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Preparation and Evaluation of Nonionic Amphiphilic
Phenolic Biocides in Urethane Hydrogels
James H. Wynne,^1 Ramesh R. Pant, 2 Joanne M. Jones-Meehan,^2 J. Paige Phillips 3
(^1) Chemistry Division, Naval Research Laboratory, 4555 Overlook Avenue SW, Code 6100, Washington, DC 20375 (^2) Office of National Laboratories, Science and Technology Directorate, Department of Homeland Security,
Washington, DC 20528 (^3) Department of Chemistry and Biochemistry, University of Southern Mississippi, 118 College Drive, Hattiesburg,
Mississippi 29406
Received 10 July 2007; accepted 27 August 2007 DOI 10.1002/app. Published online 1 November 2007 in Wiley InterScience (www.interscience.wiley.com).
ABSTRACT: Phenols are rarely used in the preparation of polyurethanes because of the inherent competitive reaction of the phenolic moiety with isocyanates. This work repre- sents a successful application of the combination of phenols with isocyanates toward the development of phenolic-based antimicrobial urethane coatings for niche applications. In this effort, a series of nonionic amphiphilic phenolic mole- cules were prepared by condensation of 4-hexylresorcinol with the corresponding hydroxyl-terminated monomethyl poly(ethylene glycol) in the presence of a catalytic amount of acid in refluxing toluene. These new molecules were eval- uated against a variety of Gram-positive and Gram-negative bacteria for their antimicrobial activity in minimum inhibi- tory concentration solution testing. The same amphiphilic molecules were also incorporated into a hydrophilic poly-
urethane hydrogel and dispensed as films for evaluation of surface activity with a newly developed protocol. All sam- ples possessed some degree of surface antimicrobial activity, which was expressed as a log kill reduction in colony-form- ing units starting from an initial bacterial concentration of 10^7 CFU, and structural features of the phenolic com- pound were found to contribute significantly to the observed antimicrobial activity. The highest activity was observed in samples containing the phenolic compound with the shortest ethylene oxide polar structural feature and therefore highest mobility in the highly polar urethane resin. Ó 2007 Wiley Peri- odicals, Inc.y^ J Appl Polym Sci 107: 2089–2094, 2008
Key words: additives; biomaterials; hydrogels; polyur- ethanes; self-organization
INTRODUCTION
Phenolic compounds are common, naturally occur- ring, biologically active agents and often represent a large fraction of the extracts obtained from natural products. 1 These compounds are now finding appli- cations as antimicrobial additives to coatings and films for a variety of markets, such as the food industry. A specific example includes a clever intro- duction of essential oil extracts of oregano to an algi- nate-based film applied to beef muscle slices for enhanced preservation.^2 In addition to their broad applications as biocides and as synthetic intermedi- ates in the preparation of pesticides, phenolic-based materials are also added liberally to coatings as anti- oxidants, such as the monomer 2,4-di-tert-butylphe- nol and oligomeric compound nonylphenol disulfide
(Ethanox 323), which are available commercially. 3 Recent studies by Boudjouk et al. 4 and Yoon et al. 5 have reported the use of phenolic biocides in the preparation of active antimicrobial coatings. Specifi- cally, the Thomas group incorporated Triclosan [5-chloro-2-(2,4-dichlorophenoxy)phenol] as a func- tional pendant group into the silicone backbone for use in the preparation of new antifouling coatings, and Yoon’s group polymerized vinyl monomers hav- ing phenol pendant groups to produce antimicrobial polymers. As a matrix material, versatile, inexpensive, and readily available polyurethane has found wide use in the preparation of antimicrobial films and coat- ings, and several excellent recent studies exist. A polyether-type polyurethane film containing well- dispersed silver nanoparticles was prepared by Hsu et al. 6 and found to impart increased biostability when implanted in a rat subcutaneous model. Simi- larly, Piozzi 7 developed a dual-antimicrobial agent polymer system composed of a new silver-complex- ing polyurethane and ciprofloxacin antibiotic addi- tive for coating medical devices. Most recently, the Piozzi group^8 reported the preparation of antibiotic- releasing polyurethane coatings for central venous catheters, which may ultimately prevent bacterial
Correspondence to: J. H. Wynne (james.wynne@nrl. navy.mil). Contract grant sponsor: Defense Advanced Research Projects Agency-Defense Science Office (DARPA-DSO). Contract grant sponsor: Office of Naval Research.
