WO2013102795A1

WO2013102795A1 - Antimicrobial polymer compounds, process for the preparation and uses thereof

Info

Publication number: WO2013102795A1
Application number: PCT/IB2012/050012
Authority: WO
Inventors: Jean DUHAMEL; Jamie YIP
Original assignee: Sas Spirience
Priority date: 2012-01-02
Filing date: 2012-01-02
Publication date: 2013-07-11

Abstract

The present invention relates to an antimicrobially active polymer compound defined as follows : K-S-A where : - K is a polymer or copolymer of one or more alpha-olefins; - S is a cyclic dicarboxylic acid anhydride moiety; - and A is a molecule comprising at least one free NH2 group, or a substituent with a free amine group, when not bound to S; wherein S is covalently bound to K via one of its uncarboxylated ring carbon atoms, and A is covalently bound to S via an imide bond in replacement of the "oxy" functional group of the cyclic dicarboxylic acid anhydride, and S-A together form a substitution unit having antimicrobial activity.

Description

ANTIMICROBIAL POLYMER COMPOUNDS, PROCESS FOR THE PREPARATION

AND USES THEREOF

The present invention relates to antimicrobial polymer compositions, processes for their preparation and uses of said antimicrobial polymers.

Antimicrobial polymer compositions have been known for a while, but traditionally these have been obtained by blending polymers or copolymers with antimicrobial agents or compounds. Examples of such compositions obtained by blending are described for example in several patent publications.

For example, in Japanese patent application published under the number JP 2158644, a composition is described, allegedly having excellent anti-fungal and antimicrob al properties, and having sufficient heat and impact resistance. This composition is obtained by blending crystalline propylene-ethylene copolymer with an inorganic and specific antimicrob al agent, in particular by blending :

- (A) 70-99 weight percent of crystalline propylene-ethylene copolymer having .1-30 weight percent of eibyienic unit, content, and 0.1-1 OOg/10mm melt flow rate;

- (B) 30-1 weight percent of inorganic fi ller having a particle diameter less than or equal to 20 micrometers, and preferably less than or equal to 10 micrometers;

- (C) a thiazole compound in an amount of 0.01-3 parts, and/or di henyl ether compound in. an amount of 0.01-3 parts, by weight based on 100 parts by weight of the total amount of the components (A) and (B).

In a similar vein, US patent application published under the number US2002/0065340, describes a microbe inhibiting compound incorporated into a biodegradable polymer composition, preferably a starch based polymer. The compositions described herein can be used in food packaging or fruit shock absorber nets.

One of the problems with blended compositions like those described above is that despite blending, the antimicrobial agents tend to leach out after time, or else, depending on the environment conditions in which they are used, may possibly react or be degraded and/or destroyed, thereby reducing their efficacy. In recent years, considerable attention has been paid to the preparation of micro- and nanofibrous materials having antibacterial and antimycotic activity. An example of such a fibrous material is described in the article entitled "Electrospun Mats from Styrene/Maleic Anhydride Copolymers: Modification with Amines and Assessment of Antimicrobial

Activity", by Milena Ignatova et al, Macromol. Biosci. 2010, 10, 944-954. This article mentions that "previous attempts involved different approaches for the preparation of such materials, including electrospinning of polymers with intrinsic antimicrobial properties such as chitosan or its quaternized derivatives, poly( vinyl pyrrolidone)/iodine complex (PVP- iodine), sulfonated poly( vinyl phenol), hyaluronic acid, etc, and preparation of coatings from polymers with intrinsic antimicrobial properties on the surface of electrospun micro- and nanofibres. In most cases, to impart antimicrobial properties to the micro- and nanofibrous materials, the antibacterial and antimycotic agents, such as 8-hydro-xyquinoline derivatives, cefazolin, itraconazole, mefoxin, chlorhexidine (CHX) or silver nanoparticles were added to the electrospinning solution and thus were simply physically blended with the polymer matrix. The main disadvantage of incorporation of the antimicrobial agent in this way, either on the surface or in the bulk, was its rapid release from the electrospun mats and the subsequent loss of antimicrobial activity. Consequently, the work described in this article related to an attempt to create a covalent binding of 5-amino-8-hydroxy-quinoline (5NH28Q), chlorhexidine (CHX) or Jeffamines ® by using the reactive anhydride groups of the fibers of styrene/maleic anhydride copolymers. Indeed, one of the examples describes the immersion of P(St-alt-MA)/P(St-co-MA) mats in lOmL solution of 5NH28Q dihydrochloride in distilled water which then left to react for 24 hours at room temperature. After this, the obtained modified mats were repeatedly rinsed with distilled water to remove any non-reacted

5NH28Q and freeze-dried. Although this article demonstrates the feasibility of obtaining covalently bound anti-microbial moieties to a styrene-maleic anhydride (SMA) copolymer, both the reaction scheme and the FTIR results demonstrate that (i) not all of the reactive MAH groups are converted, since there is residual absorbance, and (ii) the covalent bonds formed with the SMA are amide linkages. Such amide linkages tend to suffer in aqueous conditions from a certain degree of hydrolysis, and can thus be broken down, leading to antimicrobial moiety release, and thereby diminished antimicrobial activity of the polymer.

