WO2008156636A1

WO2008156636A1 - Antimicrobial coatings for conversion of spores into their bacterial vegetative form for decontamination

Info

Publication number: WO2008156636A1
Application number: PCT/US2008/007338
Authority: WO
Inventors: Steven N. Kaganove
Original assignee: Michigan Molecular Institute
Priority date: 2007-06-12
Filing date: 2008-06-12
Publication date: 2008-12-24

Abstract

The present invention provides an antimicrobial surface polymer coating capable of killing or deactivating bacterial spores which comprises: (1) hyperbranched polymers having (a) at least one heterocyclic N- halamine terminal group, or (b) at least one quaternary ammonium terminal group, or (c) a mixture comprising at least one each of quaternary ammonium and heterocyclic N-halamine terminal groups; or (2) polyamidoamine dendrimers having at least one heterocyclic N- halamine terminal group, or (3) linear PEI with hydantoin and quaternary ammonium groups at each repeat unit. The coating is preferred where small molecule germinants are encapsulated within it. The substrate that is coated can be sterilized prior to applying the coating, but it is not required to do so.

Description

ANTIMICROBIAL COATINGS FOR CONVERSION OF SPORES INTO THEIR BACTERIAL VEGETATIVE FORM FOR DECONTAMINATION

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to antimicrobial coatings for conversion of spores into their bacterial vegative form for decontamination. These antimicrobial surface coatings are effective in killing both vegetative microorganisms and microbiological spores.

Description of Related Art

The decontamination of various foods or surfaces with which foods come in contact poses problems of effectiveness without toxicity. Also many uses for antimicrobial products are found by consumers, including antimicrobial soaps and cleansers, fabric and air cleaners.

Military uses for decontamination occur because of concerns for the safety of water supplies and air intake systems throughout buildings where such organisms could pose serious threats. There is a need for disinfection of medical devices and surfaces in healthcare facilities to counteract the spread of nosocomial infections.

All these products are used for elimination of odors or disease-causing organisms.

The ability to form an impervious spore is the key to survival for certain species of bacteria including Bacillus and Clostridium, as well as several other microorganisms. Vegetative bacteria form a metabolically dormant spore in response to environmental stresses, such as nutrient deprivation. The result of spore formation is a highly resistant cell than can endure a variety of environmental stresses, including heat, pressure, radiation, and toxic chemicals. Bacillus and Clostridium are the causative agents for a variety of maladies including anthrax, tetanus, botulism and gas gangrene. Consequently, reliable and versatile treatments for the disinfection of these bacteria and also their spores are needed.

In the context of the threat of bioterrorism attack, the United States Environmental Protection Agency (EPA) has approved the use of several chemical treatments for the decontamination of B. anthracis, including hydrogen peroxide vapor, methyl bromide, hydrogen peroxide, peroxyacetic acid, ethylene oxide, paraformaldehyde, bleach (sodium hypochlorite), and chlorine dioxide. However, all of these agents are moderate to severely caustic, and they should not be used in rooms that are occupied by people. In addition to this reason, these agents are most appropriately used for disinfection after contamination has occurred. Since most of these chemical agents also have limited shelf lifetimes, they should be recently formulated prior to application. Consequently, there is a need for versatile antimicrobial materials and coatings that will convey biocidal properties to coated articles prior to a contamination event and that also have a long shelf life.

Although there are numerous products and potential products that have been used or described as antimicrobial agents, none describe the present invention. Some of these products are described by the following references:

US Patent 6,812,298 teaches hyperbranched polyureas, polyurethanes, polyamidoamines, polyamides, and polyesters. Their use as a component in the present invention is not disclosed. Published US 2005/0136522 describes surfaces for protection from toxins but not biocidal materials or germinants.

Published US 2005/0136523 describes surfaces of textiles with a PEM outer layer for protection from bacteria.

Published US 2005/0152955 describes a wound dressing having an antimicrobial coating within the dressing. The layers each can release the antimicrobial material.

US Patent 6,656,919 describes decontamination of surfaces after spores are present.

Published US 2007/0062884 describes the use of N-halamine in 1-10 member rings with N atoms.

Published US 2005/0271780 describes the use of a coating for protecting foods. The organic polymer matrix is very broad and does not teach the present polymers.

Published US 2008/0050410, published after the present US provisional filing date, describes a self-decontaminating surface coating with a partially hydrophobic surface that is resistant to spores. Key aspects of the coating include a curable hydrophilic resin, hydrophobic particulate additives, and small molecule germinating additives and biocidal chemicals. Importantly, the germinants and biocidal additives are not covalently bonded to the coating and will leach out over time. As a consequence of this result, these coatings have a limited operational lifetime, and they cannot easily be regenerated in situ. Furthermore, they do not have an inherently large carrying capacity for the germinating and biocidal additives, which also limits their operational lifetime. This is demonstrated in Example 4 of the citation where activity against B. anthracis rapidly declines after repeated spore challenges.

