Composition containing alginate and polyimidazolium salt

Abstract

Described herein is a composition comprising an alginate salt and a polyimidazolium salt, the polyimdazolium salt forms crosslinks in the alginate salt. The composition may be used a composite material, a gel, an aqueous solution, and a sheet. Methods of preparing the composition are described as well.

Description

COMPOSITION CONTAINING ALGINATE AND POLYIMIDAZOLIUM SALTREFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to Singapore patent application number 10202400622P with a filing date of 7 March 2024 and titled “Alginate- polyionene: A Green Slow-release Antimicrobial Composite” and is incorporated herein by reference.FIELD OF THE INVENTION

[0002] The present application relates to a polyimidazolium crosslinked alginate and its use.BACKGROUND OF THE INVENTION

[0003] Alginate is a non-toxic, sustainable, low-cost and biodegradable naturalbased polymer, and has been widely used in the food packaging, biomedical, textile and paper industries. Particularly, alginate is continually used in wound dressing for tissue regeneration and healing owing to its excellent absorption property and ability to load and slow-release of drugs to promote healing.

[0004] Alginic acid (schematic structure shown in FIG. 1 ) is a linear copolymer polysaccharide with homopolymeric blocks of (1 — >4)-linked [3-D-mannuronic acid and (1 — >4)-linked a-L-guluronic acid. Alginic acid may contain sequential and / or alternating blocks of p-D-mannuronic acid and a-L-guluronic acid. Alginate is the salt of alginic acid, typically the sodium salt but potassium and calcium salts may be formed too.

[0005] Alginate is an anionic polysaccharide and its ion-induced gelation is the most distinct property. The general and most common gelation mechanism is through crosslinking of polysaccharide chains with high valence metal cations, such as Ca2+, widely known as the “egg-box” model (schematic structure shown in FIG. 2). Recently, alginate has been modified through various crosslinking strategies to enhance its functionalities for wider applications. For example, alginate-basedhydrogel with antibacterial and magnetic properties was successfully prepared by incorporating ferroferric oxide (FesC ) and silver (Ag) nanoparticles. Alginate-based polyelectrolyte hydrogels were also formed through crosslinking with polycations such as chitosan, gelatin, poly (ethylene imine) etc for various desired properties and applications.

[0006] Polyimidazolium (PIM) salts are polycationic and impart good antimicrobial functionalities against a wide range of micro-organisms.SUMMARY OF THE INVENTION

[0007] In a first aspect there is provided, a composition comprising an alginate salt and a polyimidazolium salt, wherein the polyimdazolium salt forms crosslinks in the alginate salt.

[0008] Preferably, the polyimidazolium salt is of Formula 1 or Formula 2.ormu a ,

[0009] wherein R2and R3if present is each independently a divalent hydrocarbon moiety having 2 to 20 carbon atoms which is optionally substituted with one or more heteroatoms or functional groups, X is an anion, and n is from 5 to 10. In an example, X may be a chloride, a bromide, an iodide, a tosylate, a mesylate, or a triflate. Preferably, X is a chloride, a bromide, or an iodide.

[0010] Preferably, R2and R3if present is each independently selected from the group consisting of a saturated unsubstituted divalent aliphatic moiety, a saturated substituted divalent aliphatic moiety, an unsaturated unsubstituted divalent aliphatic moiety, an unsaturated substituted divalent aliphatic moiety, a unsubstituted divalent aryl moiety, a substituted divalent aryl moiety, a unsubstituted divalent aralkyl moiety, and a substituted divalent aralkyl moiety, the aliphatic moiety having 2 to 10 carbon atoms, the aryl moiety and the aralkyl moiety having 6 to 16 carbon atoms.

[0011] Preferably, R2and R3if present is each independently comprises two of the following groups: an allyl moiety, an alkenyl moiety, a benzyl moiety, a napthylmethyl moiety, and combinations thereof. More preferably, R2and R3if present each comprises two of the same moiety, in other words, two of the same functional groups.

[0012] In an embodiment, R2comprises a diallyl moiety and R3comprises a - dibenzyl moiety. In an embodiment, R2comprises %R, R5and R6are each independently selected from the group consisting of a hydrogen, a methyl, an ethyl, and a propyl, preferably R5and R6are hydrogen. The alkene may be the E or Z isomer or a mixture. In an embodiment, R3comprisesselected from the group consisting of a fluoride, a chloride, a bromide, an iodide, a methyl, an ethyl, a propyl, a CF3, a hydroxyl group, an amine, a nitro group, an ester, an amide, an ether, a ring component and any combinations thereof, m is from 0 to 4, the ring component has are at least two adjacent R4connected to form a saturated or unsaturated ring having at least 2 carbon atoms and 1 or 2 additional atoms selected from carbon, oxygen, nitrogen, and sulfur. The dibenzyl moiety may be atthe 1 ,2, 1 ,3, or 1 ,4 positions. The R4substituent / s if present may be at any suitable position and may be used to tune the properties of the benzyl if needed.

[0013] In an embodiment, R2and R3if present each independently comprises a polyether, a polycarbonate, a polyamide, and combinations thereof.

[0014] In an embodiment, the polyimidazolium salt is of Formula 3,wherein R4is selected from the group consisting of a fluoride, a chloride, a bromide, an iodide, a methyl, an ethyl, a propyl, a CF3, a hydroxyl group, an amine, a nitro group, an ester, an amide, an ether, a ring component and any combinations thereof, m is from 0 to 4, the ring component has are at least two adjacent R4connected to form a saturated or unsaturated ring having at least 2 carbon atoms and 1 or 2 additional atoms selected from carbon, oxygen, nitrogen, and sulfur, and R5and R6are each independently selected from the group consisting of a hydrogen, a methyl, an ethyl, and a propyl, and n is from 5 to 10.

