Nitric oxide releasing articles with enhanced albumin affinity and methods for making and using the same

Abstract

Described herein are articles comprising a nitric oxide releasing compound and dextran bonded to at least one surface of the article, wherein a sulfonated triazine is covalently bonded to dextran. In one aspect, the articles bind to albumin, which can reduce clotting at the surface of the article and the risk of thrombosis.

Description

ATTORNEY DOCKET NO.: 222105-2300 NITRIC OXIDE RELEASING ARTICLES WITH ENHANCED ALBUMIN AFFINITY AND METHODS FOR MAKING AND USING THE SAME STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under R01HL134899 awarded by the National Institutes of Health. The government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to co-pending U.S. Provisional Patent Application No.63 / 651,610, filed on May 24, 2024, the contents of which are incorporated by reference herein in their entireties. BACKGROUND

[0003] Blood-contacting medical devices such as catheters, stents, and vascular grafts are commonly used in clinics for short, extended, and lifelong treatment periods. However, artificial surfaces are subject to inferior blood compatibility when compared to native endothelium membranes. Foreign body reaction-induced thrombosis is one of the most critical problems associated with device failure that can lead to severe complications in patient treatments [1]. In addition, different device usage durations can also contribute to the degree of thrombosis. For example, the central venous catheter (CVC) is commonly used in intensive care units for treatment access to the superior vena cava in patients [2]. It is estimated that every year, more than 5 million CVCs are inserted in patients within the United States alone [3]. Concurrently, CVCs are responsible for over 70% of the deep vein thrombosis reported [4].

[0004] Along with thrombosis-related complications, the biomaterial can also initiate intrinsic coagulation pathways and inhibit physiological hemostasis, contributing to the formation of fibrin plugs with aggregated platelets, red blood cells, and leukocytes [5]. Therefore, regulating host response upon exposure to foreign devices is a promising strategy to prevent thrombus formation and maintain device functionality. In an attempt to reduce device-related clotting, systematic administration of antiplatelet / anticoagulant drugs such as heparin and warfarin has been used in clinical practice. However, such systematic therapy of anti-clotting drugs can often induce the risk of excessive bleeding, where its long-term administration can even lead to adverse effects such as hemorrhage [6]. Therefore, recent research endeavors haveATTORNEY DOCKET NO.: 222105-2300 focused on modifying the surfaces of blood-contacting devices in an attempt to ameliorate device-associated thrombosis. SUMMARY

[0005] Described herein are articles comprising a nitric oxide releasing compound and dextran bonded to at least one surface of the article, wherein a sulfonated triazine is covalently bonded to dextran. In one aspect, the articles bind to albumin, which can reduce clotting at the surface of the article and the risk of thrombosis.

[0006] Other compositions, apparatus, methods, features, and advantages will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional compositions, apparatus, methods, features and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Further aspects of the present disclosure will be readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

[0008] FIG. 1 shows a NO-releasing silicone rubber surface conjugated with dextran and Cibacron Blue 3G-A dye (NOBD) that displays selective albumin affinity in a physiological environment.

[0009] FIG.2 shows Cibacron blue F3G A (CB) covalently attached to dextran.

[0010] FIG.3 shows the fabrication of NOBD films. SR surface was first solvent-swelled with 25 mg mL-1SNAP before amination of the surface. Simultaneously, 200k dextran solution was oxidized with sodium (meta)periodate and dialyzed overnight against D.I. water. The NO-SR samples were then placed in oxidized dextran solution to form a Schiff’s base, followed by the covalent attachment of Cibacron Blue 3GA dye in basic condition. After 6 hr incubation, samples were retrieved and rinsed with D.I. water and stored in -20 C° between usage.

[0011] FIGS.4A-4C show the surface characterization of materials produced herein. (A) FTIR characterization of sample surfaces via VariGATR grazing angle accessory. NOBD Surface shows characteristic bond of dextran and Cibacron blue F3GA structure. (B) Static contact angle measurement. D.I. water droplet image showing representative contact angle on the surface (N > 3). (C) Surface attached dextran stability in physiological condition (N > 3).ATTORNEY DOCKET NO.: 222105-2300

[0012] FIGS.5A-5B show BD characterization. (A) Absorbance spectra of Cibacron blue dye in 0.01 M PBS showing λmaxof 601 nm. (B) The cumulative amount of Cibacron blue dye leaching out of the BD control and NOBD polymer surface within 24 h incubation in the dark at 37 °C (N > 3).

[0013] FIGS.6A-6B show NO release characterization. (A) SNAP leaching out of the polymer within 24 h incubation in physiological conditions (37 °C, pH 7.4). (B) Average surface fluxes of NO generated from sample films over a 24 hr incubation study under physiological conditions (N > 3).

[0014] FIGS. 7A-7D show the biocompatibility evaluation of surfaces. . (A) Indirect contact cytotoxicity screening of sample groups against BJ human fibroblast cells (24 h) showed the viability of cells across all sample groups tested (N > 5). (B) Surface adsorbed bovine serum albumin after 90 mins incubation at physiology conditions (N > 4). (C) Percent hemolysis of the film samples (N > 4).2-5% hemolytic activity is considered slightly hemolytic, and > 5% activity is considered hemolytic according to ASTM F756. ****represents statistical significance (p < 0.0001). (D) The antibacterial efficacy of NOBD films was evaluated against Escherichia coli 25922 (E.coli). The polymer reduced the viability of surface-adhered E.coli by 93.52%, 71.01% less than NO ctrl.

[0015] FIGS. 8A-8B show anti-fouling characteristics of NOBD films. (A) Human serum fibrinogen adsorption after 90 mins incubation in BSA + Fg solution to show competitive binding (N > 6). (B) Representative image of FITC-Fibrinogen adhesion on surface after 90 mins incubation. * represents statistical significance (p < 0.05), ****(p < 0.0001).

[0016] FIGS.9A-9C show (A) Surface-adhered porcine platelets were placed on the sample surface (N > 6). (B) Degree of platelet activation and platelet count on representative SEM image (C) after whole blood exposure. * represents statistical significance (p < 0.05), **(p < 0.01), ***(p < 0.001), ****(p < 0.0001).

[0017] FIGS. 10A-10B show clotting time assay. (A) Representative sample surface clot formation at different time point (N = 2). (B) Representative clotting SEM images. DETAILED DESCRIPTION

[0018] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to beATTORNEY DOCKET NO.: 222105-2300 included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0019] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0020] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0021] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0022] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0023] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.

[0024] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methodsATTORNEY DOCKET NO.: 222105-2300 belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0025] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure. Definitions

[0026] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

[0027] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polysiloxane” include, but are not limited to, mixtures or combinations of two or more such polysiloxanes, and the like.

[0028] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0029] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in theATTORNEY DOCKET NO.: 222105-2300 disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0030] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0031] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0032] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance and instances where it does not.ATTORNEY DOCKET NO.: 222105-2300

[0033] As used herein, the term “biocompatible,” with respect to a substance or fluid described herein, indicates that the substance or fluid does not adversely affect the short-term viability or long-term proliferation of a target biological particle within a particular time range.

[0034] The terms “antimicrobial” and “antimicrobial characteristic” refer to the ability to kill and / or inhibit the growth of microorganisms. A substance having an antimicrobial characteristic may be harmful to microorganisms (e.g., bacteria, fungi, protozoans, algae, and the like). A substance having an antimicrobial characteristic can kill the microorganism and / or prevent or substantially prevent the growth or reproduction of the microorganism.

[0035] The term “antimicrobial effective amount” as used herein refers to that amount of the compound being administered which will kill microorganisms or inhibit growth and / or reproduction thereof to some extent (e.g. from about 5% to about 100%). In reference to the compositions or articles of the disclosure, an antimicrobial effective amount refers to that amount which has the effect of diminishment of the presence of existing microorganisms, stabilization (e.g., not increasing) of the number of microorganisms present, preventing the presence of additional microorganisms, delaying or slowing of the reproduction of microorganisms, and combinations thereof.