Finally, other attempts to add antimicrobial moieties directly to polymer chains have generally involved : addition of polyoxyethylene (POE) moieties to the polymer;

- addition of ammonium cation groups with known antibacterial activity to the polymer;

- addition or complexation of multivalent metal ions, such as copper, silver, gold, and the like, to the polymer structure.

All of these approaches have their drawbacks, most notably because they are all release the antimicrobial moiety over time, or the polymeric compounds can undergo crosslinking to a certain extent over time, which makes them more difficult to work with.

The present invention aims to solve such drawbacks by proposing antimicrobial polymer compounds and compositions that do not leach or degrade the antimicrobial moiety in aqueous environments, a process for making said compounds and compositions, and uses thereof which provide antimicrobial activity.

Insofar as the expression "antimicrobial activity" is concerned, this relates to the capacity of the polymers of the invention to kill or inhibit the growth of microorganisms such as bacteria, fungi, or protozoans.

Accordingly, the present invention provides a antimicrobially active polymer compound defined as follows :

K-S-A where :

- K is a polymer or copolymer of one or more alpha-olefms;

- S is a cyclic dicarboxylic acid anhydride moiety;

- and A is a molecule comprising at least one free NH₂ group, or a substituent with a free amine group, when not bound to S, wherein S is covalently bound to K via one of its uncarboxylated ring carbon atoms, and A is covalently bound to S via an imide bond in replacement of the "oxy" functional group of the cyclic dicarboxylic acid anhydride, and S-A together form a substitution unit having antimicrobial activity.

The polymer compounds defined as above are insoluble in polar solvents and they preserve their antimicrobial activity to a much greater extent compared to the prior art modified antimicrobial polymers, which can not be used in most food, medical or cosmetic packaging or storage applications, since they degrade very quickly and lose their antimicrobial activity.

In the compounds of the present invention, however, the covalent imide bond created between the polymer chain grafted with cyclic dicarboxylic acid anhydride moieties and the antimicrobial moiety decreases its solubility in polar solvents. Furthermore, there are no free or active cyclic dicarboxylic acid anhydride groups remaining to cause the polymer to react further or degrade in environments that would traditionally be the source of problems with the known modified polymers of the prior art, meaning that the polymer compounds according to the present invention are extremely stable over time and under various conditions of use. Any free or active dicarboxylic acid anhydride groups that are left in the polymer would cause an increase in solubility of the polymer in water, which would therefore require the polymer to be stabilised in some way, for example, by reticulation, for use in aqueous environments or where the polymer would come into contact with water or polar solvents.

This is not the case for the compounds of the present invention, where no reticulation is required, the polymers are sufficiently stable and resistant to such environments without any further required stabilisation steps. Most notably, the antimicrobial polymer compounds of the present invention remain heat stable at temperatures greater than 120°C, which makes them suitable, among others, for the use in polymer extrusion, polymer film drawing and polymer moulding processing. This increased heat stability also means that the polymer compounds of the invention can be steam sterilised.

Preferably, the polymer or copolymer of one or more alpha-olefms suitable for use in preparing the compounds of the present invention has a structure as follows :

-(CH₂)_n-(CH2-CR¹R²)_m- where :

- R¹ is H, methyl, or alkyl C₂-Ci₀, optionally branched, and/or substituted; R² is methyl, or alkyl C₂-C₁0, optionally branched, and/or substituted

- n is the molar fraction of methylene units, defined as being from 0 to 1 ; and

- m is the molar fraction of the alpha-olefin units, defined as being from 1 to 0,

- where n+m = 1.

Even more preferably, the polymer or copolymer is chosen from the group consisting of polyethylene (PE), polypropylene (PP), their copolymers (EP), and polyisobutylene.

The addition of a cyclic dicarboxylic acid anhydride moiety to such polyolefms introduces reactive groups into these polymers which are otherwise normally relatively inert. The cyclic dicarboxylic acid anhydride moieties are bound to the polymer chains via one of the ring's uncarboxylated carbon atoms. They can however still react with other molecules, such as, in the case of the present invention, free -NH₂ groups, or a substituent with a free amine group, for example, with primary amines.

In a particularly preferred embodiment of the present invention, the cyclic dicarboxylic acid anhydride S grafted to the polymer is maieic acid anhydride (MAH) which, when covalentiy bound to the polymer , forms a cyclic succinic acid anhyride group, or "Su" moiety. An example of such a grafted polymer is Ethylene-Propylene Maieic Acid Anhydride or EPMAH. Most maleated polyolefms are usually polydisperse in length, the succinic acid anhydride pendants being randomly located along the backbone, and oligoMAH as well as single MAH units can attach onto the polyolefin backbone. Such EPMAH polymers are available commercially, for example sold under the tradename Fusabond MD353D by Dupont.

Preferably, the cyclic dicarboxylic acid anhydride S is present in the polymer in amounts comprised between 50 micromoles per gram to 500 micromoles per gram of polymer, and even more preferably is present in an amount equal to about 200 micromoles per gram of polymer.