The literature teachings are to general aspects of components of the present invention, but not that they should be used together or even how one could make the present coating having all the functional groups present. Thus this above literature has limited relevance.

Clearly, it would be desirable to provide a biocidal product that has a broad spectrum of antimicrobial activity, that has the biocidal entity bound to a substrate to avoid leaching, that has a useful longer shelf life, and that could be regenerated with simple chemical agents.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antimicrobial coatings that convert bacterial spores into their more vulnerable vegetative form, where they are subsequently deactivated or killed. More specifically, this invention provides an antimicrobial surface polymer coating capable of killing or deactivating bacterial spores which comprises:

(1) hyperbranched polymers having (a) at least one heterocyclic N- halamine terminal group, or (b) at least one quaternary ammonium terminal group, or (c) a mixture comprising at least one each of quaternary ammonium and heterocyclic N-halamine terminal groups; or

(2) polyamidoamine dendrimers having at least one heterocyclic N- halamine terminal group, or (3) linear PEI with hydantoin and quaternary ammonium groups at each repeat unit.

These polymer coatings may optimally be crosslinked and/or grafted to surfaces for increased durability and/or may form the top layer of a polyelectrolyte multilayer (PEM).

Any chemical that induces activation and/or germination of bacterial endospores can be utilized. Amino acids from the group consisting of L-alanine, glycine, L-valine, L- leucine, L-isoleucine, L-praline, L-serine, L-threonine, L-methionine, L-cysteine, L-tyrosine, L-phenylalanine, L-tryptophan, L-asparagine, L-glutamine, L-aspartic acid, L-glutamic acid, L-lysine, L-arginine and L-histidine are preferred. Also other nutrient germinants are possible such as taurine, etc. More preferred is the combination of L-alanine and inosine. Non-nutrient germinants, such as calcium (I⁺) dipicolinate, and dodecylamine are also preferred. Sodium taurocholate is preferred for the germination of C. difficile. Mixtures of some or all of the preceding germinants are envisioned. The coating of the present invention is applied to a surface before it is exposed to spores or bacteria of the types desired or intended to be killed by use of this coating. The methods for applying the solution are anything that permits the coating to be applied and dried to the substrate, such as a solution of the polymer in a polar organic solvent or water, and then dipping the substrate into the solution one or more times, spraying the solution onto the substrate, spin coating the solution onto the substrate, or wiping the solution onto the substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the use of PAMAM dendrimers or hyperbranched polyamide polymers (HBP) as biocidal coatings applied to surfaces for the purpose of decontaminating bacterial spores, and which have at least one each of N-chlorohydantoin and alkyl quaternary ammonium terminal groups. These dendritic polymers contain internal "cargo space" that enables encapsulation of small molecular weight germinants.

Figure 2 illustrates the use of both (a) PAMAM dendrimers or hyperbranched polymers, which contain internal "cargo space" and (b) PEM, which readily accommodates charged ions and small molecules within available interstitial sites in formation of the antimicrobial coating of this invention. Figure 2 shows a schematic representation of a biocidal PEM composed of alternating layers of (a) PDADMAC; (b) PSS; (c) hyperbranched polymer top layer containing at least one each of N-chlorohydantoin and alkyl quaternary ammonium terminal groups; and (d) small molecule germinants adsorbed within interstitial sites of the PEM, and also within the "cargo space" of the hyperbranched polymer.

Figure 3 shows a schematic representation of a biocidal PEM composed of alternating layers of (a) PDADMAC; (b) PSS; (c) linear PEI top layer with pendant N- chlorohydantoin and alkyl quaternary ammonium groups; and (d) small molecule germinants adsorbed within interstitial sites of the PEM.

Figure 4 illustrates the use of both (a) PEM, which readily accommodate charged ions and small molecules within available interstitial sites with (b) dendritic polymers,

-A- which contain internal "cargo space", as a component of the PEM, in the formation of the antimicrobial coating of this invention. Fig. 4 shows a schematic representation of a spore- killing PEM composed of alternating layers of (a) PDADMAC; (b) PSS; (c) PAMAM dendrimer layer near top; (d) linear PEI top layer with pendant N-chlorohydantoin and alkyl quaternary ammonium groups; and (e) small molecule germinants adsorbed within the interstitial sites of the PEM, and within the PAMAM dendrimer layer "cargo space".

DETAILED DESCRIPTION OF THE INVENTION

Glossary The following terms as used in this application are to be defined as stated below and for these terms, the singular includes the plural.