[0015] In an embodiment, the polyimidazolium salt is of Formula 4,formula 4, n is from 5 to 10. In an embodiment, the polyimidazolium salt of Formula 4 may have a number average molecular weight of 2000 to 3000. In another embodiment, it may be the weight average molecular weight.

[0017] In an embodiment, the alginate salt is sodium alginate and has at least one of the following properties: a molecular weight of 100,000 to 250,000 g / mol or a 1 wt% aqueous solution of the sodium alginate has a viscosity of 10 - 30 centipoise,

[0018] In an embodiment, a weight ratio of the alginate salt to the polyimidazolium salt is at least 2:1 or a ratio of cations in the alginate salt to anions in the polyimidazolium salt is at least 1 :0.19. Preferably, a weight ratio of the alginate salt to the polyimidazolium salt is at least 10:1 or a ratio of cations in the alginate salt to anions in the polyimidazolium salt is at least 5:0.19. Preferably, the weight ratio of the alginate salt to the polyimidazolium salt is at most 50:1 or the ratio of cations in the alginate salt to anions in the polyimidazolium salt is at most 25:0.19. The weight ratio of the alginate salt to the polyimidazolium salt may be from 2:1 to 50:1 , preferably from 10:1 to 50:1 .

[0019] In an embodiment, the composition further comprises a benzalkonium salt of Formula 5, BnN(CH3)2(R1), where R1is selected from the group consisting of a C8 alkyl, a C10 alkyl, a C12 alkyl, a C14 alkyl, a C16 alkyl, a C18 alkyl, and combinations thereof. Preferably, a weight ratio of the alginate salt to the benzalkonium salt is at least 2:1 . More preferably, the weight ratio of the alginate salt to the benzalkonium salt is at least 10:1 . Even more preferably, the weight ratio of the alginate salt to the benzalkonium salt is at most 50:1 . The weight ratio of the alginate salt to the benzalkonium salt may be from 2:1 to 50:1 or from 10:1 to 50:1 . Advantageously, the addition of the benzalkonium salt provides the composition with antiviral activity.

[0020] In an embodiment, the composition further comprises a calcium cation, wherein the calcium cation forms crosslinks in the alginate salt. Advantageously, the calcium cation further crosslinks the alginate chains to strengthen it.

[0021] In an embodiment, the composition consists essentially of the alginate salt and the polyimidazolium salt, and optionally the benzalkonium salt of Formula 5 and the calcium cation. In an embodiment, the composition consists of the alginate salt and the polyimidazolium salt, and optionally the benzalkonium salt of Formula 5 and the calcium cation.

[0022] In an embodiment, a method of preparing the composition comprises mixing in water an alginate salt and a polyimidazolium salt to allow the polyimidazolium salt to form crosslinks in the alginate salt; and drying the mixture to form the composition. The alginate salt and polyimidazolium salt are preferably added as solutions. The alginate salt aqueous solution may be prepared from 1 -3 wt% of the alginate salt. The polyimidazolium salt may be prepared from 1 -10 wt% of the polyimidazolium salt.

[0023] Preferably, a 1 -10 wt% of the benzalkonium salt of Formula 5 is mixed with the alginate slat and the polyimidazolium salt.

[0024] Preferably, the method further comprises placing the dried composition in an aqueous solution of Ca2+cations to form a second composition; and drying the second composition. The second composition further contains crosslinks of the alginate chains formed by the Ca2+cations and is further strengthened.

[0025] A composition containing the alginate salt and the polyimidazolium salt may be used to form a gel which may be used as a wound dressing. A composition according to the first aspect may be for use as medicament or in therapy. The composition may be for use as a wound dressing or to heal a wound or to kill bacteria. In an embodiment, use of the composition according to the first aspect in the manufacture of a medicament for the treatment of a wound. The composition may be used in various forms including as a composite material, a gel, and an aqueous solution.

[0026] In a second aspect, there is provided an aqueous solution comprising the composition according to the first aspect.

[0027] In a third aspect, there is provided an article comprising a surface and a surface coated with the composition according to the first aspect.

[0028] In a fourth aspect, there is provided a method of coating a surface of an article, the method comprises coating the surface of the article with the aqueous solution according to the second aspect; and allowing the coating to dry. Preferably, the method further comprises applying a second coating of an aqueous solution of calcium ions and allowing the second coating to dry.

[0029] In a fifth aspect, there is provided a sheet comprising the composition according to the first aspect and a calcium cation, wherein a ratio of the alginate salt to the calcium cation is from 0.9 g of the alginate salt to 0.5 - 5 mmol of the calcium cation and a second weight ratio of the alginate salt to the polyimidazolium salt is at least 2:0.1 .

[0030] Preferably, the second weight ratio of the alginate salt to the polyimidazolium salt is at least 2:0.1 -5. More preferably, the second weight ratio of the alginate salt to the polyimidazolium salt is 2:0.1 -3. The sheet containing alginate and the polyimidazolium salt has antimicrobial properties and is useful as a disinfectant.

[0031] In an embodiment, the sheet further comprises a benzalkonium salt of Formula 5, BnN(CH3)2(R1), where R1is selected from the group consisting of a C8 alkyl, a C10 alkyl, a C12 alkyl, a C14 alkyl, a C16 alkyl, a C18 alkyl, and combinations thereof. Preferably, a third weight ratio of the alginate salt to the benzalkonium salt of Formula 5 is at least 2:0.1 . Advantageously, the addition of the benzalkonium salt provides the sheet with antiviral properties as well improving the effectiveness of the sheet as a disinfectant.

[0032] In an embodiment, the sheet consisting essentially of the composition and the calcium cation, and optionally the benzalkonium salt of Formula 5. In an embodiment, the sheet consists of the composition and the calcium cation, and optionally the benzalkonium salt of Formula 5.