[0036] The terms “bacteria” or “bacterium” include, but are not limited to, gram positive and gram negative bacteria. Bacteria can include, but are not limited to, Abiotrophia, Achromobacter, Acidaminococcus, Acidovorax, Acinetobacter, Actinobacillus, Actinobaculum, Actinomadura, Actinomyces, Aerococcus, Aeromonas, Afipia, Agrobacterium, Alcaligenes, Alloiococcus, Alteromonas, Amycolata, Amycolatopsis, Anaerobospirillum, Anabaena affinis and other cyanobacteria (including the Anabaena, Anabaenopsis, Aphanizomenon, Camesiphon, Cylindrospermopsis, Gloeobacter Hapalosiphon, Lyngbya, Microcystis, Nodularia, Nostoc, Phormidium, Planktothrix, Pseudoanabaena, Schizothrix, Spirulina, Trichodesmium, and Umezakia genera) Anaerorhabdus, Arachnia, Arcanobacterium, Arcobacter, Arthrobacter, Atopobium, Aureobacterium, Bacteroides, Balneatrix, Bartonella, Bergeyella, Bifidobacterium, Bilophila Branhamella, Borrelia, Bordetella, Brachyspira, Brevibacillus, Brevibacterium, Brevundimonas, Brucella, Burkholderia, Buttiauxella, Butyrivibrio, Calymmatobacterium, Campylobacter, Capnocytophaga, Cardiobacterium, Catonella, Cedecea, Cellulomonas, Centipeda, Chlamydia, Chlamydophila, Chromobacterium, Chyseobacterium, Chryseomonas, Citrobacter, Clostridium, Collinsella, Comamonas, Corynebacterium, Coxiella, Cryptobacterium, Delftia, Dermabacter, Dermatophilus, Desulfomonas, Desulfovibrio, Dialister, Dichelobacter, Dolosicoccus, Dolosigranulum, Edwardsiella, Eggerthella, Ehrlichia, Eikenella, Empedobacter, Enterobacter, Enterococcus, Erwinia, Erysipelothrix, Escherichia, Eubacterium, Ewingella,ATTORNEY DOCKET NO.: 222105-2300 Exiguobacterium, Facklamia, Filifactor, Flavimonas, Flavobacterium, Francisella, Fusobacterium, Gardnerella, Gemella, Globicatella, Gordona, Haemophilus, Hafnia, Helicobacter, Helococcus, Holdemania Ignavigranum, Johnsonella, Kingella, Klebsiella, Kocuria, Koserella, Kurthia, Kytococcus, Lactobacillus, Lactococcus, Lautropia, Leclercia, Legionella, Leminorella, Leptospira, Leptotrichia, Leuconostoc, Listeria, Listonella, Megasphaera, Methylobacterium, Microbacterium, Micrococcus, Mitsuokella, Mobiluncus, Moellerella, Moraxella, Morganella, Mycobacterium, Mycoplasma, Myroides, Neisseria, Nocardia, Nocardiopsis, Ochrobactrum, Oeskovia, Oligella, Orientia, Paenibacillus, Pantoea, Parachlamydia, Pasteurella, Pediococcus, Peptococcus, Peptostreptococcus, Photobacterium, Photorhabdus, Phytoplasma, Plesiomonas, Porphyrimonas, Prevotella, Propionibacterium, Proteus, Providencia, Pseudomonas, Pseudonocardia, Pseudoramibacter, Psychrobacter, Rahnella, Ralstonia, Rhodococcus, Rickettsia Rochalimaea Roseomonas, Rothia, Ruminococcus, Salmonella, Selenomonas, Serpulina, Serratia, Shewenella, Shigella, Simkania, Slackia, Sphingobacterium, Sphingomonas, Spirillum, Spiroplasma, Staphylococcus, Stenotrophomonas, Stomatococcus, Streptobacillus, Streptococcus, Streptomyces, Succinivibrio, Sutterella, Suttonella, Tatumella, Tissierella, Trabulsiella, Treponema, Tropheryma, Tsakamurella, Turicella, Ureaplasma, Vagococcus, Veillonella, Vibrio, Weeksella, Wolinella, Xanthomonas, Xenorhabdus, Yersinia, and Yokenella. Other examples of bacterium include Mycobacterium tuberculosis, M. bovis, M. typhimurium, M. bovis strain BCG, BCG substrains, M. avium, M. intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans, M. avium subspecies paratuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus equi, Streptococcus pyogenes, Streptococcus agalactiae, Listeria monocytogenes, Listeria ivanovii, Bacillus anthracis, B. subtilis, Nocardia asteroides, and other Nocardia species, Streptococcus viridans group, Peptococcus species, Peptostreptococcus species, Actinomyces israelii and other Actinomyces species, and Propionibacterium acnes, Clostridium tetani, Clostridium botulinum, other Clostridium species, Pseudomonas aeruginosa, other Pseudomonas species, Campylobacter species, Vibrio cholera, Ehrlichia species, Actinobacillus pleuropneumoniae, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Legionella pneumophila, other Legionella species, Salmonella typhi, other Salmonella species, Shigella species Brucella abortus, other Brucella species, Chlamydi trachomatis, Chlamydia psittaci, Coxiella burnetti, Escherichia coli, Neiserria meningitidis, Neiserria gonorrhea, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Yersinia pestis, Yersinia enterolitica, other Yersinia species, Escherichia coli, E. hirae and other Escherichia species, as well as other Enterobacteria, Brucella abortus and other Brucella species, Burkholderia cepacia, Burkholderia pseudomallei, Francisella tularensis, Bacteroides fragilis,ATTORNEY DOCKET NO.: 222105-2300 Fudobascterium nucleatum, Provetella species, and Cowdria ruminantium, or any strain or variant thereof. The gram-positive bacteria may include, but is not limited to, gram positive Cocci (e.g., Streptococcus, Staphylococcus, and Enterococcus). The gram-negative bacteria may include, but is not limited to, gram negative rods (e.g., Bacteroidaceae, Enterobacteriaceae, Vibrionaceae, Pasteurellae and Pseudomonadaceae).

[0037] The term “antimicrobial effective amount” as used herein refers to that amount of the compound being administered / released that will kill microorganisms or inhibit growth and / or reproduction thereof to some extent (e.g. from about 5% to about 100%). In reference to the compositions or articles of the disclosure, an antimicrobial effective amount refers to that amount which has the effect of diminishment of the presence of existing microorganisms, stabilization (e.g., not increasing) of the number of microorganisms present, preventing the presence of additional microorganisms, delaying or slowing of the reproduction of microorganisms, and combinations thereof. Similarly, the term “antibacterial effective amount” refers to that amount of a compound being administered / released that will kill bacterial organisms or inhibit growth and / or reproduction thereof to some extent (e.g., from about 5% to about 100%). In reference to the compositions or articles of the disclosure, an antibacterial effective amount refers to that amount which has the effect of diminishment of the presence of existing bacteria, stabilization (e.g., not increasing) of the number of bacteria present, preventing the presence of additional bacteria, delaying or slowing of the reproduction of bacteria, and combinations thereof.

[0038] As used herein, the term “subject” includes humans, mammals (e.g., cats, dogs, horses, etc.), birds, and the like. Typical subjects to which embodiments of the present disclosure may be administered will be mammals, particularly primates, especially humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like. Additionally, for in vitro applications, such as in vitro diagnostic and research applications, body fluids and cell samples of the above subjects will be suitable for use, such as mammalian (particularly primate such as human) blood, urine, or tissue samples, or blood, urine, or tissue samples of the animals mentioned for veterinary applications. In some embodiments, a system includes a sample and a host. The term “living host” refers to the entire host or organism and not just a part excised (e.g., a liver or other organ) from the living host.ATTORNEY DOCKET NO.: 222105-2300

[0039] A residue of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species. Thus, an ethylene glycol residue in a polyester refers to one or more -OCH2CH2O- units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a sebacic acid residue in a polyester refers to one or more -CO(CH2)8CO- moieties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester.

[0040] As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).

[0041] The term "alkyl" refers to the radical of saturated aliphatic groups, including straight- chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.

[0042] In some embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30for straight chains, C3-C30for branched chains), 20 or fewer, 12 or fewer, or 7 or fewer. Likewise, in some embodiments cycloalkyls have from 3-10 carbon atoms in their ring structure, e.g. have 5, 6 or 7 carbons in the ring structure. The term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl,ATTORNEY DOCKET NO.: 222105-2300 carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.

[0043] Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above having from one to ten carbons, or from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. In embodiments described in the present application, preferred alkyl groups are lower alkyls. In some embodiments, a substituent designated herein as alkyl is a lower alkyl.

[0044] It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include halogen, hydroxy, nitro, thiols, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like. Cycloalkyls can be substituted in the same manner.

[0045] The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is a type of cycloalkyl group as defined above, and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0046] “Aryl”, as used herein, refers to C5-C10-membered aromatic, heterocyclic, fused aromatic, fused heterocyclic, biaromatic, or bihetereocyclic ring systems. Broadly defined, “aryl”, as used herein, includes 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles” or “heteroaromatics”. The aromatic ring can beATTORNEY DOCKET NO.: 222105-2300 substituted at one or more ring positions with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN; and combinations thereof.

[0047] The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (i.e., “fused rings”) wherein at least one of the rings is aromatic, e.g., the other cyclic ring or rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and / or heterocycles. Examples of heterocyclic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3- oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4- thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. One or more of the rings can be substituted as defined above for “aryl”.

[0048] The terms "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, and tert-butoxy. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl, and -O- alkynyl. Aroxy can be represented by –O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined below. The alkoxy and aroxy groups can be substituted as described above forATTORNEY DOCKET NO.: 222105-2300 alkyl. The term “herteroalkoxy" is an alkoxy group substituted with one or more heteroatoms such as, for example, oxygen, nitrogen, or sulfur.

[0049] The terms “cycloalkoxy” and “cycloalkoxyl” as used herein refer to a cycloalkyl group bonded through an ether linkage; that is, a “cycloalkoxy” group can be defined as —OA1where A1is cycloalkyl as defined above. “Cycloalkoxy” also includes polymers of cycloalkoxy groups as just described; that is, a cycloalkoxy can be a polyether such as —OA1—OA2or —OA1— (OA2)a—OA3, where “a” is an integer of from 1 to 200 and A1, A2, and A3are cycloalkyl groups.

[0050] The terms “aryloxy” and “aryloxyl” as used herein refer to an aryl group bonded through an ether linkage; that is, an “aryloxy” group can be defined as —OA1where A1is aryl as defined above. “Aryloxy” also includes polymers of aryloxy groups as just described; that is, an aryloxy can be a polyether such as —OA1—OA2or —OA1—(OA2)a—OA3, where “a” is an integer of from 1 to 200 and A1, A2, and A3are aryl groups.

[0051] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:wherein R9, R10, and R'10each independently represent a hydrogen, an alkyl, an alkenyl, - (CH2)m-R8or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R8represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In some embodiments, only one of R9or R10can be a carbonyl, e.g., R9, R10and the nitrogen together do not form an imide. In still other embodiments, the term “amine” does not encompass amides, e.g., wherein one of R9and R10represents a carbonyl. In additional embodiments, R9and R10(and optionally R’10) each independently represent a hydrogen, an alkyl or cycloalkyl, an alkenyl or cycloalkenyl, or alkynyl. Thus, the term "alkylamine" as used herein means an amine group, as defined above, having a substituted (as described above for alkyl) or unsubstituted alkyl attached thereto, i.e., at least one of R9and R10is an alkyl group.