AFGK means a combination of L-asparagine, D-fructose, D-glucose, and K⁺ ions

CaDPA means calcium (I⁺) dipicolinate

DMF means dimethylformamide DMSO means dimethylsulfoxide

HBP means hyperbranched polymer, and can be a polyamide, a polyurea, a polyurethane, a polyethyleneimine or a polyamidoamine

NMP means l-methyl-2-pyrrolidinone

PAA means poly(acrylic acid) PAH means poly(allyl amine hydrochloride)

PAMAM dendrimer means poly(amidoamine) dendrimer

PDADMAC means poly(diallyldimethylammonium chloride)

PEI means poly(ethyleneimine) in its linear or branched form unless specified as a dendrimer PEM means polyelectrolyte multilayers

PSS means poly(sodium 4-styrenesulfonate)

QACs means quaternary ammonium compounds

Room temperature means ambient temperature, about 20 to about 25⁰C

The present invention provides versatile coatings containing strongly biocidal chemical entities that convey broad spectrum antimicrobial activity against mold, viruses, bacteria and bacterial spores. The coatings are preferably polymers, and the biocidal entities are chemically grafted to or encapsulated within these polymers so that they cannot leach out over time, and are resistant to repeated wear and tear. Some biocidal entities may be repeatedly regenerated by treatment with commercial cleaning products that contain bleach. These features will convey extended operational lifetimes.

Additionally, the present invention provides for the incorporation of chemical entities that on contact trigger the germination of bacterial spores into vegetative bacteria, which are then more easily killed by the biocidal entities. The amount of incorporated "germinants" is optimized through the use polymer architectures that have a large carrying capacity for these small molecule "guests".

The present invention provides a series of structurally related, potent antimicrobial coatings that can be applied to a variety of common porous and nonporous surfaces, including metal, glass, plastic, fabrics, and fibers; thereby conveying protection to useful items such as clothing, equipment, and air filtration systems. Particular focus is on formulations that can be applied to the fibers of typical air filters for the protection of HVAC systems, thereby providing an effective means of making buildings resistant to attack from biological warfare agents. These coatings can be used on articles typically found in hospitals or long-term care facilities such as bed rails, tray tables and bathroom fixtures in order to prevent the spread of hospital-acquired infections, such as Clostridium difficile, which is also a preferred use.

It is known that bacterial spores are more difficult to kill than corresponding vegetative cells [e.g., Russell, A. D., Clin. Microbiol. Rev. 3, 99-119 (1990); Rode, L. J. et al., Nature 188, 1132-1134 (I960)], and hence, an antimicrobial coating intended for protection against the most serious biological warfare agents is of little value if it cannot efficiently kill spores. Conversely, a coating that is sufficiently potent to kill bacterial spores would also likely have broad spectrum activity and be effective against less hardy microorganisms, such as vegetative bacteria and viruses.

The present invention incorporates a combination of two biocidal chemistries with a cocktail of one or more small molecules that are known to initiate germination of B. subtilis, B. anthracis, or Clostridium difficile spores. It has long been known that germinated spores are more susceptible to biocidal chemistries than dormant spores, and hence, the spore killing efficiency of the coatings is strongly enhanced by the incorporation of germinants. Nutrient germinants include amino acids, such as those from the group consisting of L- alanine, glycine, L-valine, L-leucine, L-isoleucine, L-praline, L-serine, L-threonine, L- methionine, L-cysteine, L-tyrosine, L-phenylalanine, L-tryptophan, L-asparagine, L- glutamine, L-aspartic acid, L-glutamic acid, L-lysine, L-arginine and L-histidine, as well as other known nutrient germinants such as glucose, taurine, inosine, or AFGK, which is a combination of L-asparagine, D-fructose, D-glucose, and K⁺ ions. A particularly active formulation for germination is the combination of L-alanine and inosine. Non-nutrient germinants include calcium (2^*) dipicolinate (CaDPA), surfactants (in particular dodecylamine) and treatment at high pressure. Sodium taurocholate is the preferred germinant for C. difficile. For B. subtilis and B. anthracis the preferred germinants are CaDPA and L-alanine/inosine combination respectively.

The biocidal chemical entities to be tested will include quaternary ammonium compounds (QACs) [Domagk, G., Deut. Med. Wochenschr. 6_i, 829 (1935); Isquith, A. J. et al, Appl. Microbiol. 24, 859-863 (1972)] and N-halamines [Sun, G. et al, J. Chem. Educ. 82, 60-64 (2005)], which are both known for their antimicrobial activity. The activity of QACs against bacteria stems from their ability to disrupt the permeability of the cell wall, due to electrostatic interactions; while the activity of N-halamines stems from their ability to oxidize the cell wall and other cellular components on contact.

Although N-halamines have been shown to be more strongly biocidal than QACs in certain side-by-side tests, it has nevertheless been argued that the combination of both chemistries conveys certain advantages since the reactive N-X functionality (where X = Cl or Br) slowly decomposes or is consumed over an extended period of time, while in contrast QAC functional groups are not consumed when they kill bacteria. However, it is also possible to repeatedly restore the activity of "spent" N-halamine functional groups through treatment with commercial bleach solution.

