WO2017099668A1

WO2017099668A1 - Functional cationic derivatives of saccharides as antimicrobial agents

Info

Publication number: WO2017099668A1
Application number: PCT/SG2016/050593
Authority: WO
Inventors: Yi Yan Yang; Shrinivas Venkataraman; Ashlynn L. Lee
Original assignee: Agency For Science, Technology And Research
Priority date: 2015-12-07
Filing date: 2016-12-07
Publication date: 2017-06-15

Abstract

A method for synthesizing a derivative of a saccharide such as starch, substituted with quaternary ammonium cations, forming a cationic polysaccharide. The method comprises subjecting a saccharide to a mixture of an optionally substituted halo-epoxide such as epichlorohydrin and one or more optionally substituted amines. The method may be prepared in a one-pot synthesis via the formation of a glycidyl derivative of a tertiary amine as an in situ intermediate. The starch polymers formed are biodegradable, have antimicrobial properties and can be further formed into antimicrobial hydrogels by photo-crosslinking of the starch derivative.

Description

Functional cationic derivatives of saccharides as antimicrobial agents

Technical Field

The present invention generally relates to a method of making saccharide derivatives substituted with quaternary ammonium cations by chemical reaction in the presence of a mixture of an optionally substituted halo-epoxide and one or more optionally substituted amines. The reaction products and hydrogels made therefrom are biodegradable materials that possess favorable antimicrobial properties.

Background Art

Cationic polysaccharides are an important class of materials, used in diverse applications in paper, textile, food, cosmetics and petroleum industries as key additives. One of the major advantages of cationic polymers is their antimicrobial character. These materials can be synthesized with tailored cationic charge and amphiphilic balance to impart broad antimicrobial activity. It has been demonstrated that most of the cationic polymers work against the microbes by binding to their cell membranes, causing membrane-lysis. Owing to the membrane-lytic nature of the mechanism, there will be less chance of drug resistance development. With the emergence of "super bugs" with resistance against multiple drugs, development of antimicrobial polymers from inexpensive starting materials such as polysaccharides will have broad societal and environmental impact.

Depletion of petroleum based resources and growing environmental concerns have motivated scientists to develop eco-friendly sustainable materials. Biopolymers obtained from renewable sources such as plants offer a great platform for developing sustainable materials. Starch is one of the readily available and abundant biopolymers, which has been used as a platform to develop new materials for numerous industrial applications. From chemistry perspective, starch is complex biopolymer comprised of anhydroglucose units (AGU) with both linear and branched linkages. In a typical starch sample, depending upon the actual molecular weight and the branching pattern, there are two different types of starch molecules present: amy lose (linear) and amylopectin (branched). Although the relative percentage of amylose and amylopectin varies depending upon the actual starch bio-source, typically they consist of around 20 to 25% amylose and 75 to 80% amylopectin by weight.

Despite of all the recent efforts to utilize polysaccharides for the making of antimicrobial materials there is still a need to find a way to make them using facile reactions to form crosslinking or late-stage derivatization. Further there is a need to achieve a right balance of charge and amphiphilicity for use in various applications. A new method ideally would therefore provide an easy way to change these features. Embedded reactive groups should facilitate the rapid development of formulations in different forms such as solution, gel and surface coating by completely eliminating the need to painstakingly optimize and characterize the polymer composition to suit different applications.

There is still a need for such approach which will be particularly time and effort saving for the development of antimicrobials with broad spectrum of action as multiple parameters (amphiphilicity, cationic charge and charge density, etc.) require optimization to achieve high potency and selectivity (for instance between mammalian cells and microbes). Furthermore there is still an increasing need for designing and developing antimicrobial materials for surfaces that aimed at offering effective antibacterial capabilities, while avoiding the use of disinfectants that pose potential risks of residual toxicity, environmental contamination, and promotion of bacterial resistance.

Summary of Invention

According a first aspect of the invention a method for making a saccharide derivative substituted with a quaternary ammonium cation, the method comprising subjecting a saccharide to a mixture of an optionally substituted halo-epoxide and one or more optionally substituted amines.

Advantageously, this method uses the actual microstructure of the polysaccharide molecules and their relatively high molar mass dispersity. The hydroxyl groups present in these molecules serve as a platform to append functionalities. According to the invention the hydroxyl functional groups are used to append cationic charges via the reaction with via an in situ intermediate formed from an epoxy derivative of tertiary amine. The method allows appending functionalities that have the right balance of charge and amphiphilicity. It further allows for facile reactions to form crosslinking or late-stage derivatization. The later can be achieved by the introduction of specific functional groups, such as for instance alkenes. The simple a and easy process from reaction with a mixture and in-situ existants of intermediates provides means of time- and effort saving for the development of antimicrobials with broad spectrum of action as multiple parameters (amphiphilicity, cationic charge and charge density, etc.) can be influenced. The method is especially well- sited for such developments as it can be run as one pot method without prior isolation of intermediates to react with the saccharide.

Advantageously saccharide derivative substituted with quaternary ammonium cations made by the method show enhanced potency to inhibit microbes with selectivity for mammal cells.

An optional alternative to the main invention is provided wherein a mixture of an optionally substituted halo-epoxide and two or more different optionally substituted amines are used. Using several amines provides an even better ability to balance charge and amphiphilicity. This allows to tailor-make saccharides with a high potency to inhibit microbes combined with a good selectivity to avoid adverse effects to mammalian cells.

In another embodiment of the invention one or more optionally substituted amines are selected from the group consisting of optionally substituted tertiary amines. Preferably at least one tertiary amine is selected wherein 2 of the 3 substituents of the tertiary amine are selected from Ci_6 alkyl and the third substituent is a C₂-6 alkenyl group. Advantageously embedding such reactive alkene groups will facilitate the rapid development of formulations in different forms such as solutions, (hydro)gels and surface coatings. Such solutions, (hydro)gels and surface coatings may be formed by crosslinking.

According to a second aspect of the invention there is provided a saccharide derivative substituted with quaternary ammonium cation groups which can be obtained according to the method of the invention and which is itself a part of the invention. The saccharide derivative substituted with quaternary ammonium cation groups is a saccharide that comprises hydroxy groups as a saccharide functionality which in part are substituted by one or more optionally substituted quaternary ammonium cations linked via a crosslinker and the wherein degree of substitution of the hydroxy groups of the saccharide is about 20% to 80.

Advantageously such saccharide derivatives show a high potency in inhibiting microbes and are still selective with regard to action on mammal cells.

According to an optional embodiment the saccharide derivative is a substituted soluble starch wherein the ammonium cation groups are selected from quaternary N,N-dimethyl-arylalkyl ammonium and Ν,Ν-dimethyl-allyl ammonium groups in a molar ratio of 90 to 10 to about 60 to 40. Advantageously, such substituted starch is a biodegradable antimicrobial material with improved characteristics with regard to amphiphilicity, cation charge and charge density.

According to a third aspect of the invention the use of a saccharide derivative made according to the inventive process or a saccharide derivative according to the second aspect of the invention for inhibiting microbes is provided.

According to a fourth aspect of the invention, there is provided a method for synthesizing a hydrogel comprising the steps of mixing a saccharide derivative made according to the method of the invention with a photoinitiator in a solvent and exposing the mixture to UV light. Advantageously hydrogels made with the inventive method show excellent antimicrobial action.

According to one embodiment a soluble starch wherein the ammonium cation groups are selected from quaternary Ν,Ν-dimethyl-benzyl ammonium and N,N-dimethyl-allyl ammonium groups in a molar ratio of 90 to 10 to about 60 to 40 is used as the starting material. Advantageously the hydrogels obtained with the method of the invention are able to eradicate S. aureus, E. coli, P. aeruginosa, and C. albicans effectively upon contact and show high antimicrobial activity.

A further aspect of the invention therefore relates to the use of the saccharide derivatives and hydrogels made or provided according to the invention as antimicrobials in consumer care products or food packagings, as antimicrobial agents, viscosity modifiers, surface modifiers or interfacial stabilizers. In these applications the combination of biodegradability, material strength and antimicrobial activity can be advantageously used.