[0052] The terms “treat”, “treating”, and “treatment” are an approach for obtaining beneficial or desired clinical results. Specifically, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (e.g., notATTORNEY DOCKET NO.: 222105-2300 worsening) of disease, delaying or slowing of disease progression, substantially preventing spread of disease, amelioration or palliation of the disease state, and remission (partial or total) whether detectable or undetectable.

[0053] The term “prevent” or “preventing” as used herein is defined as eliminating or reducing the likelihood of the occurrence of one or more symptoms of a disease or disorder (e.g., biofilm formation) when using the compositions as described herein when compared to a control where the composition is not used. Nitric Oxide Releasing Articles and Methods of Making and Uses Thereof

[0054] Described herein are articles comprising a nitric oxide releasing compound and dextran bonded to at least one surface of the article, wherein a sulfonated triazine is covalently bonded to dextran. In one aspect, the articles bind to albumin, which can reduce clotting at the surface of the article and the risk of thrombosis.

[0055] In one aspect, the article is produced the method comprising: (a) attaching linker to at least one surface of the article, wherein the article comprises a nitric oxide releasing compound to produce a pre-functionalized article; (b) contacting the pre-functionalized article with oxidized dextran, wherein the linker covalently bonds to the oxidized dextran to produce a dextran functionalized article; and (c) contacting the dextran functionalized surface with a sulfonated triazine, wherein the sulfonated triazine forms a covalent bond with the oxidized dextran.

[0056] The articles described herein include a nitric oxide releasing compound. In one aspect, the nitric oxide releasing compound is applied to at least one surface of thew article using techniques such as, for example, spraying or brushing a solution of the nitric oxide releasing compound on the surface of the article. In another aspect, the article is submerged in a solution of the nitric oxide releasing, where the nitric oxide releasing compound is incorporated in the article. Depending upon the selectin of the material used to produce the article, the nitric oxide releasing compound can be partially or completely incorporated or distributed throughout the article.

[0057] In one aspect, the nitric oxide releasing compound is an S-nitrosothiol (RSNO) compound. In a further aspect, the nitric oxide compound is S-nitroso-N-acetylpenicillamine, S-nitroso-glutathione, S-nitroso-N-acetylcysteine, S-nitrosocysteine, S-nitrosopenicillamine, S-nitroso-B,D-glucose, S-nitrosocaptopril, S-nitrosocysteamine, S-nitroso-3-mercapto- propanoic acid, S-nitroso-N-acetyl-l-cysteine ethyl ester (SNACET), S-nitroso-N-acetyl-L- methionine, S-nitrosomercaptoethanol, or any combination thereof. In another aspect, theATTORNEY DOCKET NO.: 222105-2300 nitric oxide releasing compound can be S-nitrosothiol conjugated polymers, S-nitrosothiol modified-dendrimers, S-nitrosothiol modified polysaccharides, S-nitrosothiol modified nano / microparticles, or S-nitrosothiol modified-proteins. The nitric oxide releasing compound can also include other NO-donors such as, for example, nitrates and N-diazeniumdiolates (NONOates).

[0001] In other aspects, the nitric oxide releasing compound includes a modified antibiotic compound including a nitric oxide release agent covalently attached to an antibiotic molecule. Having a single molecule with the combined functionalities of both of a stable NO donor and an antibiotic can be a very efficient approach for combating and preventing biofilm related infections. The modified antibiotic compound can be a synthetic RSNO covalently attached to an antibiotic molecule to create a novel dual functional antimicrobial agent, also referred to as a modified antibiotic compound. In other aspects, the nitric oxide releasing agent is S-nitroso- N-acetylpenicillamine (SNAP), S-nitroso-glutathione, S-nitroso-N-acetylcysteine, S- nitrosocysteine, S-nitrosopenicillamine, S-nitroso-B,D-glucose, S-nitrosocaptopril, S- nitrosocysteamine, S-nitroso-3-mercapto-propanoic acid, or any combination thereof. In other aspects, the antiobiotic molecule can be ampicillin, vancomycin, gentamicin, cephalexin, or any combination thereof. In further aspects, the modified antibiotic compound includes SNAP covalently bonded to ampicillin, referred to herein as SNAPicillin. SNAP can be represented by the following structure:

[0058] SNAPicillin can be represented by the following structure:ATTORNEY DOCKET NO.: 222105-2300

[0059] In one aspect, the modified antibiotic compound can be formed by covalently attaching a nitric oxide release agent to an antibiotic molecule. The attachment can be formed by mixing the nitric oxide release agent and the antibiotic molecule in a solvent and then nitrosating the mixture. The nitrosation can occur through the excess addition of t-butyl nitrite or an acidified sodium nitrite solution to the mixture. The excess addition can be about a 3 times molar excess of t-butyl nitrate with respect to the quantity of the antibiotic molecule, such as ampicillin. Methods for producing the modified antibiotic compound useful as nitric oxide releasing compounds are described in US Patent No.11,220,516, which is incorporated by reference in its entirety.

[0060] The amount of nitric oxide releasing compound present in the article can vary depending upon the application or use of the article. The Examples provide non-limiting procedures for incorporating the nitric oxide releasing compound into the articles described herein.

[0061] In one aspect, the article with the nitric oxide releasing compound can be pre- functionalized so that dextran can be bonded to the surface of the article. In one aspect, a linker is applied to at least one surface of the article to produce a pre-functionalized article. In one aspect, the surface of the article can be contacted with one or more compounds possessing a nucleophilic group. Depending upon the material of the article, the compound with the nucleophilic group can be covalently bonded to the surface of the article via a number of different techniques. For example, the compound with the nucleophilic group can include a functional group that reacts with the material of the article to form a covalent bond.

[0062] In one aspect, the surface of the article is pre-functionalized with a silane compound comprising a nucleophilic group. In another aspect, the surface of the article is pre functionalized with a silane compound comprising at least one hydroxyl group, thiol group, or amino group. In another aspect, the surface of the article is pre-functionalized with anATTORNEY DOCKET NO.: 222105-2300 aminosilane. In another aspect, the surface of the article is pre-functionalized with a compound having the structure I R1R1Si(CH2)oNH2R1I wherein each R1is independently an alkoxy group, cycloalkoxy group, or aryloxy group, and o is an integer from 1 to 10. In one aspect, each R1is the same alkoxy group (e.g., methoxy, ethoxy), and o is 1, 2, or 3.

[0063] In one aspect, the aminosilane is 3-aminopropyltrimethoxysilane, 3- aminopropyltriethoxysilane, 4-aminobutyltriethoxysilane, N-methyl-3-amino-2- methylpropyltrimethoxysilane, N-ethyl-3-amino-2-methylpropyltrimethoxysilane, N- ethyl-3-amino-2-methylpropyldiethoxymethylsilane, N-ethyl-3-amino-2- methylpropyltriethoxysilane, N-ethyl-3-amino-2-methylpropylmethyldimethoxysilane, N-butyl-3-amino-2-methylpropyltrimethoxysilane, 3-(N-methyl-2-amino-1-methyl-1- ethoxy)-propyltrimethoxysilane, N-ethyl-4-amino-3,3-dimethyl- butyldimethoxymethylsilane, N-ethyl-4-amino-3,3-dimethylbutyltrimethoxy-silane, N- (cyclohexyl)-3-aminopropyltrimethoxysilane, N-(2-aminoethyl)-3- aminopropyltrimethoxysilane, N-(2-aminoethyl)-3-aminopropyltriethoxy-silane, N-(2- aminoethyl)-3-aminopropylmethyldimethoxysilane, aminopropyltriethoxysilane, bis-(3- trimethoxysilyl-2-methylpropyl)amine and N-(3'-trimethoxysilylpropyl)-3-amino-2- methylpropyltrimethoxysilane.

[0064] The surface of the article with the nitric oxide releasing compound can be contacted with the linker by techniques known in the art including dipping, coating, or spraying the compound on the surface of the article. Exemplary non-limiting methods for pre-functionalizing the surface of the article are provided in the Examples.

[0065] After the article with the nitric oxide releasing compound has been pre-functionalized, the article is contacted with oxidized dextran, wherein the linker covalently bonds to the oxidized dextran to produce a dextran functionalized article. Dextran is a hydrophilic, hemocompatible oligosaccharide with flexible alpha (1^6) linkage within branched glucose monomers, which acts as a steric barrier against non-specific protein adsorption.ATTORNEY DOCKET NO.: 222105-2300

[0066] In one aspect, dextran is oxidized to convert hydroxyl groups to aldehyde groups. Non- limiting procedures for producing oxidized dextran are provided in the Examples. In one aspect, when the article is pre-functionalized with an aminosilane, the free amino groups can react with the aldehyde groups of the oxidized dextran to form new imine bonds (i.e., Schiff bases). In one aspect, the pre-functionalized article with the nitric oxide releasing compound is heated in a solution composed of the oxidized dextran.

[0067] In certain aspects, after the oxidized dextran has reacted with the linker to covalently bond the dextran to the article, a borohydride such as, for example, sodium cyanoborohydride, can be added to stabilize the formation of the imine groups when formed by the reaction between aldehyde groups of the oxidized dextran and amino groups of the linker. Non-limiting procedures for performing this step are provided in the Examples. In one aspect, dextran has an average molecular weight of about 50 kDa to about 1,000 kDa, or 50 kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, or 1,000 kDa, where any value can be a lower and upper endpoint of a range (e.g., 100 kDa to 300 kDa).