[0033] Advantageously, the products with the alginate salt and the polyimidazolium salt provides an antimicrobial material which may be used in different forms including as a surface coating, a wound dressing material, and as a film material. By the addition of a benzalkonium salt, antiviral property may be added as well making the material an effective material at killing both bacteria and virus providing time and cost savings.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure (FIG.) 1 shows a schematic structure of alginate.

[0035] FIG. 2 shows a schematic structure of Ca2+ crosslinked alginate.

[0036] FIG. 3 shows a schematic structure of polyimidazolium crosslinked alginate with an example of the polyimidazolium salt.

[0037] FIG. 4 panel (a) shows alginate-PIM solutions coated on glass slides before and after rinsing. FIG. 4 panel (b) shows alginate-PIM solutions of various ratios.

[0038] FIG. 5 shows a graph of the wound healing study with alginate-PIM gel.

[0039] FIG. 6 shows the antimicrobial activity of Alg-1 % PIM sheets (0.5 cm) against a) E. coli (30s exposure) and b) S. aureus (24 h exposure) using 100-fold dilution media. ‘Represents no colony was observed at 102dilution.

[0040] FIG. 7 shows a graph of the evaluation of the control release of PIM from different alginate-PIM sheets.

[0041] FIG. 8 shows the antimicrobial activity of Alg-1% PIM sheets (0.5 cm) after 10 cycles of washing using 100-fold dilution media with the bars from left to right being for 0 hour, control and Alg-1 % PIM respectively. ‘Represents no colony was observed at 102dilution.

[0042] FIG. 9 shows the regeneration of Alg-0.1% PIM sheets of various thickness in PIM solutions.

[0043] FIG. 10 shows a general synthetic scheme to prepare the compounds of Formulas 1 to 4.DETAILED DESCRIPTION OF THE INVENTION

[0044] In the following description, numerous specific details are set forth in order to provide a thorough understanding of various illustrative embodiments of the invention. It will be understood, however, to one skilled in the art, that embodiments of the invention may be practiced without some or all of these specific details. Embodiments described in the context of one of the methods or devices are analogously valid for the other methods or devices. Similarly, embodiments described in the context of a method are analogously valid for a device, and vice versa.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs.

[0046] The phrase “at least one of A and B” means it requires only A alone, B alone, or A and B, i.e. only one of A or B is required.

[0047] As used herein, the articles “a”, “an” and “the” as used with regard to a feature or element include a reference to one or more of the features or elements. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. As used herein, the terms “first,” “second," and “third,” etc. are used merely as labels, and are not intended to impose numerical requirements on their objects. As used herein, the terms “top”, “bottom”, “left”, “right”, “side”, “vertical” and “horizontal” are used to describe relative arrangements of the elements and features. As used herein, the term “each other" denotes a reciprocal relation between two or more objects, depending on the number of objects involved.

[0048] Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the invention. Where a value being discussed has inherent limits, for example where a component can be present at a concentration of from 0 to 100%, or where the pH of an aqueous solution can range from 1 to 14, those inherent limits are specifically disclosed. Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the invention, as are ranges based thereon.

[0049] Although each of these terms has a distinct meaning, the terms “comprising", “consisting of” and “consisting essentially of" may be interchanged for one another throughout the instant application. The term “having” has the same meaning as “comprising” and may be replaced with either the term “consisting ofor “consisting essentially of’. The expression "consisting essentially of" means allows the presence of other components in addition to the components mandatory in the claim, provided that the essential characteristics of the claimed composition were not materially affected by their presence.

[0050] The term “aliphatic group” or “aliphatic” refers to a moiety that may be saturated (e.g. single bond) or contain one or more units of unsaturation, e.g., double and / or triple bonds, and in particular refers to the carbon atom forming the bond. An aliphatic group may be straight chained, branched or cyclic, contain carbon, hydrogen or, optionally, one or more heteroatoms and may be substituted or unsubstituted. Non-limiting examples of substituents include a halogen, a hydroxyl, an ether, an amine, a carbamate, a carbonate ester, a urea, an aryl group (e.g. benzyl, phenyl ethyl and the like), a carbonyl, a carboxylic acid, an ester, an amide, a cyano, a nitro, a thiol, a sulfoxide, and a sulfone. It is understood that the substituent may be further substituted.

[0051] The term "alkyl" as used herein is a branched or unbranched saturated monovalent hydrocarbon radical of 1 to 24 carbon atoms, such as methyl, ethyl, n- propyl, isopropyl, nbutyl, isobutyl, s-butyl, f-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A "lower alkyl" group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms. A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0052] The term “aryl group” refers to a monovalent aromatic radical which includes carbocyclic aromatic rings and heteroaryl rings (nitrogen, oxygen, and sulphur and the like), and in particular refers to the atom forming the bond being part of the ring structure. The term “aromatic group” may be used interchangeablywith the terms “aryl”, “aryl ring” “aromatic ring”, “aryl group” and “aromatic group”. The aryl group may be substituted at any one or more substitutable ring atom. Nonlimiting examples of substituents include a halogen, a hydroxyl, an ether, an amine, a carbamate, a carbonate ester, a urea, an aliphatic group (e.g. tolyl, mesityl) an aryl group (e.g. biphenyl), a carbonyl, a carboxylic acid, an ester, an amide, a cyano, a nitro, a thiol, a sulfoxide, and a sulfone. It is understood that the substituent may be further substituted.

[0053] The term “aralkyl" or “arylalkyl” refers to a monovalent alkyl radical substituted with one or more aryl groups. In certain embodiments, the aralkyl has from 7 to 30 (C7-30), from 7 to 20 (C7-20), or from 7 to 16 (C7-16) carbon atoms. Examples of aralkyl groups include, but are not limited to, benzyl, 2-phenylethyl, and 3-phenylpropyl. In certain embodiments, aralkyl are optionally substituted with one or more substituents.

[0054] The term “substituted" shall mean the replacement of one or more hydrogen atoms in a given structure with a substituent including, but not limited to, halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, or aliphatic. It is understood that the substituent may be further substituted.