These heterocyclic N-halamine terminal group moieties are selected from the following structures:

(A) (B) (C)

( D) (E )

wherein Ri, R₂, R₃ and R₄ are independently selected from a Ci -C₄ alkyl, aryl, or hydroxymethyl group; and wherein X is Cl or Br.

In order to fabricate these biocidal functional groups into robust coatings, they are grafted onto linear and highly branched "dendritic" polymers, as either pendant groups on the former, or as peripheral end-groups on the latter. In addition to the clear advantage of durability, polymer-based QACs have also been shown to exhibit enhanced antimicrobial activity over their small molecule counterparts. Both chemistries are expected to function as contact biocides because these active functional groups should stay bound to the polymer surfaces and not leach out. Optional chemical grafting of the polymers to surfaces may also be expected to improve coating durability. A particularly convenient method of synthesizing durable crosslinkable dendritic polymers from commercially available precursors is through attachment of hydrolysable alkoxysilane functionality to their terminal groups. This is described for PAMAM and PPI dendrimers in US Patents 5,739,218 and 5,902,863, and hyperbranched polyureas, polyurethanes, polyamodiamines, polyamides and polyesters in US Patent 6,534,600. These crosslinkable functional groups also chemically attach to the surfaces of a variety of substrates including glass, quartz, silicon wafers, paper, cloth, etc.

A key technical challenge is to provide "carrying space" for the small molecule germinants and to be able to deliver them to the surface of spores that land on the polymer coated surface. This result is accomplished through the use of polymer architectures that have inherently large carrying capacities, including: (a) dendritic and/or hyperbranched polymers (HBP), which contain internal "cargo space"; (b) PEM, which readily accommodates charged ions and small molecules within available interstitial sites; or (c) a combination of both architectures. Some combinations of polymer architectures are illustrated in Figures 1-4. Figure 1 illustrates a biocidal coating composed solely of a PAMAM dendrimer or hyperbranched polymer with N-chlorohydantoin and alkyl quaternary ammonium terminal groups, and small molecule germinants are encapsulated within its "cargo space". Figure 2 illustrates a similar PAMAM dendrimer or hyperbranched polymer (HBP), but which is deposited as the top layer of a PEM. Small molecule germinants are encapsulated both within the "cargo space" of the hyperbranched polymer top layer and within available interstitial sites of the PEM below it.

Figure 3 illustrates a biocidal PEM similar to Figure 2, but its top layer is linear PEI with pendant N-chlorohydantoin and alkyl quaternary ammonium groups, and small molecule germinants are only encapsulated with the interstitial sites of the PEM.

A more complex biocidal architectural construct is illustrated in Figure 4, which is composed of a PEM and linear PEI with pendant N-chlorohydantoin and alkyl quaternary ammonium groups as a top layer. One constituent of the PEM is a dendritic polymer, which functions as one or more of the positively charged layers. Small molecule germinants are encapsulated within available charged interstitial sites of the PEM, and also within the cargo space of the dendritic polymer layers. The amount of small molecule guests that could be accommodated within the coatings should be controllable based on the selected thickness of the PEMs, and the size/generation of the dendritic polymers.

PEMs are becoming increasingly popular in current polymer and materials science research because they are relatively easy to fabricate, and they are comprised of charged polymers that are usually commercially available and relatively inexpensive. Their thickness is precisely controllable through rational manipulation of a variety of parameters including the number of deposited layers, concentration of coating solutions, time of immersion, polymer molecular weight, and the concentration of added salts, if present. When two weakly ionized polyelectrolytes such as PAA and PAH are used, the thickness can also be controlled by precise adjustment of the coating solution pH for the deposition of each layer. In addition to this property, high porosity can be introduced through brief immersion of PAA/PAH multilayers in low pH solutions, which is described in US Published Patent 2006/0029634 and the references mentioned therein. These structures can be crosslinked with thermal treatment, thereby permanently locking in the porosity. The antimicrobial polymers described herein can be used as topmost layers on porous PAA/PAH multilayers, and can also be chemically grafted to these layers provided that at least a small number primary or secondary amine functional groups are available for reaction with underlying PAA layers. The underlying porous structure provides additional cargo space for small molecule germinants.

Hyperbranched polymers as described in US Patent 6,812,298 can be used with QACs and heterocyclic N-halamine end-groups in this invention. The synthesis of linear polymers with pendant QACs or dendrimers with QAC end- groups has been reported the literature [e.g., Ikeda, T et al, Makromol. Chem. 184, 869 (1984); Chen, C. Z., et al, Biomacromolecules X, 473-480 (2000)]. In addition to this, the synthesis of crosslinked polymers containing both pendant QACs and N-halamines (hydantoins) has also been reported [see Liang, J. et al, Biomaterials 27, 2495-2501 (2006)]. However, no literature reports of the synthesis of dendritic polymers with N- halamine end-groups are known. The concept of sequestering small molecule germinants in the cargo space of dendritic polymers or the interstitial sites of PEMs was not known prior to this invention.