Definitions

The following words and terms used herein shall have the meaning indicated: Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The term "antibacterial" refers to a capability of a material to destroy bacteria or suppresses their growth or their ability to reproduce.

The term "antimicrobial" refers to a capability of a material to inhibit or destroy microorganism or microbes or suppresses their growth or their ability to reproduce.

The term "microbe" refers to a microscopic living organism, which may be single-celled or multicellular. Microbes are very diverse and include all bacteria, archaea and most protozoa.

The term "amphophilic" refers to a capability of a molecule having both hydrophilic and hydrophobic parts.

The term "non-hemolytic" refers to a feature of a molecule/polymer to not cause the rupturing (lysis) of red blood cells (erythrocytes) and the release of their contents (cytoplasm) into surrounding fluid (e.g. blood plasma in either vivo or in vitro applications). Non-hemolytic behaviour is considered important for an antimicrobial entity to be compatible with mammalian cell usage (selectivity).

The term "cationic" refers to molecule that comprises an ion or group of ions having a positive charge and characteristically moving toward the negative electrode in electrolysis. In the context of the instant invention the cationic group may specifically relate to a protonated ammonium group or a quaternary ammonium group in some embodiments.

As used herein, the term "alkyl" includes within its meaning monovalent ("alkyl") and divalent ("alkylene") straight chain or branched chain saturated aliphatic groups having from 1 to 6 carbon atoms, e.g., 1, 2, 3, 4, 5 or 6 carbon atoms. For example, the term alkyl includes, but is not limited to, methyl, ethyl, 1 -propyl, isopropyl, 1 -butyl, 2-butyl, isobutyl, tert -butyl, amyl, 1 ,2-dimethylpropyl, 1,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1- methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2- dimethylbutyl, 1,3-dimethylbutyl, 1 ,2,2-trimethylpropyl, 1 , 1 ,2-trimethylpropyl and the like. Alkyl groups may be optionally substituted.

The term "aryl", or variants such as "aromatic group" or "arylene" as used herein refers to monovalent ("aryl") and divalent ("arylene") single, polynuclear, conjugated and fused residues of aromatic hydrocarbons having from 6 to 10 carbon atoms. Such groups include, for example, phenyl, biphenyl, naphthyl, phenanthrenyl, and the like. Aryl groups may be optionally substituted.

The term "optionally substituted" as used herein means the group to which this term refers may be unsubstituted, or may be substituted with one or more groups other than hydrogen provided that the indicated atom's normal valency is not exceeded, and that the substitution results in a stable compound. Such groups may be, for example, halogen, hydroxy, oxo, cyano, nitro, alkyl, alkoxy, haloalkyl, haloalkoxy, arylalkoxy, alkylthio, hydroxyalkyl, alkoxyalkyl, cycloalkyl, cycloalkylalkoxy, alkanoyl, alkoxycarbonyl, alkylsulfonyl, alkylsulfonyloxy, alkylsulfonylalkyl, arylsulfonyl, aryisulfonyloxy, arylsulfonylalkyl, alkylsulfonamido, aikylamido, alkyl sulfonamidoalkyl , alkylamidoalkyl, arylsulfonamido, arylcarboxamido, arylsulfonamidoalkyl, arylcarboxamidoaJkyl, aroyl, aroyl-4-alkyl, arylalkanoyl, acyl, aryl, arylalkyl, alkylaminoalkyl, a group R¾^yN-, R^xOCO(CH₂)_m, R^xCON(R^y)(CH₂)_ffi, R^xR^yNCO(CH₂)_m, R^xR^yNS0₂(CH₂)_m or R^xSQ₂NR^y(CH₂)_rjl (where each of R^x and R^y is independently selected from hydrogen or alkyl, or where appropriate R"R^y forms part of carbocylic or heterocyclic ring and m is 0, 1 , 2, 3 or 4), a group R^xR^yN(CH₂)_P~ or R^xR^yN(CH₂)_pO- (wherein p is 1 , 2, 3 or 4); wherein when the substituent is R^xR^yN(CH₂)_P~ or R^xR^y (CH₂)_pO, R^x with at least one CH₂ of the (CH₂)_P portion of the group may also form a carbocyciyl or heterocyclyl group and R^y may be hydrogen, alkyl. In these substituents all aikyi and aryl groups etc. are of the type defined above.

Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Certain embodiments may also be described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Detailed Disclosure of Embodiments

Non-limiting embodiments of the invention will be further described in greater detail by reference to specific examples, which should not be construed as in any way limiting the scope of the invention.

According to a first aspect, there is provided a method for synthesizing a saccharide derivative substituted with a quaternary ammonium cation, the method comprising subjecting a saccharide to a mixture of an optionally substituted halo-epoxide and one or more optionally substituted amines. The saccharide, the optionally substituted halo-epoxide and the one or more optionally substituted amines form a reaction mixture to produce the saccharide derivative substituted with a quaternary ammonium cation.

In one embodiment, the saccharide is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides and polysaccharides. The saccharide may consist of a plurality of monomer units. Polysaccharides may be preferred. The saccharide may be selected from saccharides which are substantially insoluble or soluble in water. Preferable the saccharide is soluble in water. Additionally or alternatively, it is selected from sugars, starch and cellulose. Soluble starch as polysaccharide may be especially mentioned. Preferably the saccharide is a starch. The starch may be a mixture selected from amylose and amylopectin, optionally in a relative percentage of about 10% amylose and 90% amylopectin to about 40% amylose and 60% amylopectin, or about 20 to 25% amylose to about 75% to 80% amylopectin. The starch may be a naturally occurring potato starch. About 20 % amylose and about 80 % amylopectin may be specifically mentioned. The molecular weight of the starch can be varied in large ranges and many native starches are suitable to be used in the invention. Typical weight-average molecular weights may be in the area of about 0.5 to 1.1 x 10⁶ for amylose and about 65 to 400 x 10⁶ for amylopectin. Starches of a weight-average molecular weight of about 0.1 to 900 x 10⁶ may be mentioned as suitable weights to be used in the method.

The optionally substituted halo-epoxide may be selected from an epoxy moiety having an aliphatic backbone, wherein one terminal unit of the aliphatic backbone is an epoxide and the other terminal unit is a halide. Epoxy alkyl halides with 3 to 7 carbon atoms may be mentioned. The halide may be selected from chloride, bromide or iodide. The aliphatic backbone may have 1 to 5 carbon atoms. It may have 1 to 3 carbon atoms. The halo-epoxide may be epichlorohydrin or other glycidyl halides, such as l-bromo-2,3-epoxypropane.

In yet another embodiment, the one or more optionally substituted amines are selected from the group consisting of optionally substituted tertiary amines. Preferred may be tertiary amines wherein 2 of the 3 substituents are Ci_₆ alkyl, i.e. methyl, ethyl, propyl, butyl, pentyl or hexyl. The remaining third substituent may be saturated or unsaturated.

The saturated substituent may be selected from an aliphatic C₂-io alkyl group, i.e. from ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl. In particular, it may be selected from an aliphatic C₄_₈ alkyl group. Alternatively, it may be selected from a cyclic C₃_i₀ alkyl group, i.e. from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclononyl or cyclodecyl, e.g. it may be cyclohexyl. Alternatively, it may be selected from a cyclic C₃_i₀ alkyl group bonded to a Ci_₃ alkyl group, i.e. from cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, cyclononylmethyl or cyclodecyl methyl, e.g. it may be cyclohexylmethyl .

The unsaturated substituent may be selected from an optionally substituted C₂-6 alkenyl or C₃_₆ alkynyl, such as ethylene, propylene, butylene, pentylene, hexylene, propargyl, butynyl, pentynyl or hexynyl. Preferably it is an optionally substituted C₃_₆ alkenyl. The optionally substituted amine may be selected from a dialkylalkenylamine. Accordingly, the dialkylalkenylamine may be Ν,Ν-dimethylallyl amine. Alternatively, it may comprise an aromatic moiety selected from aryl or arylalkyl, such as for instance phenyl, benzyl, naphthyl or naphthylmethyl. The optionally substituted amine may be selected from a dialkylbenzylamine. Accordingly, the dialkylbenzylamine may be N,N- dimethylbenzylamine .

