Antibacterial surfaces via multicomponent chitosan conjugates

Abstract

Compositions comprising dual-conjugated chitosan polymers or oligomers and functionalized metal workpieces comprising dual-conjugated chitosan polymers or oligomers are disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 223,969, filed July 20, 2021, and U.S. Provisional Patent Application No. 63 / 054,719, filed July 21, 2020, each of which is incorporated herein by reference for all purposes.

[0002] The present disclosure is directed to methods and compositions for the use of dopamine, polydopamine, and / or derivatives thereof in multipart chitosan conjugates and as attachment motifs for surface modification. [Background technology]

[0003] Bacterial contamination of surfaces is an ongoing problem in both medical and non-medical fields. Surfaces with colonized bacteria can cause illness and even death. A variety of surface treatment compositions and methods have been developed to improve the antimicrobial properties of such surfaces. Surface modification using a variety of chemical techniques, such as organothiol metals, enediol oxides, silane oxides, phosphonic acids and derivatives, has been developed. Surface modification generally involves the inclusion of active end groups that are firmly attached to the modified surface via linking groups. The active end groups impart desired functionality to the modified surface.

[0004] Polydopamine is one of the most versatile and widely used motifs for surface functionalization. Inspired by the study of marine bivalves, particularly mussels, and their ability to bind to a wide variety of surfaces, early researchers recognized the predominance of catechol (3,4-dihydroxybenzene) in the form of 3,4-dihydroxy-L-phenylalanine (DOPA) and primary and secondary amines in the form of lysine and histidine in mussel adhesive proteins. Early catechol / amine systems were used to create biomimetic adhesive systems, including DOPA-Lys polyamino acids and catechols tethered to polyamines such as chitosan.

[0005] Chitosan is a natural polysaccharide obtained by partial or complete deacetylation of chitin. As described by Dutta et al. (Chitin and chitosan: chemistry, properties and applications, J. Sci. Ind. Res., vol. 63, pp. 20-31 (2004)), chitin is the most naturally abundant polysaccharide after cellulose. Chitin exists as a mixed polymer of glucosamine and N-acylated glucosamine monomers and is characterized by the degree of deacetylation of the C-2 amine. Traditionally, chitosan is defined as having >50% deacetylation. Together with chain length and molecular weight, the degree of deacetylation (DDA) is a key determinant of chitosan's chemical and physical behavior. The glucosamine monomer unit of chitosan has the following structure:

[0006] [ka]

[0007] where n is the number of monomer units present in the oligomer or polymer. It has.

[0008] Each monomer unit has three readily available sites for chemical modification: a C-2 primary amine, a C-3 secondary OH, and a C-6 primary OH. These sites allow for easy modification and customization of chitosan for biomedical applications. In addition, chitosan is biocompatible, biodegradable, and its degradation products do not induce inflammatory responses.

[0009] Chitosan has a wide range of applications in the medical field, for example, as a vehicle for drug delivery, tissue engineering, as an antithrombogenic agent, in bone regeneration, and as an antibacterial material. In addition, chitosan has been used in cosmetics, wastewater treatment, and as a feed and food additive. Other uses of chitosan are described, for example, in Vunain et al., Fundamentals of Chitosan for Biomedical Applications (Chitosan Based Biomaterials: Volume 1: Fundamentals, Jennings, JA, Bumgardner, JD ed., pp. 3-30 (2017)).

[0010] Antibacterial chitosan is well known in the art and has been recently reviewed (Freitas et al., An Overview of Current Knowledge on the Properties, Synthesis and Applications of Quaternary Chitosan Derivatives, Polymers, vol. 12 (12), p. 2878 (2020)). Unmodified chitosan has weak antibacterial properties, but the introduction of quaternary ammonium moieties enhances both the antibacterial activity of chitosan and its water solubility. Chitosan is insoluble in neutral to basic aqueous conditions.

[0011] The most common quaternization involves N,N,N-trimethylchitosan followed by N-[(2-hydroxy-3-trimethylammonium)propyl], commonly known as HTCC. Insertion of phosphonium and pyridinium salts is also becoming increasingly common.

[0012] In addition to enhanced solubility and antimicrobial activity, mucoadhesion and permeability are significantly improved by the introduction of permanent cationic moieties on the polymer or oligomer chain. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Dutta et al. (Chitin and chitosan: chemistry, properties and applications, J. Sci. Ind. Res., vol. 63, pp. 20-31 (2004)) [Non-patent document 2] Vunain et al., Fundamentals of Chitosan for Biomedical Applications (Chitosan Based Biomaterials: Volume 1: Fundamentals, Jennings, JA, Bumgardner, JD ed., pp. 3-30 (2017) [Non-patent document 3] Freitas et al., An Overview of Current Knowledge on the Properties, Synthesis and Applications of Quaternary Chitosan Derivatives, Polymers, vol. 12 (12), p. 2878 (2020) Summary of the Invention [Means for solving the problem]

[0014] An inventive aspect of the present application is the construction of a composition comprising a quaternized chitosan polymer or oligomer, which is further modified by the attachment of dopamine or an analogue to form a three-part conjugate.

[0015] A second aspect of the invention is the further modification of these conjugates through binding to / reaction with metals, their alloys, oxides, or nanoparticle forms, and methods for effecting said binding.

[0016] Current methods for dopamine deosition often require prolonged immersion in aqueous buffers under basic conditions. An exemplary embodiment of this application is directed to a composition comprising a doubly conjugated chitosan polymer or oligomer, where the conjugate is a quaternary ammonium, phosphonium, or pyridinium compound attached to at least some of the available reactive sites (-NH, -OH) of the chitosan molecule, followed by attachment of dopamine or an analog thereof to at least some of the remaining available sites of the chitosan molecule.

[0017] Another exemplary embodiment of the present application is directed to compositions obtained by the attachment of these dual-modified chitosans via their pendant catechol (or analog) groups to surfaces, including metals, their alloys, oxides, or nanoparticles, ceramics, metalloids, polymers, etc.

[0018] <Summary> Applicants have developed a rapid and efficient method for the anodic deposition and attachment of chitosan conjugates bearing pendant catechols, modified catechols and analogs, including dopamine, to metal and metal oxide surfaces under basic or neutral conditions, in aqueous or organic media.

[0019] Anodic deposition, as well as the addition of a chemical oxidant used to promote dopamine binding and polymerization, creates a constant oxidative environment at the dopamine (DP) / surface interface. This method contrasts with other electrodeposition techniques that use lower voltages and cycle between oxidizing and reducing conditions at the DP / surface interface.

[0020] Additionally, the chitosan conjugates are bonded to the metal and / or metal oxide surfaces by heat applied by any conventional technique, such as in an oven, through microwave irradiation, or by induction.

[0021] Photografting has also been utilized to attach quaternized doubly modified chitosan conjugates via their pendant catechol or similar groups.

[0022] Where a document, act, or article of knowledge is referenced or discussed herein, this reference or discussion is not an admission that the document, act, or article of knowledge, or any combination thereof, was publicly available, publicly known, part of the common general knowledge, or otherwise constitutes prior art under applicable statutory provisions or was known as relevant to any attempt to solve any problem to which this specification is directed.

[0023] While certain aspects of the prior art are discussed to facilitate disclosure of the present invention, the inventors of the present application have not in any way disclaimed these technical aspects, and it is intended that the claimed invention may encompass or include one or more of the conventional technical aspects discussed herein.

[0024] None of the known surface functionalization methods include the attachment of chitosan and / or modified chitosan described herein to metal surfaces via dopamine or dopamine derivative linkers to render them bactericidal. In the medical device area, elution systems potentially have systemic effects and can lead to the development of resistance in bacterial organisms, while attached antibacterial surfaces are believed to avoid these drawbacks. For non-medical surfaces, attached surface treatments are potentially longer-lasting and have a longer window of effectiveness relative to commonly applied cleaning. This application describes unique compositions and rapid and convenient methods for creating naturally-based antibacterial surfaces that may have a high potential for biocompatibility. Furthermore, the method of modifying polydopamine polymers described herein does not require the use of specific derivatives of dopamine or specific pre-synthesized compounds and various steps.

[0025] Such modified surfaces can be used in a variety of applications, including but not limited to medical devices.

[0026] One inventive aspect of the present application is directed to addressing shortcomings associated with conventional methods for attaching dopamine, polydopamine, and related compounds to metal surfaces. The present application also addresses novel coatings and novel devices comprising modified surfaces.

[0027] One exemplary embodiment is a method for preparing a modified metal surface by attaching catechol or a derivative thereof to the surface of a metal, the method comprising the steps of: preparing an aqueous solution of catechol or a derivative thereof in a buffer solution, including, but not limited to, a phosphate buffer; immersing at least a portion of a metal workpiece in the aqueous solution of catechol or a derivative thereof; applying voltage or UV radiation for a duration of time to obtain a functionalized metal workpiece; and chemically bonding quaternized chitosan to the functionalized metal workpiece.

[0028] Another exemplary embodiment is a method for preparing a metal surface modified by the attachment of a quaternized chitosan dual conjugate with catechol or a derivative thereof, the method comprising the steps of: preparing an aqueous solution of chitosan or a derivative thereof in a buffer solution, including, but not limited to, phosphate buffer; immersing at least a portion of a metal workpiece in the aqueous solution of chitosan or a derivative thereof; applying voltage or UV radiation for a duration of operation to obtain a functionalized metal workpiece; and applying the chemically attached modified chitosan to the metal workpiece.