[0055] There are currently no known studies on alginate-PIM crosslinked films for antimicrobial applications. Described herein is the use of polyimidazolium salts as a dual functional polycationic crosslinker which imparts antimicrobial property to alginate materials for various applications. A schematic structure of the alginate- PIM compound is shown in FIG. 3.

[0056] Any suitable PIM may be used to crosslink the alginate. In an embodiment, the PIM has a general structure of Formula 1 or Formula 2.o u a

[0057] In Formulas 1 and 2, R2and R3if present is each independently a divalent hydrocarbon moiety having 2 to 20 carbon atoms which is optionally substituted with one or more heteroatoms or functional groups, X is an anion, and n is from 50 to 100. R2and R3may be aliphatic, aromatic, or a combination of both. R2and R3may each independently be saturated, unsaturated, substituted, un substituted, or in any combinations thereof. In an embodiment, each of R2and R3may each independently be aliphatic and contain 2 to 10 carbon atoms. In an embodiment, each of R2and R3may each independently be aromatic, or a mixture of aromatic and aliphatic, and may contain 6 to 16 carbon atoms. In Formulas 1 , 2 and 3, X may be a chloride, a bromide, an iodide, a tosylate, a mesylate, or a triflate. Preferably,X is a chloride, a bromide, or an iodide.

[0058] In an embodiment, each of R2and R3may independently contain at least two of the following: an allyl moietyan alkenyl moietycombinations thereof. These moieties are divalent to allow it to join to the other parts of the PIM. The moieties may be substituted or unsubstituted and may be bonded in any suitable position of the moiety. These functional groups may allow for the construction of the polyimdazolinium structure, for example to polymerise the monomers. In an embodiment, R2has a (divalent) diallyl moiety and R3has a (divalent) dibenzyl moiety

[0059] In an embodiment, R2and R3if present may each independently contain a polyether, a polycarbonate, a polyamide, and combinations thereof.

[0060] In an example, the PIM salt is of Formula 3.Formula 3.

[0061] The methylene imidazole may be attached to benzene ring at any position, for example, 1 ,2-disubstituted, 1 ,3-disubstituted or 1 ,4-disubstituted. The phenyl ring may be optionally further substituted with 1 to 4 substituents (R4) selected from a fluoride, a chloride, a bromide, an iodide, a methyl, an ethyl, a propyl, a CFs, a hydroxyl group, an amine, a nitro group, an ester, an amide, an ether, a ring component and any combinations thereof. The ring component may be made up of at least two adjacent R4 substituents connected to form a saturated or unsaturated ring having at least 2 carbon atoms and 1 or 2 additional atoms selected from carbon, oxygen, nitrogen, and sulfur. R5and R6are each independently selected from the group consisting of a hydrogen, a methyl, an ethyl, and a propyl. Thealkene may be the E or Z isomer or a mixture depending on the method of preparation.

[0062] Materials and methods

[0063] Preparation of alginate-PIM-BZK solution for coating

[0064] Separate stock solutions of 1 wt% alginate (Alginic acid sodium salt, molecular weight 120,000-190,000 g / moL, 15-25 centipoise (cps), 1 wt% in distilled water, purchased from Sigma-Aldrich), 1 wt% PIM and 1 wt% benzalkonium chloride (BZK) in water were prepared accordingly. The alginate salt may be purchased commercially or prepared in-situ. 1 to 3 wt% of the alginate may be used. The stock solutions of the PIM and BZK is preferably at least 1 wt% for more durable activity.

[0065] An example of the PIM used herein is the compound of Formula 4 shown below where n is from 5 to 10 and a number average molecular weight of 2000 to 3000. In another embodiment, the molecular weight may be the weight average molecular weight. Both embodiments of the molecular weight allow the PIM salt to form the crosslinks in the alginate and possess antibacterial property.The compounds of Formulas 1 to 4 may be prepared by any suitable synthesis methods. For example, the compounds of Formulas 1 to 4 may be prepared by following a similar procedure as that described in the literature (Liu et al., Biomaterials, 2013, 34, 1018, and Liu et al., Biomaterials, 2012, 33, 8625) or as outlined in the synthetic scheme in FIG. 10. The leaving group in FIG. 10 may be any typical leaving group in organic chemistry, common examples include chloride, bromide, iodide, tosylate, mesylate and triflate. In an example, the compound of Formula 4 may be prepared by the reaction of 1 ,2-bis((1H-imidazole-1-yl)methyl)benzene with the appropriate amount of (2E)-1 ,4-dibromobut-2-ene by heating in DMF as shown in FIG. 10 to produce the PIM salt. Similar methods may be used to produce the compounds of Formulas 1 to 3. The compound of Formula 3 may be produced on other methods depending on the polymerization technique used.

[0066] BZK is alkyldimethylbenzylammonium chloride where the alkyl is a mixture of even numbered C8 to C18 alkyl chains (PhCH2N(CH3)2(R1), where R1is selected from the group consisting of a 08 alkyl, a C10 alkyl, a C12 alkyl, a C14 alkyl, a 016 alkyl, a C18 alkyl, and combinations thereof). PhCFh- is also commonly known as benzyl (Bn). The average molecular weight of the BZK used herein is 395. The BZK used herein is purchased from Sigma-Aldrich which is approximately 70% benzyldimethyldodecylammonium chloride (R1is a C12 alkyl) and 30% benzyldimethyltetradecylammonium chloride (R1is a C14 alkyl) as measured by HPLC. Any BZK with antimicrobial activity may be used.

[0067] In a 2 mL tube, mixtures of alginate-PIM-BZK were prepared in specific ratios discussed herein. The unit molecular weight for alginate, PIM and BZK is about 176, 454 and 395 respectively.