In one embodiment at least one tertiary amine is selected wherein 2 of the 3 substituents of the tertiary amine are selected from Ci_₆ alkyl and the third substituent is a C₂-e alkenyl group or a C₃_6 alkynyl group. The third substituent may be a C₃_₆ alkenyl group. This tertiary amine can introduce alkenyl or alkynyl groups into the reaction product. Tertiary allyl amines may be preferred.

In another embodiment at least one tertiary amine is selected wherein 2 of the 3 substituents of the tertiary amine are selected from Ci_₆ alkyl and the third substituent is an aryl or arylalkyl group. This tertiary amine can introduce aryl moieties into the reaction product to influence the hydrophobicity of the reaction product. Benzyl amines may be preferred.

In one embodiment at least two different amines are used in the reaction mixtures. The optionally substitutes amines can e.g. be selected from two different amines which are preferably tertiary amines. The mixing ratio of the amines may be in a molar ratio from about 10 to 90 to about 50 to 50, or about 15 to 85 to about 40 to 60, or about 20 to 80 to about 30 to 70. It may be about 20 to 80 or about 50 to 50. Such mixtures may be those of an N,N- dimethylarylalkylamine and Ν,Ν-dimethylalkylene amine, such as e.g. N,N- dimethylbenzylamine and Ν,Ν-dimethylallylamine. For these the following mixing ratios can be mentioned about 5 : 95, about 10 : 90, about 15:95, about 20:80, about 25:85, about 30 to 70, about 35:65, about 40 to 60 and about 45 to 55.

The method wherein the one or more optionally substituted amines comprise a N,N- dimethylallylamine and N,N-dimethylarylalkylamine in a molar ratio of about 90 to 10 to about 60 to 40 may be specifically mentioned.

The method according to the invention can be performed as a one pot method. In such one -pot synthesis is characterized by a chemical reaction whereby all reactants are subjected to successive chemical reactions in just one reactor. Intermediates are reacted in-situ. In the reaction according to the invention any formation of an epoxy derivative of the amine (such as e.g. a glycidyl derivative of a tertiary amine) can be mentioned as intermediate that is reacted in- situ. In one embodiment, the reaction product of the halo-epoxide functions as a cross-linker between the saccharide and the optionally substituted amines in the final reaction product.

In one embodiment the mixture of the optionally substituted halo-epoxide and one or more optionally substituted amines comprises a solvent, optionally a polar solvent. The preferred solvent may be water or water in admixture with other water miscible solvents (aqueous medium). The saccharide is then reacted in an aqueous medium with the epoxide and the amine(s). In a preferred method the saccharide is first dissolved in water and then the reactants are added simultaneously. Alternatively, the epoxide is added first and then the amine(s) are added. In such embodiment the saccharide, preferably a native starch, is first dissolved in water or an aqueous medium at elevated temperatures. Typical temperatures may be about 60 to 95 °C. The reaction may then be started by heating to a temperature higher than 50°C, or higher than 80°C, or to about 90°C. Then the reaction is finalized at elevated temperatures of about 30 to 60 °C for about 10 to 48 hours and optionally under nitrogen protection. The reaction product can be purified by dialysis with a molecular weight cut off (MWCO) (e.g. with an MCWO of 2,000 to 5,000). Lyophilisation can be used to obtain the final product of the reaction in pure form.

The halo-epoxide and the amine(s) may be used in about equimolar amounts to each other or, preferably, the amine(s) are used in slight excess. In one embodiment the amine(s) are used in about 1 : 1.01 to about 1 : 1.2 molar excess compared to the halo-epoxide.

According to another embodiment 1 g of starch is reacted with about 0.01 to 0.5 mol of optionally substituted halo-epoxide and about 0.01 to 0.5 mol of one or more optionally substituted amines. Preferred molar amounts of the halo-epoxide and/or amines used that can be mentioned are about 0.03 to 0.3 mol, about 0.04 to 0.1 mol.

In yet another embodiment, the molar mixing ratio of the saccharide (as anhydroglucose unit of saccharide) to the optionally substituted halo-epoxide and the sum of one or more optionally substituted amines may be in the range of about 1:5-15:5-15, or about 1 :8-12:8-12, or about 1 : 10: 11. The molar mixing ratio may be calculated on basis of one monomer or anhydroglucose unit within the saccharide. Other mixing ratios that can be mentioned include 1 :3-18:3-18, 1 : 1- 50: 1-60 or 1 : 1-20: 1-20.

In another embodiment, there may not be any additional base, such as an inorganic base or an additional nitrogen base present in the reaction mixture. The amine(s) may be the only base used in the method according to the invention.

The saccharide derivative substituted with quaternary ammonium cations made by the method according to the invention is also a part of the instant invention. In an optional embodiment of the second aspect, the degree of substitution of the hydroxy groups of the saccharide is about 20% to 80%, or about 29% to about 74%.

According to the second aspect of the invention there is provided a saccharide derivative substituted with quaternary ammonium cation groups, which is a saccharide that comprises hydroxy groups as a saccharide functionality which in part are substituted by one or more optionally substituted quaternary ammonium cations linked via a cross-linker and the wherein degree of substitution of the hydroxy groups of the saccharide is about 20% to 80%, or about 29% to about 74%.

Accordingly there is provided a saccharide derivative comprising saccharide functionality, a cross-linker and one or more optionally substituted quaternary ammonium cations. The saccharide functionality may comprise hydroxy groups that may be partially substituted with via the cross-linker.

In one embodiment, the saccharide derivative is selected from the group consisting of modified monosaccharides, disaccharides, oligosaccharides and polysaccharides. The saccharide derivative may consist of a plurality of monomer units. Modified polysaccharides may be preferred. The saccharide derivative may be selected from substituted saccharides which are substantially insoluble or soluble in water. Preferable the unsubstituted saccharide is soluble in water. Additionally or alternatively, the saccharide derivative is selected from substituted sugars, starch and cellulose. Modified soluble starch as polysaccharide derivative may be especially mentioned. Advantageously the substituted saccharide is based on starch. The starch may be a mixture selected from amylose and amylopectin, optionally in a relative percentage of about 10% amylose and 90% amylopectin to about 40% amylose and 60% amylopectin, or about 20 to 25% amylose to about 75% to 80% amylopectin. The starch may be a naturally occurring potato starch. About 20 % amylose and about 80 % amylopectin may be specifically mentioned. The molecular weight of the starch can be varied in large ranges and many native starches are suitable to be used in the invention. Typical weight-average molecular weights may be in the area of about 0.5 to 1.1 x 10⁶ for amylose and about 65 to 400 x 10⁶ for amylopectin. Starches of a weight- average molecular weight of about 0.1 to 900 x 10⁶ may be mentioned as suitable weights to be used in the method.

The cross-linker may be present between the saccharide functionality and the one or more quaternary ammonium cations. The cross-linker may be a hydroxy-aliphatic moiety. The cross- linker may be a hydroxy-C₃_₈ alkyl group, i.e. propanolyl, butanolyl, pentanolyl, hexanolyl, heptanolyl or octanolyl. The alkyl chain of the hydroxy-C_{3 8} alkyl group may be branched or linear. The hydroxy group may be a primary or a secondary alcohol. The alkyl chain of the hydroxy-C₃_g alkyl group may be covalently bond to the saccharide via an ether-bond. The crosslinker may be linked via an ether link to the saccharide hydroxyl functionality. The quaternary ammonium cation may be bound to one end of the alipaphatic part and the ether link to the other end (see also Figure 2). The cross-linking group is preferably a hydroxy-substituted C_{3 7} alkyl moiety. The crosslinking unit may therefore represent a C₃_₇ alkyl linker which is substituted by hydroxyl. Preferably the linker is a -CH₂-CH(OH)-CH₂- or a - CH(CH₂(OH))-CH₂- group. This linker may be obtained from the reaction of a glycidyl group with the hydroxy groups of the saccharide.