[0029] In another embodiment of preparing a modified metal surface having catechol or a derivative thereof bound to the surface of the metal, the method includes the steps of preparing a solution of catechol or a derivative thereof in a non-aqueous protic solvent in the presence of a base; immersing at least a portion of a metal workpiece in the solution of catechol or a derivative thereof; applying a voltage or UV radiation for a duration of time to obtain a functionalized metal workpiece; and chemically bonding the modified chitosan to the functionalized metal workpiece.

[0030] In another embodiment of preparing a modified metal surface having catechol or a derivative thereof bound to the surface of the metal, the method includes the steps of preparing a solution of doubly conjugated chitosan or a derivative thereof in a non-aqueous protic solvent in the presence of a base; immersing at least a portion of a metal workpiece in the solution of chitosan or a derivative thereof; and applying voltage or UV radiation for a working period to obtain a functionalized metal workpiece having modified chitosan chemically bound to the metal workpiece.

[0031] In another exemplary embodiment, the non-aqueous protic solvent comprises any organic solvent, including, but not limited to, alcohol, which may be selected from, but not limited to, methanol, ethanol, propanol, butanol, and the like.

[0032] In another exemplary embodiment, the base can be any organic base that is immiscible with organic solvents.

[0033] In another exemplary embodiment, the base can be piperidine.

[0034] In another exemplary embodiment, a metal workpiece coated with a thin layer of dual-conjugated chitosan in an aqueous or non-aqueous solution can be exposed to ultraviolet light or heat to form a functionalized metal workpiece.

[0035] In another exemplary embodiment, the method includes exposure to ultraviolet light (photografting), where a metal workpiece coated with a thin layer of doubly conjugated chitosan in a non-aqueous protic solution is photografted at a wavelength (λ) of about 100 to about 400 nm. max In another exemplary embodiment, the wavelength may be about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm, about 225 nm, about 250 nm, about 251 nm, about 252 nm, about 253 nm, about 254 nm, about 255 nm, about 256 nm, about 257 nm, about 258 nm, about 259 nm, about 260 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, or any wavelength between about 100 nm and about 400 nm.

[0036] In another exemplary embodiment, photografting can be carried out for a time period of about 0.1 seconds to 10 minutes. In another exemplary embodiment, photografting can be carried out for about 0.1 seconds, about 0.5 seconds, about 1 second, about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 60 seconds, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, or any time period between about 0.1 seconds and about 10 minutes.

[0037] In another exemplary embodiment, catechol or a derivative thereof is covalently bonded to the surface of a functionalized metal workpiece.

[0038] In another exemplary embodiment, the surface of a metal workpiece functionalized with dopamine or a derivative thereof is activated by the addition of diimidazole, a derivative thereof, or any reagent designed and used to couple, bond, or attach molecules, oligomers, or polymers to one another.

[0039] In another exemplary embodiment, the coupling agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI).

[0040] In another exemplary embodiment, the metal-bound chitosan-dopamine or similar conjugate is further modified by the attachment of a quaternary ammonium, pyridinium, or phosphonium compound.

[0041] In another exemplary embodiment, the catechol or derivative thereof includes any substituted or unsubstituted catechol, which can be represented by the following formula: [ka] wherein each of R1, R2, R3, R4, and R5 is independently selected from the group consisting of thiol, primary amine, secondary amine, nitrile, aldehyde, imidazole, azide, halide, polyhexamethylene dithiocarbonate, hydrogen, hydroxyl, carboxylic acid, carboxylic acid ester, or carboxamide, with the proviso that at least one of R1, R2, R3, R4, and R5 is not a hydrogen atom, a ranges from 0 to 10, and b ranges from 0 to 10, with the proviso that a or b is at least 1. Catechol or its derivatives include, but are not limited to, catecholamines, phenolic aldehydes, tyrosol and its derivatives, including hydroxyl, methoxy, and mixed methoxy-hydroxy derivatives of tyrosol, hydroxylated tyramine, tyramine and its derivatives, including dopamine and its derivatives, and the like. In another exemplary embodiment, other hydroxyl-containing compounds, both substituted and unsubstituted, including, but not limited to, phenol, can be used as linkers. In another exemplary embodiment, the catechol or its derivative is dopamine or polydopamine. In another exemplary embodiment, the dopamine derivative may be a methoxy derivative, a 4-ethanol derivative, or the like.

[0042] In another exemplary embodiment, the doubly conjugated chitosan can be represented by the following formula: [ka] wherein each of R1, R2, R3, and R4 is independently selected from the group consisting of thiol, alkane, alkene, alkyne, secondary amine, nitrile, aldehyde, imidazole, azide, halide, polyhexamethylene dithiocarbonate, hydrogen, hydroxyl, carboxylic acid, carboxylic acid ester, acetyl, carboxamide, urea, and catechol or analogs thereof, provided that at least one of R1, R2, R3, and R4 is not a hydrogen atom. In the above formula, n represents the number of monomer units in the chitosan chain and can range from about 1 to about 500. The C-2 nitrogen must be at least 50% deacetylated (by definition for chitosan), and each monomer unit can be attached to one of two conjugating moieties.

[0043] The most amenable attachment points for conjugates are at the deacetylated amines, with secondary attachments at the C-3 and C-5 hydroxyls being less common. In doubly conjugated chitosan, one conjugate will react with a subset of the available deacylated monomers, while the second conjugate will react with a subset of the remaining unconjugated deacylated monomers.

[0044] Chitosan can undergo a deacetylation reaction such that any percentage of the acetyl groups between about 50% to about 100%, about 55% to about 95%, about 60% to about 90%, about 65% to about 85%, about 70% to about 80%, or about 50% to about 100% can be removed, and further such that at least two of the R1 to R4 groups can be modified.

[0045] At least one of the R1-R4 groups may have a quaternary ammonium, phosphonium, or pyridinium functional group, and the chitosan may have between about 1% and about 99%, between about 5% and about 90%, between about 10% and about 80%, between about 15% and about 75%, between about 20% and about 70%, between about 25% and about 65%, between about 30% and about 60%, between about 35% and about 55%, between about 40% and about 50% quaternization, or any percentage of quaternization between about 1% and about 99%.

[0046] At least one of the R1-R4 groups may be a catechol analog that provides a pendant dihydroxybenzyl group for future immobilization onto a substrate.

[0047] A composition comprising a doubly conjugated chitosan polymer or oligomer having the formula: [ka] wherein each of R1, R2, R3, R4, R5, and R6 is independently selected from the group consisting of thiol, alkane, alkene, alkyne, secondary amine, nitrile, aldehyde, imidazole, azide, halide, polyhexamethylene dithiocarbonate, hydrogen, hydroxyl, carboxylic acid, carboxylic acid ester, carboxamide, urea, and catechol or analogs thereof, with the proviso that at least one of R1, R2, R3, R4, R5, and R6 is not a hydrogen atom, n represents the number of monomer units available for conjugates and is from about 1 to about 500, and m represents the number of monomer units available for a second conjugate and is from about 1 to about 500.

[0048] In certain embodiments, at least two of R1-R6 are independently selected from the group consisting of thiol, alkane, alkene, alkyne, secondary amine, nitrile, aldehyde, imidazole, azide, halide, polyhexamethylene dithiocarbonate, hydrogen, hydroxyl, carboxylic acid, carboxylic acid ester, carboxamide, urea, and catechol or an analog thereof.

[0049] In certain embodiments, at least one of R1-R6 is selected from the group consisting of a quaternary ammonium, quaternary phosphonium, or quaternary pyridinium functional group, and the degree of quaternization of the doubly conjugated chitosan is from about 1% to about 99%.

[0050] Chitosan may undergo a deacetylation reaction such that any percentage of acetyl groups between about 50% and about 100%, about 55% and about 95%, about 60% and about 90%, about 65% and about 85%, about 70% and about 80%, or about 50% and about 100% may be removed, and further, at least two of the R1 to R4 groups may be modified (denatured).

[0051] At least one of the R1-R6 groups may have a quaternary ammonium, phosphonium, or pyridinium functional group, and the chitosan may be quaternized to any percentage between about 1% and about 99%, between about 5% and about 90%, between about 10% and about 80%, between about 15% and about 75%, between about 20% and about 70%, between about 25% and about 65%, between about 30% and about 60%, between about 35% and about 55%, between about 40% and about 50%, or between about 1% and about 99%.

[0052] At least one of the R1-R6 groups may be a catechol analog that provides a pendant dihydroxybenzyl group for possible future immobilization onto a substrate.

[0053] At least one of the R1-R6 groups is catechol or a derivative or analog thereof that provides a pendant dihydroxybenzyl group for immobilizing the doubly conjugated chitosan on a substrate.

[0054] In certain embodiments, the doubly conjugated chitosan polymer or oligomer has the formula: [ka] wherein the catechol and quaternary ammonium moieties are randomly distributed among the available C-2 amine linkage positions of the doubly conjugated chitosan polymer or oligomer, and q and n each range from about 1 to about 500. is.

[0055] In another exemplary embodiment, the metal is a non-ferrous metal. In another exemplary embodiment, the metal is selected from, but is not limited to, Ti, Zr, Hf, V, Nb, Ta, Al, Co, W, Mg, and alloys, oxides, and nanoparticle forms thereof. In another exemplary embodiment, the metal is selected from, but is not limited to, titanium, titanium oxide, titanium alloy, aluminum, aluminum oxide, and aluminum alloy. In another exemplary embodiment, the metal is stainless steel.

