N-Halamine Biocidal Materials with Superior Antimicrobial Efficacies for Wound Dressings.

This work demonstrated the successful application of N-halamine technology for wound dressings rendered antimicrobial by facile and inexpensive processes. Four N-halamine compounds, which possess different functional groups and chemistry, were synthesized. The N-halamine compounds, which contained oxidative chlorine, the source of antimicrobial activity, were impregnated into or coated onto standard non-antimicrobial wound dressings. N-halamine-employed wound dressings inactivated about 6 to 7 logs of Staphylococcus aureus and Pseudomonas aeruginosa bacteria in brief periods of contact time. Moreover, the N-halamine-modified wound dressings showed superior antimicrobial efficacies when compared to commercially available silver wound dressings. Zone of inhibition tests revealed that there was no significant leaching of the oxidative chlorine from the materials, and inactivation of bacteria occurred by direct contact. Shelf life stability tests showed that the dressings were stable to loss of oxidative chlorine when they were stored for 6 months in dark environmental conditions. They also remained stable under florescent lighting for up to 2 months of storage. They could be stored in opaque packaging to improve their shelf life stabilities. In vitro skin irritation testing was performed using a three-dimensional human reconstructed tissue model (EpiDerm™). No potential skin irritation was observed. In vitro cytocompatibility was also evaluated. These results indicate that N-halamine wound dressings potentially can be employed to prevent infections, while at the same time improving the healing process by eliminating undesired bacterial growth.

Inter- and intramolecular mechanisms for chlorine rearrangements in trimethyl-substituted N-chlorohydantoins.

The antimicrobial compounds 1-chloro-3,5,5-trimethylhydantoin and 3-chloro-1,5,5-trimethylhydantoin (1 and 2, respectively) have been synthesized and examined via a joint experimental and computational study. The measured rate of loss of oxidative chlorine in the absence and presence of exposure to UVA irradiation determined 2 to be less stable than 1. An interesting migration reaction was observed during UVA irradiation that featured the production of chlorine rearrangement and dechlorinated compounds. Two novel hydrogen atom transfer reaction (HATR) mechanisms have been proposed: (1) an intramolecular process in which a hydrogen atom undergoes a series of sigmatropic shifts, and (2) an intermolecular pathway in which a radical abstracts a hydrogen atom from a neighboring molecule. Density functional theory (DFT) calculations at the UB3LYP/6-311G++(2d,p) theory level have been employed to elucidate the preferred reaction pathway. Both proposed HATR mechanisms predicted 2 to possess a lower free energy of activation, ΔG(‡), relative to 1 in accordance with the experimental stability measurements. However, the intermolecular route had an overall lower absolute ΔG(‡) and was more consistent with measured product ratios in solution. The intermolecular reaction pathway, unlike the intramolecular route, also predicted the lack of formation of a migration product featuring a Cl covalently bonded to a methylene group at the 5-position of the hydantoin moiety, which was verified by NMR experiments.

N-halamine surface coating for mitigation of biofilm and microbial contamination in water systems for space travel.

A copolymer termed HASL produced from monomeric units of 2-acrylamido-2-methyl-1-(5-methylhydantoinyl)propane (HA) and of 3-(trimethoxysilyl)propyl methacrylate (SL) has been coated onto stainless steel and Inconel™ substrates, which upon halogenation with either aqueous oxidative chlorine or bromine, became antimicrobial. It has been demonstrated that the halogenated stainless steel and Inconel™ substrates were effective in producing 6 to 7 log inactivations of Staphylococcus aureus and Escherichia coli O157:H7 within about 10 min, and in prevention of Pseudomonas aeruginosa biofilm formation over a period of at least 72 h on the stainless steel substrates. Upon loss of halogen, the HASL coating could be re-charged with aqueous halogen. The HASL coating was easily applied to the substrates via a simple dip-coating method and was reasonably stable to contact with water. Both chlorinated substrates could be loaded with at least 6 × 1016 oxidative Cl atoms per cm2 and maintained a loading of greater than 1 × 1016 chlorine atoms per cm2 for a period of 3-7 days while agitated in aqueous solution. After loss of chlorine to a level below 1 × 1016 atoms per cm2, the substrates could be recharged to the 6 × 1016 Cl atoms per cm2 level for at least 5 times over a 28 day period. The new antimicrobial coating technology has potential for use in a variety of important applications, particularly for water treatment and storage on spacecraft.

N-halamine biocidal coatings via a layer-by-layer assembly technique.