[0068] After the dextran functionalized surface has been produced, it is contacted with a sulfonated triazine, wherein the sulfonated triazine forms a covalent bond with the oxidized dextran. Examples of sulfonated triazines include, but are not limited to, Cibacron Blu33GA, Procion Red H-E7B, Procion Green H-4G and Yellow H-E3G. Cibacron Blue 3GA dye has been known for its affinity for albumin. Its affinity originates from its structure, which is similar to that of the natural ligand of proteins with co-factor binding. CB dye has several aromatic sulfonate rings that allow it to bind with the hydrophobic part of the albumin protein.

[0069] FIG. 2 provides the structure of Cibcaron Bue 3GA covalently bonded to dextran. Cibcaron Bue 3GA is a chlorinated compound, where nucleophilic substitution between the chlorine moiety of the triazine dye and the hydroxyl groups of dextran occurs in basic aqueous media to covalently bond dextran with the dye.

[0070] The articles described herein are useful in applications where it is desirable to reduce or prevent biofouling (e.g., bacterial adhesion, platelet formation, fibrinogen adhesion, etc.) of implantable medical devices. Implantable medical devices are a leading cause of infection such as nosocomial infections. The articles described herein can reduce or prevent biofouling in a subject when the device is introduced into the subject. In one aspect, the articles described herein can reduce or prevent bacterial growth on a surface of an implantable device. In another aspect, the articles described herein can reduce or prevent biofilm formation on a surface of an implantable device.ATTORNEY DOCKET NO.: 222105-2300

[0071] By varying the relative amount of the nitric oxide releasing compound in the articles described herein, the rate of release of the nitric oxide from the article can be modified. In certain applications, it is desirable to have sustained release of nitric oxide from the polysiloxane under physiological conditions. In one aspect, nitric oxide is released from the article for at least 24 hours at 37oC.

[0072] In another aspect, the articles described herein can reduce or prevent fibrinogen formation on a surface of an implantable device. In one aspect, the articles described herein have an affinity for albumin. Not wishing to be bound by theory, when placed in a blood environment, the surface-bounded albumin layer will reduce clotting by minimizing the intrinsic coagulation pathway contributed by the fibrinogen adsorbed (FIG. 1). Concurrently, nitric oxide released from the article will mediate activated platelet contribution to surface clot formation.

[0073] In one aspect, the article is an implantable device such as a urinary catheter, artificial heart valve, a vascular catheter, a graft, or a stent. In other aspects, the device is intended to contact human blood or tissue. In one aspect, the device is a hemodialysis device or a component thereof. In another aspect, the article s composed of a rubber material such as, for example, a silicone rubber. Aspects

[0074] Aspect 1. An article comprising a nitric oxide releasing compound and dextran bonded to at least one surface of the article, wherein a sulfonated triazine is covalently bonded to dextran.

[0075] Aspect 2. The article of Aspect 1, wherein the nitric oxide releasing compound is an S-nitrosothiol conjugated polymer, an S-nitrosothiol modified-dendrimer, an S-nitrosothiol modified polysaccharide, an S-nitrosothiol modified nano / microparticle, an S-nitrosothiol modified-protein, a nitrate, an N-diazeniumdiolates (NONOate), or an S-nitrosothiol (RSNO).

[0076] Aspect 3. The article of Aspect 1, wherein the nitric oxide release agent is S-nitroso- N-acetylpenicillamine, S-nitroso-glutathione, S-nitroso-N-acetylcysteine, S-nitrosocysteine, S-nitrosopenicillamine, S-nitroso-B,D-glucose, S-nitrosocaptopril, S-nitrosocysteamine, S- nitroso-3-mercapto-propanoic acid, S-nitroso-N-acetyl-l-cysteine ethyl ester (SNACET), S- nitroso-N-acetyl-L-methionine, or S-nitrosomercaptoethanol.

[0077] Aspect 4. The article of Aspect 1, wherein the nitric oxide releasing compound is a modified antibiotic compound comprising a nitric oxide release agent covalently attached to an antibiotic molecule.ATTORNEY DOCKET NO.: 222105-2300

[0078] Aspect 5. The article of Aspect 4, wherein the antibiotic molecule is ampicillin, vancomycin, gentamicin, or cephalexin.

[0079] Aspect 6. The article of Aspect 4, wherein the modified antibiotic compound comprises S-nitroso-N-acetylpenicillamine covalently attached to ampicillin.

[0080] Aspect 7. The article of Aspects 1-6, wherein the nitric oxide releasing compound is on the surface of the article.

[0081] Aspect 8. The article of Aspects 1-6, wherein the nitric oxide releasing compound is on the surface of the article is incorporated throughout the article.

[0082] Aspect 9. The article of Aspects 1-8, wherein dextran is bonded to the at least one surface of the article by a linker.

[0083] Aspect 10. The article of Aspect 9, wherein the linker comprises a silane compound comprising a nucleophilic group.

[0084] Aspect 11. The article of Aspect 9, wherein the linker comprises a silane compound comprising at least one hydroxyl group, thiol group, or amino group.

[0085] Aspect 12. The article of Aspect 9, wherein the linker comprises an aminosilane.

[0086] Aspect 13. The article of Aspect 9, wherein the linker comprises a compound having the structure I R1R1Si(CH2)oNH2R1I wherein each R1is independently an alkoxy group, cycloalkoxy group, or aryloxy group, and o is an integer from 1 to 10.

[0087] Aspect 14. The article of Aspects 1-13, wherein the dextran is bonded to the at least one surface of the article by an aminosilane, wherein the aminosilane forms an imine group with dextran.

[0088] Aspect 15. The article of Aspects 1-14, wherein the dextran has an average molecular weight of about 50 kDa to about 1,000 kDa.

[0089] Aspect 16. The article of Aspects 1-15, wherein the sulfonated triazine is selected from the group consisting of Cibacron Blue F3GA, Procion Red H-E7B, Procion Green H-4G and Yellow H-E3G.ATTORNEY DOCKET NO.: 222105-2300

[0090] Aspect 17. The article of Aspects 1-15, wherein the sulfonated triazine is Cibacron Blue F3GA.

[0091] Aspect 18. An article produced by the method comprising (a) attaching linker to at least one surface of the article, wherein the article comprises a nitric oxide releasing compound to produce a pre-functionalized article; (b) contacting the pre-functionalized article with oxidized dextran, wherein the linker covalently bonds to the oxidized dextran to produce a dextran functionalized article; and (c) contacting the dextran functionalized surface with a sulfonated triazine, wherein the sulfonated triazine forms a covalent bond with the oxidized dextran.

[0092] Aspect 19. The article of Aspect 18, wherein the article comprising the nitric oxide releasing compound is produced by soaking the article in a solution of the nitric oxide releasing compound.

[0093] Aspect 20. The article of Aspect 18 or 19, wherein step (a) comprises heating the article in a solution comprising the linker.

[0094] Aspect 21. The article of Aspect 20, wherein the linker comprises an aminosilane.

[0095] Aspect 22. The article of Aspects 18-21, wherein step (b) comprises heating the pre- functionalized article in a solution comprising the oxidized dextran.

[0096] Aspect 23. The article of Aspect 22, wherein a borohydride is added after step (b) and prior to step (c).

[0097] Aspect 24. The article of Aspect 22, wherein a borohydride is sodium cyanoborohydride.

[0098] Aspect 25. The article of Aspects 18-24, wherein step (c) comprises contacting the dextran functionalized surface with a solution comprising the sulfonated triazine.

[0099] Aspect 26. The article of Aspects 1-25, wherein the article comprises a medical device.

[0100] Aspect 27. The article of Aspect 26, wherein the device is an implantable device.

[0101] Aspect 28. The article of Aspect 26, wherein the device is selected from the group consisting of: a vascular catheter, a urinary catheter, other catheters, a coronary stent, a wound dressing, and a vascular graft.

[0102] Aspect 29. The article of Aspects 1-28, wherein the article comprises silicone rubber.

[0103] Aspect 30. The article of Aspects 1-29, wherein the article has a contact angle of about 50 degrees to 65 degrees.ATTORNEY DOCKET NO.: 222105-2300

[0104] Aspect 31. The article of Aspects 1-30, wherein the article releases nitric oxide for at least 24 hours.

[0105] Aspect 32. The article of Aspects 1-31, wherein the article binds to albumin.

[0106] Aspect 33. The article of Aspects 1-32, wherein the article reduces or prevents fibrinogen adhesion to the article.

[0107] Aspect 34. The article of Aspects 1-33, wherein the article reduces or prevents platelet adhesion to the article.

[0108] Aspect 35. The article of Aspects 1-34, wherein the article reduces or prevents bacterial growth on the article.

[0109] Aspect 36. The article of Aspects 1-35, wherein the article reduces or prevents biofilm formation on the article. EXAMPLES

[0110] Now having described the embodiments of the present disclosure, in general, the following Examples describe some additional embodiments of the present disclosure. While embodiments of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.

[0111] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations and are set forth only for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiments of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure. MATERIALS AND METHODS

[0112] Materials

[0113] Tetrahydrofuran (THF) anhydrous, ethanol, methanol, hydrochloric acid, sulfuric acid, Hexamethyldisilazane (HMDS), phosphate-buffered saline (0.01M PBS, pH 7.4), Fibrinogen from bovine plasma, and ethylenediaminetetraacetic acid (100 μM EDTA) were purchased from Sigma Aldrich (St. Louis, MO, USA). S-nitrso-N-acetylpenicillamine (SNAP) was purchased from PharmaBlock (Hatfield, PA, USA). The high-purity, high-temperature siliconeATTORNEY DOCKET NO.: 222105-2300 rubber (SR) sheet was purchased from McMaster-Carr (Elmhurst, IL, USA). S. aureus (ATCC 6538), E. coli (ATCC 25922) were obtained from the American Type Culture Collection (Manassas, VA). Lysogeny broth (L.B.), tryptic soy (T.S.) agar, and broth were both obtained from Fischer Bioreagents (Fair Lawn, NJ, USA). Porcine whole blood was obtained from the University of Georgia Swine Unit for hemolysis study. Nitrogen and oxygen gas were purchased from Airgas (Atlanta, GA, USA). BJ fibroblast cells (BJ CRL-2522) were derived from stocks previously acquired from the American Type Culture Collection. Fetal bovine serum (FBS), and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from VWR (Radnor, PA, USA). Yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich.