As mentioned above, the polymers of the present invention also contain a further molecule A, which, when bound to S, forms a substitution unit S-A having antimicrobial activity. Molecule A represents a molecule which, in the unbound state, in other words, when not bound to the polymer via S, comprises at least one free NH₂ group, or a substituent with a free amine group.

Preferably, molecule A is a free NH₂ group, or a substituent with a free amine group as mentioned above, but more preferably is selected from the group consisting of 5 -amino- 1,10- phenanthrolene, aka l,10-phenanthrolin-5 -amine; 5-aminoquinoline; 2-aminopyridine; 3- aminopyridine; 4-aminopyridine; 4-amino methylpyridine; 1 -amino anthraquinone; 2-amino- 1 ,3,4-thiadiazole; N,N-dimethyl-l ,3-diaminopropane; N,N-dibutyl-l ,3-diaminopropane;

chlorhexidine; hexamethylenedioxy-4,4'-dibenzamidine-bis-hydroxy-2-ethanesulfonate;

imidocarbonimidic diamide; and dequalinium.

All of the above molecules will form a covalent imide bond with the cyclic dicarboxylic acid anhydride moieties, replacing the "oxy" functional group of the cyclic dicarboxylic acid anhydride with the nitrogen atom from the free amine group, and causing water to be formed and eliminated as the leaving group from the reaction. The reactive acid anhydride moieties are all converted to imide bonds, leaving no reactive groups to affect future stability of the antimicrobial polymer compounds or compromise their use in aqueous environments.

In still yet another preferred embodiment of the present invention, the polymer compounds further contain one or more metal ions also known to exert an antimicrobial activity, bound to the polymer molecule by coordination complexation. Such metal ions are known per se in the art and generally comprise monovalent or bivalent metal ions, such as those of iron, cobalt, nickel, copper, zinc, ruthenium, rhodium, palladium, silver, cadmium, platinum, gold, mercury, magnesium, and the like.

The above mentioned metal ions can form coordinated complexes with the polymer by several known methods of complexation, wherein a complex forming agent is bound to both the polymer and a metal ion as described above. One such preferred example in accordance with the present invention of a complex forming agent is phenanthrolene, which forms a pincer- like chelate with the modified antimicrobial polymer compounds of the present invention. This can be achieved by reacting the polymers of the invention with silver nitrate and phthalimide in the presence of base in toluene at high temperature in order to yield the desired product.

In addition to the antimicrobial polymer compounds per se, the invention also provides a process for their preparation. This process comprises the steps of :

- (a) reacting a polymer or copolymer K of one or more alpha-olefms having a graft S consisting of a cyclic dicarboxylic acid anhydride moiety with a molecule A comprising at least one free NH₂ group, or a substituent with a free amine group, to cause the "oxy" functional group of the cyclic dicarboxylic acid anhydride to be replaced by the nitrogen atom of the free amine group and thereby form a covalent imide bond, wherein the imidation reaction takes place in an apolar solvent at a temperature comprised between 140°C and 210°C for a duration comprised between 1 hour and 24 hours;

- (b) precipitating out the resulting imidated reaction product by addition of the hot reaction mixture to a polar solvent in the liquid state;

- (c) filtering and drying the precipitated reaction product.

Step (a) can optionally be preceded by a dehydration step, which takes place at a temperature greater than 100°C and less than 200°C, and for a period comprised between 1 hour and 24 hours. Most preferably, if used, the dehydration step preceding step (a) is carried out at a temperature of 170°C for a period of 6 hours.

In yet another preferred embodiment of the process according to the present invention, the solvent used in the imidation reaction of step (a) is an apolar solvent selected from the group consisting of xylene, dodecane, paraffinic oil, and biphenyl, and most preferably the apolar solvent for this step is biphenyl.

Preferably, the polymer or copolymer of one or more alpha-olefms suitable for use in the process according to the invention is chosen from the group consisting of polyethylene (PE), polypropylene (PP), their copolymers (EP), and polyisobutylene. Such polymers can even more preferably have a structure as follows :

-(CH₂)_n-(CH2-CR¹R²)_m- where :

- R¹ is H, methyl, or alkyl C₂-Ci₀, optionally branched, and/or substituted;

- R² is methyl, or alkyl C₂-Ci₀, optionally branched, and/or substituted; n is the molar fraction of methylene units, defined as being from 0 to 1 ; and

- m is the molar fraction of alpha-olefm units, defined as being from 1 to 0,

- where n+m = 1.

In a particularly preferred embodiment of the process according to the present invention, the cyclic dicarboxylic acid anhydride is succinic acid anhydride resulting from the maleation of a polyolefin with maieic acid anhydride (MAH).

Most preferably, the grafted polymer used in the process of the present invention is ethylene- propylene maieic acid anhydride, also known as EPMAH. Such EPMAH polymers are available commercially, for example sold by DSM (Netherlands) or Dupont.

In accordance with another preferred embodiment of the process of the present invention, molecule A comprises at least one free NH₂ group, or a substituent with a free amine group, for example, a primary amine.