Two N-halamine copolymer precursors, poly(2,2,6,6-tetramethyl-4-piperidyl methacrylate-co-acrylic acid potassium salt) and poly(2,2,6,6-tetramethyl-4-piperidyl methacrylate-co-trimethyl-2-methacryloxyethylammonium chloride) have been synthesized and successfully coated onto cotton fabric via a layer-by-layer (LbL) assembly technique. A multilayer thin film was deposited onto the fiber surfaces by alternative exposure to polyelectrolyte solutions. The coating was rendered biocidal by a dilute household bleach treatment. The biocidal efficacies of tested swatches composed of treated fibers were evaluated against Staphylococcus aureus and Escherichia coli. It was determined that chlorinated samples inactivated both S. aureus and E. coli O157:H7 within 15 min of contact time, whereas the unchlorinated control samples did not exhibit significant biocidal activities. Stabilities of the coatings toward washing and ultraviolet light exposure have also been studied. It was found that the stability toward washing was superior, whereas the UVA light stability was moderate compared to previously studied N-halamine moieties. The layer-by-layer assembly technique can be used to attach N-halamine precursor polymers onto cellulose surfaces without using covalently bonding tethering groups which limit the structure designs. In addition, ionic precursors are very soluble in water, thus promising for biocidal coatings without the use of organic solvents.

Why does Kevlar decompose, while Nomex does not, when treated with aqueous chlorine solutions?

Kevlar and Nomex are high-performance polymers which have wide varieties of applications in daily life. Recently, they have been proposed to be biocidal materials when reacted with household bleach (sodium hypochlorite solution) because they contain amide moieties which can be chlorinated to generate biocidal N-halamine functional groups. Although Nomex can be chlorinated without any significant decomposition, Kevlar decomposes under the same chlorination conditions. In this study, two mimics for each of the polymers were synthesized to simulate the carboxylate and diaminophenylene components of the materials. It was found that the p-diaminophenylene component of the Kevlar mimic is oxidized to a quinone-type structure upon treatment with hypochlorous acid, which then decomposes. However, such a mechanism for the Nomex mimic is not possible. In this paper, based upon these observations, a plausible answer will be provided to the title question.

N-halamine/quat siloxane copolymers for use in biocidal coatings.

A series of copolymers incorporating N-halamine siloxane and quaternary ammonium salt siloxane units has been prepared. The primary function of the quat units was to render the siloxane copolymers soluble in water. The copolymers have been coated onto cotton swatches and evaluated for biocidal efficacy against Staphylococcus aureus and Escherichia coli O157:H7. It was determined that both N-halamine and quat functional groups were effective against S. aureus, but only the N-halamine units were effective against Escherichia coli O157:H7. The copolymers should be useful for applications for which aqueous media is preferred over organic solvents to be used during coating procedures.

Synthesis and antimicrobial applications of 5,5'-ethylenebis[5-methyl-3-(3-triethoxysilylpropyl)hydantoin].

A novel, durable, long lasting, N-halamine siloxane monomer precursor, 5,5'-ethylenebis[5-methyl-3-(3-triethoxysilylpropyl)hydantoin] has been prepared and characterized by (1)H-NMR and FTIR for the purpose of functionalizing the surfaces of various materials. In this work, the precursor N-halamine moiety was attached by siloxane covalent bonding to surfaces of cotton fibers. Simulated laundering tests indicated that the chlorinated N-halamine structure could survive many repeated home launderings. The materials were rendered biocidal after exposure to oxidative halogen solutions, i.e. dilute household bleach. Once chlorinated, these materials were biocidal against Staphylococcus aureus and Escherichia coli. Upon loss of the halogen from either long-term use or consumption by the microbes on the surfaces, they could be simply recharged by further exposure to dilute bleach to regain biocidal activity.

N-Halamine-modified antimicrobial polypropylene nonwoven fabrics for use against airborne bacteria.

Disinfecting, nonbleaching compound 1-chloro-2,2,5,5-tetramethyl-4-imidazolidinone (MC) was uniformly coated onto polypropylene melt-blown nonwoven fabrics having basis-weights of 22 and 50 g/m(2) in order to impart antimicrobial properties via a pad-dry technique. The antimicrobial efficacies of the tested fabrics loaded with MC compound were evaluated against bioaerosols of Staphylococcus aureus and Escherichia coli O157:H7 utilizing a colony counting method. It was determined that both types of coated fabrics exhibited superior antimicrobial efficacy upon exposure to aerosol generation for 3 h. The effect of the coating on air permeability was found to be minimal. Samples were stable for a 6 month time period when they were stored in darkness. However, when the fabrics were exposed to fluorescent light, partial chlorine loss was observed. The MC-coated fabrics exhibited great potential for use in protective face masks and air filters to combat airborne pathogens.

Electronic structures of some antimicrobial N-chloramines. Possible existence of intramolecular hydrogen bonding and its effect on germicidal efficiency.