[0114] Fabrication of BD-incorporated Surfaces

[0115] Dextran and Cibacron blue immobilization were performed via a modified version based on a previously described method.

[0020] The SR surface was treated with low-pressure air plasma for 5 minutes at 75 W to facilitate hydroxyl group coverage on the surface. Immediately afterward, plasma-treated surfaces were incubated in APTMS solution at 1:1 volumetric ratio to anhydrous ethanol for 1 hr and stirred. Samples were then rinsed with deionized (D.I.) water and dried under a vacuum overnight. Once deposited, APTMS forms a layer of primary amines on the surface of the SR.

[0116] Concurrently, 2.5 g of 200k MW dextran is dissolved in 50 mL of D.I. water, followed by the addition of 2.5 mg of sodium metaperiodate (NaIO4) to oxidize the dextran. The solution was incubated in the dark for 2 hr, followed by 24 hr dialysis against D.I. water to remove any residue NaIO4.The SR films were then incubated with shaking in the oxidized dextran solution at 150 rpm for 1 hr. 3 mg mL-1sodium cyanoborohydride was added to the solution and incubated at 150 rpm to stabilize the Schiff's base formation between aldehydes of oxidized dextran and amino groups on SR surface. After 2 hr, samples were removed and rinsed with D.I. water. Cibacron blue dye was then covalently coupled to the SR-dextran polymer in basic condition. The solution consists of 6.5 mM Cibacron blue dye, 0.3 M NaCl, and 0.7 M NaHCO3. The blue dextran (BD) containing samples were removed after 6 hr incubation and rinsed with D.I. water.

[0117] SNAP-containing SR was first prepared for the fabrication of NO-releasing blue dextran SR (NOBD). A 25 mg mL-1SNAP swelling solution was prepared by dissolving recrystallized SNAP in anhydrous THF according to previously optimized NO release kinetics.

[0021] After swelling, the SR was removed from the SNAP-THF solution, briefly washed in D.I. water, and dried for an additional 48 hr under a fume hood and desiccator to allow any excess THF to evaporate. After complete evaporation of the excess THF, the films were brieflyATTORNEY DOCKET NO.: 222105-2300 washed with methanol and D.I. water to remove any SNAP crystals from the surface. The NOBD films then undergo the same covalent attachment steps as the control BD films.

[0118] Surface characterization of films

[0119] FTIR

[0120] FTIR measurements were performed and recorded using Spextrum 3 spectrometer (PerkinElmer Greenville, SC, USA). FTIR analysis of the fabricated films was carried out using VariGATR grazing angle accessory (Harrick Scientific Products). Kbr pellet loading method was also used to collect absorbance readings across the infrared spectra from 4000-650 cm-1with a Spectrum Two spectrometer from Perkin Elmer (Greenville, SC, USA). A total of 128 scans were collected for each prepared sample with a resolution of 4cm-1.

[0121] Static Contact Angle

[0122] The static contact angle measurements were conducted using an Ossila Contact Angle Goniometer (Sheffield, UK) and Ossila Contact Angle (v3.0.3.0) software.5 µL of deionized water droplets was dropped on three different locations on the film, and the droplet was allowed to be set for 10 s before measurement. The static contact angle was calculated using sessile drop approximation via frame capture. Final data are reported as the mean static contact angle ± standard deviation (S.D.) (N = 9).

[0123] Amine Quantification

[0124] Surface amine quantification was carried out via previously described fluorescence labeling quantification methods

[0022] . Briefly, a 0.001 mg mL-1FITC solution in carbonated buffer was prepared. Afterward, 300 uL of the FITC solution was added to each centrifuge tube containing individual samples. After overnight incubation, samples were washed 3x each in PBS and D.I water and read at λmax= 495 nm (ex: 490, em: 525).

[0125] Coating stability

[0126] To determine the stability of the dextran coating on the SR surface, a modified version of the previously described procedure was used.

[0023] The dextran-coated surface was incubated with 1:1 D.I. water and 95% sulfuric acid containing 0.1 wt% anthrone. Samples remained incubated at 95 C° for 7 min and followed by centrifuge at 12000 rpm for 10 min. The samples were then read at 625 nm. The standard curve of anthrone solution was prepared using known anthrone concentration dissolved in sulfuric acid and D.I. water to interpolate the absorbance measurement recorded from the BioTek Cytation 5 Cell Imaging Multimode Reader (Santa Clara, CA, USA).

[0127] Surface release characterization

[0128] LeachingATTORNEY DOCKET NO.: 222105-2300

[0129] Quantification of SNAP leaching into PBS was performed by measuring the absorbance at 340 nm, SNAP's maxima absorbance spectra (εSNAP= 1075 M-1cm-1), using a UV-vis spectrophotometer (Thermo-scientific Genesys 10S UV-vis). Samples were submerged in 1 mL PBS containing 0.1 mM EDTA at physiological temperature (37 °C) between all measurements. The amount of SNAP leached from the films was measured by absorbance recorded at different time points. The standard curve of SNAP was prepared using known SNAP concentrations dissolved in 1 mL of PBS to interpolate the absorbance measurement recorded from the UV-vis converted to SNAP concentration in the solution. The final data are shown as percent of total SNAP leached cumulatively within 24 h ± S.D. (N = 3 independently prepared films per sample type).

[0130] NO release

[0131] The NO flux from NO-incorporated films was measured and recorded using Sievers chemiluminescence NO analyzers (NOA 280i, G.E. Analytical, Boulder, CO, USA) at a physiological temperature (37 °C). NO-incorporated SR films (N > 3) were measured. Samples were shielded from light and submerged in 3 mL of PBS containing 0.1 mM EDTA during real- time release. Baseline measurements of NO release in ppb were first established without the presence of films in the reaction chamber. Then, NO released from the polymer films was purged from the reaction chamber by a continuous supply of high-purity nitrogen gas maintained at a constant flow rate of 200 mL min-1into a chemiluminescence reaction chamber. NO measurement in the reaction chamber is based on a gas-phase chemiluminescent reaction between NO and ozone, which produces an excitable photon from NO2that passes through the photomultiplier tube to generate a voltage signal. NO release measured in ppb was normalized according to the sample surface area, and a calibration constant (mol ppb-1min-1) was used to calculate NO flux (x10-10mol cm-2min-1).

[0132] Nitric oxide analyzer (NOA) data were collected over the measurement period. Samples were submerged in PBS and kept at physiological temperature (37 °C) between measurements. PBS was replaced after each NO measurement to avoid saturation. The final data are shown as mean ± S.D. (N > 3 independently prepared films per sample type).

[0133] Cibacron blue leaching

[0134] Cibcaron blue leaching was quantified in PBS by measuring the absorbance at 610 nm via a UV-vis spectrophotometer. Samples were submerged in 1mL PBS containing 0.1 mM EDTA at physiological temperature (37 °C) between all measurements. The concentration of Cibacron blue dye leaching from the surface was measured by absorbance recorded at various different time points. The standard curve of Cibacron blue was prepared using a known concentration of Cibacron blue dissolved in PBS to interpolate the absorbance measurement recorded from the UV-vis concerted to Cibacron blue concentration in the leachate. UV-ATTORNEY DOCKET NO.: 222105-2300 absorption spectrum of Cibacron blue obtained in PBS was prepared using 0.0625 mM, 0.03125 mM, and 0.015625 mM. The final data are shown as percent of total Cibacron blue leached cumulatively within 48 h ± S.D. (N = 3 independently prepared films per sample type).

[0135] In Vitro Assessment of Mammalian Cell Cytotoxicity

[0136] Mouse fibroblast 3T3 cells were revived from cryo-stocks and cultured in DMEM media supplemented with 10% FBS and 1% P&S. Cells were incubated at 37 °C under a 5% CO2atmosphere. Cells were grown up to 70% monolayer confluency before passaging. For experiments, cells were grown to 70% confluency, then detached (Trypsin-EDTA 0.05%), and collected via centrifugation (200 RCF, 5 min). Cells were resuspended in media and counted using an EVETMautomated cell counter from NanoEntek (Waltham, MA). Cells were then seeded on 96-well plates at 10,000 cells / well density. In parallel, leachate samples were prepared for each sample according to ISO 10993-5 standards for the evaluation of medical devices

[0024] . Circular coupons of each polymer type (~1.11 cm2) were incubated in supplemented DMEM media (1 mL) for 24 h at 37 °C under a 5% CO2atmosphere.