Even more preferably, molecule A comprising at least one free NH₂ group, or a substituent with a free amine group, is selected from the group consisting of 5 -amino- 1,10- phenanthrolene, aka l,10-phenanthrolin-5 -amine; 5-aminoquinoline; 2-aminopyridine; 3- aminopyridine; 4-aminopyridine; 4-amino methylpyridine; 1 -amino anthraquinone; 2-amino- 1 ,3,4-thiadiazole; N,N-dimethyl-l ,3-diaminopropane; N,N-dibutyl-l ,3-diaminopropane; chlorhexidine; hexamethylenedioxy-4,4'-dibenzamidine-bis-hydroxy-2-ethanesulfonate; imidocarbonimidic diamide; and dequalinium.

In a most preferred embodiment of the process according to the invention, step (a) is carried out at a temperature of 190°C for a duration of 12 hours. In a further preferred embodiment of the process according to the present invention, said process comprises the additional step of :

- (d) reacting the polymer product obtained in step (c) with silver nitrate and

phthalimide in the presence of base in toluene at high temperature ;

- (e) precipitating the hot product obtained in step (d) into a polar solvent in the liquid state.

Still yet another embodiment of the present invention is an antimicrobially active article manufactured from or containing the antimicrobial polymer compounds of the present invention. Such articles can for example be films, extrusion products, moulded products, all made from the antimicrobial polymer compounds of the invention. In one preferred embodiment, the article is an antimicrobially active film, obtained by melting the polymer compounds of the present invention and then extruding or drawing said melted compound into a film. Said film can be used by itself, i.e. it is free standing, or in association with other polymer films, for example, through co-extrusion or co-drawing, or through hot press welding or lamination of the antimicrobial polymer films to another film layer. In a similar manner, the antimicrobial polymers can be used on their own or blended with other polymers and moulded into articles through known techniques such as injection or cast moulding.

In a preferred alternative process, the antimicrobially active polymers of the invention can be synthesized in a reactive extruder, whereby the reaction between the maleated polymer and moiety A occurs in an extruder without the use of solvents, thereby replacing the separate steps outlined above involving solvents. The reaction product can then be output as an extruded form of polymer directly.

Brief Description of the Figures

There now follows a brief description of the Figures in which :

- Figure 1 represents the general structure of a silver-labeled phenanthrolene succinimyl ethylene-propylene copolymer (Ag-P-Su-EP); - Figure 2A represents the general structure of a maleated ethylene-propylene copolymer (EPMAH);

- Figure 2B represents the general structure of a phenanthrolene-labeled succinimyl ethylene-propylene copolymer (P-Su-EP, Figure 2B);

- Figure 3 A represents a FT-IR spectrum of EPMAH before dehydration;

- Figure 3B represents a FT-IR spectrum of EPMAH after dehydration, where the

maleic acid anhydride moiety has been converted into the cyclic form of succinic acid anhydride (Su-EP);

- Figure 4 represents a FT-IR spectrum of P-Su-EP;

- Figures 5A, 5B, 5C, and 5D, respectively represent UV-Vis absorption spectra of EPMAH, P-Su-EP, aminophenanthrolene (P), and a model comparative compound P-Su;

- Figure 6 represents the general structure of a model comparative compound P-Su.

Examples

For the purposes of demonstrating the antimicrobial effects of the polymers according to the present invention, an example was chosen which made use of ethylene -propylene maleic acid anhydride grafted polymers as the starting material. This grafted polymer was used to synthesize a silver- labeled ethylene-propylene copolymer (Ag-P-Su-EP). The general structure of Ag-P-Su-EP is shown in Figure 1.

First, a maleated ethylene-propylene copolymer (EPMAH, Figure 2A) was reacted with an amine, 5-amino-l,10-phenanthrolene, to yield a phenanthrolene-labeled succinimyl ethylene- propylene copolymer (P-Su-EP, Figure 2B). The polymer compound P-Su-EP was then reacted with silver nitrate and phthalimide to yield Ag-P-Su-EP, the structure of which is given in Figure 1.

Maleated ethylene-propylene copolymer (EPMAH) was obtained from DSM. ReagentPlus 99.5% biphenyl, ACS reagent > 99% silver nitrate, and > 99%> phthalimide were obtained from Sigma- Aldrich. HPLC grade toluene, methanol, and acetone were obtained from

Caledon. 5-amino-l,10-phenanthrolene was obtained from Polysciences, Inc. All

commercially available products identified herein were used as received unless otherwise indicated.

Throughout the present description of the examples, FT-IR spectra were obtained on a Bruker Vector 22 FT-IR Spectrophotometer. Spectra were acquired from 700 to 4000 cm^"1 with a resolution of 1 cm^"1 and a total of 16 scans per spectrum. Polymer samples were first dissolved in either THF or hexanes, as specified. A few drops of the polymer solution were then placed on a clean NaCl salt plate, and the solvent (hexanes) was allowed to dry under a stream of nitrogen, or in the vacuum oven, leaving a polymer film on the salt plate. Hexanes were the preferred solvent for this procedure since they were easier to remove than tetrahydrofuran (THF). Spectra were then acquired. If the absorbance peaks were too low in the FT-IR spectrum, another few drops of polymer solution were added until a decent signal was obtained. If the signal was too high, i.e. where absorbance was greater than 1, the salt plate was cleaned, the polymer solution diluted, and a new film was created.