Journal of Applied Polymer Science, Vol. 107, 2089–2094 (2008) VVC (^) 2007 Wiley Periodicals, Inc. yThis article is a US Gov- ernment work and, as such, is in the public domain in the United States of America.
colonization and the emergence of bacterial resist- ance. In an elegant application of surface-active bio- cides, block urethane polymers were synthesized by the Wynne group containing pendant hydantoin groups that are readily converted to the active bioci- dal compound when treated with hypochlorite. 9 Our interest lies in the synthesis, characterization, and evaluation of custom antimicrobial compounds designed for both high biocidal activity at the sur- face against a variety of pathogens and compatibility with commercial carrier liquids and common poly- mer resins. 10 In this work, a polyurethane hydrogel was chosen as the polymer matrix because of its exceptional versatility and unique water absorption characteristics, and the specific urethane hydrogel employed is capable of absorbing 5–25% water by weight. The phenolic biocidal compounds possess polar polyether segments to promote water solubility and resin compatibility along with nonpolar alkyl chains to promote orientation and present the phe- nolic OH at the air–surface interface. Coatings having these unique characteristics are suitable for eventual use in food service and storage areas and in hazardous waste containment and temporary stor- age vessels to deter bacterial growth.
RESULTS AND DISCUSSION
We report the synthesis and evaluation of a novel se- ries of amphiphilic phenolic biocides. Each biocide was first evaluated for the minimum inhibitory con- centration (MIC) with a standard aqueous solution test protocol. 11 Each of the biocides was then blended into a hydrophilic urethane hydrogel in tet- rahydrofuran, and coatings were solvent-cast and analyzed for antimicrobial activity. This is the first
report of a series of amphiphilic phenolic biocides blended within a urethane hydrogel. Biocidal activity results from film studies were obtained with a new film testing protocol and subsequently compared to solution MIC values. Because of their broad spectrum of antimicrobial activity, benign environmental impact, and current commercial utility, a nonionic amphiphilic phenol biocidal moiety was selected for this study. The amphiphilic biocides were synthesized by condensa- tion of 4-hexylresorcinol with the corresponding hydroxyl-terminated monomethyl poly(ethylene gly- col) (PEG) in the presence of a catalytic amount of acid in refluxing toluene (Fig. 1). Although this reac- tion was predicted to afford a mixture of products, the desired product ( 3 ) was obtained as the major product along with the disubstituted and alternate trisubstituted products in greatly diminished quanti- ties. It is believed that the steric effects of the n-hexyl substituent of the resorcinol selectively directed con- densation to the one position, resulting in the forma- tion of 3 almost quantitatively. The byproducts, acid catalyst, and unreacted starting materials were removed by flash column chromatography, and this resulted in the isolation of the desired product in significant purity. All newly prepared amphiphilic phenolic biocides [2-hexyl-5-(2-methoxy-ethoxy)-phenol (3a), 2-hexyl-5- [2-(2-methoxy-ethoxy)-ethoxy]-phenol (3b), 2-hexyl- 5-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-phenol (3c), 2-hexyl-5-(2-{2-[2-(2-methoxy-ethoxy)-ethoxy]- ethoxy}-ethoxy)-phenol (3d), and 2-hexyl-5-[poly(ethy- lene glycol) monomethyl ether]-phenol (3e)] were subjected to MIC evaluations against both Gram- positive and Gram-negative bacteria (Table I). Although in general all were effective in the lysing of bacteria, none of the phenols (3a–3e) were out- standing performers when directly compared to pub- lished MIC data for commercial phenolic biocides. 12 However, recent findings in our laboratory suggest that one must be cautious in dismissing the utility of an apparently high-MIC biocide, as there is often a poor correlation of solution antimicrobial activity and the effectiveness of the same biocide in a film/ coating. 13 All phenols are inactivated by inclusion in
Figure 1 Synthetic scheme for the amphiphilic phenol biocide series.
TABLE I MIC (lg/mL)a^ of Aqueous Phenolic Biocides
Entry 1 Product
Yield (%)
S. aureus (Gram-positive)
B. anthracis (Sterne; Gram-positive)
E. coli (Gram-negative)
S. typhimurium (Gram-negative) 1 n 5 1 3a 73 124 170 52 173 2 n 5 2 3b 78 113 165 67 186 3 n 5 3 3c 64 75 167 47 143 4 n 5 4 3d 55 83 189 81 132 5 n 5 16 3e 48 102 174 94 129 a (^) Milligrams of biocide required to neutralize 1 mL of the respective bacteria at 10 (^5) CFU/mL.