[0137] Afterward, the media for each well was replaced with a corresponding experimental group of the leachate-containing media for each material tested, along with positive and negative controls on each plate. Following an additional 24 hr of incubation, the media was aspirated and replaced with DMEM supplemented with 10% CCK-8 reagent. Plates were incubated for an additional 1.5 h, then read for absorbance at 450 and 650 nm wavelengths. The relative cellular viability of cells treated with leachates with respect to untreated cells was calculated according to Equation 1. Final values are reported as the mean ± S.D. (N = 3 treatment sets across passages). (ODRelative Cellular Viability = 450 − OD650)treatment group(OD × 100% (1)450 − OD650)control group

[0138] In vitro Blood Compatibility Screening

[0139] The hemocompatibility of the material was further demonstrated via a hemolysis assessment using the NAMSA ASTM F756 protocol. The plasma hemoglobin count of fresh porcine blood was analyzed using an Element HT5 Veterinary Hematology Analyzer. Using the known plasma hemoglobin count, the whole blood was diluted to achieve a final hemoglobin concentration of 10 mg mL-1in calcium- and magnesium-free (CMF) PBS (Corning, Corning, NY, USA). Each sample was individually incubated in diluted whole blood for 3 h at 37 °C with periodic inversion every 30 mins. Positive and negative controls were prepared with 1 mL of the diluted whole blood incubated in 7 mL of D.I. water and CMF-PBS, respectively. After incubation, the samples were centrifuged to leave a supernatant containing any freed hemoglobin.ATTORNEY DOCKET NO.: 222105-2300

[0140] This supernatant was combined with Drabkin's reagent 1:1 and allowed to react for 15 min before reading the absorbance at 540 mm. The percent hemolysis was calculated using the following equation:Where AbsDilutedblood and AbsBlankwere the absorbance of positive and negative controls, and Abs sample was the absorbance of the sample incubated.

[0141] Vitro Evaluation of Bacteria Surface Adhesion

[0142] To determine the antimicrobial effect of NOBD surface, viable adhered bacteria were quantified based on a previously established protocol

[0025] . S. aureus (ATCC 6538) and E. coli (ATCC 6538) were used to assess the antibacterial activity of candidate films against Gram- positive and Gram-negative bacterial strains. LB agar and broth, TSA and TSB medium, and PBS were prepared according to the manufacturer's instructions. All were sterilized in the autoclave for 30 min prior to usage. Petri dishes (60 x 15 mm) containing autoclaved Tryptic Soy Agar / LB Agar were prepared according to the manufacturer's instructions. A single colony was inoculated in medium and allowed to grow at 37 °C and 150 rpm in a shaker incubator for 12 h or until reaching the log phase. After 12 h, the bacterial culture was rinsed and centrifuged at 4400 rpm for 7 min twice, and the supernatant was discarded. After washing, the optical density (O.D.) of the bacteria culture was measured using a UV-vis spectrophotometer (Thermo-Scientific Genesys 10S UV-Vis) to ensure the bacterial growth is in log phase. The resulting bacterial solution was then diluted to 1× 108CFU mL-1in PBS for the bacterial adhesion assay. Using a 24-well plate, each film sample went under 15 mins UV sterilization on both sides and was then incubated with 1 mL of the final bacterial solution for 24 h at 37 °C at 150 rpm (N = 5).

[0143] To evaluate the reduction in viable adhered bacteria, samples were extracted from bacterial solution after 24 h exposure and rinsed with 0.1 M 1x PBS to remove any loosely attached bacteria. Adhered bacteria were then detached by homogenization (Omni-TH ultrasonic homogenizer) at 25,000 rpm and vortexed for 60 s each. The resulting solution containing detached bacteria was then serially diluted in the range of 10-1- 10-3and plated on agar plates for 24 h at 37 °C. The colony-forming units (CFUs) were counted, and the number of viable bacteria on the control films with the same dilution factor was compared to the SNAPicillin films. The percentage of reduction in bacterial viability was determined by Equation 1, where C is the number of CFUs normalized to surface area (CFU cm-2) for a specific group.ATTORNEY DOCKET NO.: 222105-2300 %Reduction in Bacterial Viability (%) =C× 100% (2)Control

[0144] The final data are shown as mean ± S.D. (N = 4 independently prepared films per sample type) with results provided in FIG.7D.

[0145] Albumin adhesion

[0146] Surface albumin adhesion was quantified via PerceTMBCA Protein Assay Kits (Thermo Scientific). Briefly, films were preincubated with 35 mg mL-1Bovine Serum Albumin (BSA) for 90 mins. The film was then gently rinsed with CMF-PBS to remove any loosely bound albumin. A working solution of 200 µL was added to each well, and the well plate was placed on a shaker for 30 seconds. The 96-well plate containing samples was shielded from light and allowed to incubate at 37 C° for 30 mins. The well plate was then cooled to room temperature. Albumin was quantified by measuring the absorbance at 562 nm and interpolated using a 1:10 dilution factor and a standard curve of BSA.

[0147] Fibrinogen Adhesion

[0148] Protein adsorption on the fabricated surfaces was quantified through a modified version of a previously reported method.

[0026] Briefly, fluorescein isothiocyanate (FITC)-labeled human fibrinogen was diluted with unlabeled fibrinogen in CMF-PBS solution at a ratio of 1:10. Polymer samples were incubated in phosphate buffer solution at 37 C for 60 minutes in a 96- well plate to reach surface saturation. Background reading of the plate was collected before the addition of the diluted fibrinogen solution. The film surface was exposed to a final physiological concentration of 2mg ml-1of fibrinogen solution. The 96-well plate was subsequently incubated for 90 mins in static physiological conditions at 37 C°. After the incubation period, the sample surface exposed to fibrinogen solution was heavily diluted with CMF-PBS to remove any unbounded protein from the surface. The remaining adsorbed fibrinogen was quantified by measuring the excitation / emission of the sample at 495 / 519 nm and interpolated using a 1:10 dilution factor and a standard curve of FITC-labeled fibrinogen.

[0149] Platelet Adhesion Assessment

[0150] The fabricated surface was exposed to porcine blood plasma with a standardized concentration of platelets to assess surface platelet adhesion. All protocols related to the use of whole blood were approved by the University of Georgia Institutional Animal Care and Use Committee.

[0151] Freshly drawn porcine blood was anticoagulated with sodium citrate at a ratio of 9:1. Anticoagulated whole blood was then centrifuged at 277 RCF for 12 min. The platelet-rich plasma (PRP) portion was pipetted out without disturbing the buffy coat layer of the leukocytes. The remaining blood was then spun again at 3082 RCF for 20 mins to collect platelet-poorATTORNEY DOCKET NO.: 222105-2300 plasma (PPP). The total platelet count of the PRP was determined using a HESKA Element- HT5 Hematology Analyzer (Loveland, CO, USA). The PRP was then diluted to 2 × 108platelets mL–1with the PPP solution obtained earlier. Right before polymer incubation in normalized platelet solution, calcium chloride (2.5 mM) was added to reverse the anticoagulant (sodium citrate), hence the working solution. Individual polymer (6mm diameter) was exposed to 1mL of the working platelet solution. Samples were then incubated at 37 C° for 90 mins on a rocker. Samples were manually rocked every 30 min to ensure the surfaces were fully submerged.

[0152] Following the incubation period, the samples were removed and rinsed with PBS. The surface-adhered platelets were then lysed with a 2% v / v Triton X-100 solution for 30 minutes. Afterward, the degree of platelet adhesion was quantified by lactate dehydrogenase released during lysis using a Roche Cytotoxicity Detection Kit (λ=492 nm with λref=620 nm). A calibration curve was constructed using known dilutions of the final platelet solution. The amount of platelets adhered to the sample surface was interpolated from the calibration curve.

[0153] Whole blood Exposure

[0154] To evaluate the thrombotic activity in vitro, samples were exposed to porcine whole blood for 60 min. Samples were then rinsed with CMF-PBS and fixed in 2% glutaraldehyde. Scanning Electron Microscopy (SEM) imaging of whole blood exposed films was performed using a Thermo Fisher Scientific Teneo Field Emission SEM. The surface was fixed via a series of methanol and chemical drying. Specimens were then mounted onto aluminum specimen mounts using double-sided black carbon adhesive. Mounts were sputter-coated with 10 nm Au−Pd via a Leica EM ACE200 sputter coater (Buffalo Grove, IL). Surface imaging of films was conducted with an accelerating voltage of 5 kV.

[0155] Coagulation Time Assay

[0156] The samples were placed in 96-well plates. Porcine whole blood was obtained and diluted with 1:9 V / V (sodium citrate / blood). The anticoagulant in the whole blood was reversed with calcium chloride (0.1 M) right before usage. For the coagulation assay, 50 µL of mixed whole blood was added to the individual wells in the 96-well plate. D.I. water was introduced at various time intervals (5,10,15 mins) to terminate the clotting process. The films were carefully removed from the well and rinsed with CMF-PBS. The surface was then fixed using the same method described in section 2.4.10. before SEM imaging.

[0157] Statistical Analysis of Data

[0158] All data obtained are expressed as mean ± S.D. One-way ANOVA with post-hoc analysis via Tukey's Test for correction of multiple comparisons and unpaired t-tests were calculated to determine any significant difference between the material groups and at differentATTORNEY DOCKET NO.: 222105-2300 time points. GraphPad Prism Software v9.1 (San Diego, CA, USA) was used for all analyses. Values of p < 0.05 were deemed statistically significant. RESULTS AND DISCUSSION

[0159] Material Characterization

[0160] Surface Preparation

[0161] The surface design goal was to fabricate a synergistic material that can minimize foreign body response while posing broad-spectrum antibacterial efficacy. The NO-releasing blue dextran (NOBD) surface was fabricated via a modified version based on previous literature

[0020] (FIG. 3). NO donor, SNAP, was incorporated into SR films via the solvent- swelling method in THF. The surface was then aminated via APTMS, and Schiff's base reaction was formed with the oxidized dextran solution. Sodium cyanoborohydride was later added to facilitate the reductive amination reaction to a secondary amine. When the aldehyde group on dextran reacts with primary amine on the SR surface, it can be reversed by hydrolysis in an aqueous solution. The addition of sodium cyanoborohydride stabilized the formation of Schiff's base. Afterward, dextran-coated SR films were incubated in Cibcaron Bue 3GA dye solution to allow for nucleophilic substitution between the chlorine moiety of the triazine dye and the hydroxyl groups of dextran substrate in basic aqueous media.