[0068] 0.6 mL of the mixture was dropping cast onto a 2.5 cm x 2.5 cm glass slide, dried in an oven overnight, rinsed with water for 10 seconds thrice and dried in the oven. The coated glass slides were soaked in 1 wt% CaCl2 solution for 10 mins and left to dry in oven. The post treatment with calcium chloride further solidifies the coating by forming additional cross-links across the alginate chains. The calcium chloride is not added in the initial mixing step as it may precipitate the alginate.

[0069] Surface contact killing assay Antimicrobial property was determined using the Japanese Industrial Standard (JIS Z 2801 : 2010) protocol (Japanese Industrial Standard JIS Z 2801. Test for antibacterial activity and efficacy. www.jsa.or.jD. Japanese Standards Association. Tokyo. Japan. 2010). Briefly, bacteria (E. coli or S. aureus) were suspended in 5 mL of Tryptic Soy Broth (TSB) nutrient broth and adjusted to ODeoo = 0.07, corresponding to 3 x 108CFU / mL. The solution was further diluted 100 times to 106CFU / ml. 100 pL of cell suspensionswere placed on the surface of the samples for 30 sec. The respective cell suspensions were collected with 9.9 ml_ of TSB and plated on LB agar plates, following appropriate dilutions to compute for colony forming units per mL (CFU / mL) using standard plate counts techniques. All experiments were carried out in three replicates.

[0070] Antiviral testing against MHV coronavirus

[0071] Surfaces were tested for their antiviral activity according to ISO 27102 (ISO-21702:2019. Measurement of antiviral activity on plastics and other non- porous surfaces, 2019.). Stock concentration of MHV was pre-determined to be 107PFU / mL, and 100 pL aliquot was introduced onto the square surfaces measuring 2.5 cm x 2.5 cm. The droplet was immediately covered with a plastic polyethylene sheet measuring 2 cm x 2 cm, and the samples were incubated at room temperature for a pre-determined duration. Experiments were carried out in triplicate, and on at least two independent occasions. Viable viral particles were recovered by the addition of 5 mL of DMEM, and determined via standard plaque assays, with NCTC 1469 as host cells.

[0072] Wound healing method: Rats (Wistar, male / female, 7-8 weeks) were selected as models in animal experiments. The infected wound model was made by two main steps: i) removal of a full thickness of skin (0 0.8 cm) on the part of rat dorsal thoracic with the biopsy punch; ii) introduction of 100 pL of SA suspension (1 x 108CFU / mL) and incubation for 1 day. After that, 100 pL gel was added to treat the infected wound topically. A commercial antibacterial plaster with BZK (bought from Watsons) was used for comparison of the effectiveness of wound healing. After 6 days, the treated wounds were cut off and fixed with 10% formalin solution for hematoxylin and eosin (H&E) staining and histological analysis.

[0073] Preparation of alginate-PIM sheet PIM was synthesized using the method described above. CaSO4.2H2O (0.135 g, 0.784 mmol,) was suspended in water (15 mL) and poured onto a weighing boat. 3 wt% of alginate in water (0.9 g, 30 mL) was mixed with CaSC>4 slurry. The gelated mixture was then transferred onto an 8 cm x 8 cm square petri dish to form a 0.5 cm pre-gel, kept in a fridge overnight and placed in a dry box for several days until a thin, dry film was formed. The amountof calcium sulphate may be used from 0.5 mmol to 0.8 mmol. The dried pre-gel was soaked in 20 ml_ of 100 mM CaCIs (control) or x % of PIM solution (samples) overnight. The solutions were removed and subsequently soaked in 20 mL of 200 mM CaCk overnight to form additional crosslinks in the alginate chains and strengthen the alginate-PIM sheet. Finally, the film was rinsed thoroughly with water and left to dry at room temperature. The final alginate-PIM film is crosslinked with a ratio of approximately 0.9g of alginate to 0.5-4.8 mmol of Ca2+.

[0074] PIM-release profile of alginate-PIM sheet The PIM-release profile in Phosphate-buffered saline (PBS) was measured based on the literature method. The concentration of released PIM was determined using spectrophotometric method. UV spectrum scan was carried out for PIM in solution. Maximum absorption (A max) at 258 nm was observed for the material and subsequently used for the semi-quantitative assay. Briefly, the alginate-PIM sheet (100 mg) was placed in 1 mL of phosphate-buffered saline (PBS) in a 6-well plate and incubated at 300 rpm at room temperature for 1 hour. The material was sequentially and periodically transferred to a fresh well containing 1 mL PBS. The resultant PBS solution containing the released PIM was subsequently used for semi-quantitative assay. The total amount of PIM in alginate-PIM sheet was pre-determined.

[0075] Cell Viability Assay The cell cytotoxicity assessment was conducted using an L929 mouse fibroblast cell viability assay according to ISO 10993-5 method (Direct Contact). The L929 mouse fibroblast cells (NCTC clone 929; CCL- 1 ) were cultured in complete DMEM media (containing 10% (v / v) heat-inactivated fetal bovine serum (FBS), 100 U / mL penicillin and 100 pg / mL streptomycin) at 37 °C in 5% CO2 incubator. L929 fibroblasts were grown in a 96 well plate at 10,000 cell / well (100 pl) in complete media for 24h. The test materials (5 mg and 10 mg) were then added to the cells and incubated for another 24 h. Following the 24h incubation / treatment, materials were removed, and media was replaced with MTT reagent (in complete DMEM media) and incubated for 2h at 37aC in 5%CO2 incubator. The formed formazan crystals were dissolved in 100 ul of DMSO at room temperature for 3 h (in dark). The absorbance was read at 570 nm with reference to 620 nm. The percentage of cell viability was calculated as the ratio of theabsorbance of treated cells to the absorbance of the control groups (untreated). All experiments were performed in triplicate in three independent experiments.