2090 WYNNE ET AL.
which place limits on solution biocidal activity or in this case aggregate and concentrate at the urethane– air interface, thus inhibiting coating surface activity. In the challenge with the Gram-negative bacteria, E. coli, optimum results were obtained in films con- taining 0.5 and 1% loadings of an amphiphilic phe- nolic biocide. Diminished activity resulted from both higher and lower weight percentage loadings. Com- pound 3a showed superior overall activity among the group of biocidal compounds tested. Three com- mon pathogenic Gram-positive bacteria were also evaluated, including the spore former B. anthracis (D Sterne). The amphiphilic phenol biocides were more active against both Staphylococcus aureus and Salmo- nella typhimurium than B. anthracis. This is to be expected because phenols are known to be less active toward spore-forming bacteria. Although the results from the exposure of films to S. typhimurium were slightly diminished compared with those of S. aureus, trends were similar and the effects of load- ing were comparable for all samples examined. Structural features of the amphiphilic phenolic bio- cides, such as the length of the tethered PEG chain, were found to significantly affect the antimicrobial ac- tivity of the resulting biocide. Biocidal testing results indicate that a lengthening of the PEG chain results in significantly diminished antimicrobial activity. This reduced activity was attributed to agent mobility and solubility: specifically, the ability of the phenol to remain mobile within the curing resin and present its active phenolic OH subunit at the surface. The longer, more hydrophilic PEG chain, as observed in com- pound 3e, is believed to remain deeper in the bulk of the coating rather than self-stratifying to the air–coat- ing interface, as was desired for making surface- active biocidal coatings. The Yoon group observed a
similar effect in a biocidal system, and the relative biocidal activity among a series of structurally similar agents was attributed at least in part to the com- pound’s hydrophilic nature and ease of diffusion in media. 5 The hydrophobic hexyl alkyl chain substitu- ent is believed to assist in the mobilization of the bio- cidal moiety to the coating–air interface, thus result- ing in the increased bioactivity. Less hydrophilic teth- ers, as observed in phenols 3a–3d, provide more flexibility and consequently greater freedom for movement within the coating upon application and curing. This increased flexibility allows for maximum orientation of the active functional group with respect to surface-residing contamination/bacteria and thus may actually promote self-concentration at the sur- face, ultimately resulting in a more viable and effec- tive antimicrobial coating.
EXPERIMENTAL
General methods Moisture-sensitive reactions were conducted in oven- dried glassware under a nitrogen atmosphere. Ana- lytical thin-layer chromatography was performed on precoated silica gel sheets, and flash column chroma- tography was accomplished with silica gel (60 A˚ , 200–400 mesh). External elemental analyses were per- formed by Atlantic Microlab, Inc. (Norcross, GA). All melting points are uncorrected. Unless otherwise noted, 1 H-NMR and 13 C-NMR spectra were taken in CDCl 3 at 300 and 75 MHz, respectively, with a TMS internal standard. Chemical shifts are reported in units downfield from tetramethyl silane (TMS). Cou- pling constant (J) values are reported in hertz. The polyurethane hydrogel, Hydrothane, was purchased from Cardiotech International.
Film forming and characterization Films were prepared by the combination of 0.80 g of Hydrothane with 25 mL of freshly distilled tetrahy- drofuran and stirring for 4 h, at which time the polymer completely dissolved. To the dissolved Hydrothane, a solution consisting of 0.008 g of 3 dis- solved in 1 mL of di-H 2 O was added dropwise, resulting in a final loading of 1 wt % (w/w) with respect to polymer solids. The final solution was allowed to stir for an additional 30 min, and films were solvent-cast by the addition of a 1-mL solution via a pipette to a precleaned microscope glass slide. The glass slide was held overnight in a sterilized, covered Petri dish to slow the rate of evaporation. The resulting films were rinsed with 5 mL of di-H 2 O to clean the surface of any unincluded ammonium salt before subsequent examination and antimicro- bial testing.
Figure 2 Antimicrobial action versus challenge for amphi- philic phenolic biocide 3a in a polyurethane hydrogel.
2092 WYNNE ET AL.
General antimicrobial testing
The general procedure for the preparation of growth media was as follows. To a 1-L Erlenmeyer flask equipped with a stirring bar were added 25.7 g of Letheen broth (Difco Laboratories, Detroit, MI) and 1 L of Milli-Q filtered water. The mixture was stirred over low heat for 30 min. Aliquots (4.5 mL) of the resulting solution were added to autoclavable cul- ture tubes ( 200) to be used in subsequent serial dilutions. The test tubes were covered with plastic lids and autoclaved at 121 8 C (and 15 psi) for 25 min. Letheen broth was selected for its ability to neutral- ize the biocidal effect of phenols with sorbitan monooleate, so that continued antibacterial action would not occur after the serial dilution step. For the preparation of bacteria, S. aureus (ATCC 12598), E. coli (ATCC 0157 : H7), S. typhimurium (ATCC 14028), and B. anthracis (ATCC 34F2) cells were each grown in our laboratory according to standard microbiological techniques. Bacteria were harvested from an agar plate by the removal of a single col- ony-forming unit with a sterile inoculating loop and its placement in Letheen broth. The culture was incubated at 28–30 8 C overnight in a shaking incuba- tor. The cells were then pelleted by centrifugation at 3000 rpm and 18 8 C. The cells were then resuspended in a 0.5% saline solution to achieve a density of about 10 9 CFU/mL as determined by McFarland tur- bidity standards.