The photoelectron spectra of eight N-chloramines and N,N-dichloramines derived from either alpha-aminoisobutyric acid or 2-amino-2-methylpropanol have been measured. The lone-pair ionization potentials obtained from the photoelectron spectra have been interpreted to indicate that a substantial intramolecular interaction exists between tne N-H function and the various oxygen lone pairs of the N-chloramines. Such an intramolecular interaction for the N-chloramines can explain at least in part why these molecules are less potent as antimicrobial agents than are the N,N-cichloramine analogues for which a similar intramolecular interaction is impossible.

Inactivation of rotavirus by new polymeric water disinfectants.

Two new insoluble polymeric materials were evaluated for their efficacies in inactivating rotavirus in flowing water in a biocidal filter application. The two polymers are N-chloro and N-bromo derivatives of a poly-styrene hydantoin prepared from commercial poly-styrene. The studies were conducted for rotavirus in halogen demand-free water at pH 7.0, 25 degrees C and Environmental Protection Agency (EPA) Test Water no. 2 at pH 9.0, 4 degrees C which contained heavy halogen demand. The range of flow rates studied was 0.16-1.22 ml s-1 corresponding to contact times in the range of 4-24 s. Both of the polymers were effective in inactivating rotavirus, the N-bromo derivative providing a 4-6 log reduction under the test conditions. The materials may be useful as supplemental filters for hand-held water purification units.

Antimicrobial activity of N-chloramine compounds.

Cellular mechanisms of action of two representative N-chloramines were studied. Both compounds, 3-chloro-4,4-dimethyl-2-oxazolidinone (I) and N-chlorosuccinimide (III), inhibited bacterial growth and exerted profound inhibition of bacterial DNA, RNA, and protein synthesis at a concentration of 10(-5) M. Enzymes containing sulfhydryl groups generally were significantly inhibited by these chloramines at 10(-4) M. Dihydrofolate reductase, which contains no sulfhydryl groups, also was inhibited but at much higher chloramine concentrations (10(-2) M); ribonuclease, which also contains no sulfhydryl groups, was unaffected. All of these inhibitory effects of the chloramines could be prevented if sulfhydryl-containing reagents (mercaptoethanol or dithiothreitol) were added before or together with the chloramine. Once inhibition was produced by the chloramine, it was not reversible by later addition of the sulfhydryl reagents. These results suggest that these chloramines act at sulfhydryl sites as well as at other sites in both cells and purified enzymes.

Syntheses and antibacterial activity of new N-halamine compounds.

The syntheses of three new N,N'-dihalamine compounds in the class of tetramethylimidazolidinones are described. The stabilities and disinfection efficacies against Staphylococcus aureus of the compounds in water solution are compared with those for 3-chloro-4,4-dimethyl-2-oxazolidinone which has been studied extensively in recent years in these laboratories and elsewhere. The new compounds exhibit considerable promise as general-purpose disinfectants, particularly in applications for which long-term stability is necessary.

Interaction of methyl benzoate as a model odorant with a series of free-base amino acids and some amino-acid hydrochlorides.

The interactions of methyl benzoate as a model odorant with a series of free-base amino acids: lysine, tryptophan, arginine, proline, histidine, cysteine, leucine, threonine and phenylalanine, were studied by gas-phase adsorption on solid amino-acid samples. DL-, D- and L-isomers were investigated for all of the amino acids with the exception of cysteine where only DL- and D- were studied. Langmuir adsorption isotherms show that the strongest interactions are with lysine. Correlation of the relative interaction strength with the chemical structures suggests that binding is strongest to the remote amino groups in the amino-acid side-chain and involves a nucleophilic attack of a lone pair of electrons on the epsilon-nitrogen of lysine to the carbon of the carbonyl group of the methyl benzoate. This suggestion is supported by studies on hydrochlorides of lysine where the side-chain amino groups are protonated and thus cannot participate in a lone-pair interaction. Arginine mono-hydrochloride was found to adsorb more methyl benzoate than even the lysine free-base, an observation which is not fully understood.

Infection-resistant nonleachable materials for urologic devices.

Bacterial colonization is the primary clinical problem faced by the surgeon and medical device innovator. Despite the absence of effective systemic treatment, medical implants and devices have been deployed with increasing success over the past five decades. Infection-resistant materials (IRMs) are a relatively recent addition to the science of implant and device development. The first IRM utilized leachable antimicrobial agents. Nonleachable technologies are being developed, some of which have the potential to make organ replacement even more successful in the future.

Potential uses of combined halogen disinfectants in poultry processing.

Five organic N-halamine compounds (combined halogen disinfectants) were compared for their bactericidal activities against Salmonella typhimurium under controlled pH and temperature. All five compounds were effective as bactericides in demand-free buffers ranging from pH 5.0 to 9.0 and treatment temperatures from 4 to 48 C. The range of contact times necessary for a 99.9999% inactivation of viable cells was from .22 to 29 min, depending on the halogen concentration, temperature, and pH of the demand-free buffer. Two of the compounds (3-chloro-4,4-dimethyl-2-oxazolidinone and 1,3-dichloro-4,4,5,5-tetramethyl-2-imidazolidinone) were found to have considerable promise in high-temperature applications, and a third compound (1-bromo-3-chloro-4,4,5,5-tetramethyl-2-imidazolidinone) was more suitable for low-temperature treatments.