[0162] Fourier-Transform infrared spectroscopy (FTIR) analysis

[0163] The surface of the sample films was assessed via FTIR analysis (FIG.4A). shows the FTIR spectra of the films. After evaluation, FTIR spectra of dextral ctrl, BD ctrl, and NOBD sample groups exhibited a stretching band characteristic of O-H, hydrogen-bonded alcohol, at ~ 3380 cm-1. The bands with moderate intensity at 1565 cm-1and 1624 cm-1are the spectral evidence that characterizes the structure's C=C and C=O stretching vibration. The bands at 1120 cm-1and 1230 cm-1indicate the presence of S=O stretching vibrations, a characteristic bond of the Cibacron blue F3GA. Another potential signature band between 850-700 cm-1frequency possibility indicates the C-Cl stretching vibration of the dye. The result of FTIR confirmed the successful immobilization of blue dextran onto the SR surface.

[0164] Surface Static Contact Angle

[0165] The surface wettability of a material is an important parameter to consider when designing a biomaterial. Surface wettability can have consequences on protein adsorption, bacterial attachment, cell adhesion, and so on. Moreover, wettability also plays a role in the water uptake of the material, and low water uptake can facilitate more extended drug diffusion. For instance, SR has a rigid cross-linked structure that translates to a reduced rate of NO release and component migration within the resin but a longer therapeutic window

[0027] . Static contact angles for SR Ctrl and NO Ctrl were 109.1± 0.97° and 106.2 ± 4.51°, respectively. Previous studies have also reported that SNAP incorporation does not alter the surfaceATTORNEY DOCKET NO.: 222105-2300 wettability of the polymer. After immobilization of blue dextran on the surface, a significant drop in contact angle was observed for the BD control and NOBD films, with an average of 52.91 ± 5.84° and 58.26 ± 8.25°, respectively (FIG. 4B). This stable drop of ~50°on the BD immobilized group indicates the presence of dextran on the surface. The persistence of dextran is due to the ability of the molecule to bind to multiple amine groups on the SR surface, thus preventing both migration of unoxidized PDMS to the surface and polar group rotation

[0028] .

[0166] Coating Stability

[0167] The anthrone method was used to determine the concentration of dextran. Dextran is dehydrated by concentrated H2SO4to form furfural. Anthrone's enol tautomer, an active form of anthranol, reacts by condensing with furfural to yield a green / blue color, which can read at 620 nm

[0023] . BD Ctrl and NOBD samples were incubated in PBS at physiological conditions for 24 hr before coating stability assessment. Results demonstrated that after 24 hr, surface- immobilized dextran concentration was not altered significantly. At the end of the measuring period, surface-immobilized dextran concentration for BD control was reduced by 32.38%, while NOBD only lost 28.18% of the immobilized dextran (FIG.4C).

[0168] Cibacron blue immobilization on NOBD surfaces was quantified via a spectrophotometric method. UV-absorption spectrum of CB obtained in PBS was prepared using 0.0625 mM, 0.03125 mM, and 0.015625 mM. Absorption spectra of CB in a PBS solution exhibited a broad maximum spectra adsorption centered at 610 nm (FIG.5A). The standard curve of CB concentration was also recorded. BD Ctrl and NOBD samples were incubated in PBS at physiological conditions (37 C°), and the amount of CB leaching was quantified within 48 hr. As illustrated in FIG. 5B, CB leaching started approaching the plate at hour 8 of the measurement. At the end of the leaching experiment, both BD-containing samples had minimal cumulative CB leaching of less than 0.01 µM. Collectively, these results demonstrated the stability of the blue dextran on the SR surface after covalent immobilization under physiological conditions (37 C°).

[0169] NO release under physiological condition

[0170] NO Release Characteristics

[0171] NO's therapeutic role strongly depends on the surface flux concentration and localized release. Therefore, investigating the NO elution behavior from the polymer-solution interface will be a valuable metric for predicting the surface-environment interaction. NO is released from SNAP under the stimulus of light, heat, moisture, and metal ions. As a class of RSNOs, SNAP undergoes hemolytic cleavage of the S-N bond and decomposes into a disulfide dimer of NAP or (NAP)2to release NO.ATTORNEY DOCKET NO.: 222105-2300

[0172] NO release measurements from the NO Ctrl and NOBD films were carried out using a chemiluminescence-based nitric oxide analyzer. PBS with EDTA (pH 7.4) was used during NO release measurement to chelate the presence of trace metal ions, which can catalyze the NO release.

[0173] FIG.6B shows NO surface flux released from NO Ctrl and NOBD over 24 hr. NOBD films had the initial release of 4.43 ± 0.67 × 10-10mol min-1cm-2in hour 0, and this elevated flux was sustained till hour 24 when the samples were still able to release at 3.37 ± 0.83 × 10-10mol min-1cm-2. In comparison, NO Ctrl had lower flux at both 0h and 24 h time points, of 1.62 ± 0.21 × 10-10mol min-1cm-2and 0.68 ± 0.16 × 10-10mol min-1cm-2, respectively. The higher NO flux of NOBD can be mainly attributed to the enhanced surface hydrophilicity compared to the NO Ctrl. The surface wettability greatly increased after the immobilization of the BD, which facilitated a faster rate of hydrolysis into the polymer matrix. The elevated NO release from NOBD samples can prevent and eradicate infection during the 24 hr timeframe, which is a critical window to prevent hospital-acquired infections after device insertion.

[0174] SNAP Leaching

[0175] Excess SNAP diffusion can lead to undesirable side effects in the physiological system. In this study, NO ctrl and NOBD films were incubated in PBS under physiological conditions. SNAP diffusion out of the samples was monitored for up to 60 h via UV-vis spectroscopy. As shown in FIG.6A, SNAP diffused out of the polymer matric rapidly within the first 20 h and approached the plateau after 24 h. NOBD had a higher SNAP diffusion of 0.10 ± 0.03 mg mL-1SNAP compared to NO Ctrl's 0.03 ± 0.01 mg mL-1. The greater surface wettability of the NOBD facilitated this enhanced SNAP leaching profile. Previous literature has reported that hydrophilic polymers tend to diffuse more SNAP over time

[0027] .

[0176] In Vitro Biocompatibility Evaluation

[0177] Enhanced Affinity for Albumin

[0178] Before further testing the sample surface's biocompatibility and antifouling capabilities, it is important to validate that NOBD surface have an enhanced affinity for the plasma protein. As shown in FIG. 7A, BD Control increased surface absorbed albumin by 6-fold when compared to SR Ctrl. This enhanced absorbed albumin was more abundant on the NOBD surfaces, resulting in a significant 10-fold increase compared to the SR Ctrl. A slightly enhanced affinity was observed in the NO-releasing sample groups when compared to their SR Ctrl and BD Ctrl counterparts. The results demonstrated here are one of the first investigations into albumin-NO surface interaction. Nonetheless, it is evident that BD- immobilized surface has enhanced affinity for albumin at physiological concentrations.

[0179] In Vitro Screening Evaluation of CytocompatibilityATTORNEY DOCKET NO.: 222105-2300

[0180] Ensuring the NOBD surfaces do not elicit cytotoxicity to cells is critical before further in vitro evaluation of the material. Therefore, the relative cell viability of BJ Fibroblast cells were assessed against exposure to the leachate extracts from all control samples and NOBD in accordance with the ISO 10993-5 standard of biological evaluation of medical devices

[0024] . FIG. 7B shows the cell viability of the surface against BJ Fibroblast cells compared to the controls (BJ Fibroblast grown in DMEM media in the absence of any abstracts). All sample groups were above the 80% viability threshold, indicating their cytocompatibility. A slightly enhanced cell viability can be seen in the NO Ctrl group when compared to the other two sample types. Studies have shown in the past that the NO-releasing polymer tends to proliferate cell viability slightly. Low concentrations of NO have been observed to enhance fibroblast collagen synthesis

[0030] .

[0181] In Vitro Screen Evaluation of Hemocompatibility

[0182] Another important parameter to consider during the design of the medical device is its material-blood compatibility. Similar to how coating can elicit unwanted toxicity toward cells, surface design can also lower compatibility with blood and cause lysis of the red blood cell. In this study, samples were evaluated for their hemolysis activity according to the NAMSA ASTM F756 protocol. Per protocol definition, samples with 2-5% hemolytic activity are considered slightly hemolytic, while >5% are considered hemolytic. As shown in FIG. 7C, all tested samples are nonhemolytic after subtracting the negative Ctrl (HDPE).

[0183] In vitro Antifouling Assessment

[0184] Resistance to Fibrinogen Adhesion

[0185] In this study, the fabricated NOBD surface has a higher binding affinity to albumin. When placed in the blood environment, the surface-bounded albumin layer will reduce clotting by minimizing the intrinsic coagulation pathway contributed by the fibrinogen adsorbed.

[0186] The samples were co-incubated in a solution containing physiologically relevant concentrations of albumin (35 mg mL-1) and fibrinogen (2 mg mL-1) to mimic the in vivo environment. As a result, a general trend towards resistance to fibrinogen fouling was observed (FIG.8A). NO control surface showed a 95.63% increase in fibrinogen absorption when compared to untreated SR control. Meanwhile, BD control itself was capable of reducing the fibrinogen absorption by 17.37%. Remarkably, when combined with NO, NOBD surfaces reduced fibrinogen by 62.75% when compared to untreated SR control and 80.96% when compared to NO control. Fluorescent images of FITC-Fg on surfaces also showed that fibrinogen fouling was largely prevented by BD control and NOBD surfaces (FIG.8B).ATTORNEY DOCKET NO.: 222105-2300

[0187] Platelet Attenuation Capability of NOBD Surfaces

[0188] To verify the translational antifouling capability of the BD surface and platelet attenuation from the NO release, in vitro platelet adhesion test was carried out. After 90 min exposure to fresh porcine PRP, BD control, and NOBD surface demonstrated a significant reduction in adhered platelet when compared to SR control and NO control (FIG.9A). NOBD group showed the highest and statistically significant reduction of 68.49% and 80.77% when compared to NO control and SR control, respectively. Interestingly, even without the platelet modulating effects from the NO release, the BD Ctrl group was able to reduce 47.28% and 67.83% of the platelet reduction when compared to NO control and pristine SR control, respectively.