Procedure for coating challenge tests
This method of evaluation is a well-established serial dilution screening that has been employed previ- ously. 16,17^ A 1-lL aliquot was taken from a solution of each bacterium (concentration 5 10 9 ) and applied directly to the coating on a microscope slide, resulting in the delivery of 10 7 CFU/cm 2. The slides were placed in sterile Petri dishes with a piece of hydrated filter paper in the bottom of each dish. The use of the hydrated filter paper prevented the death of the bacte- ria by desiccation. After the slides were allowed to incubate for 2 h, the coating surface was thoroughly swabbed with two sterile swabs and vortexed in a 4.5-mL solution of previously sterilized Letheen broth. Letheen broth was selected because it contains sorbi- tan monooleate, which would neutralize the antimi- crobial activity of any phenol that may have been extracted by aggressive swabbing, thus preventing additional kill once recovered. The initial tube was then serially diluted by the extraction of 0.5 mL and its placement into the subsequent tube for a total of seven tubes. The tubes were allowed to incubate at 358 C for 24 h before they were read. Positive growth was indicated by the presence of stringlike, filamen- tous growth of colonies of bacteria in solution, not
mere murkiness, which may result from other forms of contamination. All data reported are averages of triplicates, and data are reported as a log reduction from a starting concentration of 10 7 CFU/mL.
General procedure for preparation of 3
In a 25-mL, round-bottom flask equipped with a magnetic stirring bar, Dean–Stark trap, and con- denser were placed 4-hexyl-benzene-1,3-diol (4-hexy- lresorcinol; 0.59 g, 6.25 mmol), an ethylene oxide monomethyl ether (6.25 mmol), p-toluenesulfonic acid (0.01 g, 0.008 mmol), and 20 mL of toluene. An additional 7 mL of toluene was placed in the Dean– Stark trap to prevent the pot volume from becoming too low. The solution was allowed to reflux vigo- rously for 24 h in an oil bath. The resulting solution was allowed to cool to room temperature and con- centrated with the rotary evaporator. The resulting oil was eluted through a silica gel column with a hexane/EtOAc (1 : 1) solvent system, and the desired product eluted in the first fraction.
3a Fourier transform infrared (FTIR): 3362, 2950, 2930, 2858, 1606, 1519, 1463, 1376, 1297, 1221, 1162, 1114, 1055, 972 cm^21. 1 H-NMR (CDCl 3 ): 6.91 (d, J 5 9, 1H), 6.34 (d, J 5 2, 1H), 5.76 (d, J 5 5, 2H), 3.54 (d, J 5 5, 2H), 3.37 (s, 3H), 2.48 (t, J 5 8, 2H), 1.55–1. (m, 2H), 1.34–1.25 (m, 6H), 0.87 d (t, J 5 7, 3H). 13 C- NMR (CDCl 3 ): 154.3, 154.2, 130.7, 121.3, 107.5, 102.9, 73.4, 61.5, 58.7, 31.7, 29.2, 29.1, 22.6, 14.1 d. A NAL. Calcd for C 15 H 24 O 3 : C, 71.39%; H, 9.59%. Found: C, 71.68%; H, 9.31%.
3b FTIR: 3346, 2961, 2922, 2858, 1622, 1519, 1459, 1376, 1301, 1225, 1166, 1118, 968 cm^21. 1 H-NMR (CDCl 3 ): 6.92 (d, J 5 9, 1H), 6.36 (d, J 5 2, 1H), 6.32 (d, J 5 6, 1H), 5.37 (bs, 1OH), 3.78 (t, J 5 6, 2H), 3.66 (t, J 5 5, 2H), 3.63–3.59 (m, 4H), 3.42 (s, 3H), 2.50 (t, J 5 6, 2H), 1.55–1.51 d (m, 2H). 13 C-NMR (CDCl 3 ): 154.6, 154.4, 130.6, 120.9, 107.4, 102.8, 72.2, 71.9, 69.9, 61.8, 58.9, 31.7, 30.0, 29.2, 29.1, 22.6, 14.1 d. A NAL. Calcd for C 17 H 28 O 4 : C, 68.89%; H, 9.52%. Found: C, 68.73%; H, 9.44%.
3c FTIR: 3345, 2950, 2913, 2851, 1626, 1601, 1519, 1459, 1380, 1348, 1301, 1217, 976 cm^21. 1 H-NMR (CDCl 3 ): 6.91 (d, J 5 9, 1H), 6.41 (d, J 5 2, 1H), 6.33 (d, J 5 6, 1H), 3.77–3.73 (m, 2H), 3.70–3.56 (m, 10H), 3.38 (s, 3H), 2.50 (t, J 5 6, 2H), 1.55–1.49 (m, 2H), 1.33–1.
NONIONIC AMPHIPHILIC PHENOLIC BIOCIDES 2093