The one or more optionally substituted quaternary ammonium cations may have 4 substituents, one of which may be covalently bound to the cross-linker. 2 of the 3 remaining substituents may be Ci-6 alkyl, i.e. methyl, ethyl, propyl, butyl, pentyl or hexyl. The remaining substituent may be saturated or unsaturated. The saturated substituent may be selected from an aliphatic C₂-io alkyl group, i.e. from ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl. In particular, it may be selected from an aliphatic C₄_₈ alkyl group. Alternatively, it may be selected from a cyclic C₃_io alkyl group, i.e. from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclononyl or cyclodecyl, e.g. it may be cyclohexyl. Alternatively, it may be selected from a cyclic C₃_io alkyl group bonded to a C_{i 3} alkyl group, i.e. from cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, cyclononylmethyl or cyclodecyl methyl, e.g. it may be cyclohexylmethyl.

The unsaturated substituent may be selected from an optionally substituted C_{2 6} alkenyl or C_{3 6} alkynyl, such as ethylene, propylene, butylene, pentylene, hexylene, propargyl, butynyl, pentynyl or hexynyl. Preferably it is an optionally substituted C_{3 6} alkenyl. The one or more optionally substituted quaternary cation may be selected from a dialkylalkenylammonium cation. Accordingly, the dialkylalkenylammonium cation may be N, N-dimethylallyl ammonium.

Alternatively, the substituent may comprise an aromatic moiety selected from aryl or arylalkyl, such as for instance phenyl, benzyl, naphthyl or naphthylmethyl. The one or more optionally substituted quaternary ammonium cation may be selected from a dialkylbenzylamino cation. Accordingly, the dialkylbenzylamine cation may be N, N-dimethylbenzylammonium. In one embodiment at least one quarternary ammonioum cation is present in the saccharide derivative wherein 2 of the 4 substituents of the ammonium are selected from Ci_₆ alkyl and a third substituent is a C₂-6 alkenyl or C_{3 6} alkynyl group. Preferably it is a C_{3 6} alkenyl group. This cationic group carries alkenyl groups of the saccharide derivative. Quarternary dialkylallyl ammonium groups as quarternary ammonioum cations are specifically mentioned.

In one embodiment at least one quarternary ammonium cation is present in the saccharide derivative wherein 2 of the 4 substituents of the ammonium are selected from C ₆ alkyl and the third substituent is an aryl or arylalkyl group. This cationic group introduces aryl moieties into the saccharide derivative to influence the hydrophobicity of the reaction product. Benzyl amines may be preferred.

The counter anion of the quaternary ammonium groups may be selected from halogen anions, such as chloride or bromide.

In some embodiments, the one or more optionally substituted quaternary ammonium cations comprise two different quaternary ammonium cations. The ratio of the two ammonium groups may be in a molar ratio from about 10 to 90 to about 50 to 50 or about 15 to 85 to about 40 to 60, or about 20 to 80 to about 30 to 70. It may be about 12 to 88 or about 45 to 55. Such mixtures may be those of an N,N-dimethylarylalkylammonium and N,N-dimethylalkylene ammonium, such as e.g. N,N-dimethylbenzylammonium and N,N-dimethylallylammonium. Other ratios that can be mentioned include about 5 to 95, 8 to 92, 15 to 85, 25 to 75 and 30 to70.

The saccharide derivative of the second aspect of the invention may be made according to the method provided in this invention. All embodiments and preferred conditions of the method then also relate to the obtainable product of the method.

The saccharide derivatives according to the invention show no self-assembly behavior; specifically they have a CAC value which is at least above 50 mg/L.

According to a third aspect of the invention there is provided the use of a saccharide derivative made according to the inventive process or provided according to the second aspect of the invention for inhibiting microbes. The inhibition of the microbes can be described as an antimicrobial activity of the saccharide derivatives. The antimicrobial activity is especially strong against bacteria and yeast. The bacteria may be selected from gram negative and gram positive bacteria. The microbes may be selected from bacteria and funghi.

In a particular embodiment, the saccharide derivative substituted with quaternary ammonium cations has inhibitory activity against S. aureus, E. coli, P. aeruginosa and C. albicans. The saccharide derivatives according to the invention show a broad antimicrobial spectrum and are non-hemolytic.

The antimicrobial use can be achieved by simple mixing of the saccharide derivatives with a medium of the microbes or by exposing the microbes to a surface coated with a film of the saccharide derivative.

According to a fourth aspect of the invention, there is provided a method for making a hydrogel comprising the steps of mixing a saccharide derivative made according to the method of the invention or a saccharide derivative of the second aspect of the invention with a photoinitiator in a solvent and exposing the mixture to UV light. The saccharide derivative can be any saccharide derivative as described above and is used as starting material for the photo-initialized hydrogel forming. According to one embodiment a soluble starch wherein the ammonium cation groups are selected from quaternary Ν,Ν-dimethyl-benzyl ammonium and N,N-dimethyl-allyl ammonium groups in a molar ratio of 90 to 10 to about 60 to 40 is used as the starting material.

The photoinitiator may be any photo-initiator selected from free-radical initiators such as, methyl benzoylformate, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone or mixtures thereof. In one embodiment, the photoinitiator may be 2-hydroxy-4'-(2-hydroxyethoxy)-2 methylpropiophenone. The light which activates the photoinitiator (typically ultraviolet light) may have a wavelength in the range of about 320 to 500 nm. The photo-initiator concentration in the reaction solution is typically about 0.1 to 5 wt. %, preferably about 0.3 to 0.7 wt. % of the whole solution. The photoinitiator is preferably added after adding the other components and the solvent.

In another embodiment, the solvent may be selected from polar solvents. The solvent may be water or a mixture of water with water miscible solvents (aqueous medium).

In another embodiment of the fourth aspect, the method may further comprise a thiol-ene photopolymerization step. A PEG hydrogel is formed after adding a PEG additive to the mixture. The PEG additive may be a linker between the saccharide derivatives. It may form a hydrogel network structure. The PEG additive is preferably a multiarm PEG additive. 4-arm PEG thiols may be especially mentioned. This PEG additive may be of the formula (I)

wherein n is selected so that the molecular weight is in the range of about 5,00 to 20,000. The number of ethylene oxide units in the PEG chain may not be equal for all arms. The total molecular weight for the multi-arm PEG is the sum of the PEG molecular weights of each arm. The additive may be a 4-arm PEG(lOk) thiol. The PEG additive may be added in a concentration of about 1 to 10 wt. % or about 4 to 8 wt. %, preferably about 3 to 5 wt. % of the solution in the solvent. The overall concentration of saccharide derivative used in the method is between about 5 and 45 wt. %, or about 7 and 30 wt. %, preferably about 10 and 25 wt. % of the total solution in the solvent.

The multiarm PEG additive may also be replaced by other small molecule multi-arm thiols according to further embodiments of the invention. Such other thiols include, but are not limited to pentaerythritol tetrakis(3-mercaptopropionate), pentaerythritol tetrakis(2-mercaptoacetate), trimethylolpropane tris(3-mercaptopropionate), trimethylolpropane tris(3-mercaptopropionate), and tris[2-(3-mercaptopropionyloxy)ethyl] isocyanurate

The hydrogel may have a hydrogel network structure formed during the photo reaction of the fourth aspect of the invention, especially when a PEG additive is being used. The hydrogel can be formed as a film on a surface using the method of the invention. The hydrogels made with the inventive method show excellent antimicrobial action comparable to that of the saccharide derivatives according to the first and second aspect of the invention. Hydrogels made according to the method of the fourth aspect of the invention and their use for inhibiting microbes is another aspect of the invention. The hydrogels obtained with this method of the invention are able to eradicate S. aureus, E. coli, P. aeruginosa, and C. albicans effectively upon contact and therefore show high antimicrobial activity. The hydrogels can therefore be used to coat wound care materials or other consumer products that require anti-microbial surfaces. The hydrogels show a excellent adherence on fibres, such as cellulose fibres.

The antimicrobial use can be achieved by contacting a medium of the microbes with the hydrogels or by exposing the microbes to a surface coated with a film of the hydrogels.