[0056] In another exemplary embodiment, the concentration of the catechol or derivative thereof in the solution is about 0.1% to about 20% by weight, or about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, or any percentage between about 0.1% and about 20%.

[0057] In another exemplary embodiment, the applied voltage can range from about 1 V to about 100 V. In another exemplary embodiment, the applied voltage can range from about 1 V, about 2 V, about 3 V, about 4 V, about 5 V, about 6 V, about 7 V, about 8 V, about 9 V, about 10 V, about 11 V, about 12 V, about 13 V, about 14 V, about 15 V, about 16 V, about 17 V, about 18 V, about 19 V, about 20 V, about 21 V, about 22 V, about 23 V, about 24 V, about 25 V, about 26 V, about 27 V, about 28 V, about 29 V, about 30 V, about 31 V, about 32 V, about 33 V, about 34 V, about 35 V, about 36 V, about 37 V, about 38 V, about 39 V, about 40 V, about 41 V, about 42 V, about 43 V, about 44 V, about 45 V, about 46 V, about 47 V, about 48 V, about 49 V, about 50 V, about 51 V, about 52 V, about 53 V, about 54 V, about 55 V, about 56 V, about 57 V, about 58 V, about 59 V, about 60 V, about 61 V, about 62 V, about 63 V, about 64 V, about 65 V, about 66 V, about 67 V, about 68 V, about 69 V, about 70 V, about 71 V, about 72 V, about 73 V, about 74 V, about 75 V, about 76 V, about 77 V, about 78 V, about 79 V, about 80 V, about 81 V, 5V, about 26V, about 27V, about 28V, about 29V, about 30V, about 31V, about 32V, about 33V, about 34V, about 35V, about 36V, about 37V, about 38V , about 39V, about 40V, about 41V, about 42V, about 43V, about 44V, about 45V, about 46V, about 47V, about 48V, about 49V, about 50V, about 51V, about 5 2V, approximately 53V, approximately 54V, approximately 55V, approximately 56V, approximately 57V, approximately 58V, approximately 59V, approximately 60V, approximately 61V, approximately 62V, approximately 63V, approximately 64V, approximately 65V , about 66V, about 67V, about 68V, about 69V, about 70V, about 71V, about 72V, about 73V, about 74V, about 75V, about 76V, about 77V, about 78V, about It can be 79V, about 80V, about 81V, about 82V, about 83V, about 84V, about 85V, about 86V, about 87V, about 88V, about 89V, about 90V, about 91V, about 92V, about 93V, about 94V, about 95V, about 96V, about 97V, about 98V, about 99V, about 100V, or any voltage between about 1V and about 100V.

[0058] In another exemplary embodiment, the time is from about 1 second to about 60 minutes, or from about 1 second, about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 60 seconds, about 65 seconds, about 70 seconds, about 75 seconds, about 80 seconds, about 85 seconds, about 90 seconds, about 95 seconds, about 100 seconds, about 105 seconds, about 110 seconds, about 115 seconds, about 120 seconds, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, or about 7 minutes. , about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or any time between about 1 second and about 60 minutes.

[0059] In other exemplary embodiments, the pH of the aqueous buffer is about 6.0 to about 9.5, about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0, about 8.0, about 8.5, about 9, about 9.5, or any pH between about 6.0 and about 9.5.

[0060] In another exemplary embodiment, the dopamine solution comprises about 1% to about 50% dopamine by weight with about 1% to about 20% piperidine by weight in ethanol or methanol, preferably about 5% to about 45% dopamine by weight with about 5% to about 15% piperidine by weight, about 10% to about 40% dopamine by weight with about 10% to about 15% piperidine by weight, or any amount of dopamine between about 1% to about 50% by weight with about 1% to 20% piperidine by weight within these ranges, inclusive of the endpoints of these ranges.

[0061] In other exemplary embodiments, UV irradiation is carried out for about 1 minute to about 60 minutes, about 5 minutes to about 55 minutes, about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, about 25 minutes to about 35 minutes, or any time between about 1 minute and about 60 minutes (including the endpoints of these ranges), or any time less than about 3 hours.

[0062] In other exemplary embodiments, heat is applied for about 30 minutes to about 1 hour, about 1 hour to about 2 hours, about 2 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to about 10 hours, or any time between 30 minutes and 10 hours, including the endpoints of these ranges.

[0063] In another exemplary embodiment, the modified chitosan solution comprises an oligo-chitosan.

[0064] In another exemplary embodiment, oligo-chitosan may be modified at the C-2 amine by the addition of a quaternary ammonium, phosphonium, or pyridinium compound, and the modified (modified) chitosan may be further modified at the C-6 hydroxyl, C-3 hydroxyl, or previously unreacted C-2 amine by the attachment of dopamine (polydopamine), through which the oligo-chitosan is immobilized on a surface.

[0065] In another exemplary embodiment, oligo-chitosan may be modified at the C-2 amine by the addition of a quaternary ammonium, phosphonium, or pyridinium compound, and the modified (modified) chitosan may be further modified at the C-6 hydroxyl, C-3 hydroxyl, or previously unreacted C-2 amine by the attachment of 3,4-dihydroxyhydrocinnamic acid, through which the oligo-chitosan is immobilized on a surface.

[0066] In another exemplary embodiment, the modified chitosan solution comprises an oligo-chitosan modified in the glucosamine monomer with a quaternary phosphonium compound.

[0067] In another exemplary embodiment, a modified chitosan solution containing oligo-chitosan is modified at available sites with a quaternized phosphonium conjugate, and the degree of conjugation is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85 ... 0%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to 90%, about 90% to about 100%, or any value within these ranges (including the endpoints of these ranges).

[0068] In another exemplary embodiment, the modified chitosan solution comprises a quaternized oligo-chitosan further modified at the remaining available sites with dopamine or an analog thereof, and the degree of conjugation is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75 ... about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to 80%, about 80% to about 90%, about 90% to about 100%, or any value within these ranges (including the endpoints of these ranges).

[0069] In another exemplary embodiment, the modified chitosan solution comprises an oligo-chitosan modified in glucosamine monomers with a quaternary ammonium compound.

[0070] In another exemplary embodiment, the modified chitosan solution comprises oligo-chitosan modified at available sites with quaternary ammonium, phosphonium, or pyridinium groups, the percentage of available sites being about 1%, about 2%, about 3%, about 4%, about 5%, about 5 to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, 25% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, or any value within these ranges, including the endpoints of these ranges.

[0071] In another exemplary embodiment, the modified chitosan solution comprises a quaternized chitosan further modified with dopamine or an analog thereof, wherein the dopamine or analog thereof is conjugated to the modified chitosan at the remaining sites available for conjugation, the percentage of available sites (not conjugated to a quaternary ammonium, phosphonium, or pyridinium group) being about 1%, about 2%, about 3%, about 4%, about 5%, about 5 to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, 25% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50 to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, or any value within these ranges (including the endpoints of these ranges).

[0072] In another exemplary embodiment, the oligo-chitosan or modified oligo-chitosan is about 5 monomers in length, about 5 to about 10 monomers in length, about 10 to about 20 monomers in length, about 20 to about 30 monomers in length, about 30 to about 40 monomers in length, about 40 to about 50 monomers in length, about 50 to about 60 monomers in length, about 60 to about 70 monomers in length, about 70 to about 80 monomers in length, about 80 to about 90 monomers in length, 90 to about 100 monomers in length, about 100 to about 200 monomers in length, about 200 to about 300 monomers in length, about 300 to about 400 monomers in length, about 400 to about 500 monomers in length, or any value within these ranges (including the endpoints of these ranges).

[0073] In another exemplary embodiment, the method further comprises preparing a modified chitosan-containing solution.

[0074] In another exemplary embodiment, a method for preparing a modified chitosan-containing solution includes the steps of: (a) preparing an aqueous solution of chitosan in acetic acid and hydrogen peroxide; (b) heating the aqueous solution of chitosan; (c) adjusting the pH of the aqueous solution of chitosan to pH=9; and (d) recovering the oligo-chitosan product.

[0075] In another exemplary embodiment, the weight average molecular weight of the oligo-chitosan is from about 100 to about 5000. In another exemplary embodiment, the weight average molecular weight of the oligo-chitosan is from about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3100, about 3200, about 3300, about 3400, about 3500, about 3600, about 3700, about 3800, about 3900, about 4000, about 4100, about 4200, about 4300, about 4400, about 4500, about 4600, about 4700, about 4800, about 4900, about 5000, about 5100, about 5200, about 5300, about 5400, about 5500, about 5600, about 5700, about 5800, about 5900, about 6000, about 6100, about 6200, about 6300, about 6400, about 6500, about 6600, about 6700, The weight average molecular weight is 0, about 2700, about 2800, about 2900, about 3000, about 3100, about 3200, about 3300, about 3400, about 3500, about 3600, about 3700, about 3800, about 3900, about 4000, about 4100, about 4200, about 4300, about 4400, about 4500, about 4600, about 4700, about 4800, about 4900, about 5000, or any weight average molecular weight between about 100 and about 5000.

[0076] In another exemplary embodiment, the pH of the aqueous solution of chitosan is about 7.5 to about 10. In another exemplary embodiment, the pH of the aqueous solution of chitosan is about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, or any pH between about 7.5 and about 10.

[0077] In another exemplary embodiment, quaternized chitosan can be prepared by the addition of glycidyltrimethylammonium chloride to allow for permanent quaternary ammonium functionality. Alternatively, a quaternized molecule can be attached to the C-2 amine of the chitosan monomer, and alternative molecules can be attached to the same location for additional functionality; different attachment chemistries can be used, such as between the chitosan and the linker.