Combined halogen disinfectants in poultry processing.

Three organic N-halamine compounds (combined halogen disinfectants) were compared with free chlorine (as calcium hypochlorite) as bactericides against Salmonella typhimurium and unidentified normal poultry bacterial flora under controlled conditions of pH, temperature, and halogen demand similar to those encountered in poultry processing. Two of the compounds (3-chloro-4,4-dimethyl-2-oxazolidinone and 1,3-dichloro-4,4,5,5-tetramethyl-2-imidazolidinone) at a concentration of 50 mg/L were found to cause a 99.9999% decline in viable organisms in less than 1 min at 48 C, whereas a third compound (1-bromo-3-chloro-4,4,5,5-tetramethyl-2-imidazolidinone) was found to be less suitable (5.6 min to 99.9999% decline under the same conditions).

Antimicrobial N-halamine modified chitosan films.

The inherent antimicrobial properties and biodegradability of chitosan make it an ideal candidate for antimicrobial materials. In this study, N-halamine precursor 3-glycidyl-5,5-dimethylhydantoin (GH) was synthesized and bonded onto chitosan by a ring opening reaction between chitosan and GH. The chitosan film modified with the N-halamine precursor could be rendered biocidal after exposure to a dilute household bleach solution. Syntheses routes, characterization data, and antimicrobial test results are presented. The chlorinated films with 2.60 × 10(18) atoms/cm(2) of active chlorine were challenged with Staphylococcus aureus (ATCC 6538) and Escherichia coli O157:H7 (ATCC 43895) and showed good efficacy against these two bacterial species with log reductions of 7.4 and 7.5 within 10 and 5 min of contact time, respectively. These films may serve as potential materials for food packaging and biomedical applications.

Polymeric antimicrobial N-halamine epoxides.

A new N-halamine copolymer has been prepared, characterized, and evaluated for antimicrobial efficacy, stability toward hydrolyses, and stability toward UVA degradation when covalently bound to cellulose fibers. A copolymer of 3-chloro-2-hydroxypropylmethacrylate and glycidyl methacrylate was coated onto cotton, and, after curing, was treated with an aqueous solution containing the potassium salt of 5,5-dimethylhydantoin to form a coating which became antimicrobial upon exposure to househod bleach (sodium hypochlorite). The coating inactivated S. aureus and E. coli O157:H7 within minutes of contact time and was quite stable toward washing and UVA photodegradation.

N-halamine copolymers for use in antimicrobial paints.

A series of copolymers containing units of a novel hydantoinylacrylamide and the sodium salt of 2-(acrylamido)-2-methylpropanesulfonic acid have been synthesized. The homopolymer of the hydantoinylacrylamide compound was insoluble in water, while the copolymers with the sulfonic acid sodium salt were water-dispersible/soluble, with the solution becoming completely transparent when the feed ratio for the copolymer contained 7 parts of the hydantoin moiety to 3 parts of the sodium sulfonate moiety. The polymers were added into a commercial water-based latex paint, and upon drying, the painted surfaces treated with the water-miscible copolymers were rendered antimicrobial following chlorination with dilute household bleach. The chlorinated homopolymer failed to provide an antimicrobial property for the paint because of its tendency to isolate into aggregates in the paint, while the completely miscible copolymers were capable of 6-log inactivation of Staphylococcus aureus and Escherichia coli O157:H7 within 5 min of contact time.

Mechanism of photolytic decomposition of N-halamine antimicrobial siloxane coatings.

Generally, antimicrobial N-halamine siloxane coatings can be rehalogenated repetitively upon loss of their biocidal efficacies, a marked advantage over coatings containing other antimicrobial materials. However, the N-halamine materials tend to slowly decompose upon exposure to ultraviolet irradiation as in direct sunlight. In this work the mechanism of photolytic decomposition for the N-halamine siloxanes has been studied using spectroscopic and theoretical methods. It was found that the N-chlorinated coatings slowly decomposed upon UVA irradiation, whereas the unhalogenated coatings did not. Model compound evidence in this work suggests that upon UVA irradiation, the N-Cl bond dissociates homolytically, followed by a Cl radical migration to the alkyl side chain connected to the siloxane tethering group. An alpha and/or beta scission then occurs causing partial loss of the biocidal moiety from the surface of the coated material, thus precluding complete rechlorination. NMR, FTIR, GCMS, and computations at the DFT (U)B3LYP/6-311++G(2d,p) level of theory have been employed in reaching this conclusion.