A further aspect of the invention relates to the use of the saccharide derivatives and hydrogels made according to the invention as antimicrobials in consumer care products or food packagings, as antimicrobial agents, viscosity modifiers, surface modifiers or interfacial stabilizers. In these applications the combination of biodegradability, material strength and antimicrobial activity can be used.

Consumer care products include, but are not limited to healthcare products, such as wound care products.

Examples

Non-limiting examples of the invention and a comparative example will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

Materials

Soluble starch was purchased from Sigma Aldrich (# 33615, lot # SZBE0520V) with potato as the biological source of the starch. Unless, specifically mentioned, all other reagents were purchased from Sigma-Aldrich or TCI and all other solvents were of analytical grade, purchased from Fisher Scientific or J. T. Baker and used as received.

Examples, analytical methods and microbial activity

Example 1: General procedure for the synthesis of starch derivatives (Figure 2)

Representative example - Starch- Allyl-Bn (20-80)

In a scintillation vial (20 mL) with magnetic stir bar, soluble starch (1.0 g) was dispersed in deionized (DI) water (10 mL) and heated at 90 °C for 15 min with magnetic stirring on. With the heat treatment, the starch solution became translucent solution and was transferred to a round bottom flask (100 mL). The vial was rinsed with additional DI H₂0 (10 mL), and added to the flask. To this starch solution (0.05 g/mL), epichlorohydrin (4.84 mL), N,N- dimethylbenzylamine (8.00 mL) and N,N-dimethylallylamine (1.62 mL) were added. The reaction mixture was allowed to stir under nitrogen, maintained at 40 °C for 24 hours, followed by purification by dialysis (MWCO - 3500 Da) against 2 L of 0.1 Ν HC1 (one time), and 2 L of DI H₂0 (four times). The aqueous solution was lyophilized to result in white solid. Isolated yield (average of three independent synthesis runs): ~ 2.2 ± 0.3 g; degree of substitution (DS, average of three independent synthesis runs): ~ 0.26 ± 0.02.

The modification of starch was accordingly conducted in aqueous condition by using a mixture of epichlorohydrin and functional tertiary amine (acting both as the base and also as the reagent) via the formation of glycidyl derivative of tertiary amine as an in situ intermediate (see Figure 2; and Nichifor, M.; Stanciu, M. C; Simionescu, B. C. Carbohydrate Polymers 2010, 82, 965). For all the experiments (even with the use of two different of tertiary amines), the following feed ratio was used: anhydroglucose unit (monomer unit; 162.15 g/mol) : epichlorohydrin : tertiary amine = 1 : 10 : 11. The reaction was allowed to proceed at 40 °C for 24 h, followed by which the polymer was purified by extensive dialysis to get rid of the small molecule impurities. Upon lyophilisation, the final product was obtained as white powder.

Nuclear magnetic resonance (NMR) spectroscopy

The 'Pi- spectra of starch polymers were recorded at 65 °C with OMSO-d₆ (l g ampoules from Cambridge Isotope Laboratories, Inc.) as the solvent, using a Bruker Avance 400 spectrometer, and operated at 400 MHz with the solvent proton signal as the internal reference standard. Samples for NMR was prepared by first heating ~ 4 mg of starch or starch derivatives in 1 g DMSO- 6, followed by heating the mixture at 65 °C for 1 h, followed by allowing the solution to cool back to room temperature naturally. Trifluoroacetic acid- (Sigma Aldrich, 20 μί.) was added directly into the NMR tube just before the measurement (see Tizzotti, M. J.; Sweedman, M. C; Tang, D.; Schaefer, C; Gilbert, R. G. Journal of Agricultural and Food Chemistry 2011, 59, 6913).

NMR of starch derivatives were conducted by using DMSO-ife as the solvent. In order to tackle the self-associative interactions of the starch backbone, primarily arising from hydrogen- bonding interactions, all NMR spectra were acquired at 65 °C. Starch-based materials have tendency to absorb water. Extensive drying of the polymeric sample and also the use of dry DMSO-ife (ampoule) did not eliminate the peak associated with water at 3.3 ppm. So, addition of deutrated trifluoroacetic acid (TFA-cf) was necessary to obtain spectra that could be used for quantifying the degree of substitution (DS, the average number of substituted units per glucose monomer). Gilbert et al., have demonstrated that the addition of small quantities of TFA as a straightforward procedure to characterize starch derivatives. By this approach all the exchangeable protons and water can be shifted to higher frequency, thereby enabling quantification of DS. In the ¾ NMR experiments, ~ 4 mg of modified starch, 1 g of dry DMSO-ife (ampoule) and 20 μL· of TFA-d were typically used. To avoid any potential complication arising from the presence of organic acid, TFA was added just prior to the NMR experiment, minimizing the exposure of polymer to acidic environment for only limited time.

NMR spectrum was found to better resolve with the addition of TFA and with the given ratio of polymer to acid, it was possible to consistently shift all the water and other exchangeable protons to > 10 ppm (Figure 1).

For the starch polymers modified with tertiary amine and epichlorohydrin, the NMR spectra were found to have all the relevant spectral features, expected for that particular amine (SI). The degree of substitution values were found to be in the range of 0.29 - 0.74 for the series of modified cationic starched modified with one tertiary amine (entries 1-5 in Table 1). Such variation in DS values with respect to the changes in the actual choice of tertiary amine has been observed before. This might be partly due to the factors including the changes in the solvent quality, the addition of different amines, causing differences in the conformation of starch chains. Also, since the tertiary amine used to actually functionalize is also acting as the base, the inherent differences in the relative basicity of the amines might also be contributing to the observed differences in DS. These tertiary amines were chosen based on their ability to introduce simultaneously both charge and hydrophobicity to the polymer.

For antimicrobial polymers tailoring of properties it was found that the properties can be tuned according to the method of the invention by addressing the cationic/hydrophobic group, necessitating use of two different components to tune. According to the invention polymers with different ratios of Ν,Ν-dimethylallylamine and Ν,Ν-dimethylbenzylamine were prepared. Feed- ratios of about 50:50 and 20:80 molar ratio of Ν,Ν-dimethylallylamine and N,N- dimethylbenzylamine were used.

For the starch polymers modified with epichlorohydrin, Ν,Ν-dimethylbenzylamine and N,N- dimethylallylamine, the NMR spectra were found to have all the relevant spectral features, expected for that particular amine (Figure 1). In the final polymer it was found that the two different amines were incorporated in the 45:55 and 12:88 ratio of Ν,Ν-dimethylallylamine and Ν,Ν-dimethylbenzylamine respectively (entries 6 and 7 in Table 1).

[S&F comment: Change made to express allyhbenzyl here too]

Also, experiments were conducted to check the reproducibility of the synthesis by repeating the synthesis of sample 7 (entries 8 and 9 in Table 1), illustrating that these materials can be accessed with high reproducibility. By using a one -pot procedure, soluble starch can be effectively modified to incorporate a wide variety of cationic groups by either using a single or multiple tertiary amines in a reproducible manner.

Table 1. Degree of substitution of cationically modified starch polymers

[Table 1]

s.

Polymer R¹N(Me)₂ R²N(Me)₂ DS^a R*-R^{2 a}

No.

1 Starch-C4 NA^b 0.55 NA

2 Starch-Cyclohexyl NA 0.56 NA

3 Starch-C8 N NA 0.74 NA

4 Starch-Bn NA 0.42 NA

5 Starch-Allyl 1 NA 0.29 NA

6 Starch-Allyl-Bn(50-50) 1 0.16 45-55 7 Starch-Allyl-Bn(20-80) I 0.24 12-88

8 Starch-Allyl-Bn(20-80) I 0.28 12-88

9 Starch-Allyl-Bn(20-80) I 0.27 12-88 ^a - as determined by ¾ NMR spectroscopy with DMSO- ¾ and TFA-d at 65 °C; ^b - not applicable

Method determination of degree of substitution (DS) by *H NMR (see Figure 1)

DS was calculated by using the following formula:

DS = Non exchangeable (NE) proton per anhydroglucose units (AGU)/(3*[I₂.6 s o - U)

NE proton per AGU = 7; 3 is used to account for the possibility that there are 3 reactive sites per AGU. In our calculation, contribution from degree of branching - i.e., the change in the possible reactive sites from 3 to 2 at the branching site, has been ignored; I₂.6 s o = Integral value corresponding to all the protons within 2.6 - 8.0 ppm range; I_s = Integral value corresponding to the NE protons from the substitution reaction within 2.6 - 8.0 ppm range.