[0078] In another exemplary embodiment, the quaternized chitosan is further modified (also called "modified") by conjugation with dopamine or an analog thereof.

[0079] In another exemplary embodiment, quaternized chitosan conjugated with dopamine or an analog thereof is conjugated to a metal, its alloy, oxide, or nanoparticle form.

[0080] In another exemplary embodiment, dopamine or its analogue-linked chitosan or oligo-chitosan bound to metals, alloys, oxides, or nanoparticles can be quaternized by conjugation with quaternary ammonium, pyridinium, or phosphonium compounds.

[0081] In another exemplary embodiment, any of the solutions described herein may comprise a solvent selected from, but not limited to, methanol, ethanol, tetrahydrofuran (THF), dimethylformamide (DMF), and mixtures thereof.

[0082] Details of other exemplary embodiments of the present disclosure are included in the following detailed description and accompanying drawings. [Brief explanation of the drawings]

[0083] [Figure 1] FIG. 1 is an infrared spectrum of a metal surface modified with dopamine / polydopamine according to an example embodiment. [Figure 2]FIG. 2 is an infrared spectrum of a metal surface modified with dopamine / polydopamine attached by application of ultraviolet radiation. [Figure 3] FIG. 3 is an infrared spectrum of a chitosan-modified surface, according to an example embodiment. [Figure 4] FIG. 4 is an infrared spectrum of a doubly conjugated chitosan oligomer. [Figure 5] FIG. 5 shows titer data for the number of bacterial species based on the results of the experimental examples of the present application. DETAILED DESCRIPTION OF THE INVENTION

[0084] The advantages and features of the present disclosure, as well as methods for achieving the same, will be more clearly understood from the exemplary embodiments described below with reference to the accompanying drawings. However, the present disclosure is not limited to the following exemplary embodiments and can be embodied in different forms. The exemplary embodiments are provided solely for the purpose of completing the disclosure and fully conveying the scope of the present invention to those skilled in the art to which the disclosure pertains, and the present disclosure is defined by any appended claims and combinations thereof.

[0085] Shapes, sizes, ratios, angles, numbers, and the like shown in the accompanying drawings are merely examples, and the present disclosure is not limited thereto. Numerical references and the like generally indicate elements and the like throughout this specification. Furthermore, in the following description, detailed descriptions of well-known related art may be omitted to avoid unnecessarily obscuring the subject matter of the present disclosure. As used herein, terms such as "including," "having," "comprising," and "consisting of" are generally intended to allow other components to be included, unless these terms are used in conjunction with the term "only." Any reference to the singular can also include the plural, unless a statement expressly states otherwise.

[0086] Components are to be construed as including normal tolerances, even if not expressly stated. For example, with respect to the embodiments described herein, a tolerance of up to plus or minus ten percent (±10%) is considered normal tolerance.

[0087] As used herein, the singular forms "a," "an," and "the" in foreign language specifications are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the use of "or" is intended to include "and / or" unless the context clearly indicates otherwise.

[0088] When the positional relationship between two parts is described using terms such as "on," "above," "below," and "next," one or more parts may be located between the two parts, unless these terms are used in conjunction with the terms "immediately adjacent" or "directly."

[0089] When an element or layer is referred to as being "on" another element or layer, the element or layer can be directly on the other element or layer, or there can be intervening elements or layers.

[0090] Although the terms "first," "second," and the like are used to describe various components, these components (components) are not limited by these terms. These terms are used simply to distinguish one component (component) from another, and the first component (component) may be the second component (component) in the technical concept of the present disclosure.

[0091] The size and thickness of each structure illustrated in the drawings are depicted for convenience of explanation, and the drawings are not necessarily drawn to scale.

[0092] Whenever a range of numerical values ​​is given herein, it is meant to include any recited numerical value (fractional or integer) within the given range. The phrases "ranging from / between" a first given number and a second given number, and "ranging from / to" a first given number to a second given number, are used interchangeably herein and are meant to include the first given number and the second given number, and all fractional and integer numbers therebetween.

[0093] The dimensions and values ​​disclosed herein are not to be understood as being strictly limited to the exact numerical values ​​stated. Rather, unless otherwise specified, each such dimension is intended to mean both the stated value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "10%" is intended to mean "about 10%."

[0094] A number and / or numerical range preceded by the term "about" should not be considered limiting to the stated range, but rather should be understood to include the range that would be accepted by one of ordinary skill in the art for configuration in accordance with the subject invention.

[0095] The features of the various embodiments of the present disclosure can be partially or fully combined or combined with one another, can be connected and operated in various technical ways, and the embodiments can be practiced independently of one another or in conjunction with one another.

[0096] Various exemplary embodiments of the present disclosure will now be described in detail with reference to the accompanying drawings.

[0097] One exemplary embodiment is a method for preparing a modified metal surface by attaching catechol or a derivative thereof to the surface of a metal, the method comprising the steps of: preparing an aqueous or non-aqueous protic solution of catechol or a derivative thereof in a buffer or organic solvent; adjusting to a basic pH or adding a base; immersing at least a portion of a metal workpiece in the solution of catechol or a derivative thereof; applying a voltage such that the workpiece is an anode in an electrochemical circuit for a treatment time to obtain a functionalized metal workpiece; and chemically attaching modified chitosan to the functionalized metal workpiece.

[0098] One exemplary embodiment is a method for preparing a modified metal surface by attaching a doubly modified (modified) chitosan or a derivative thereof to the surface of a metal, the method comprising the steps of: preparing an aqueous or non-aqueous protic solution of chitosan or a derivative thereof in a buffer or organic solvent; adjusting to a basic pH or adding a base; immersing at least a portion of a metal workpiece in the solution of the doubly modified chitosan or a derivative thereof; and applying a voltage such that the workpiece is an anode in an electrochemical circuit during the treatment time to obtain a functionalized metal workpiece.

[0099] An exemplary embodiment is a method for preparing a modified metal surface by attaching a catechol- or derivative-modified (modified) chitosan or derivative thereof to the surface of a metal, the method comprising the steps of: preparing an aqueous or non-aqueous protic solution of the modified chitosan or derivative thereof in a buffer or organic solvent; adjusting to a basic pH or adding a base; immersing at least a portion of a metal workpiece in the solution of the modified chitosan or derivative thereof; applying a voltage such that the workpiece is an anode in an electrochemical circuit during the treatment time to obtain a functionalized metal workpiece; and chemically attaching a quaternary ammonium, pyridinium, or phosphonium molecule to obtain a functionalized metal workpiece.

[0100] Another exemplary embodiment is a method for preparing a modified metal surface by bonding catechol or a derivative thereof to the surface of a metal, the method comprising the steps of preparing an aqueous or non-aqueous protic solution of catechol or a derivative thereof in a buffer or organic solvent; adjusting to a basic pH or adding a base; spraying to cover the surface of the metal workpiece; exposing the workpiece to ultraviolet light for a treatment time to ensure chemical bonding to the workpiece to create a functionalized metal workpiece; and chemically bonding the modified chitosan to the functionalized metal workpiece.

[0101] Another exemplary embodiment is a method for preparing a modified metal surface by bonding a doubly modified (modified) chitosan or its derivative to the surface of a metal, the method comprising the steps of preparing an aqueous or non-aqueous protic solution of chitosan or its derivative in a buffer or organic solvent; adjusting to a basic pH or adding a base; spraying to cover the surface of the metal workpiece; and exposing the workpiece to ultraviolet light for a time sufficient to ensure chemical bonding to the workpiece to create a functionalized metal workpiece.

[0102] Another exemplary embodiment is a method for preparing a modified metal surface by attaching a catechol- or derivative-modified (modified) chitosan or its derivative to the surface of a metal, the method comprising the steps of preparing an aqueous or non-aqueous protic solution of the modified chitosan or its derivative in a buffer or organic solvent; adjusting to a basic pH or adding a base; spraying to cover the surface of the metal workpiece; exposing the workpiece to ultraviolet light for a period of time to ensure chemical bonding to the workpiece to create a functionalized metal workpiece; and chemically bonding a quaternary ammonium, pyridinium, or phosphonium molecule to the functionalized metal workpiece.

[0103] Another exemplary embodiment is a method for preparing a modified metal surface by attaching a modified chitosan or a derivative thereof to the surface of a metal, the method comprising the steps of: preparing an aqueous or non-aqueous solution of a doubly modified chitosan having a quaternary ammonium, pyridinium, or phosphonium compound and a catechol or a derivative thereof attached thereto; spraying the solution onto the metal surface so as to cover it; and baking in an oven for a time and at a temperature sufficient to cause chemical bonding of the quaternized chitosan conjugate to the surface.

[0104] Another exemplary embodiment is a method for preparing a modified metal surface by attaching a modified chitosan or a derivative thereof to the surface of a metal, the method comprising the steps of: preparing an aqueous or non-aqueous solution of a doubly modified chitosan having a quaternary ammonium, pyridinium, or phosphonium compound and a catechol or a derivative thereof attached thereto; spraying the solution onto the metal surface so as to cover it; and exposing the surface to ultraviolet light for a time sufficient to cause the quaternized chitosan conjugate to chemically bond to the surface.

