading obtained was about 0.35% by weight Cl+; in this case a 6 log reduction of both of the bacterial species occurred in a contact time interval of 5-30 min. Chlorine Stabilities and Regeneration. Possibly the most significant advantages of antimicrobial coatings containing N-halamine moieties over other materials are that ones with structural features such as I and TS are very stable to loss of halogen through hydrolyses and that they can be regenerated by exposure to free halogen in aqueous solution. Table 4 illustrates these points for silica gel coated with chlorinated TS and PTS. The samples were packed into a column filter and exposed to flowing unchlorinated water (5 mL/min) for 168 h. It is evident that this treatment caused very minimal losses in weight % Cl+ loadings over the 168 h and that most of the

wt % Cl+ c 2.68 2.57

a

The materials were packed into a glass column filter and exposed to flowing distilled water at 5 mL/min. b Prechlorinated materials. c Materials rechlorinated after 168 h. Table 5. Washing Tests for Cotton Swatches Coated with TS, TS-Cl, PTS, and PTS-Cl

coating material

machine washesa

chlorination after washing (wt % Cl+)

TS TS TS TS PTS PTS PTS PTS

0 5 10 50 0 5 10 50

0.80 0.40 0.30 0.04 0.80 0.33 0.22 0.05

chlorination before washing (wt % Cl+)

chlorination before and after washing (wt % Cl+)

0.80 0.57 0.49 0.29 0.80 0.54 0.49 0.31

0.80 0.66 0.60 0.42 0.80 0.72 0.55 0.40

a AATCC Test Method 61 (Test 2A Procedure). In this standard method each washing cycle is equivalent to five household machine washings followed by rinsing and drying at 65 °C for 15 min.

loadings could be regenerated by exposure to further diluted bleach for both materials. The latter fact indicates that the siloxanes themselves were very stable to the removal by hydrolyses. The results of washing tests on cotton swatches coated with chlorinated and unchlorinated TS and PTS are given in Table 5. The fact that the weight % Cl+ loading declined steadily from a maximum of 0.80% for unwashed material to 0.04-0.05% for coated material washed the equivalent of 50 times by a standard procedure (see Experimental Section) before chlorination is evidence that the siloxane bond to the cellulose is broken by hydrolysis over time under severe washing conditions. However, the loss was much less drastic (to 0.29-0.31%) for prechlorinated coated material. This probably indicates that the chlorinated coatings (TS-Cl and PTS-Cl), being more hydrophobic than their unchlorinated counterparts, are resistant to the hydrolysis process. Also, the data indicate that chlorine can be partially restored to the prechlorinated material following a washing process. These stability results are superior to those reported previously for I.9 In parallel studies for swatches coated with TS, designed to simulate more realistic washing conditions in which about 200 ppm of Cl+ was added to the wash water (pH varied from 10.0 to 10.4) for each washing cycle, followed by drying in a machine dryer for 15 min at 65 °C, the weight % Cl+ determined after the equivalent of 5, 10, 25, and 50 machine washings was 0.42 ( 0.05. This concentration is equivalent to 4200 ppm of Cl+ on the cloth which would provide excellent antimicrobial activity throughout the lifetime of a coated fabric. Conclusion Compounds TS and PTS have been synthesized and coated onto silica gel and cellulose, and TS has been copolymerized with a commercial polyurethane formulation. Upon exposure to dilute household bleach, the coatings became effectively biocidal against S. aureus and E. coli O157:H7 bacterial challenges. The coatings were demonstrated to be quite stable

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to loss of oxidative chlorine and could be partially rechlorinated after the chlorine was finally lost. These new biocidal materials have two advantages over prior N-halamine coatings: two nitrogen sites for chlorination and a very stable amine N-Cl bond. The two materials will be somewhat more expensive to produce than is I, but they should find application because of their higher and more stable chlorine loadings. Acknowledgment The authors acknowledge the support of the USAF through Contract FO8637-01-C-6004 for this work and helpful conversations with and assistance in data collection by Hasan Kocer and Eric Funderburg. Literature Cited (1) Lambert, J. L.; Fina, G. T.; Fina, L. R. Preparation and Properties of Triiodide-, Pentaiodide-, and Heptaiodide-Quaternary Ammonium Strong Base Anion-Exchange Resin Disinfectants. Ind. Eng. Chem. Prod. Res. DeV. 1980, 19, 256-258. (2) Tiller, J. C.; Liao, C. J.; Lewis, K.; Klibanov, A. M. Designing Surfaces That Kill Bacteria on Contact. Proc. Natl. Acad. Sci. 2001, 98, 5981-5985. (3) Fuchs, A. D.; Tiller, J. C. Contact-Active Antimicrobial Coatings Derived from Aqueous Suspensions. Angew. Chem., Int. Ed. 2006, 45, 6759-6762. (4) Wynne, K. J.; Pinar, K.; Brunson, K.; Duan, B.; Wood, L.; Ohman, D. Contact Biocidal Polymeric Surface Modifiers: Polyurethanes Containing 2,2-substituted-1,3-propylene Oxide Soft Blocks with Alkylammonium Side Chains. Am. Chem. Soc. Natl. Meet. San Francisco, Sep 2006, Abs. POLY055. (5) Worley, S. D.; Sun, G. Biocidal Polymers. Trends Polym. Sci. 1996, 4, 364-370. (6) Kanazawa, A.; Ikeda, T.; Endo, T. Polymeric Phosphonium Salts as a Novel Class of Cationic Biocides. III. Immobilization of Phosphonium Salts by Surface Photografting and Antibacterial Activity of the SurfaceTreated Polymer Films. J. Polym. Sci., Part A: Polym. Chem. 1993, 31, 1467-1472. (7) Kanazawa, A.; Ikeda, T.; Endo, T. Polymeric Phosphonium Salts as a Novel Class of Cationic Biocides. VII. Synthesis and Antibacterial Activity of Polymeric Phosphonium Salts and Their Model Compounds Containing Long Alkyl Chains. J. Appl. Polym. Sci. 1994, 53, 1237-1244. (8) Engel, R.; Cohen, J. L. I.; Fincher, K. M. Antimicrobial Surfaces. U.S. Patent Appl. 0060199758, May 2006. (9) Worley, S. D.; Chen, Y.; Wang, J. W.; Wu, R.; Cho, U.; Broughton, R. M.; Kim, J.; Wei, C. I.; Williams, J. F.; Chen, J.; Li, Y. Novel

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ReceiVed for reView December 8, 2006 ReVised manuscript receiVed February 8, 2007 Accepted February 10, 2007 IE061583+