Example calculation using a representative example - Starch-Allyl-Bn(20-80): DS = 7/(3*[29.99 - l*(5+6+7) + 0.13(5+6+5)]) = 0.24.

The phenyl region I_7.0 _ s o = 5.0, corresponding to 5H of benzene ring within 7.0 - 8.0 ppm range, was used to normalize the N,N-dimethylbenzylamine substitution reaction and 15.95 - 6.20 = 0.13, corresponds to 1H proton from the ally lie -alkene proton within 5.95 - 6.20 ppm range, was used to estimate the N,N-dimethylallylamine substitution reaction (ΝΕ protons from glyceryl-subunit = 5; ΝΕ protons from dimethyl groups= 6; ΝΕ protons from benzyl-subunit = 7; ΝΕ protons from allyl-subunit = 5).

Ratio of substitution of two different amines were calculated as follows by using the integration values per 1H obtained from I₇.„ ._{8 0} and I₅.₉₅ - 6.20: R¹ : R² = 0.13/(1+0.13) : 1/(1+0.13) = 12 : 88.

Fluorescence measurements

The critical aggregation concentrations (CACs) of the polymers in DI water were determined by fluorescence spectroscopy using pyrene as the probe (see Wilhelm, M.; Zhao, C. L.; Wang, Y.; Xu, R.; Winnik, M. A.; Mura, J. L.; Riess, G.; Croucher, M. D. Macromolecules 1991, 24, 1033). The fluorescence spectra were recorded using a Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon) at 25 °C. The polymer samples were equilibrated for 10 min before taking measurements. Aliquots of pyrene in acetone solution (6.16 X 10⁵ M, 10 μΕ) were added to glass vials and air-dried to remove the acetone. All polymers were dissolved directly in HPLC grade water. Subsequently, polymer solutions of varying concentrations were then added to the pyrene at 1 mL each, and left to stand for 24 h. The final pyrene concentration in each vial is 6.16 x 10⁷ M. The excitation spectra were scanned at wavelength from 300 to 360 nm with an emission wavelength of 395 nm. Both the excitation and emission bandwidths were set at 1.0 nm. The intensity (peak height) ratio of I340/I337 from the excitation spectra was analysed as a function of polymer concentration. The CAC was taken at the point of intersection between the tangent to the curve at the inflection and tangent of the points at low concentrations. In aqueous solutions, amphiphilic molecules spontaneously self-assemble to form aggregates of various morphologies above a critical concentration. The CAC is an important parameter to characterize aggregation behaviour, both for small surfactant molecules and for amphiphilic polymers (Prazeres, T. J.; Beija, M.; Fernandes, F. V.; Marcelino, P. G.; Farinha, J. P. S.; Martinho, J. Inorganica Chimica Acta 2012, 381, 181). The self-assembly of the modified starch polymers in aqueous solution was investigated by determining their critical aggregation concentrations. Pyrene, a hydrophobic molecule that preferentially resides in the hydrophobic portions of the aggregates, was used as the photo probe because of its unique photophysical characteristics. Amongst the polymers, only starch-C8 was found to self-assemble with a CAC value of 40 mg/L. The other polymers would most likely exist as individual molecules in an aqueous environment.

Minimal inhibitory concentration (MIC) measurements

Bacteria (5. aureus, E. coli, and P. aeruginosa) and yeast (C. albicans) obtained from ATCC were reconstituted from their lyophilized form according to the manufacturer's protocol. Bacterial samples were cultured in Mueller Hinton Broth (MHB) at 37 °C, and yeast cells were cultured in Yeast Mannitol Broth (YMB) at room temperature under constant shaking of 100 rpm. The MICs of the polymers were measured using the broth microdilution method. Briefly, 100 μL· of the respect broth medium containing a polymer at various concentrations (15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000 mg/L) was placed into each well of a 96-well tissue culture plate. An equal volume of microbial suspension (3 x 10^s CFU mL ¹) was added into each well. Prior to mixing, the microbial sample was first inoculated overnight to enter its log growth phase. The concentration of microbial solution was adjusted to give an initial optical density (O.D.) reading of approximately 0.07 at 600 nm wavelength on a microplate reader (TECAN, Switzerland), which corresponds to the concentration of Mc Farland 1.0 solution (3 x 10^s CFU mL ¹). The microbial solution was further diluted by 1000 times to achieve an initial loading of 3 x 10^s CFU mL ¹ before mixing with the polymer solutions. The bacterial samples were kept in an incubator at 37 °C for 18 h, while the yeast samples were kept at room temperature for 42 h under constant shaking of 100 rpm. The MIC was determined as the concentration of the polymer at which no increase in O.D. measurements at the end of the respective incubation time. Broth containing microbial cells alone was used as negative control, and each condition was tested in 6 replicates.

The modified starch polymers were tested for their ability to impede growth of several common microbes, including 5. aureus, E. coli, P. aeruginosa and C. albicans. Gram positive 5. aureus was found to be most susceptible to the starch polymers with MIC ranging between 15.6 to 250 mg/L (Table 2). The growth of E. coli and P. aeruginosa can also be effectively inhibited using the polymers with a minimum MIC of 31.3 mg/L. Broad spectrum antimicrobial activity was observed for Starch-C8 and Starch-Allyl-Bn(80-20) where the growth of all the microbes was successfully impeded.

Table 2. Minimum inhibitory concentrations (MIC) and haemolytic concentrations of modified starch polymers.

[Table 2]

MIC (mg/L)

Polymer SA PA EC CA HC_sft (mg/L) HC_2ft (mg/L) Starch-C4 15.6 31.3 >1000 500 >1000 >1000

Starch-Cyclohexyl 31.3 31.3 >1000 500 >1000 >1000

Starch-C8 31.3 250 125 500 250 62.5

Starch-Bn 31.3 31.3 62.5 >1000 >1000 500

Starch-Allyl 31.3 >1000 >1000 500 >1000 >1000

Starch-Allyl-Bn(20-80) 15.6 31.3 31.3 500 >1000 >1000

Starch-Allyl-Bn(50-50) 15.6 31.3 >1000 500 >1000 >1000

Killing kinetics and killing efficiency studies of starch-based polymer solutions

To investigate if the starch-based polymers are microbicidal, the killing kinetics and killing efficiency of the polymers were tested using agar plating assay. The microbes were inoculated and prepared according to the same procedure in the MIC measurement described above. Subsequently, the microbes were treated with antimicrobial polymer at various concentrations (0.5 x MIC, MIC, 2 x MIC and 4 x MIC) and incubated at 37 °C under constant shaking of 100 rpm. At regular time intervals (20 min, 1 h, 2 h, 4 h, 6 h), samples were taken for a series of tenfold dilutions, and plated onto LB agar plates. The plates were incubated for 24 h at 37 °C and counted for colony-forming units (CFU). For killing-efficiency determination, the samples were taken after 18 h incubation and plated using the same protocol for viable counts. Broth containing microbial cells alone was used as negative control, and each condition was tested in triplicates.

Starch-C4 and Starch-Cyclohexyl were selected to conduct killing kinetics and efficiency tests on 5. aureus and P. aeruginosa. Varying concentrations of 0.5x, lx, 2x and 4x MIC were used From Figure 3 it can be seen that lx MIC of each of the polymers were adequate in killing more than 99.9% of the microbes by 18 h post-treatment. Both polymers displayed similar killing kinetics (Figure 4), and were able to achieve a 3-log reduction (99.9% killing) of 5. aureus by 4 h and 6 h using 2x and lx MIC respectively. Starch-Cyclohexyl was more efficient in killing P. aeruginosa and 2x MIC was able to result in 3-log reduction of the microbes by 4 h post- treatment while Starch-C4 required 6 h to achieve the same killing efficiency.