A showing has been provided by this invention of the features: (1) that germinants can be sequestered in the cargo space of dendritic polymers, or the interstitial sites of PEMs, which are coated on glass substrates; (2) that germination of bacterial spores are effected that come into contact with the fabricated coatings; and (3) that the tested biocidal chemistries kill bacterial spores either with or without added germinants, e.g. with at least 2- 3 log reduction of viability. Surface Groups.

The number of surface groups present as terminal groups will depend upon the polymer used and the generation of the dendrimer. At least one heterocyclic N-halamine terminal group and/or quaternary ammonium group must be present in the polymer. To obtain better efficacy it is desirable that more of these groups are present as terminal groups; preferably these terminal groups are of a number of groups sufficient to occupy from about 20% to about 100% of the terminal groups; and more preferably these terminal groups are of a number of groups sufficient to occupy from about 50% to about 100% of the terminal groups. Preparation of the antimicrobial coating.

The polymer coating is made from a polymer having the terminal groups on its surface and possible encapsulated small molecule germinants by dissolving, suspending or emulsifying the polymer with a suitable solvent such as water or a polar organic solvent, including but not limited to methanol, ethanol, DMSO and DMF. This is preferably done at room temperature, although the need for temperatures slightly above room temperature may be necessary. The liquid is then applied to the substrate by dipping the object into the liquid (such as cloth, glass slides, objects) where one or more coats of the polymer coating of this invention is applied, or sprayed on the object, or wiped on the object, or spin coated on the object, or any other means that applies the liquid to the desired site. Upon drying (using air, heat, etc.) the antimicrobial coating is formed. The Figures provided illustrate the present coating as formed.

Uses.

The antimicrobial coating has as one of its purposes that spores are made subject to easier destruction by causing them to change from their spore form to their vegetative form. Thus when spores come into contact with the present polymer coating they are more easily killed or render harmless. These polymer coatings are useful in a variety of settings for hazard reduction caused by such spores, such as i) in hospitals, for example to coat bed handrails, call buttons, disposable gowns, instruments, bedpans, and other surfaces that require a longer spore clearance than is usually available from a disinfectant, ii) in air systems, for example by coating the filters and/or conduits to remove spores from the air stream of an HVAC system, iii) in water systems, for example for by coating pipes as outlets from water supplies, or filters, or iv) any place that can be coated for longer treatment to eliminate spores and reduce the hazard from such spores. Also the substrate that is coated can be sterilized to spores or bacteria prior to using the present polymer coating for additional longer sterilization. This polymer coating is not intended for use in diagnostic applications or assays.

The invention will be further clarified by a consideration of the following examples, which are intended to be purely exemplary of the present invention. Example 1: Preparation of linear PEI with pendant hydantoin functional groups, and quaternary ammonium functionality attached to most, or all, of the repeat units

Hydantoin is attached to the PEI backbone (Oxazogen) as shown in Scheme 1 below, using the known reaction of 3-hydroxymethyl-5,5-dimethylhydantoin with secondary amines. 3-Hydroxymethyl-5,5-dimethylhydantoin is found to react with PEI to a high degree of conversion. Quaternary ammonium functionality is generated in a subsequent step by exhaustive alkylation of the resulting tertiary amine with 1-bromohexane.

Scheme 1

Example 2: Preparation of G4 PAMAM dendrimer with 100% 5,5-dimethylhydantoin functionalized end-groups The potassium salt of 5,5-dimethylhydantoin was prepared according to procedures outlined in US Patents 4,412,078 and 6,969,769.

To a round-bottomed flask equipped with a magnetic stirring bar and reflux condenser was added a mixture of ethanol (40 mL), 5,5-dimethylhydantoin (5.00 g; 39.0 mmol), and potassium hydroxide (2.20 g; 39.2 mmol). The mixture was heated in an oil bath until the temperature reached 75°C, at which point the solution became clear. The ethanol was removed by rotary evaporation, and the solid potassium salt was dried under vacuum at 60⁰C for 2 days. The flask was covered with a rubber septum, and anhydrous NMP (40 mL) was added to the flask via syringe through the septum. This solution was stored at room temperature for several days for subsequent reaction with the dendrimer. The G4 PAMAM dendrimer (Dendritech) was lyophilized under high vacuum overnight in a round-bottomed flask equipped with a magnetic stirring bar, and then weighed (1.696 g; 0.1192 mmol; 7.628 mmol Of-NH₂ groups). The flask was covered with a rubber septum, and anhydrous NMP (25 mL) was added to the flask via syringe through the septum. When the dendrimer had dissolved, the rubber septum was replaced with a pressure-equalized addition funnel. A small stoichiometric excess of 2-chloroethyl isocyanate (0.95 g, 9.00 mmol) was dissolved in anhydrous NMP (8 mL) and added dropwise to the dendrimer solution at room temperature. The solution was stirred overnight at room temperature. The potassium salt of 5,5-dimethylhydantoin in NMP was briefly heated to 60⁰C until a clear solution formed, then transferred to the pressure-equalized addition funnel, and then added dropwise to the stirred reaction mixture over 30 minutes. The reaction mixture was slowly heated up to 8O⁰C and stirred for 24 hours. The hydantoin- PAMAM derivative was purified by ultrafiltration in water (Millipore YMl; MWCO 1000) in six passes. The water was removed by rotary evaporation, and the dendrimer was lyophilized under high vacuum overnight, yielding a brown, glassy solid (2.668 g; 0.0994 mmol; 83% yield)and its spectra are as follows:

¹H NMR (DMSO-dβ) δ 1.26 ppm (s; =C(CH₃)₂); 2.20-3.41 ppm (PAMAM dendrimer protons); 5.96-6.02 ppm (-NH-CO-NH-); 7.81-8.00 ppm (-CO-NH-CH₂-); 8.18 ppm (s; hydantoin N-H); and 1³C NMR (CD₃OD) δ 25.04 ppm (=C(CH₃)₂); 34.72 ppm (-CH₂-CO-NH-); 38.62 and 39.20 ppm (-CO-NH-CH₂CH₂-NH-CO-NH-); 40.01 ppm (-CO-NH-CH₂-); 40.58 and 40.88 ppm (-NH-CH₂CH₂-hydantoin); 51.06 ppm (=N-CH₂CH₂-CO-); 53.39 ppm (-CH₂=N- CH₂CH₂-CO); 59.65 ppm (-C(CH₃)₂); 157.90 ppm (-N-CO-NH-); 160.83 ppm (-NH-CO- NH-CH₂CH₂-); 174.61 and 175.02 ppm (-CH₂CH₂-CO-NH-); 179.91 ppm (-N-CO- C(CH₃)₂)

Example 3: Preparation of G4 PAMAM dendrimer with approximately 50% 5,5- dimethylhydantoin functionalized end-groups and 50% quaternary ammonium functionalized end-groups The potassium salt of 5,5-dimethylhydantoin was prepared according to procedures outlined in US Patents 4,412,078 and 6,969,769. To a round-bottomed flask equipped with a magnetic stirring bar and reflux condenser was added a mixture of ethanol (5 mL), 5,5-dimethylhydantoin (0.30 g; 2.3 mmol), and potassium hydroxide (0.139 g; 2.47 mmol). The mixture was heated in an oil bath until the temperature reached 75°C, at which point the solution became clear. The ethanol was removed by rotary evaporation, and the solid potassium salt was dried under vacuum at 60⁰C for 2 days. The flask was covered with a rubber septum, and anhydrous NMP (5 mL) was added to the flask via syringe through the septum. This solution was stored at room temperature for several days for subsequent reaction with the dendrimer. The G4 PAMAM dendrimer (Dendritech) was lyophilized under high vacuum overnight in a round-bottomed flask equipped with a magnetic stir bar, and then weighed (0.85 g; 0.060 mmol; 3.8 mmol Of-NH₂ groups). The flask was covered with a rubber septum, and anhydrous NMP (17 mL) was added to the flask via syringe through the septum. When the dendrimer had dissolved, the rubber septum was replaced with a pressure-equalized addition funnel. A small stoichiometric excess of 2-chloroethyl isocyanate (0.45 g, 4.74 mmol) was dissolved in anhydrous NMP (5 mL) and added dropwise to the dendrimer solution at room temperature. The solution was stirred overnight at room temperature. N,N-dimethyldecylamine (0.36g; 1.9 mmol) was dissolved in anhydrous NMP (5 mL), combined with the potassium salt of 5,5-dimethylhydantoin in NMP, then transferred to the pressure-equalized addition funnel, and then added dropwise to the stirred reaction mixture over 1 hour. The reaction mixture was stirred for an additional 1 hour at room temperature, and then slowly heated up to 80⁰C and stirred for 72 hours. After the first 2 hours of stirring at 80⁰C, additional N,N-dimethyldecylamine (1.50 g; 8.09 mmol) was added to the reaction mixture. The hydantoin-PAMAM derivative was purified by ultrafiltration in methanol (Millipore YMl; MWCO 1000) in six passes. The methanol was removed by rotary evaporation, and the dendrimer was lyophilized under high vacuum overnight, yielding a brown, glassy solid (1.407 g) and its spectra are as follows:

¹H NMR (DMSO-de) δ 0.84-0.85 ppm (-(CH₂)₉-CH₃); 1.24 ppm (=C(CH₃)₂ and - (CH₂)-C₈Hi₇); 2.16-3.38 ppm (PAMAM dendrimer protons); 6.09-6.53 ppm (-NH-CO-NH-); 7.93-8.10 ppm (-CO-NH-CH₂- and hydantoin N-H). Example 4: Fabrication of polyelectrolyte multilayers on substrates and exposure to germinant solutions