[0076] Mechanical property measurement Tensile stress-strain experiments were carried out to characterize the mechanical strength of the silicone material. The mechanical property of the samples was evaluated on 3340 Series Single Column Table Frames (INSTRON 3344 universal testing machine (Instron, Norwood, Massachusetts). The 25 mmx 10 mm dimension PDMS material was clamped to expose 0.14 mm mid-section of the material. The test samples were mounted on two mechanical grips with one grip attached to the cross head where the load cell is mounted, and the other grip attached to the fixed end. The initial length of the sample was recorded, and the device subsequently stretched the samples vertically at a constant speed of 10mm / min. Data was collected and processed using the respective software. All tests were conducted at room temperature and at least in triplicates for each tested material. Relationship between the stress and strain, load (N), ultimate tensile strength (MPa), elongation (mm) and modulus (MPa) were determined at end point at maximum elongation.

[0077] Discussion

[0078] Alginate-PIM solution for coating

[0079] To obtain a durable coating on the glass surface, different alginate to PIM ratios (Alg:PIM) were mixed and their durability observed. For 1 :2 and 1 :1 Alg:PIM weight ratios, the coatings were hygroscopic and easily rinsed off with water. With 2:1 Alg:PIM weight ratio, the coating was much more durable (FIG. 4 panel a). Coating solutions of these three ratios were also centrifuged, rinsed, and dried in oven. Only the solution with 2:1 Alg:PIM weight ratio showed that a continuous film was formed (FIG. 4 panel b). Hence, the ratio of alginate must be at least 2 times that of PIM for a durable film to form. The unit molecular weight for alginate, PIM and BZK is about 176, 454 and 395 respectively. Hence, a Alg:PIM weight ratio of 2:1 represents the anion / cation ratio of approximately 1 :0.19. Higher ratio of PIM may result in non-homogeneous crosslinking and a hygroscopic coating layer.

[0080] After further optimization, a coating solution with a Alg:PIM:BZK weight ratio of 5:0.5:0.5 was prepared and coated onto a 2.5 cm x 2.5 cm glass slide. Thecoating was further strengthened by soaking in 1 wt% CaCh solution for 10 minutes. An equal amount of PIM and BZK without Alg has also been coated for comparison. Both the surfaces of Alg:PIM:BZK and PIM:BZK coated glass slides were wiped manually with wet wipes 100 times and antimicrobial tests were carried out to determine their efficacy against E. coli (Gram-negative bacteria), S. aureus (Grampositive bacteria), C. albicans (fungi) and mouse hepatitis virus (MHV). The alginate-PIM solution alone have antimicrobial properties, however the addition of BZK provides additional antivirus activity and makes the coating solution more widely applicable.

[0081] Table 1 shows that Alg:PIM:BZK coated glass slides retained its efficacy with complete killing against all the tested microbes, while the PIM:BZK coated glass slides had lost its efficacy with zero killing. This showed that the polycationic crosslinking of PIM and BZK with alginate improved the durability of the coating tremendously.

[0082] Table 1. Log reduction against microbes on Alg:PIM:BZK and PIMzBZK coated glass slides.Log reduction (CFU / ml)Coating Solution 0 wipesRati0S. C. MHVE. coli aureus albicansAlg-PIM-BZK +CaCI27.83 7.14 6.49 5.925 : 0.5 : 0.5Log reduction (CFU / ml)Coating Solution 100x wipesRatio s QE. coli MHV aureus albicansAlg-PIM-BZK +CaCI27.53 7.14 6.49 6.455 : 0.5 : 0.5PIM-BZK0.01 0 0 01 : 1

[0083] Alginate-PIM solution for wound healing

[0084] The in vivo application of the alginate-PIM gel to treat bacterial infection was tested on rats using the S. aureus infected wound model. Hemolysis study of the active ingredient, PIM, was proven to be safe with minimal hemolysis of ~1% even at a concentration of 5000 pg / mL. Wounds on rat skin were created and infected with S. aureus. PIM solution (P1 , 80 pg / ml in H2O) and alginate-PIM solution (AP1 , 80 pg / ml in 1 wt% Alg / HsO) were prepared and 100 pl of P1 and AP1 were applied onto the wound topically. Commercial antibacterial plaster with BZK as the active ingredient was used for comparison. Both the control and commercial plaster showed a much slower healing rate compared to AP1 and P1 (FIG. 5). AP1 and P1 showed similar healing rates, suggesting alginate is compatible with PIM and does not alter the efficacy of PIM. The alginate offers a thicker and more viscous texture that may be preferred under and more suitable for topical applications. The alginate-PIM gel is generally antibacterial and BZK may be added in the preparation of the gel to provide antiviral property as well.

[0085] Alginate-PIM Sheet

[0086] Alginate films are generally soft and have relatively low mechanical strength, thus limiting their applications. A densely interconnected alginate hydrogel through a reconstruction method via drying and subsequent rehydration with ionic crosslinking has been previously reported. An alginate-PIM sheet based on the reported method with slight modifications was achieved. After obtaining the pre-gel by mixing CaSC and alginate, it was placed in the dry box for anisotropic drying until a thin, dry film was formed. The dried alginate pre-gel was crosslinked with 0.1 - 2 wt% of PIM solutions and subsequently soaked in CaCh solution for furtherstrengthening. Different pre-gel thickness (0.1 , 0.3 and 0.5 cm) was also fabricated. Antimicrobial tests against E. coli were done on these alginate-PIM sheets and the results are shown in Table 2.