UV-Vis absorbance spectra were obtained on a Cary 100 UV- Visible Spectrophotometer. Spectra were acquired from 200 to 500 nm with a 1 nm resolution using a 1 cm path length.

The samples of P-Su-EP and Ag-P-Su-EP obtained underwent elemental analysis. Samples were analyzed for carbon, hydrogen, and nitrogen content. The resultant was burnt into ash which was further analyzed to yield the silver content assuming that silver was all that remained.

The first step in the synthesis of the silver-labeled ethylene-propylene copolymer (Ag-P-Su- EP, Figure 1) was to react the maleated ethylene -propylene copolymer (EPMAH, Figure 2 A) with 5-amino-l,10-phenanthrolene P to yield a phenanthrolene-labeled EPMAH (P-Su-EP, Figure 2B).

An optional dehydration reaction was carried out in order to ensure that all succinic anhydride pendants were closed, i.e. are in the cyclic, ring-bound form. In this way, all succinic anhydride pendants were in the closed anhydride form and were able to react with the amine during the imidation reaction to give the desired succinimide product. As an example of such a dehydration step, 1.33 g of EPMAH and 40 g biphenyl were heated to 170°C for 6 hours. Dehydration of succinic acid generally starts at 170°C and becomes rapid at temperatures of 190-210°C.

In order to assess the completeness of the dehydration reaction, a small amount of the polymer was precipitated from hot biphenyl into acetone. The FT-IR spectra of EPMAH before and after dehydration are shown in Figures 3A and B, respectively. For the sake of clarity, spectra are only shown from 1000 to 2000 cm . Peaks outside of this range are irrelevant for this discussion.

In Figure 3 A, the carbonyl peak is located at 1713 cm . This carbonyl peak location is characteristic of the succinic acid carbonyl group. In Figure 3B, the carbonyl peak has shifted to 1785 cm⁴. This carbonyl peak location is characteristic of the succinic acid anhydride carbonyl group. The shift in the location of the carbonyl peak indicates that the dehydration reaction was successful.

After the optional dehydration step, phenanthrolene amine P was added to the system. The amine was dissolved in THF and then the P/THF solution slowly added to the polymer solution. A two-fold excess of phenanthrolene was added in order to maximize the level of labeling of the succinic anhydride pendants. Once P is transferred to the hot biphenyl solution, it dissolves, and so in effect can be added either in solution as described, or even completely in the solid state, without the aid of a solvent.

After addition of P, the mixture is allowed to react at 190°C overnight. Once completed, the polymer was precipitated from hot biphenyl into acetone and then four further times from hexane into methanol. The polymer was then filtered and dried in a vacuum oven overnight.

P-Su-EP was characterized using three techniques. First, FT-IR spectra of P-Su-EP were obtained. One such spectrum is shown in Figure 4. The location of the carbonyl peak in Figure 4 had now shifted from 1785 cm⁴ for EPMAH where the pendants were present in the anhydride form (Figure 3B) to 1714 cm⁴, characteristic of the carbonyl peak observed for a succinimide group. This shift in peak location indicates that the reaction proceeded as expected. However, since the succinic acid carbonyl peak was also present at 1710 cm⁴, the peak observed at this point in Figure 4 could be due to open succinic acid groups or a combination of unreacted succinic acid and N-5 -amino- 1,10-phenanthrolene succinimide groups. In order to ensure the presence of the desired N-5 -amino- 1,10-phenanthrolene succinimide group in the polymer, UV-Vis absorbance spectra were acquired.

UV-Vis absorption spectra of EPMAH, P-Su-EP, aminophenanthrolene (P), and a model comparative compound P-Su (Figure 6) are shown in Figures 5A, B, C, and D, respectively. Synthesis of the model compound P-Su is described hereafter. The spectra shown in Figures 5 A and B are clearly different. Figure 5 A shows no peaks in the 250 - 300 nm region, while Figure 5B shows a distinct peak centred at 267 nm which is different from that obtained for aminophenanthrolene shown in Figure 5C. Interestingly, the absorption spectrum of P-Su-EP shown in Figure 5B is nearly identical to that of the P-Su model comparative compound shown in Figure 5D, proving that the reaction was indeed successful.

In order to characterise the addition of P onto the EPMAH copolymer, a model comparative compound was synthesised. The structure of the model comparative compound is shown in Figure 6. The comparative compound was obtained by reacting phenanthrolene as described above and succinic anhydride in an acetic acid/sodium acetate buffer. The solution was refluxed at 170°C overnight. A 2x molar excess of succinic anhydride was added in order to drive the reaction towards the end products. In an example reaction, 200 mg of P was reacted with 200 mg of succinic anhydride in a buffer made up of 0.5 g of sodium acetate and 10 mL of glacial acetic acid. After reaction, the solution was transferred to a 125 mL separatory funnel and 40 mL of 1M sodium carbonate solution was slowly added. The product was then recovered through multiple extractions using dichloromethane, for example, 10 extractions with 20 mL of dichloromethane per extraction. The resulting dichloromethane/crude product solution was then concentrated down to a volume of around 20 mL. This concentrated solution was washed with 2 x 30 mL 1 M sodium carbonate followed by 2 x 30 mL water. The organic phase was then dried over sodium hydroxide, the dichloromethane evaporated off, and the product dried in a vacuum oven. This procedure gave approximately 150 mg of product, corresponding to a yield of approximately 54%.