Hemolysis Assay

Fresh rat blood was subjected to 25 times dilution with PBS to reach a concentration of 4% v/v. The blood was then mixed with antimicrobial polymers at varying polymer concentrations from 3.9 to 1000 mg/L. After incubation at 37 °C for 1 h, the non-hemolysed red blood cells were separated by centrifugation at 13000 g for 5 min. Aliquots (100 μί) of the supernatant were transferred into a new 96-well plate, and the hemoglobin release was represented by absorbance readings at 576 nm using a microplate reader (TEC AN, Switzerland). Two controls were used in this assay: an untreated red blood cell suspension in PBS as the negative control, and a solution containing red blood cells lysed with 0.1% Triton -X as the positive control. The results were expressed as a percentage of the hemolytic activity of the positive control.

As listed in Table 2, all polymers were found to be non -hemolytic (HC20 > 1000 mg/L) with the exception of Starch-C8 (HC50 and HC20 = 250 and 62.5 mg/L). Starch-Bn exerts mild hemolytic activity with HC20 = 500 mg/L. Example 2: Starch-based Hydrogels and Rheological Experiments

To form the hydrogels, Starch-allyl was weighed out in the same sample tubes as either Starch- allyl-bn (20-80) or Starch-allyl-bn (50-50) to produce the S-l and S-2 and S-3 (Table 3 for exact concentrations). PEG additive was always added and mixed with the starch derivatives prior to gelation under UV radiation.

0.5 wt.% solution of photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone was then added to each of these mixtures and sonicated for 10 min to generate a homogeneous solution. 200 μΕ of each of these solutions was then transferred to the cut-out caps of 1.7 mL microcentrifuge tubes (Eppendorf, Germany) that serve as molds for the hydrogel formation. The mixtures were then exposed to UV light (320-500 nm, 900 mJ cm^-2) for 10 min to allow for photo crosslinking. The rheological analysis of the hydrogels was performed on an ARES-G2 rheometer (TA Instruments, USA) equipped with a plate -plate geometry of 8 mm diameter. Measurements were taken by equilibrating the gels at 25 °C between the plates at a gap of 2.0 mm. The data were collected under controlled strain of 2.0% and a frequency scan of 1.0 to 100 rad/s. Gelation properties of the polymer suspension was monitored by measuring the shear storage modulus (G'), as well as the loss modulus (G"), at each point.

Starch polymers were mixed in compositions as listed in Table 3 and cross-linked via UV radiation. From Figure 5, the addition of cationic Starch-Allyl-Bn(80-20) and Starch-Allyl- Bn(50-50) to the mixture resulted in slightly lower storage modulus (G') compared to hydrogel that is made up of Starch-Allyl alone. For instance, the G' value of Sample 3 was 256 Pa at 20 rad/s, while the G' values of Sample 1 and 2 were 177 and 158 Pa respectively.

Table 3. Compositions of starch-based hydrogels

[Table 3]

Sample 1 2 3

Starch-Ally (wt.%) 10 10 10

Starch-Allyl-Bn(80-20) (wt.%) 10 0 0

Starch-Allyl-Bn(50-50) (wt.%) 0 10 0

4arm PEG(lOk) SH (wt.%) 4 4 8

Killing efficiency studies of starch-based polymer hydrogels

For killing efficiency studies, Starch-allyl was weighed out in the same sample tubes as either Starch-allyl-bn (20-80) or Starch-allyl-bn (50-50) to produce the S-l and S-2 and S-3 (Table 3 for exact concentrations).

0.5 wt.% solution of photoinitiator 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone was then added to each of these mixtures and sonicated for 10 min to generate a homogeneous solution. 100 μΕ of each of these solutions was then transferred to 96-well plate and then exposed to UV light (320-500 nm, 900 mJ cm^-2) for 10 min to allow for photocrosslinking. The hydrogels were then washed three times using 100 μΕ to remove any excess photoinitiator. 100 μΕ of the microbial suspension containing 3 x 10^s CFU mL ¹ and was added into each well. The plates were then incubated at 37 °C under constant shaking of 100 rpm. 18 h later, the microbial samples were taken for a series of tenfold dilution, and plated onto LB agar plates. The plates were then incubated for 24 h at 37 °C and counted for colony -forming units (CFU). Broth containing microbial cells alone was used as negative control, and each condition was tested in triplicates.

The killing efficiency of starch-based hydrogels were tested using the polymer compositions listed in Table 3 (see Figure 6). Sample 3, which consisted solely of Starch-Allyl showed limited effectiveness against E. coli and P. aeruginosa with killing efficiency that fell below 99.9%. Samples 1 and 2, which consisted of Starch-Allyl and Starch-Allyl-Bn mixtures, displayed broad spectrum killing properties where > 99.9% killing was achieved in all the tested microbes

Use Example: Killing efficiency studies of starch-based polymer hydrogels coated onto cotton pads

Ultraviolet (UV)-cross-linking of polymers onto cotton pads

Polymers were weighed and mixed with 4-arm-PEG-thiol (10k) in varying weight ratios to form hydrogels S-l, S-2 and S-3. 0.5 wt.% solution of photoinitiator 2-Hydroxy-4'-(2- hydroxyethoxy)-2-methylpropiophenone was then added to each of these mixtures. The polymer and 4-arm-PEG-thiol were dissolved via ultrasonication (Ultrasonic Bath, Elmasonic S60H, Germany) for 30 minutes. A 1 cm x 1 cm cotton pad was placed in a 48 -well plate. 100

of the polymer solution was coated onto the upper surface of the cotton pad in the well using a micropipette, and gelled using a UV-cross-linker (Spectronics Corporation, U.S. A). The UV radiation was set at an energy setting of 9999 MJ cm ², and radiation time was 5 minutes. After cross-linking, the cotton pad was flipped onto its other side using forceps and the above process was repeated to formulate a fully-coated cotton pad.

Antibacterial tests of hydrogel-coated cotton pads for the determination of killing efficiency

Without removing the cotton pads from the 48 -well plate after undergoing UV -cross-linking, 200 of sterile water was added the control cotton pad. The bacterial concentration was adjusted to give a reading of 0.07 at 600 nm wavelength on a microplate reader. Then, 0.5

of the bacterial solution was mixed with 5 mL of MHB to dilute the bacterial concentration to 3xl0⁴ CFU/mL. 100

of the diluted solution was pipetted onto the hydrogel-coated cotton pad in the well, and this was repeated for the control. The 48 -well plate was incubated in a shaker (VWR Incubating Mini Shaker VWR 12620-944, U.S.A.) at 37°C for 4 hours. After 4 hours, 100 of MHB was added into each well containing the cotton pads to elute the bacteria from the sample surface. The solutions were pipetted from the respective wells into clean microfuge tubes and underwent serial dilution, before being spread onto LB agar. They were incubated for 24 hours, and the colonies were counted afterwards.

Calculation of killing efficiency

The percentage reduction of bacteria can be calculated using the following formula:

B - A

R =—— X 100%

B

where R is the % reduction in bacteria after treatment with the hydrogel-coated cotton pad, A is the number of number of colonies of bacteria recovered from the hydrogel-coated cotton pad, and B is the number of colonies of bacteria recovered from untreated cotton pad. The hydrogels were successfully cross-linked onto both sides of the cotton pads to mimic wound-dressing material, by using the formulations at 2 x the scale of composition listed in Table 4. The hydrogels permeated the entirety of the cotton pads, and formed a thin and well- defined layer on the exterior. The material is more elastic and is visibly firmer than an unmodified cotton pad. Thereafter, the materials were subjected to antibacterial tests to determine the killing efficiency of each hydrogel. All three hydrogel samples (Table 4) exhibited antibacterial properties (killing efficiency > 99.5%). They also displayed higher killing efficiencies for Gram-positive 5. aureus than Gram-negative P. aeruginosa and E. coli. Hydrogel sample 3, generally displayed lower killing efficiencies, and this is likely due to the absence of the hydrophobic benzyl functional groups which are responsible for disrupting the lipid component of the bacterial membrane, demonstrating the importance of tailoring compositions of the polymers to achieve a more ideal hydrophobic/hydrophilic balance. The antibacterial tests show that the hydrogel-coated materials formulated can inhibit the growth of bacteria on surfaces which the hydrogels are in contact with, hence showing potential for the hydrogels to be developed into wound-dressing materials.