[0105] Another exemplary embodiment is a method for preparing a modified metal surface by bonding a modified chitosan or a derivative thereof to the surface of a metal, the method comprising the steps of: preparing an aqueous or non-aqueous solution of a doubly modified chitosan having attached thereto a quaternary ammonium, pyridinium, or phosphonium compound, and a catechol or a derivative thereof; immersing a metal workpiece in the solution; placing it in contact with the anode of an electrochemical cell; and impregnating the cathode and applying a voltage sufficient to achieve chemical bonding of the chitosan conjugate to the metal workpiece.

[0106] In another exemplary embodiment, catechol or a derivative thereof is covalently bonded to the surface of a functionalized metal workpiece.

[0107] In another exemplary embodiment, the workpiece functionalized with catechol or its derivatives is activated by the addition of coupling agents known to those skilled in the art to achieve carbon-carbon bond formation, carbon-oxygen bond formation, carbon-sulfur bond formation, carbon-nitrogen bond formation, carbon-phosphorus bond formation, carbon-silicon bond formation, etc., and to achieve the attachment or immobilization of further functional chemical entities, such as modified and unmodified chitosan oligomers.

[0108] In another exemplary embodiment, the fully formed chitosan conjugate is attached to the surface of a metal, alloy, oxide, or nanoparticle.

[0109] In another exemplary embodiment, the catechol or derivative thereof includes any substituted or unsubstituted catechol, which can be represented by the following formula: [ka] wherein each of R1, R2, R3, R4, and R5 is independently selected from the group consisting of thiol, primary amine, secondary amine, nitrile, aldehyde, imidazole, azide, halide, polyhexamethylene dithiocarbonate, hydrogen, hydroxyl, carboxylic acid, carboxylic acid ester, or carboxamide, with the proviso that at least one of R1, R2, R3, R4, and R5 is not a hydrogen atom, a ranges from 0 to 10, and b ranges from 0 to 10, with the proviso that a or b is at least 1. Catechol or its derivatives include, but are not limited to, catecholamines, phenolic aldehydes, tyrosol and its derivatives, including hydroxyl, methoxy, and mixed methoxy-hydroxy derivatives of tyrosol, hydroxylated tyramine, tyramine and its derivatives, including dopamine and its derivatives, and the like. In another exemplary embodiment, other hydroxy-containing compounds, including, but not limited to, substituted or unsubstituted phenols, may be used as linkers. In another exemplary embodiment, the catechol or its derivative is dopamine or polydopamine. In another exemplary embodiment, the dopamine derivative may be a methoxy derivative, a 4-ethanol derivative, or the like.

[0110] In another exemplary embodiment, the metal is a non-ferrous metal, including its respective oxide, alloy, and nanoparticle forms. Metals include, but are not limited to, titanium and its oxides and alloys, aluminum and its oxides and alloys, vanadium and its oxides and alloys, zirconium and its oxides and alloys, hafnium and its oxides and alloys, niobium and its oxides and alloys, tantalum and its oxides and alloys, tungsten and its oxides and alloys, magnesium and its oxides and alloys, stainless steel, cobalt chrome, etc. However, the materials to which catechol or its derivatives are bound are not limited and include polymers, biological polymers, ceramics, etc.

[0111] In another exemplary embodiment, the concentration of the catechol or derivative thereof in the solution is about 0.1% to about 20% by weight, or about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, 19%, about 20%, or any percentage between about 0.1% and about 20%.

[0112] In another exemplary embodiment, the applied voltage can range from about 1 V to about 100 V. In another exemplary embodiment, the applied voltage can range from about 1 V, about 2 V, about 3 V, about 4 V, about 5 V, about 6 V, about 7 V, about 8 V, about 9 V, about 10 V, about 11 V, about 12 V, about 13 V, about 14 V, about 15 V, about 16 V, about 17 V, about 18 V, about 19 V, about 20 V, about 21 V, about 22 V, about 23 V, about 24 V, about 25 V, about 26 V, about 27 V, about 28 V, about 29 V, about 30 V, about 31 V, about 32 V, about 33 V, about 34 V, about 35 V, about 36 V, about 37 V, about 38 V, about 39 V, about 40 V, about 41 V, about 42 V, about 43 V, about 44 V, about 45 V, about 46 V, about 47 V, about 48 V, about 49 V, about 50 V, about 51 V, about 52 V, about 53 V, about 54 V, about 55 V, about 56 V, about 57 V, about 58 V, about 59 V, about 60 V, about 61 V, about 62 V, about 63 V, about 64 V, about 65 V, about 66 V, about 67 V, about 68 V, about 69 V, about 70 V, about 71 V, about 72 V, about 73 V, about 74 V, about 75 V, about 76 V, about 77 V, about 78 V, about 79 V, about 80 V, about 81 V, 5V, about 26V, about 27V, about 28V, about 29V, about 30V, about 31V, about 32V, about 33V, about 34V, about 35V, about 36V, about 37V, about 38V , about 39V, about 40V, about 41V, about 42V, about 43V, about 44V, about 45V, about 46V, about 47V, about 48V, about 49V, about 50V, about 51V, about 5 2V, approximately 53V, approximately 54V, approximately 55V, approximately 56V, approximately 57V, approximately 58V, approximately 59V, approximately 60V, approximately 61V, approximately 62V, approximately 63V, approximately 64V, approximately 65V , about 66V, about 67V, about 68V, about 69V, about 70V, about 71V, about 72V, about 73V, about 74V, about 75V, about 76V, about 77V, about 78V, about It can be 79V, about 80V, about 81V, about 82V, about 83V, about 84V, about 85V, about 86V, about 87V, about 88V, about 89V, about 90V, about 91V, about 92V, about 93V, about 94V, about 95V, about 96V, about 97V, about 98V, about 99V, about 100V, or any voltage between about 1V and about 100V.

[0113] In another exemplary embodiment, the voltage is applied for a duration of about 1 second to about 60 minutes, or about 1 second, about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 60 seconds, about 65 seconds, about 70 seconds, about 75 seconds, about 80 seconds, about 85 seconds, about 90 seconds, about 95 seconds, about 100 seconds, about 105 seconds, about 110 seconds, about 115 seconds, about 120 seconds, about 130 seconds, about 140 seconds, about 150 seconds, about 160 seconds, about 170 seconds, about 180 seconds, about 190 seconds, about 200 seconds, about 210 seconds, about 220 seconds, about 230 seconds, about 240 seconds, about 250 seconds, about 260 seconds, about 270 seconds, about 280 seconds, about 290 seconds, about 300 seconds, about 310 seconds, about 320 seconds, about 330 seconds, about 340 seconds, about 350 seconds, about 360 seconds, about 370 seconds, about 380 seconds, about 390 seconds, about 400 seconds, about 410 seconds, about 420 seconds, about 430 seconds, about 440 seconds, about 450 seconds, about 460 seconds, about 470 seconds, about 480 seconds, about 490 seconds, about 500 seconds, about 510 seconds, about 520 seconds, about 530 seconds, about 540 seconds, about 550 seconds, about 560 seconds, about 570 seconds, about 580 seconds, about 590 seconds, about 60 seconds, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or any time between about 1 second and about 60 minutes.

[0114] In other exemplary embodiments, the pH of the buffer is about 7.5, about 8.0, about 8.5, about 9, about 9.5, or any pH between about 7.5 and about 9.5.

[0115] In another exemplary embodiment, the method further comprises cleaning and drying the functionalized surface.

[0116] In another exemplary embodiment, the method further comprises drying the functionalized surface under vacuum or under an inert or non-reactive atmosphere, hi another exemplary embodiment, the inert atmosphere is nitrogen or argon.

[0117] In another exemplary embodiment, the functionalized metal workpiece in the imidazole solution is stirred for about 1 hour to about 10 hours, hi another exemplary embodiment, the stirring is carried out for about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours, about 10 hours, or any duration between about 1 hour and 10 hours.

[0118] In another exemplary embodiment, the solution of modified chitosan comprises oligo-chitosan.

[0119] In another exemplary embodiment, oligo-chitosan can be modified at the C-2 amine of its monomer unit by the addition of a quaternary ammonium, pyridinium or phosphonium compound, and the modified chitosan can be further modified at the C-6 hydroxyl, C-3 hydroxyl or C-2 amine by the attachment of dopamine (polydopamine) or its analogs, through which it is immobilized on a surface.

[0120] In another embodiment, the solution of modified chitosan comprises an oligo-chitosan modified in glucosamine monomers with a quaternary phosphonium compound.

[0121] In another embodiment, the modified chitosan solution comprises oligo-chitosan modified (modified) at available sites with a quaternary phosphonium conjugate, the degree of conjugation being about 1%, about 2%, about 3%, about 4%, about 5%, about 5 to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, 25% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, or any value within these ranges, inclusive of the endpoints of these ranges.

[0122] In another embodiment, the solution of modified chitosan comprises oligo-chitosan modified in glucosamine monomers with quaternary ammonium, phosphonium, or pyridinium compounds.

[0123] In another embodiment, the modified chitosan solution comprises oligo-chitosan modified (modified) at available sites with quaternary ammonium, phosphonium, or pyridinium conjugates to the extent of about 1%, about 2%, about 3%, about 4%, about 5%, about 5 to about 10%, about 10 to about 15%, about 15 to about 20%, about 20 to about 25%, 25 to about 30%, about 30 to about 40%, about 40 to about 50%, about 50 to about 60%, about 60 to 70%, about 70 to about 80%, about 80 to about 90%, about 90 to about 100%, or any value within these ranges, including the endpoints of these ranges.