An NMR spectrum of the crude product was obtained. The aromatic protons on the phenanthrolene group were present in the 7.7 - 9.2 ppm range and integrated to 7 protons, as expected. The protons of the succinimide group were observed in the 2.8 - 3.1 ppm range and integrated to roughly 4 protons, also as expected. The additional peaks at 5.7, 2.5, 2.2, 2.0, and 1.2 ppm indicated the presence of residual impurities. As mentioned above, the UV-Vis absorbance spectrum of the model compound (Figure 5D) matched that of P-Su-EP (Figure 5B), providing qualitative evidence that the phenanthrolene labeling reaction had indeed proceeded as expected.

Once it was established that P had been successfully added to Su-EP, the final step was to label P-Su-EP with silver to yield Ag-P-Su-EP, the structure of which is given in Figure 1. The reaction to obtain this product involves reacting the P groups of the P-Su-EP copolymer with silver nitrate and phthalimide, in the presence of base in toluene at high temperature in order to yield the desired product. In an example reaction, 566 mg of P-Su-EP was reacted with 71 mg of AgN03, 83 mg of NaOH, and 58.1 mg of phthalimide in 30 mL toluene at 100°C. The solution was left to react overnight and then precipitated from hot toluene into methanol. The polymer was further purified with two additional precipitations from hot toluene into methanol. The final mass of Ag-P-Su-EP was 321.2 mg, giving a yield of approximately 50%.

Two samples of P-Su-EP and Ag-P-Su-EP underwent elemental analysis, which yielded the wt % of carbon, hydrogen, and nitrogen in the sample. The samples were then burned with oxygen and the mass of the remaining ash was determined. Table 1 describes the chemical building blocks that were used to estimate the chemical composition of the modified

EPMAHs. First, EPMAH is a copolymer of ethylene (-CH2-CH2-) and propylene (- CH(CH3)-CH2-), a few units of which were maleated and subsequently modified. In essence, these polymers can be viewed as being constituted of methylene units (two for ethylene and three for propylene) and a few maleated methylene units. Consequently, the polymers are viewed as being composed of a molar fraction x of modified methylene units and a molar fraction (1 - x) of unmodified methylene units. Whereas a methylene unit has a molar mass of 14 g.mol^"1, the modified methylene units (MMU in Table 1) of P-Su-EP and Ag-P-Su-EP have molar masses of 289 g.mol^"1 and 543 g.mol^"1 respectively.

Table 1 : Chemical composition of the modified polymers (MMU = modified methylene unit)

Based on the chemical structures shown in Table 1 , the chemical composition of the modified polymers was estimated in Table 2, knowing that EPMAH contained 200 micromoles of MAH per gram of EPMAH. The 200 micromol.g MAH content of the EPMAH sample used was confirmed in three separate determinations.

Table 2 : Elemental analysis of the modified EPMs

Considering that the MAH content of EPMAH used to determine the chemical composition of the modified EPMAHs is accurate within ±10 %, the data listed in Table 2 are in good agreement with the expected composition of the modified polymers.

Preparation of Polymer Films

Anti-microbially active polymer films according to the invention were prepared as follows. A solution of the polymer was prepared in tetrahydrofuran (THF). The solution was

concentrated by evaporating as much THF as possible. A small rubber ring was placed on a clean glass plate and the plate with the ring was placed on a hot plate. The hot plate was brought under a gentle stream of nitrogen and the plate was heated up. A few drops of the polymer solution was deposited slowly inside the ring on the glass plate. The stream of nitrogen enabled the evaporation of the THF, leaving behind a film. The process was repeated several times, until a reasonably thick film was obtained. The film was peeled off with a sharp blade and cut into small pieces which were tested for antibacterial activity.

Biological Activity of Substituted EPMAH Polymers

The bactericidal or bacteriostatic effectiveness of the substituted EPMAH polymer prepared above was tested using two different methods :

- the first, a quantitative method, consisting in placing the polymer into a peptone- supplemented buffered bacterial suspension, with 1,000,000 CFU/ml of Staphylococcus epidermidis strain, and then measuring the number of surviving colonies after incubation at 30°C and 48 hours, compared to a control in which no polymer was present ;

- the second, a qualitative method, in which fragments of polymer were applied onto a plate count agar gel inoculated with the same strain of Staphylococcus epidermidis bacteria. The 90 mm diameter petri dish containing the plate count agar gel was covered with bacterial suspension at 10,000 CFU/ml, then dried for a few minutes. The polymer fragments were then placed on top of the thus formed bacterial film.