Table 4: Killing efficiencies of different hydrogel formulations on bacteria

[Table 4]

In summary the working examples show:

Biodegradable antimicrobial starch polymers were successfully made by one -pot synthesis using a mixture of epichlorohydrin and functional tertiary amine via the formation of glycidyl derivative of tertiary amine as an in situ intermediate. The antimicrobial activity of the polymers was tuned via cationic charge/hydrophobicity balance by using a mixture of N,N- dimethylbenzylamine and N,N-dimethylallylamine to modify starch. The optimized antimicrobial starch polymer, Starch-Allyl-Bn(20-80) (feed molar ratio of N,N- dimethylallylamine and Ν,Ν-dimethylbenzylamine = 20:80), have strong activity against 5. aureus, E. coli and P. aeruginosa with high selectivity over mammalian cells. Antimicrobial hydrogels were also made by photo crosslinking of Ν,Ν-dimethylallylamine and/or N,N- dimethylbenzylamine modified starch. The hydrogels containing both N,N-dimethylallylamine and Ν,Ν-dimethylbenzylamine effectively eradicated S. aureus, E. coli, P. aeruginosa, and C. albicans upon contact. Description of Drawings

The accompanying drawings illustrate a disclosed embodiment or reaction scheme and serve to explain the principles of the disclosed embodiments. It is to be understood, however, that the drawings are designed for purposes of illustration of examples only, and not as a limitation of the invention.

Fig.l

[Fig. 1] shows the determination of degree of substitution by NMR.

Fig.2

[Fig. 2] is a schematic drawing of the general approach to access cationically modified starch in one step process by using epichlorohydrin and slight excess of functional tertiary amines.

Fig.3

[Fig. 3] shows the antimicrobial activity of modified starch polymers against (A) 5. aureus and (B) P. aeruginosa; Starch-C4 in dark and Starch-Cyclohexyl in light grey columns.

Fig.4

[Fig. 4] shows the killing kinetics study of modified starch polymers Starch-C4 and Starch- Cyclohexyl against (A and B) S. aureus and (C and D) P. aeruginosa respectively.

Fig.5

[Fig. 5] shows the mechanical properties of starch-based hydrogels with different polymer compositions. (A to C) represents data from Samples 1 to 3 in Table 3 respectively.

Fig.6

[Fig. 6] shows the killing efficiency of Starch-based hydrogels. Samples 1 to 3 correspond to those listed in Table 3.

Industrial Applicability

The method according to the first aspect of the invention allows for the tailor-made synthesis of saccharide derivatives with antimicrobial activity. They saccharide derivatives further show selectivity for mammal cells. Therefore the saccharide derivatives may find use in various applications that require such properties. They may for instance be used as additives or materials in applications such as consumer care products or food packagings, as antimicrobial agents, viscosity modifiers, surface modifiers or interfacial stabilizers.

The saccharide derivatives can further be polymerised to form hydrogels with comparable antibacterial activity. Applications of the hydrogels where a more rigid hydrogel as anti-bacterial material is used can be envisioned. The cotton materials of the use example may be mentioned in this regard. It will be apparent that various other modifications and adaptations of the invention are available to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.

Claims

1. A method for making a saccharide derivative substituted with quaternary ammonium cations, the method comprising subjecting a saccharide to a mixture of an optionally substituted halo-epoxide and one or more optionally substituted amines.

2. The method of claim 1 wherein the method is performed as one pot method.

3. The method of claim 1 wherein the saccharide is subjected to a mixture of an optionally substituted halo-epoxide and two or more different optionally substituted amines.

4. The method of claim 1 wherein the saccharide is selected from starch.

5. The method of any of claim 1 to 4 wherein the optionally substituted halo-epoxide is selected from a moiety having an aliphatic backbone, wherein one terminal unit of the aliphatic backbone is an epoxide and the other terminal unit is a halide.

6. The method of claim 5, wherein the optionally substituted halo-epoxide is epichlorohydrin.

7. The method of any of claims 1 to 6, wherein the one or more optionally substituted amines are selected from the group consisting of optionally substituted tertiary amines.

8. The method of claim 7, wherein 2 of the 3 substituents of the tertiary amines are selected from Ci_₆ alkyl and the third substituent is selected from a C₂ io alkyl group, C₂-e alkenyl, phenyl, benzyl, naphthyl or methyl -naphthyl.

9. The method of claim 7, wherein at least one tertiary amine is selected wherein 2 of the 3 substituents of the tertiary amine are selected from C ₆ alkyl and the third substituent is a C₂-6 alkenyl or C_{3 6} alkynyl group.

10. The method of claim 1 or claim 7, wherein the one or more optionally substituted amines comprise two different amines, wherein the mixing ratio of the amines are in a molar ratio from about 10 to 90 to about 50 to 50.

11. The method of claim 1 or claim 7, wherein the one or more optionally substituted amines comprise a Ν,Ν-dimethylallylamine and N,N-dimethylarylalkylamine in a molar ratio of about 10 to 90 to about 40 to 60.

12. The method of claim 1 or claim 7, wherein the one or more optionally substituted amines comprise a Ν,Ν-dimethylallylamine and N,N-dimethylbenzylamine in a molar ratio of about 10 to 90 to about 40 to 60.

13. The method of any of claims 1 to 12, wherein the mixture of the optionally substituted halo-epoxide and one or more optionally substituted amines comprises water.

14. The method of any of claims 1 to 13, wherein about 1 g of starch is reacted with about 0.01 to 0.5 mol of optionally substituted halo-epoxide and about 0.01 to 0.5 mol of one or more optionally substituted amines.

15. The method of claim 1 or 14 wherein the amines are used in about 1 : 1.01 to about 1: 1.2 molar excess compared to the halo-epoxide.

16. A saccharide derivative substituted with quaternary ammonium cation groups, comprising hydroxy groups as a saccharide functionality which in part are substituted by one or more optionally substituted quaternary ammonium cations linked via a crosslinker and the wherein degree of substitution of the hydroxy groups of the saccharide is about 20% to 80%.

17. A saccharide derivative according to claim 16 wherein the ammonium cation groups are at least two different ammonium groups.

18. A saccharide derivative according to claim 16 wherein the saccharide derivative is a modified soluble starch and wherein the ammonium cation groups are selected from quaternary N,N-dimethyl-arylalkyl ammonium and Ν,Ν-dimethyl-allyl ammonium groups in a molar ratio of 90 to 10 to about 60 to 40.

19. A saccharide derivative according to claim 16 wherein the saccharide derivative is a modified soluble starch and wherein the ammonium cation groups are selected from quaternary N,N-dimethyl4oenzyl ammonium and Ν,Ν-dimethyl-allyl ammonium groups in a molar ratio of 90 to 10 to about 60 to 40.

20. The use of a saccharide derivative made according to the method of any of claims 1 to 15 or the saccharides of any of claims 16 to 19 for inhibiting microbes.

21. A method for making a hydrogel comprising the steps of mixing a saccharide derivative made according to the method of any of claims 1 to 15 or a saccharide derivative of any of claims 16 to 19 with a photoinitiator in a solvent and exposing the mixture to UV light.

22. The method of claim 21 comprising the step of adding a PEG additive to the mixture before UV curing.

23. The use of the hydrogel made according to claim 21 or 22 for inhibiting microbes.

24. The use of the saccharide derivatives made according to the method of any of claims 1 to 15, the saccharide derivatives according to any of claims 16 to 19 or the hydrogel according to claim 21 or 22 as component of consumer care products or food packagings, as antimicrobial agent, viscosity modifier, surface modifier or interfacial stabilizer.