Polyelectrolyte multilayers were formed on I"x3" (2.54 cm x 7.62 cm) cleaned glass or quartz slides, QCM resonators, and silicon substrates by sequential immersion in respective aqueous polyelectrolyte solutions. After treatment with each polyelectrolyte solution (preferably 20 min at 1 mM), the substrates were immersed for 30 sec in deionized water, and then rinsed in a fresh stream of deionized water. Substrates were first modified with a "priming layer" of branched polyethyleneimine (BPEI) followed by deposition of alternating layers of PSS and PAH to make a BPEI/(PSS/PAH)_n assembly. After assembly of the multilayered structures, coated substrates were immersed in

0.1 mM aqueous solutions of each germinant, including calcium dipicolinate (Ca-DPA), inosine, dodecylamine, alanine, and sodium taurocholate. Their uptake was monitored by a shift in resonant frequency when QCM resonators were used as the coated substrate. QCM results for the deposition of two and three PSS-PAH bilayers, followed by treatment with Ca-DPA and L-alanine are shown in Tables I-IV below. In each case, frequency shifts ranging from 9-36 Hz indicated successful uptake of small molecule germinants into the fabricated PEM coatings.

TABLE I

QCM

Frequency

PEM Coating Shift, Hz

PEI

PEI/PSS 1

PEI/PSS/PAH 21

PEI/PSS/PAH/PSS 39

PEI/[PSS/PAH]₂ 23

PEI/[PSS/PAH]_/Ca-DPA 16

TABLE π

QCM

Frequency

PEM Coating Shift, Hz

PEI

PEI/PSS 60

PEI/PSS/PAH -13

PEI/PSS/PAH/PSS 71

PEI/[PSS/PAH]2 71

PEI/[PSS/PAH]₃ 85

PEI/[PSS/PAH]₃/Ca-DPA 36

TABLE m

QCM

Frequency

PEM Coating Shift, Hz

PEI

PEI/PSS 16

PEI/PSS/PAH 14

PEI/PSS/PAH/PSS 22

PEI/IPSS/PAHfe 16

PEI/[PSS/PAH]₂/L-alanine 32

TABLE IV

QCM Frequency

PEM Coating Shift, Hz

PEI

PEI/PSS 19

PEI/PSS/PAH 16

PEI/PSS/PAH/PSS 24

PEI/IPSS/PAHfc/PSS 26

PEI/[PSS/PAH]₃ 42

PEI/lPSS/PAHfe/L-alanine 9 Example 5: Results of exposure of Bacillus subtilis spores to encapsulated geπninant

In order to facilitate germination testing of aerobic spore-forming bacteria, the strain Bacillus subtilis 168, was used as a model for B. anthracis. Spores were prepared using either modified G or CCY medium as the sporulation broth. After three days incubation, cells were pelleted by centrifiigation at 3200 rpm, 22°C, 15 minutes in a swinging bucket rotor (Rotanta 460R). Culture supernatants were decanted, and pellets resuspended in sterile-filtered deionized water. Water washes were repeated at least six times prior to heat inactivation to kill remaining vegetative cells (65°C for 30 minutes). Approximately 95% or more refractile spores were observed in every spore prep used, and aliquots were stored at -20⁰C until use.

In order to test germination levels afforded by the formulated coatings, a 10 microliter (μL) volume of Bacillus spore suspension (approximately 10⁵ viable spores) was spotted onto the surface of coated I"x3" (2.54 cm x 7.62 cm) glass slides. During spore application each slide was placed in a humidity chamber (Petri plate that contained a wetted filter disk) to prevent dehydration of the spot while the spores incubated for one hour at ambient room temperature (~25°C). After incubation, each spot was collected from the slide in addition to three equivalent volume washes of each spot with sterile deionized water. The collected spots were then split into two volumes: one for total viable cell count, and one for viable spore count. The total viable cell count was performed by plating serial dilutions of the slide- incubated spores onto brain-heart infusion agar (Bio- World). In order to detect germinated spores, the aliquot to evaluate viable spore counts was placed into a water bath heated to 65°C and incubated for 30 minutes prior to dilution and plating. The ratio of viable cells remaining in the heat killed sample to total viable cells was used to calculate the percent Bacillus spores that germinated after one hour incubation on each slide. Table V represents the results of the testing.

TABLE V

Bacteria Viable Cells Heat Resistant

Challenge Recovered Cells Recovered Germination

Coating (CFU/10 μL) (CFU) (CFU) %

PEI/[PSS/PAH]₂ 4.60E+05 4.48E+05 2.49E+05 44.5%

PEI/[PSS/PAH]₂/DPA 4.60E-H)5 4.57E+O5 1.90E+05 58.4%

PEI/[PSS/PAH]₃/DPA 5.57E+O5 1.79E-H)5 4.77E+04 73.3% Example 6: Coating of antimicrobial polymers as thin films on bare substrates, and as top layers on polyelectrolyte multilayers

Antimicrobial polymers prepared above in Examples 1-3 are deposited on glass and on quartz slides as thin films by immersion in dilute solution. The antimicrobial polymers of Examples 1 and 3 are also deposited as a top cationic layer on polyelectrolyte multilayers. The hydantoin functional groups are chlorinated by treatment with commercial grade bleach. This may be done either prior to deposition of the polymer on the PEM or after. The amount of chlorine in the coating of selected samples is probed by colorimetric titration.