[0124] In another embodiment, the oligo-chitosan or modified oligo-chitosan is about 5 monomers in length, about 5 to about 10 monomers in length, about 10 to about 20 monomers in length, about 20 to about 30 monomers in length, about 30 to about 40 monomers in length, about 40 to about 50 monomers in length, about 50 to about 60 monomers in length, about 60 to about 70 monomers in length, about 70 to about 80 monomers in length, about 80 to about 90 monomers in length, about 90 to about 100 monomers in length, about 100 to about 200 monomers in length, about 200 to about 300 monomers in length, about 300 to about 400 monomers in length, about 400 to about 500 monomers in length, or any value within these ranges (including the endpoints of these ranges).

[0125] In another exemplary embodiment, the method further comprises preparing a solution containing modified chitosan.

[0126] In another exemplary embodiment, a method for preparing a modified chitosan-containing solution includes the steps of: (a) preparing an aqueous solution of chitosan in acetic acid and hydrogen peroxide; (b) heating the aqueous chitosan solution; (c) adjusting the pH of the aqueous chitosan solution to pH=9; and (d) recovering the oligo-chitosan product.

[0127] In another exemplary embodiment, the weight average molecular weight of the oligo-chitosan is from about 100 to about 5000. In another exemplary embodiment, the weight average molecular weight of the oligo-chitosan is from about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3100, about 3200, about 3300, about 3400, about 3500, about 3600, about 3700, about 3800, about 3900, about 4000, about 4100, about 4200, about 4300, about 4400, about 4500, about 4600, about 4700, about 4800, about 4900, about 5000, about 5100, about 5200, about 5300, about 5400, about 5500, about 5600, about 5700, about 5800, about 5900, about 6000, about 6100, about 6200, about 6300, about 6400, about 6500, about 6600, about 6700, The weight average molecular weight is 0, about 2700, about 2800, about 2900, about 3000, about 3100, about 3200, about 3300, about 3400, about 3500, about 3600, about 3700, about 3800, about 3900, about 4000, about 4100, about 4200, about 4300, about 4400, about 4500, about 4600, about 4700, about 4800, about 4900, about 5000, or any weight average molecular weight between about 100 and about 5000.

[0128] In another exemplary embodiment, the pH of the aqueous solution of chitosan is about 7.5 to about 10. In another exemplary embodiment, the pH of the aqueous solution of chitosan is about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, or any pH between about 7.5 and about 10.

[0129] In another exemplary embodiment, quaternized chitosan can be prepared by the addition of glycidyltrimethylammonium chloride to allow permanent quaternary ammonium functionality. Alternative quaternized molecules can be attached to the C-2 amine, C-3 hydroxyl, or C-6 hydroxyl of chitosan monomers; alternative molecules can be attached to the same sites for additional functionality; different attachment chemistries can be used to immobilize modified oligo-chitosans to functionalized surfaces, etc.

[0130] In another exemplary embodiment, any of the solutions described herein may include a solvent selected from, but not limited to, methanol, ethanol, tetrahydrofuran (THF), dimethylformamide (DMF), and mixtures thereof.

[0131] Catechol is a phenol derivative containing an additional hydroxyl group and has the following chemical structure: [ka]

[0132] Dopamine is a substituted catechol with an ethylamine group as a substituent and has the following chemical structure: [ka] It has.

[0133] Catechol and catechol derivatives, such as dopamine, bind to metal surfaces by forming covalent bonds with the ring oxygen. The pendant amine branches of dopamine can then participate in dopamine polymerization to form polydopamine on the surface, stabilizing the bound molecule. Several amines are available for further reaction with desired molecules to impart surface functionality. The utility of such molecules is that they can contain functional groups that can be further modified. This allows for the incorporation of various chemistries onto the distal ends of these molecules. Therefore, the covalent attachment of these molecules followed by the incorporation of tailored chemistries is equivalent to the covalent attachment of the aforementioned chemistries onto the surface.

[0134] Such functional groups include, but are not limited to, substituted or unsubstituted hydroxyl, substituted or unsubstituted amine, substituted or unsubstituted ester, substituted or unsubstituted thiol, substituted or unsubstituted ether, substituted or unsubstituted phosphonate, substituted or unsubstituted phosphinate, substituted or unsubstituted phosphate, substituted or unsubstituted carboxylate, substituted or unsubstituted acrylate, substituted or unsubstituted sulfonate, substituted or unsubstituted sulfinate, substituted or unsubstituted sulfoxide, substituted or unsubstituted silane, substituted or unsubstituted siloxane, substituted or unsubstituted polydimethylsiloxane (PDMS), substituted or unsubstituted amino ester, substituted or unsubstituted peptide, both natural and synthetic, substituted or unsubstituted oligopeptide, protein, nucleotide, oligonucleotide, polynucleotide, substituted or unsubstituted sugar, substituted or unsubstituted polysaccharide, etc., and these groups may be further modified (modified). The functional group may also be an ester, such as methacrylate, urethane, urea, alkyl halide, etc.

[0135] Manipulating the chemical presentation of a surface to the environment alters the behavior of that surface when compared to an untreated surface. For example, surfaces can be made to be hydrophobic, hydrophilic, oleophobic, lubricious, antibacterial, etc. Surfaces can also be made to either enhance or diminish adhesive effectiveness. Additionally, proteins and protein fractions, amino acids (both natural and synthetic), and peptides can be attached to enhance or diminish desired physical, chemical, or biological responses.

[0136] As described in the exemplary embodiments of this application, successful surface modification depends on the choice of linker and conjugation chemistry. While several conjugation strategies for both metallic and non-metallic surfaces have been developed, the focus has been on the use of modified catechol, primary dopamine, or 3,4-dihydroxyhydrocinnamic acid as the linker of choice. Catechol or its derivatives are attractive because of the ubiquity of the surfaces to which it can be applied and its ease of further modification.

[0137] In some embodiments, dopamine or polydopamine is covalently attached to a metal surface, leaving the amine moieties of the dopamine molecules available on the surface for further functionalization. Urethane or urea formation was chosen as the coupling chemistry for attaching modified chitosan to polydopamine. While many of the amines attached to the dopamine monomers participate in dopamine polymerization, a sufficient number remain available for further surface modification. In the studies described herein, carbonyldiimidazole (CDI) in methylene chloride was chosen for the activation chemistry, allowing the activated amines of dopamine to couple with the C-6 hydroxyl or unreacted C-2 amine of modified chitosan. Alternatively, more environmentally friendly coupling chemistries can be used.

[0138] The final step in this method is the attachment of modified chitosan to the CDI-activated polydopamine surface. Modified chitosan (HTCC) is coupled to the CDI-activated polydopamine surface, imparting antibacterial properties to the surface. The most amenable attachment site has been shown to be the C-2 amine, which was not quaternized in the formation of HTCC. This completes the construction of a uniquely structured antibacterial surface.

[0139] A first schematic representation of the modification method described herein is as follows.

[0140] [ka]

[0141] In the above illustration, n represents the number of quaternized monomer units, from about 1 to about 500, and m represents the number of remaining deacetylated, non-quaternized monomer units bound to the dopamine surface, from about 1 to about 500.

[0142] The first schematic representation above is provided for clarity and should not be construed as being limited to quaternized positions alternating with dopamine attachment points in a regular pattern, but may include different chemical environments of the C-2 amine of the chitosan monomers randomly distributed between the quaternized, dopamine-bound, acetylated, and deacetylated sites.

[0143] The second schematic representation further illustrates another exemplary embodiment of the present application.

[0144] [ka]

[0145] In the above schematic representation, n represents the number of quaternized monomer units, from about 1 to about 500; m represents the number of non-quaternized deacetylated monomer units available for conjugation, from about 1 to about 500; and q represents the number of catechol-derived monomer units, from about 1 to about 500.

[0146] The second schematic representation above is provided for clarity and should not be construed as being limited to quaternized positions alternating with cinnamic acid attachment points in a regular pattern, but may include various chemical environments of the C-2 amine of the chitosan monomers randomly distributed between the acetylated and deacetylated sites to which the quaternized cinnamic acid is attached.

[0147] In this example, the fully formed doubly conjugated chitosan is synthesized prior to immobilization onto the metal surface. The catechol-pendant HTCC is isolated and can be stored until required addition for convenience of this method. HTCC can be synthesized as described above and in the literature. The second conjugate is prepared by coupling HTCC to 3,4-dihydroxyhydrocinnamic acid via well-known EDC / NHS coupling to form an amide bond.

[0148] Immobilization on the metal surface can be achieved by the methods previously described, namely baking, anodization, or photografting.

[0149] The prepared workpieces are then analyzed using standard analytical techniques, including but not limited to infrared (IR) spectroscopy, contact angle, X-ray photoelectron spectroscopy (XPS), energy dispersive X-ray spectroscopy (EDX), atomic force microscopy (AFM), time-of-flight secondary ion mass spectroscopy (Tof-SIMS), coefficient of friction (COF) testing, and combinations thereof. [Example]

[0150] The functionalization of metal surfaces using dopamine as a linking group is illustrated in the following examples. The methods described herein result in highly potent surfaces that produce greater than 5 log kill relative to controls for several organisms while maintaining excellent biocompatibility.