Quantitative Test conditions :

- 73 mg of P-Su-EP was added to 29 micro litres of bacterial suspension ;

- 34 mg of Ag-P-Su-EP was added to 13,6 micro litres of bacterial suspension; 1 ml of the bacterial suspension was used as a control;

- the three bacterial suspensions were then incubated for 48 hours at 30°C. At the end of the incubation period, each test suspension was further diluted into a series of 10-fold decreasing dilutions using a tryptone salt solution and then placed on 90 mm diameter Petri dishes. The 10^~5 and 10^"6 dilutions were retained for result analysis, as they presented less than 150 total colonies and thus could be interpreted.

Qualitative Test conditions :

90 mm diameter Petri dishes containing plate count agar culture media were covered with a 10,000 CFU/ml suspension of Staphylococcus epidermidis suspension and then dried for a few minutes. P-Su-EP and Ag-P-Su-EP polymer fragments were placed onto the bacterial film thus formed and the dishes incubated at 30° C for 48 hours.

Quantitative Test Results

Table 3 : Quantitative Test Results of Polymer Antimicrobial Activity

Qualitative Test Results

A small halo of bacterial inhibition was observed around each of the polymer fragments. The Ag-P-Su-EP polymer fragments had a significantly greater halo than their P-Su-EP

counterparts.

As has been demonstrated, the polymer compounds of the present invention showed contact bactericidal activity, that is to say, bacteria present on a surface that is in contact with the polymer compound were oxidised and destroyed within a radius of approximately 10 nanometers. As to cross-reaction of the antimicrobially active agent or degraded fragments thereof, it has further been demonstrated in other tests that only polymer films with a thickness of less than 10 nanometers are likely to be affected in some way by the antimicrobial agent through direct contact with the antimicrobial polymer compounds of the present invention. It is to be noted however, that the antimicrobial agent can be chosen to reduce, minimise or completely avoid this phenomenon. The polymer compounds of the invention do not leach, which is a significant advantage over known solutions of the prior art. A further advantage is that the polymer is active against microbes for far longer than any of the known prior art compounds or products. Most current products are only active for a few weeks, and at most, a few months. The antimicrobially active polymer compounds of the present invention provide solid binding of the antimicrobial agents to the polymer chain, which are not released under operating conditions, and thus provide prolonged antimicrobial activity.

Claims

1) Antimicrobially active polymer compound defined as follows

K-S-A where

- K is a polymer or copolymer of one or more alpha-olefms;

- S is a cyclic dicarboxylic acid anhydride moiety;

- and A is a molecule comprising at least one free NH₂ group, or a substituent with a free amine group, when not bound to S; wherein S is covalently bound to K via one of its uncarboxylated ring carbon atoms, and A is covalently bound to S via an imide bond in replacement of the "oxy" functional group of the cyclic dicarboxylic acid anhydride, and S-A together form a substitution unit having antimicrobial activity.

2) Antimicrobially active polymer compound according to any one of claim 1 , wherein the polymer or copolymer of one or more alpha-olefms has a structure as follows :

- -(CH₂)_n-(CH₂-CR¹R²)_m-

- where :

- R2 is methyl, or alkyl C₂-Ci₀, optionally branched, and/or substituted;

- n is the molar fraction of methylene defined as being from 0 to 1 ;

- m is the molar fraction of the alpha-olefin defined as being from 1 to 0;

- where n+m = 1.

3) Antimicrobially active polymer compound according to claim 1 or claim 2, wherein the polymer or copolymer of one or more alpha-olefms is chosen from the group

1 consisting of polyethylene (PE), polypropylene (PP), their copolymers (EP), and polyisobutylene.

4) Antimicrobially active polymer compound according to any one of claims 1 to 3, wherein the cyclic dicarboxyiic acid anhydride S grafted to the polymer K is maleic acid anhydride (MAH) which, when covalentiy bound to the polymer K, forms a cyclic succinic acid anhyride group, or "Su" moiety

5) Antimicrobially active polymer compound according to any one of claims 1 to 4, wherein the grafted polymer is ethylene-propylene maleic acid anhydride (EPMAH).

6) Antimicrobially active polymer compound according to any one of claims 1 to 5, wherein molecule A, which comprises at least one free NH₂ group, or a substituent with a free amine group, when not bound to S, is selected from the group consisting of 5-amino-l,10-phenanthrolene, aka l,10-phenanthrolin-5-amine; 5-aminoquinoline; 2- aminopyridine; 3-aminopyridine; 4-aminopyridine; 4-amino methylpyridine; 1 -amino anthraquinone; 2-amino-l,3,4-thiadiazole; N,N-dimethyl-l,3-diaminopropane; N,N- dibutyl- 1 ,3-diaminopropane; chlorhexidine; hexamethylenedioxy-4,4'-dibenzamidine- bis-hydroxy-2-ethanesulfonate; imidocarbonimidic diamide; and dequalinium.

7) Antimicrobially active polymer compound according to any one of claims 1 to 6, further containing one or more metal ions known to exert an antimicrobial activity, bound to the polymer by coordination complexation.