[0189] Whole blood exposure SEM demonstrated that SR control has the most total platelet adhesion, with the majority being in the secondary activation stage (FIG.9B). Meanwhile, NO Ctrl was able to decrease the amount of platelet adhesion on the surface by 5-fold, yet it still has some adhered platelets with secondary and primary activation stages. NOBD surface had the least amount of platelet attachment onto the surface, regardless of the activation stage of the platelet (FIG.9C).

[0190] In Vitro Coagulation Effect

[0191] The fabricated surface's ability to slow the coagulation process was assessed in vitro by exposing them to porcine whole blood to evaluate their anticoagulant efficacy. D.I. water was introduced every 5, 10, and 15 mins to stop the clotting process (FIG. 10A). Under physiological conditions, clot formation often commenced within a time frame of 5-6 mins

[0032] . The SR Ctrl group needed a similar time frame to form a clot on the polymer surface. At the end of the 15 min clotting assay, the NOBD group remained the only surface free of clot formation. A similar trend can be observed on the representative SEM image of each group at the end of 15 mins (FIG.10B). SR Ctrl was covered with fibrin sheath and clot formation, while individual and activated platelet clusters can be observed on the NO and BD Ctrl surfaces. NOBD surface was covered by the least amount of foulants, including the least degree of platelet activation. These data suggest that the NO-BD films can reduce thrombus formation by attenuating platelet adhesion and activation. CONCLUSION

[0192] Successful fabrication of NO-releasing blue dextran immobilized surface enables synergistic therapeutic effect from NO and the anti-fouling surface. NOBD surface demonstrated cytocompatibility against human BJ fibroblast cells and hemocompatibility whenATTORNEY DOCKET NO.: 222105-2300 evaluated in porcine blood. NOBD polymer limited the adhesion of bacteria and eradicated 93.52% of viable E.coli on the surface (FIG.7D).

[0193] This novel surface illustrated significant prevention of platelet, albumin, and fibrinogen attachments. These results indicate the potential of NOBD surface to minimize surface-fouling from both intrinsic coagulation and activated platelet pathways.ATTORNEY DOCKET NO.: 222105-2300 REFERENCES [1] C.A. Labarrere, A.E. Dabiri, G.S. Kassab, Thrombogenic and Inflammatory Reactions to Biomaterials in Medical Devices, Front Bioeng Biotechnol 8 (2020) 123. [2] K. Albrecht, H. Nave, D. Breitmeier, B. Panning, H.D. Tröger, Applied anatomy of the superior vena cava—the carina as a landmark to guide central venous catheter placement, British Journal of Anaesthesia 92(1) (2004) 75-77. [3] D.C. McGee, M.K. Gould, Preventing complications of central venous catheterization, New England journal of medicine 348(12) (2003) 1123-1133. [4] L.B. Kreuziger, J. Jaffray, M. Carrier, Epidemiology, diagnosis, prevention and treatment of catheter-related thrombosis in children and adults, Thrombosis research 157 (2017) 64- 71. [5] T.C. Major, H. Handa, G.M. Annich, R.H. Bartlett, Development and hemocompatibility testing of nitric oxide releasing polymers using a rabbit model of thrombogenicity, Journal of biomaterials applications 29(4) (2014) 479-501. [6] D. John P. Cunha, FACOEP, HEPARIN SIDE EFFECTS CENTER, 2022. https: / / www.rxlist.com / heparin-side-effects-drug-center.htm. [7] A.J. Rabelink, [Nobel prize in Medicine and Physiology 1998 for the discovery of the role of nitric oxide as a signalling molecule], Ned Tijdschr Geneeskd 142(52) (1998) 2828-30. [8] Y. Wo, E.J. Brisbois, R.H. Bartlett, M.E. Meyerhoff, Recent advances in thromboresistant and antimicrobial polymers for biomedical applications: just say yes to nitric oxide (NO), Biomaterials Science 4(8) (2016) 1161-1183. [9] A.W. Carpenter, M.H. Schoenfisch, Nitric oxide release: Part II. Therapeutic applications, Chemical Society Reviews 41(10) (2012) 3742.

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Claims

ATTORNEY DOCKET NO.: 222105-2300 We claim:

1. An article comprising (a) a nitric oxide releasing compound and (b) dextran bonded to at least one surface of the article, wherein a sulfonated triazine is covalently bonded to dextran.

2. The article of claim 1, wherein the nitric oxide releasing compound is an S-nitrosothiol conjugated polymer, an S-nitrosothiol modified-dendrimer, an S-nitrosothiol modified polysaccharide, an S-nitrosothiol modified nano / microparticle, an S-nitrosothiol modified-protein, a nitrate, an N-diazeniumdiolates (NONOate), or an S-nitrosothiol (RSNO).

3. The article of claim 1, wherein the nitric oxide release agent is S-nitroso-N- acetylpenicillamine, S-nitroso-glutathione, S-nitroso-N-acetylcysteine, S- nitrosocysteine, S-nitrosopenicillamine, S-nitroso-B,D-glucose, S-nitrosocaptopril, S- nitrosocysteamine, S-nitroso-3-mercapto-propanoic acid, S-nitroso-N-acetyl-l-cysteine ethyl ester (SNACET), S-nitroso-N-acetyl-L-methionine, or S-nitrosomercaptoethanol.

4. The article of claim 1, wherein the nitric oxide releasing compound is a modified antibiotic compound comprising a nitric oxide release agent covalently attached to an antibiotic molecule.

5. The article of claim 4, wherein the antibiotic molecule is ampicillin, vancomycin, gentamicin, or cephalexin.

6. The article of claim 4, wherein the modified antibiotic compound comprises S-nitroso- N-acetylpenicillamine covalently attached to ampicillin.

7. The article of claim 1, wherein the nitric oxide releasing compound is on the surface of the article.

8. The article of claim 1, wherein the nitric oxide releasing compound is on the surface of the article is incorporated throughout the article.

9. The article of claim 1, wherein dextran is bonded to the at least one surface of the article by a linker.

10. The article of claim 9, wherein the linker comprises a silane compound comprising a nucleophilic group.

11. The article of claim 9, wherein the linker comprises a silane compound comprising at least one hydroxyl group, thiol group, or amino group.ATTORNEY DOCKET NO.: 222105-2300 12. The article of claim 9, wherein the linker comprises an aminosilane.

13. The article of claim 9, wherein the linker comprises a compound having the structure I R1R1Si(CH2)oNH2R1I wherein each R1is independently an alkoxy group, cycloalkoxy group, or aryloxy group, and o is an integer from 1 to 10.

14. The article of claim 1, wherein the dextran is bonded to the at least one surface of the article by an aminosilane, wherein the aminosilane forms an imine group with dextran.

15. The article of claim 1, wherein the dextran has an average molecular weight of about 50 kDa to about 1,000 kDa.

16. The article of claim 1, wherein the sulfonated triazine is selected from the group consisting of Cibacron Blue F3GA, Procion Red H-E7B, Procion Green H-4G and Yellow H-E3G.

17. The article of claim 1, wherein the sulfonated triazine is Cibacron Blue F3GA.

18. An article produced by the method comprising (a) attaching linker to at least one surface of the article, wherein the article comprises a nitric oxide releasing compound to produce a pre-functionalized article; (b) contacting the pre-functionalized article with oxidized dextran, wherein the linker covalently bonds to the oxidized dextran to produce a dextran functionalized article; and (c) contacting the dextran functionalized surface with a sulfonated triazine, wherein the sulfonated triazine forms a covalent bond with the oxidized dextran.

19. The article of claim 18, wherein the article comprising the nitric oxide releasing compound is produced by soaking the article in a solution of the nitric oxide releasing compound.

20. The article of claim 18, wherein step (a) comprises heating the article in a solution comprising the linker.ATTORNEY DOCKET NO.: 222105-2300 21. The article of claim 20, wherein the linker comprises an aminosilane.

22. The article of claim 18, wherein step (b) comprises heating the pre-functionalized article in a solution comprising the oxidized dextran.

23. The article of claim 22, wherein a borohydride is added after step (b) and prior to step (c).

24. The article of claim 22, wherein a borohydride is sodium cyanoborohydride.

25. The article of claim 18, wherein step (c) comprises contacting the dextran functionalized surface with a solution comprising the sulfonated triazine.

26. The article of any one of claims 1-25, wherein the article comprises a medical device.

27. The article of claim 26, wherein the device is an implantable device.

28. The article of claim 26, wherein the device is selected from the group consisting of: a vascular catheter, a urinary catheter, other catheters, a coronary stent, a wound dressing, and a vascular graft.

29. The article of any one of claims 1-25, wherein the article comprises silicone rubber.

30. The article of any one of claims 1-25, wherein the article has a contact angle of about 50 degrees to 65degrees.

31. The article of any one of claims 1-25, wherein the article releases nitric oxide for at least 24 hours.

32. The article of any one of claims 1-25, wherein the article binds to albumin.

33. The article of any one of claims 1-25, wherein the article reduces or prevents fibrinogen adhesion to the article.

34. The article of any one of claims 1-25, wherein the article reduces or prevents platelet adhesion to the article.

35. The article of any one of claims 1-25, wherein the article reduces or prevents bacterial growth on the article.

36. The article of any one of claims 1-25, wherein the article reduces or prevents biofilm formation on the article.

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