[0151] <Example 1: Modification of metal surfaces with dopamine> A 1% by weight aqueous solution of dopamine in pH 8.5 phosphate buffer was prepared, and clean, commercially pure (CP) titanium pieces were immersed in the buffer. Each piece was placed in contact with the anode end of a DC power supply. A voltage of 10 V was applied for 5 minutes. The pieces were then removed and sonicated in water for 15 minutes. The sonication process was repeated, followed by rinsing the pieces in copious amounts of water. This was followed by further sonication in ethanol for 5 minutes, followed by rinsing in ethanol. The pieces were dried, and the surfaces were examined using infrared (IR) spectroscopy. The IR spectrum of the surface modified using the above method is shown in Figure 1.

[0152] The spectrum in Figure 1 shows the peak at approximately 1600 cm -1 and 1490 cm -1 It shows a characteristic broad peak centered around 1428 cm, which indicates a combination of C=C and N=C stretching vibrations. The broad peak indicates a vibrating species that exists in many different chemical environments, indicative of a polymer. -1 and approximately 1268 cm -1 The peaks at indicate C=C and CNC functional groups.

[0153] Example 2: Photografting of dopamine onto titanium surface A solution of dopamine (10 mg / ml) in phosphate buffer (pH = 8.5) was prepared. The solution was sprayed onto the surface of a clean, dry titanium coupon, covering the surface. The coupon was then exposed to UV radiation (lambda max = 254 nm) for 5 minutes, followed by two additional applications and exposures of the sprayed solution. The treated coupons were then rinsed and sonicated in water for 15 minutes, followed by an EtOH rinse. They were then dried under argon. The infrared spectrum of the treated coupon is shown in Figure 2 and confirmed the presence of polydopamine on the surface.

[0154] The spectrum in Figure 2 is at approximately 1600 cm -1 and 1490 cm -1 It shows a characteristic broad peak centered around 1443 cm, which indicates a combination of C=C and N=C stretching vibrations. The broad peak indicates a vibrating species that exists in many different chemical environments, indicative of a polymer. -1 The peaks at indicate C=C and CNC functional groups.

[0155] Example 3: CDI activation of dopamine-modified metal surfaces The dopamine-bound metal coupon was immersed in a solution of 250 ml of CHCl and 3 g of CDI and stirred at room temperature for 4 hours at a stirring speed of 400-500 rpm. The coupon was removed from the CHCl / CDI solution, rinsed with acetone, and then sonicated in acetone for 10 minutes. The rinsing process was repeated. The coupon was then dried under vacuum.

[0156] <Example 4: Bonding of chitosan oligomer> The final step in this method is the attachment of modified chitosan to the CDI-activated polydopamine surface. Chitosan (average M W Modified chitosan (N-4 or HTCC) was prepared in-house by depolymerizing chitosan (N-4 or HTCC) at pH 50,000 (dd≧90%). The depolymerization product was precipitated from a solution at pH 9, washed, and isolated. The isolated product had an average M of approximately 2,000.W The isolated oligo-chitosan was quaternized by reaction between the C-2 amine of the glucosamine monomer and glycidyltrimethylammonium chloride to give a modified chitosan product, which was then coupled to a CDI-activated polydopamine surface.

[0157] A 1% by weight solution of N-4 in DMSO was prepared. Pieces with CDI-activated polydopamine surfaces were placed flat in an array, sprayed with the N-4 / DMSO solution, and heated at 100°C for approximately 10 minutes. The pieces were then turned over and the process repeated. The entire process was repeated at least two spray / heat cycles for each side. After the final spraying step, the modified pieces were heated at approximately 100°C for approximately 4 hours, followed by sonication in ethanol for 10 minutes. The properties of the surface after the attachment of modified chitosan were confirmed by infrared (IR) spectroscopy. The resulting IR spectrum is shown in Figure 3.

[0158] The spectrum in Figure 3 shows many of the peaks characteristic of chitosan and oligo-chitosan. -1 The broad peak around indicates the N-H and O-H stretching, and the C-H stretching is at 2869 cm -1 The remaining acetyl carbonyl stretch is at 1662 cm -1 and 1376 cm -1 It is shown in 1476cm -1 indicates the bending angle of the methyl group at the center of the quaternary ammonium. Another characteristic peak is at 1151 cm -1 and 1085cm -1 These correspond to the COC stretching and CO stretching, respectively.

[0159] Example 5: Formation of doubly conjugated chitosan 3,4-Dihydroxyhydrocinnamic acid (1 mmol) was dissolved in anhydrous DMSO. Next, EDC (1-ethyl-3-(dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were added to the solution in molar ratios of 1:1 and 1:2, respectively, relative to the carboxyl groups of dihydroxyhydrocinnamic acid (EDC and NHS were allowed to equilibrate to room temperature before opening). The mixture was stirred at room temperature for 3 hours. The mixture was then added dropwise to a 1% solution of quaternized chitosan (N-4 or HTCC) (w / v, 300 mg / 30 mL DMSO) with stirring. The reaction mixture was stirred overnight at room temperature in the dark. At the end of the reaction, the brown precipitate was separated by centrifugation, washed three times with acetone and DMSO, dialyzed against water using dialysis tubing (MWCO = 1 kDa, Biodesign, New York), and lyophilized. The solution was washed with acetone. Dopamine-quaternized chitosan (HyCnQ) was dried under vacuum at 25°C for 72 hours. 1 The results were confirmed by H-NMR (Bruker AC500 Spectrophotometer, Germany) and Fourier transform infrared (FT-IR) spectroscopy.

[0160] Figure 4 shows the characteristic IR spectrum of HyCnQ oligomer. -1 The broad peak around 2900 cm corresponds to the OH stretching of the many hydroxyls present. -1 The peak centered around 1733 cm corresponds to the CH stretching. The cinnamic acid bond to form an amide is observed at 1733 cm -1 This is evidenced by the presence of the C=O stretch at 1671 cm -1 Bending vibration of secondary amines at 1530cm -1 Aromatic C=C stretching at 1488cm -1 There is a methyl bending vibration peak indicating a quaternary center at

[0161] <Example 6: Surface binding of HyCnQ> A solution of dopamine-quaternized chitosan (HyCnQ) (0.1 g / 10 mL DMSO) was sprayed once onto a metal coupon (approximately 0.001 g). The coupon was placed horizontally on a plate to improve the uniformity of the coating thickness and then reacted in an oven at 100°C for 20 minutes. The coupon was removed from the oven, sprayed once more, and heated in a vacuum for an additional 4 hours. In the case of metal coupons, each side (both sides) was treated as described above. The coupon was sonicated in EtOH for 5 minutes and dried in a vacuum.

[0162] <Test example> The antibacterial properties of the surface-bound chitosan conjugates were characterized by the following test examples.

[0163] Preparation of bacterial inoculum: 10 mL of tryptic soy broth (TSB) was inoculated with a single colony of the test organism and incubated overnight at 35±2°C in a shaking incubator at 250 rpm. The next day, the overnight culture was subcultured into 3 mL of TSB and incubated at 35±2°C and 250 rpm until the optical density at 600 nm reached between 0.5 and 0.7. The subculture was diluted to an OD600 of 0.3 with sterile TSB and then further diluted to an OD600 of 0.01 with sterile equilibration buffer (0.3 mM KH2PO4, pH 7.2).

[0164] Test Specimen (Coupon) Preparation: Test specimens were prepared as described in Examples 1 to 4. Control and test specimens were sterilized in 70% EtOH for 10 minutes, dried in a laminar flow hood for 10 minutes, and transferred to wells of a 24-well plate.

[0165] <Experimental Example 1 - ASTM E2149 modified for small volume> Control and test specimens were incubated with 500 μL of bacterial inoculum at 35±2° C. and 250 rpm for 1 hour. Bacterial concentrations were determined by performing serial dilutions with equilibration buffer and using the Miles and Misra method to determine the number of colony-forming units (CFU) per milliliter. All tests were performed in triplicate, and the log reduction in mean CFU / mL was determined by the following formula: Log reduction = log10 (control CFU / mL) - log10 (test CFU / mL).

[0166] Table 1 lists the titers of treated surfaces versus controls against Staph aureus (MSSA). As shown in Table 1, in the ASTM 2149 test described above, untreated specimens had an average titer of 2.3 x 10 6 CFU / ml and for MSSA, treated specimens returned to zero bacteria, a 5+ log reduction or better than 99.999%.

[0167] [Table 1]

[0168] Figure 5 shows the titer of conjugated Dopamine-N4 against a bacterial panel. MST20-001 was assayed against a variety of bacterial species using the same method outlined in Example 1. The various species assayed against MST20-001 are listed on the left of Figure 5, with the scale below tracking the log reduction relative to the control (untreated specimens). For example, in the example above, the treated sample returned zero bacteria compared to 2.34E+6 cfu / ml in the control. Taking the initial inoculum into account, this translates to approximately 5 logs of kill, which can be attributed to the treated specimens. These data correspond to the bar (for S. aureus) across the top of the graph. The arrow to the right of the data bar indicates that zero bacteria were returned by the treated sample, and therefore the treatment titer was at least 5 logs.

[0169] In a similar manner, MRSA, S. epidermidis, and L. monocytogenes all returned zero bacteria in 2,149 evaluations. For E. faecalis, titers were less than 2 logs, or between 90 and 99 percent effective. E. faecium was reduced by more than 3 logs, or 99.9%, while P. aeruginosa and S. enterica were reduced by 99.9%, or 3 logs.

[0170] <Experimental Example 2: Autoclave Test> The control and test specimens were placed in Chex-All II instant-seal sterilization pouches and autoclaved at 121°C for 30 minutes (Model HA-300MII, Hirayama Seisakusho Co., Ltd., Tokyo, Japan). After each cycle, specimens destined for more cycles were allowed to cool for approximately 10 minutes before being transferred to new sterilization pouches. The ASTM E2149 protocol, previously described, was followed. The results are shown in Table 2, which lists sterilization test data based on the results of the experimental examples in this application.