8) Antimicrobially active polymer compound according to claim 7, wherein the metal ions are monovalent or bivalent metal ions selected from the group consisting of iron, cobalt, nickel, copper, zinc, ruthenium, rhodium, palladium, silver, cadmium, platinum, gold, mercury, magnesium, and the like.

9) Antimicrobially active polymer compound according to any one of claims 7 or 8, further comprising a complex forming agent bound to both the metal ions and the polymer compound.

10) Antimicrobially active polymer compound according to any one of claim 9, wherein the complexing agent is phenanthrolene.

2 11) Process for the preparation of an antimicrobially active polymer compound comprising the steps of :

- (a) reacting a polymer or copolymer K of one or more alpha-olefms having a graft S consisting of a cyclic dicarboxylic acid anhydride moiety, with a molecule A comprising at least one free NH₂ group or a substituent with a free amine group, to cause the "oxy" functional group of the cyclic dicarboxylic acid anhydride to be replaced by the nitrogen atom of the free amine group and thereby form a covalent imide bond, wherein the imidation reaction takes place in an apolar solvent at a temperature comprised between 140°C and 210°C for a duration comprised between 1 hour and 24 hours;

- (c) filtering and drying the precipitated reaction product.

12) Process according to claim 11 , wherein the process also comprises a dehydration step which takes place prior to step (a) at a temperature greater than 100°C and less than 200°C, and for a period comprised between 1 hour and 24 hours.

13) Process according to claims 12, wherein the dehydration step is carried out at a

temperature of 170°C for a period of 6 hours.

14) Process according to any one of claims 11 to 13, wherein step (a) is carried out in a apolar solvent selected from the group consisting of xylene, dodecane, paraffinic oil, and biphenyl.

15) Process according to any one of claims 11 to 14, wherein the apolar solvent used in step (a) is biphenyl.

16) Process according to any one of claims 11 to 15, wherein the polymer or copolymer of one or more alpha-olefms has a structure as follows :

- -(CH₂)_n-(CH2-CR¹R²)_m-

- where

3 - R¹ is H, methyl, or alkyl C₂-C₁0, optionally branched, and/or substituted;

- R² is methyl, or alkyl C₂-C₁0, optionally branched, and/or substituted;

- m is the molar fraction of alpha-olefm units, defined as being from 1 to 0;

- where n+m = 1.

17) Process according to any one of claims 11 to 16, wherein the polymer or copolymer of one or more alpha-olefms is selected from the group consisting of polyethylene (PE), polypropylene (PP), their copolymers (EP), and polyisobutylene.

18) Process according to any one of claims 11 to 17, wherein the cyclic dicarboxylic acid anhydride is maleic acid anhydride (MAH).

19) Process according to any one of claims 11 to 18, wherein the grafted polymer used in the process of the present invention is ethylene-propylene maleic acid anhydride.

20) Process according to any one of claims 11 to 19, wherein molecule A comprises at least one free NH₂ group or a substituent with a primary amine.

21) Process according to any one of claims 11 to 20, wherein molecule A comprises at least one free NH₂ group or a substituent with a free amine group selected from the group consisting of 5-amino-l,10-phenanthrolene, aka l,10-phenanthrolin-5 -amine; 5- aminoquinoline; 2-aminopyridine; 3-aminopyridine; 4-aminopyridine; 4-amino methylpyridine; 1 -amino anthraquinone; 2-amino-l,3,4-thiadiazole; N,N-dimethyl-

1 ,3-diaminopropane; Ν,Ν-dibutyl- 1 ,3-diaminopropane; chlorhexidine;

hexamethylenedioxy-4,4'-dibenzamidine-bis-hydroxy-2-ethanesulfonate;

imidocarbonimidic diamide; and dequalinium.

22) Process according to any one of claims 11 to 21, wherein step (a) is carried out at a temperature of 190°C for a duration of 12 hours.

23) Process according to any one of claims 11 to 22, further comprising the additional steps of :

4 - (d) reacting the polymer product obtained in step (c) with a metal ion and a complex forming agent, preferably silver nitrate and phthalimide respectively, in the presence of base in toluene at high temperature ;

24) Antimicrobially active article manufactured from, or containing, an antimicrobial polymer compound according to any one of claims 1 to 10 or as obtained by the process of any one of claims 11 to 23.

25) Antimicrobially active article according to claim 24, wherein said article is a film, an extrusion product, or a moulded product.

26) Antimicrobially active article according to any one of claims 24 or 25, wherein said article is an antimicrobially active film, obtained by melting the polymer compounds of the present invention and then extruding or drawing said melted compound into a film.

27) Antimicrobially active article according to any one of claims 24 to 26, wherein said film is free-standing.

28) Antimicrobially active article according to any one of claims 24 to 27, wherein said film is used in association with other polymer films.

29) Antimicrobially active article according to any one of claims 24 or 25, wherein said article is a moulded product obtained by casting or moulding said antimicrobially active polymer compound.

30) Antimicrobially active article according to claim 28, wherein said article is a moulded product obtained by casting or moulding said antimicrobially active polymer compound with one or more other polymers.

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