[0087] Table 2. Log reduction against E. coli on various Alg-PIM sheets under different nutrient broth concentrations.Log reduction (CFU / ml)Nutrient (TSB) wt% PIM _Concentration soakPre’9el ,hickness«™)0.1 cm 0.3 cm 0.5 cmPure TSB 0.1 9.65 9.09 9.900.5 9.65 9.09 9.901.0 9.76 9.09 9.902.0 3.86 9.09 9.90100x TSB 0.1 7.26 7.65 8.480.5 7.26 7.65 8.481.0 7.26 7.65 8.482.0 7.26 7.65 8.48

[0088] The surface contact killing assay based on JIS Z 2801 (Japanese Industrial Standard JIS Z 2801. Test for antibacterial activity and efficacy. www.jsa.or.jp. Japanese Standards Association. Tokyo. Japan. 2010.) employs a standard 24 h exposure time and 500-fold dilution media respectively. In this section, both non-diluted media and 100-fold dilution media to support more vigorous bacteria growth than real-world applications were used. Under these harsh conditions, the alginate-PIM sheets achieved complete killing against E. coli for all the various wt% PIM and pre-gel thickness, even under non-diluted media condition. Using 0.5 cm thick pre-gel with 1 wt% PIM soak for further testing (Alg-1 % PIM), it shows a complete killing (> 6 log reduction) against E. coli under a short exposure time of 30s (FIG. 6 panel a). The alginate-PIM sheet also has > 8 log reduction against S. aureus (FIG. 6 panel b). To test against MHV, the alginate sheets weresoaked in a mixture of 1% PIM and 0.1 % BZK solutions overnight, followed by subsequent soaking with CaCh solution, rinsed and dried before testing. The resulting sheet has complete killing (> 5 log reduction) against MHV, even after a short exposure time of 30 mins.

[0089] The release of PIM from alginate-PIM sheet (0.5 cm) in PBS was observed over 5 hours, indicating a sustained release of antimicrobial compound (FIG. 7). Alginate-PIM (0.5 cm) sheets were pre-treated with 0.1 %, 0.5%, 1%, and 2% (wt. %) of PIM solutions. The total amount absorbed into the alginate-PIM sheet (weight increment) is 21%, 23%, 26%, and 6%, respectively. The low absorption amount of alginate sheet in high concentration PIM solution is because high concentration of PIM quickly crosslinks the surface layer of alginate sheet and prevents the further absorption of PIM into the inner layer. The semi-quantitative assay further showed an initial burst of PIM component in the first hour followed by a gradual and sustained release (FIG. 7). A cumulative amount of 5.7 - 47.9 weight % of PIM component was released from the various alginate-PIM materials in 5 hours.

[0090] Recycle and regeneration of alginate-PIM Sheet

[0091] To determine the recyclability of these alginate-PIM sheets, the sheets were soaked and stirred in water for 15 minutes and the cycle repeated for 10 times. For 0.5 cm thick pre-gel with 1 wt% PIM soak, there is complete killing (> 8 log reduction) against E. coli and >99.9% killing (>3 log reduction) against S. aureus (FIG. 8).

[0092] Due to the absorbing capability of the alginate sheets, the antimicrobial component may be regenerated after it has been eventually lost as shown in FIG. 9. Table 3 shows that the alginate-PIM sheets (Alg-0.1 wt% PIM) with a lower PIM concentration lose their antimicrobial activity after one round of recycling with log reduction close to 0 (B). However, the sheets were recovered and re-soaked in 0.1% PIM solution. The sheets were then soaked in water overnight, rinsed thoroughly with water and dried at room temperature. The regenerated sheets (R) can achieve complete killing against E. coli again. The alginate-PIM sheets with different PIM concentrations may have uses in different situations and offers an effective way to tune its properties based on the PIM concentration.

[0093] Table 3. Log reduction against E. coli on Alg-0.1% PIM sheets before (B) and after regeneration (R).Pre-gel thickness (cm)% PIM soak0.1 cm 0.3 cm 0.5 cm0.1% 0.00 (B) 0.49 (B) 0.77 (B)7.62 (R) 7.62 (R) 7.62 (R)

[0094] Cell viability assay of alginate-PIM sheet

[0095] The cytotoxicity assay of the active ingredient PIM had been done and reported previously (Liu et al., Biomaterials, 2013, 34, 1018) to be safe and nontoxic. The safety of alginate-PIM has also been evaluated here using mouse fibroblast L929 cells. Table 4 shows the alginate-PIM material has no cytotoxicity effect on mouse fibroblast cells and is biocompatible.

[0096] Table 4. Cytotoxicity evaluation using mouse fibroblast L929 cells.Cell Viability (%)Alginate Alginate:PIMSheet 5 mg 85.3 84.710 mg 70.3 94.124 h extract undiluted 144.8 79.22x diluted 92.2 108.45x diluted 87.8 87.9

[0097] Mechanical properties of alginate-PIM sheet

[0098] The mechanical strength and properties of the material have been evaluated. Tensile strength / stress testing experiments indicate that the mechanical properties of the alginate-PIM polycationic crosslinked sheet are comparable to the alginate-Ca2+crosslinked product (Table 5).

[0099] Table 5. Tensile strength and modulus of alginate-Ca2+and alginate-1 % PIM sheetsModulus Maximum Tensile stress atMaterials (Automatic Force Maximum ForceYoung’s) (MPa) (N) (MPa)Alginate-Ca2+872.82 ± 122.44 26.54 ± 10.55 18.96 ± 7.54(control)Alginate-1 % PIM 753.76 ± 57.96 24.5 ± 5.61 17.50 ± 4.01

[0100] In summary, an alginate-polyionene composite material has been developed where poly-cationic polyimidazolium (PIM) salts were mixed with alginate solution or absorbed into alginate sheets. The multifunctional PIM component acts as a crosslinker to form alginate structure and as antimicrobial agent which can be released slowly from the composite material. The alginate-PIM materials have demonstrated their application as an antimicrobial coating material, wound dressing material and as a film material with durable antimicrobial activity. The antimicrobial coating material may be used to coat a surface of an article to kill bacteria and virus on the surface of the article and to ensure it remains free of bacteria and virus.

Claims

CLAIMS

1. A composition comprising an alginate salt and a polyimidazolium salt, wherein the polyimdazolium salt forms crosslinks in the alginate salt.