[0171] [Table 2]

[0172] As can be seen from the data in Tables 1 and 2, there was no decrease in potency between the non-autoclaved specimens (top) and the autoclaved specimens, demonstrating good stability of the coating.

[0173] Test Specimen Preparation: Test specimens were prepared as described in Examples 5-6. Control and test specimens were sterilized in 70% EtOH for 10 minutes, dried in a laminar flow hood for 10 minutes, and transferred to wells of a 24-well plate.

[0174] <Experimental Example 3 - ASTM E2149 modified for small volume> Control and immobilized HyCnQ specimens were incubated with 500 μL of bacterial inoculum at 35±2°C and 250 rpm for 1 hour. Bacterial concentrations were determined by performing serial dilutions with balanced buffer and using the Miles and Misra method to determine the number of colony-forming units (CFU) per milliliter. All tests were performed in triplicate, and the log reduction in mean CFU / mL was determined by the following formula: Log reduction = log10 (control CFU / mL) - log10 (test CFU / mL).

[0175] Table 3 lists the titers of treated surfaces versus controls against Staph aureus. As shown in Table 1, in ASTM 2149, described above, untreated specimens had an average of 2.3 x 10 6 CFU / ml returned, and specimens treated against MSSA returned to zero bacteria, a 6+ log reduction, or better than 99.9999%.

[0176] [Table 3]

[0177] Although exemplary embodiments of the present disclosure have been described in detail with reference to the accompanying examples and drawings, the present disclosure is not limited thereto and can be embodied in many different forms without departing from the technical concept of the present disclosure. Therefore, the exemplary embodiments of the present disclosure are provided for illustrative purposes only and are not intended to limit the technical concept of the present disclosure. The scope of protection of the present disclosure should be interpreted based on the appended claims and combinations thereof, and all technical concepts within the scope of their equivalents should be construed as falling within the scope of the present disclosure. Since various modifications can be made in the above methods and compositions without departing from the scope of the present invention, it is intended that all matters contained in the above description be interpreted as illustrative and not limiting. Other embodiments within the scope of the claims herein will be apparent to those skilled in the art from consideration of the specification or practice of the invention disclosed herein. It is intended that the specification be considered to be exemplary only, with the scope and spirit of the invention being indicated by the claims.

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Claims

1. A composition comprising a metal substrate and a chitosan polymer or oligomer having the general formula: 【Chemical 1】 (wherein n is 1 to 500, each of R1, R2, and R3 is independently selected from the group consisting of thiol, alkane, alkene, alkyne, secondary amine, nitrile, aldehyde, imidazole, azide, halide, polyhexamethylene dithiocarbonate, hydrogen, hydroxyl, carboxylic acid, carboxylic ester, carboxamide, urea, catechol, quaternary ammonium, quaternary phosphonium, and quaternary pyridinium, and combinations thereof; 1 to 99% of R1, R2, and R3 are selected from quaternary ammonium groups, quaternary phosphonium groups, and quaternary pyridinium groups; and At least one of R1, R2, and R3 is bound to a compound containing a substituted or unsubstituted catechol functional group that is bound to the surface of the metal substrate by forming a covalent bond with the ring oxygen of the catechol functional group; The compound containing the substituted or unsubstituted catechol functional group is dopamine or a derivative thereof.

2. The composition of claim 1, wherein the quaternary ammonium group is a 2-hydroxypropyltrimethylammonium group.

3. 10. The composition of claim 1, wherein the substrate comprises at least one selected from the group consisting of titanium, hafnium, cobalt chrome, stainless steel, iron, copper, zinc, aluminum, tantalum, zirconium, silicon, gold, and silver, and alloys and oxides thereof.

4. The composition of claim 3 , wherein the substrate comprises titanium, aluminum, cobalt chromium, or an alloy thereof.

5. The composition of claim 3 , wherein the substrate surface comprises a metal oxide having substituted or unsubstituted catechols attached thereto.

6. The composition of claim 3 wherein the substrate surface is stainless steel.

7. The composition of claim 3 wherein the substrate surface is titanium.

8. The composition of claim 3 wherein the substrate surface is cobalt chrome.

9. The composition of claim 3 , wherein the substrate surface is a surface of a medical device.

10. At least one of the R1 groups in the polymer or oligomer is dopamine or a derivative thereof that is bound to the surface of the metal substrate by a covalent bond to a ring oxygen of a catechol functional group; 2. The composition of claim 1, wherein the dopamine or derivative thereof is attached to the chitosan polymer or oligomer via an -NH-C(O)- group which combines with an -NH- group attached between R1 and the chitosan pyranose ring to form an -NH-C(O)-NH- group.

11. 10. A method for producing the composition of claim 1, comprising the steps of: a. reacting a quaternized chitosan polymer or oligomer with a substituted or unsubstituted catechol-containing compound, wherein said compound is dopamine or a derivative thereof, to obtain a catechol-modified doubly conjugated chitosan; b. Immersing at least a portion of a metal substrate in a non-aqueous protic solution of catechol-modified doubly conjugated chitosan; and c. Attaching catechol-modified doubly conjugated chitosan to the surface of the metal substrate; A method comprising:

12. 12. The method of claim 11, wherein the non-aqueous protic solvent of the non-aqueous protic solution is methanol or ethanol.

13. The method of claim 11 , wherein step c comprises heating, exposure to UV radiation, or anodizing.

14. 12. The method of claim 11, wherein the quaternary ammonium group is a 2-hydroxypropyltrimethylammonium group.

15. 12. The method of claim 11, wherein the substrate comprises at least one selected from the group consisting of titanium, hafnium, cobalt chrome, stainless steel, iron, copper, zinc, aluminum, tantalum, zirconium, silicon, gold, and silver, and alloys and oxides thereof.

16. The method of claim 15 , wherein the substrate comprises titanium, aluminum, cobalt chromium, or an alloy thereof.

17. The method of claim 15 , wherein the substrate surface comprises a metal oxide having substituted or unsubstituted catechols attached thereto.

18. A composition comprising a metal substrate and a doubly conjugated chitosan polymer or oligomer having the following formula (I): 【Chemistry 2】 wherein each of R 3 and R 4 is independently selected from the group consisting of an alkane, an alkene, an alkyne, a secondary amine, a nitrile, an aldehyde, an imidazole, an azide, a halide, polyhexamethylene dithiocarbonate, hydrogen, a hydroxyl, a carboxylic acid, a carboxylic acid ester, an acetyl, a carboxamide, a urea, and a thiol; each of R 1 and R 5 is independently selected from the group consisting of quaternary ammonium, quaternary phosphonium, quaternary pyridinium, alkane, alkene, alkyne, secondary amine, nitrile, aldehyde, imidazole, azide, halide, polyhexamethylene dithiocarbonate, hydrogen, hydroxyl, carboxylic acid, carboxylic ester, acetyl, carboxamide, urea, and thiol; each of R 2 and R 6 is independently selected from the group consisting of catechol or a derivative or analog thereof, alkane, alkene, alkyne, secondary amine, nitrile, aldehyde, imidazole, azide, halide, polyhexamethylene dithiocarbonate, hydrogen, hydroxyl, carboxylic acid, carboxylic acid ester, acetyl, carboxamide, urea, and thiol; At least one of R 1 and R 5 is selected from the group consisting of quaternary ammonium, quaternary phosphonium, and quaternary pyridinium; At least one of R 2 and R 6 is catechol or a derivative or analog thereof; The degree of quaternization of the doubly conjugated chitosan polymer or oligomer is 1% to 99%; the catechol or its derivative or analogue comprises dopamine or its derivative; n represents the number of monomer units available for conjugation and is 1 to 500; m represents the number of monomer units available for the second conjugate and is 1 to 500; The doubly conjugated chitosan polymer or oligomer is attached to the surface of the metal substrate by forming a covalent bond with the ring oxygen of the catechol or its derivative or analog.

19. A composition comprising a metal substrate and a doubly conjugated chitosan polymer or oligomer having formula (II): 【Chemistry 3】 wherein each of R 3 and R 4 is independently selected from the group consisting of an alkane, an alkene, an alkyne, a secondary amine, a nitrile, an aldehyde, an imidazole, an azide, a halide, polyhexamethylene dithiocarbonate, hydrogen, a hydroxyl, a carboxylic acid, a carboxylic acid ester, an acetyl, a carboxamide, a urea, and a thiol; each of R 1 and R 2 is independently selected from the group consisting of catechol or a derivative or analog thereof, quaternary ammonium, quaternary phosphonium, quaternary pyridinium, alkane, alkene, alkyne, secondary amine, nitrile, aldehyde, imidazole, azide, halide, polyhexamethylene dithiocarbonate, hydrogen, hydroxyl, carboxylic acid, carboxylic ester, acetyl, carboxamide, urea, and thiol; At least one of R 1 and R 2 is catechol or a derivative or analog thereof; The degree of quaternization of the doubly conjugated chitosan polymer or oligomer is 1% to 99%; the catechol or its derivative or analogue comprises dopamine or its derivative; n represents the number of monomer units available for conjugation and is 1 to 500; The doubly conjugated chitosan polymer or oligomer is attached to the surface of the metal substrate by forming a covalent bond with the ring oxygen of the catechol or its derivative or analog.

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