WO2004009253A1 - Surface-localized release of an anti-biofouling agent via micropatterning - Google Patents

Abstract

Methods and compositions for imparting surface-localized release of an anti-biofouling agent on a substrate, and a substrate itself. The substrate includes an anti-biofouling agent disposed on a surface of the substrate in a pre-determined pattern, wherein the anti-biofouling agent includes: (a) a nitric oxide (NO) donor moiety; and (b) a matrix adapted to release the NO in a controlled release profile.

Description

Description

SURFACE-LOCALIZED RELEASE OF AN ANTI-BIOFOULING

AGENT VIA MICROPATTERNING

Cross Reference to Related Applications

The present patent application is based on and claims priority to U.S.

Provisional Application Serial Nos. 60/397,421 and 60/401 ,105, both entitled

"SURFACE-LOCALIZED RELEASE OF AN ANTI-BIOFOULING AGENT VIA

MICROPATTERNING", which were filed July 19, 2002 and August 5, 2002, respectively, and are incorporated herein by reference in their entireties.

Technical Field The presently claimed subject matter relates to substrates adapted for both in situ and in vivo applications. More specifically, the presently claimed subject matter relates to methods and compositions for imparting surface- localized release of an anti-biofouling agent on a substrate, and to a substrate itself.

Abbreviations

ACD acid citrate dextrose

AEMP3 (aminoethylaminomethyl)-phenylethyl- trimethoxysilane

AFM atomic force microscope/microscopy

Al aluminum atm atmospheres

BTMOS isobutyltrimethoxysilane

DET3 (3-trimethoxysilylpropyl)diethylenetriamine e-beam electron beam

HMDS hexamethyldisilazane

MAHMA/N₂O₂ (Z)-1-[Λ/-methyl-Λ/-[6-(/V- methylammoniohexyl)-amino]]diazen-1- ium-1 ,2,-diolate min minute(s)

MTMOS methyltrimethoxysilane NO nitric oxide

PDMS polydimethylsiloxane ppb parts per billion

PRP platelet rich plasma SEM scanning electron microscope/microscopy

Si sila or silicon

Si-OH silanol group

UV ultraviolet (light)

Vis visible (light) Background Art

The success of certain medical devices depends upon favorable biocompatibility between the device and the environment in which it is employed. For blood-contacting devices, the interaction between the surface of the device and blood often results in protein deposition, platelet adhesion and activation, and initiation of the intrinsic coagulation cascade

(Banerjee et al., 1997; Suh, 1998; Benmakroha et al., 1995). Significant research has been devoted to developing polymeric coatings with improved thromboresistivity (i.e., the ability to resist platelet adhesion) because of its importance in designing improved bedside blood gas/electrolyte instruments, vascular blood pressure monitors, implantable catheters, dialysis units, mechanical extracorporeal circulation devices (i.e., artificial blood- oxygenation), and in vivo intravascular sensors (Espadas-Torre & Meyerhoff,

1995; Chen et al., 1998; Kyrolainen et al., 1995). Strategies reported for improving the blood compatibility of such devices include the use of more biocompatible polymer coatings (e.g., silicone rubber and/or polyurethane;

Yoda, 1998) and various surface modifications, including heparin coatings

(Weerwing et al., 1998; Walpoth et al., 1998), endothelial cell seeding (Pasic et al., 1996), albumin coatings (Marois et al., 1996), and self-assembly strategies (Ratner, 1998). The control of bacterial adhesion and inflammation at the material/tissue interface represents an equally important aspect of the biocompatibility for non-blood contacting implants such as tissue-based sensors and artificial limbs. The performance and ultimate usefulness of such devices is dependent upon materials that prevent infection. Despite sterilization and aseptic procedures, bacterial infection remains a major impediment to the extended utility of these implants. Though helpful in controlling bacterial adhesion, sterilization is often destructive (e.g., results in polymer degradation) and does not eliminate the risk of infection. In fact, over half of all hospital-acquired infections are coupled to implanted medical devices (Boelens et al., 2000). Implant-associated infections are commonly the result of microbial biofilm formation at or near the site of implantation, originating from bacterial adhesion. Once formed, biofilms are resilient against the immune system and conventional antibiotic treatments (Brown et al., 1988; Dunne et al., 1993), and cause persistent, chronic illnesses with many acute, universal symptoms.

Although the conventional (systemic) administration of antibiotics can successfully treat illness by destroying planktonic cells, drug therapy is often ineffective at reducing biofilm-associated infection (Brown et al., 1988). Poor diffusion, heterogeneous chemical conditions (e.g., pH, pO₂, ionic strength), and a mass of growth phases within the biofilm generally result in poor antibiotic activity (Potera, 1999; Costerton et al., 1999). Due to the difficulties of eliminating biofilm formation by systemic antibiotic therapies, much work has focused on developing materials that prevent the initial cellular adhesion stages of biofilm formation (Imazato et al., 1998; Elbert & Hubbell, 1998; O'Connor et al., 1998; Vacheethasanee & Marchant, 2000; Gottenbos et al., 2000; Razatos et al., 2000; Chapman et al., 2001 ). Although materials made from or integrated with poly(ethylene glycols), polyurethanes, or silicone elastiomers reduce the degree of attached cells, their ability to reduce implant-associated infection in vivo is limited. Strategies to overcome biofouling (due to platelet and bacterial adhesion) at the implant site thus represent a long felt and ongoing need in the art. The development of new approaches for improving the in vivo performance of implants such as blood-contacting and tissue-based devices is of particular interest. The presently claimed subject matter addresses this and other needs in the art.