2. The composition according to claim 1 , wherein the polyimidazolium salt is of Formula 1 or Formula 2,ormu a , wherein R2and R3if present is each independently a divalent hydrocarbon moiety having 2 to 20 carbon atoms which is optionally substituted with one or more heteroatoms or functional groups, X is an anion, and n is from 5 to 10.

3. The composition according to claim 2, wherein R2and R3if present is each independently selected from the group consisting of a saturated unsubstituted divalent aliphatic moiety, a saturated substituted divalent aliphatic moiety, an unsaturated unsubstituted divalent aliphatic moiety, an unsaturated substituted divalent aliphatic moiety, a unsubstituted divalent aryl moiety, a substituted divalent aryl moiety, a unsubstituted divalent aralkyl moiety, and a substituted divalent aralkyl moiety, thealiphatic moiety having 2 to 10 carbon atoms, the aryl moiety and the aralkyl moiety having 6 to 16 carbon atoms.

4. The composition according to claim 3, wherein R2and R3if present each independently comprises two of the following groups: an allyl moiety, an alkenyl moiety, a benzyl moiety, a napthylmethyl moiety, and combinations thereof.

5. The composition according to claim 4, wherein R2comprises a diallyl moiety and R3comprises a dibenzyl moiety.

6. The composition according to any one of claims 3 to 5, wherein R2and R3if present each independently comprises a polyether, a polycarbonate, a polyamide, and combinations thereof.

7. The composition according to any one of claims 2 to 6, wherein the polyimidazolium salt is of Formula 3,wherein R4is selected from the group consisting of a fluoride, a chloride, a bromide, an iodide, a methyl, an ethyl, a propyl, a CFs, a hydroxyl group, an amine, a nitro group, an ester, an amide, an ether, a ring component and any combinations thereof, m is from 0 to 4, the ring component has are at least two adjacent R4connected to form a saturated or unsaturated ring having at least 2 carbon atoms and 1 or 2 additional atoms selected from carbon, oxygen, nitrogen, and sulfur, andR5and R6are each independently selected from the group consisting of a hydrogen, a methyl, an ethyl, and a propyl.

8. The composition according to claim 7, wherein the polyimidazolium salt is of Formula 4,Formula 4, preferably the polyimidazolium salt of Formula 4 has a number average molecular weight from 2000 to 3000.

9. The composition according any one of claims 1 to 8, wherein the alginate salt is sodium alginate and has at least one of the following properties: a molecular weight of 100,000 to 250,000 g / mol or a 1 wt% aqueous solution of the sodium alginate has a viscosity of 10 - 30 centipoise.

10. The composition according to any one of claims 1 to 9, wherein a weight ratio of the alginate salt to the polyimidazolium salt is at least 2:1 or a ratio of cations in the alginate salt to anions in the polyimidazolium salt is at least 1 :0.19.

11. The composition according to claim 10, wherein a weight ratio of the alginate salt to the polyimidazolium salt is at least 10:1 or a ratio of cations in the alginate salt to anions in the polyimidazolium salt is at least 5:0.19.

12. The composition according to claim 10 or claim 11 wherein the weight ratio of the alginate salt to the polyimidazolium salt is at most 50:1 or theratio of cations in the alginate salt to anions in the polyimidazolium salt is at most 25:0.19.

13. The composition according to any one of claims 1 to 12, comprising a benzalkonium salt of Formula 5, BnN(CH3)2(R1), where R1is selected from the group consisting of a C8 alkyl, a C10 alkyl, a C12 alkyl, a C14 alkyl, a C16 alkyl, a C18 alkyl, and combinations thereof.

14. The composition according to claim 13, wherein a weight ratio of the alginate salt to the benzalkonium salt is at least 2:1 , preferably the weight ratio of the alginate salt to the benzalkonium salt is at least 10:1 , more preferably the weight ratio of the alginate salt to the benzalkonium salt is at most 50:1 .

15. The composition according to any one of claims 1 to 14 comprising a calcium cation, wherein the calcium cation forms crosslinks in the alginate salt.

16. The composition according to any one of claims 1 to 15 consisting essentially of the alginate salt and the polyimidazolium salt, and optionally the benzalkonium salt of Formula 5 and the calcium cation.

17. An aqueous solution comprising the composition according to any one of claims 1 to 14.

18. An article comprising a surface and a surface coated with the composition according to any one of claims 1 to 16.

19. A method of coating a surface of an article, the method comprises coating the surface of the article with the aqueous solution according to claim 17; and allowing the coating to dry, preferably the method further comprisesapplying a second coating of an aqueous solution of calcium ions and allowing the second coating to dry.

20. A sheet comprising the composition according to any one of claims 1 to 9 and a calcium cation, wherein a ratio of the alginate salt to the calcium cation is from 0.9 g of the alginate salt to 0.5 - 5 mmol of the calcium cation and a second weight ratio of the alginate salt to the polyimidazolium salt is at least 2:0.1 .

21. The sheet according to claim 20, wherein the second weight ratio of the alginate salt to the polyimidazolium salt is 2:0.1 -5, preferably the second weight ratio of the alginate salt to the polyimidazolium salt is 2:0.1 -3.

22. The sheet according to claim 20 or claim 21 , comprising a benzalkonium salt of Formula 5, BnN(CHs)2(R1), where R1is selected from the group consisting of a C8 alkyl, a C10 alkyl, a C12 alkyl, a C14 alkyl, a C16 alkyl, a C18 alkyl, and combinations thereof.

23. The sheet according to claim 22, wherein a third weight ratio of the alginate salt to the benzalkonium salt of Formula 5 is at least 2:0.1 .

24. The sheet according to any one of claims 20 to 23 consisting essentially of the composition and the calcium cation, and optionally the benzalkonium salt of Formula 5.

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