Example 7: Exposure of slides coated with antimicrobial polymers to spores of B. subtilis, B. anthracis, and E. coli to determine antimicrobial and spore killing effectiveness

In a similar manner to the experiments outlined in Examples 5 and 6 above, slides with biocidal coatings or controls are exposed to spores of B. subtilis, the Sterna strain B. anthracis, and vegetative E. coli bacteria for 0-4 hours, and samples are harvested at 30 minute intervals between 0 and 4 hours. Reactive chlorine on individual samples is quenched with sodium thiosulfate prior to harvesting of the spores. At each time interval, spores are tested for viability with BacLite™ viability stain, and others are germinated by incubation at 37°C for 30 hours in LB medium, as is routine for both B. subtilis and B. anthracis.

Although the invention has been described with reference to its preferred embodiments, those of ordinary skill in the art may, upon reading and understanding this disclosure, appreciate changes and modifications which may be made which do not depart from the scope and spirit of the invention as described above or claimed hereafter.

Claims

8. The polymer coating of Claim 1 wherein the heterocyclic N-halamine has the structure

(E) wherein Ri is independently selected from a C1-C4 alkyl, aryl, or hydroxymethyl group; and wherein X is Cl or Br.

9. The polymer coating of any one of Claims 1 or 4-8 wherein the hyperbranched polymer is a polyamide, a polyurea, a polyurethane, a polyethyleneimine or a polyamidoamine. 10. The polymer coating of any one of Claims 1 or 4-8 wherein small molecule germinants are encapsulated.

11. The polymer coating of Claim 10 wherein the germinants are selected from the group consisting of calcium (2*) dipicolinate, dodecylamine, inosine, L- alanine, L-proline, L-histidine, L-tyrosine, L-tryptophan, L-cysteine, L- serine, L-valine, L-phenylalanine, sodium taurocholate and taurine.

12. The polymer coating of Claim 1, wherein the polymer coating comprises the top layer of a polyelectrolyte multilayer (PEM).

13. The polymer coating of Claim 12 which comprises additional layers within the PEM. 14. The polymer coating of Claim 12 or 13 wherein the polyelectrolytes comprise one or more branched or linear polyethyleneimine (PEI), polyacrylic acid (PAA), poly(sodium 4-styrenesulfonate) (PSS), poly(diallyldimethylammonium chloride) (PDADMAC), poly(allyl amine hydrochloride) (PAH), and poly(amidoamine) dendrimers (PAMAM). 15. The polymer coating of Claim 12 or 13 where small molecule germinants are encapsulated both in the dendritic or hyperbranched top layer, and also within the underlying PEM structure.

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6. The polymer coating of Claim 1 where the substrate has been sterilized to spores or bacteria prior to using the coating.

-22- WHAT IS CLAIMED IS:

1. An antimicrobial surface polymer coating capable of killing or deactivating bacterial spores which comprises:

(2) polyamidoamine dendrimers having at least one heterocyclic N- halamine terminal group, or

(3) linear PEI with hydantoin and quaternary ammonium groups at each repeat unit.

2. The polymer coating of Claim 1 wherein these terminal groups are of a number of groups sufficient to occupy from about 20% to about 100% of the total terminal groups.

3. The polymer coating of Claim 2 wherein these terminal groups are of a number of groups sufficient to occupy from about 25% to about 50% of the total terminal groups.

4. The polymer coating of Claim 1 wherein the heterocyclic N-halamine has the structure

(A) wherein Ri and R₂ are independently selected from a Ci-C₄ alkyl, aryl, or hydroxymethyl group; and wherein X is Cl or Br.

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5. The polymer coating of Claim 1 wherein the heterocyclic N-halamine has the structure

(B) wherein Ri and R₂ are independently selected from a C₁-C4 alkyl, aryl, or hydroxymethyl group; and wherein X is Cl or Br.

6. The polymer coating of Claim 1 wherein the heterocyclic N-halamine has the structure

(C) wherein Ri, R₂, R3 and R₄ are independently selected from a C₁-C₄ alkyl, aryl, or hydroxymethyl group; and wherein X is Cl or Br.

7. The polymer coating of Claim 1 wherein the heterocyclic N-halamine has the structure

(D) wherein Ri, R₂, R3 and R₄ are independently selected from a Cj-C₄ alkyl, aryl, or hydroxymethyl group; and wherein X is Cl or Br.

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