Summary A method for preparing a substrate, a substrate, and an antifouling composition are disclosed. In one embodiment, the method comprises applying an anti-biofouling agent to a substrate in a pre-determined pattern, wherein the anti-biofouling agent comprises: (a) a nitric oxide (NO) donor moiety; and (b) a matrix adapted to release the NO in a controlled release profile.

Optionally, the predetermined pattern is a microarrayed pattern. The matrix can comprise a sol-gel. In this case, the sol-gel can comprise an aminosilane, an organosilane, or a combination thereof. Optionally, the aminosilane is present in an amount up to 100% of total silane.

The sol-gel can also comprise a composition having the general formula (R¹)_n-Si-(R²)_4-n, where R¹ and R² are the same or different and are selected from the group consisting of hydrogen, amino, alkyl, alkylamino, alkoxy, alkenyl, alkenylamino, alkenoxy, alkynyl, alkynlamino, alkynoxy, aryl, arylarriino, arylόxy, arid combinations thereof, and where ri is 1 , 2, 3, or 4.

The substrate can be implanted at an implant site in a subject or be used for in situ applications.

Accordingly, it is an object of the presently claimed subject matter to provide a novel substrate, and a method and composition for use in preparing the same. This and other objects are achieved in whole or in part by the presently claimed subject matter.

Some of the objects of the presently claimed subject matter having been stated hereinabove, other objects will be evident as the description proceeds, when taken in connection with the accompanying drawings as best described hereinbelow.

Brief Description of the Drawings Figures 1A-1C are schematic diagrams of sol-gel micropatterning methods based on polymeric polydimethylsiloxane (PDMS) templates created from silicon wafers prepared by photolithography (Figure 1A). The elastomeric template is placed in contact with a substrate, creating a series of microchannels on the surface (Figure 1 B). Sol droplets are placed at one end of the template and are drawn into microchannels by capillary flow. The sol-gel pattern is allowed to dry for 24 hours before template removal. Alternatively, droplets of sol solution can be placed directly on a surface.

The template is then placed on top of solution and pressure is applied to displace sol and allow for contact between template and surface (Figure 1C).

The template is removed after formation and drying of sol-gel for 24 hours.

Figures 2A-2D are contact mode atomic force microscopy (AFM) images of 20% (aminoethylaminomethyl)-phenylethyl-trimethoxysilane (AEMP3)/methyltrimethoxysilane (MTMOS; i.e., AEMP3 comprises 20% of total silane) sol-gel micropatterns on glass. Substrates in Figures 2A and 2B were fabricated using applied pressure. Substrates in Figures 2C and 2D were fabricated by capillary flow. Horizontal cross sections were used to determine feature dimensions.

Figure 3 presents a series of reaction schemes for sol-gel preparation and diazeniumdiolate formation upon exposure to high pressures of NO. Schemes 1 and 2 represent the two-step sol-gel formulation involving condensation and hydrolysis reactions, respectively. Schemes 3 and 4 illustrate formation of diazeniumdiolate structure, which upon submersion in aqueous solution decomposes to release NO and reform the diamine precursor.

Figure 4 is a plot of real-time NO (in parts per billion - ppb; time in minutes - min.) released from a 20% AEMP3/MTMOS sol-gel micropattem. Figures 5A-5C are contact mode AFM images used to characterize the stability of 20% AEMP3/MTMOS sol-gel pattern on glass before pressurizing (Figure 5A); after pressurizing to 5 atmospheres (atm) NO for 60 hours (Figure 5B); and after immersion in buffer for 3 days (Figure 5C). Figures 6A-6D are schematic representations of cell adhesion to homogeneously NO-releasing sol-gel film (Figure 6A); closely- (Figure 6B); medium- (Figure 6C); and widely spaced NO-releasing microstructures (Figure 6D).

Figures 7A and 7B are scanning electron microscopy (SEM) images of porcine platelet adhesion (light) to sol-gel micropattem control (Figure 7A) and NO-releasing sol-gel micropattem (Figure 7B). Sol-gel: 20% AEMP3/MTMOS.

Detailed Description Disclosed herein is the use of nitric oxide (NO) releasing patterns for developing improved implants that function more effectively in vivo. In one embodiment, controlled release of NO locally at the implant site addresses lingering biofouling problems that have heretofore caused the erratic performance of such implants while retaining the devices' original function.

By way of example, reducing both platelet and bacterial adhesion, and thus surface induced thrombosis and bacterial infections, can improve the biocompatibility of medical implants. The release of nitric oxide (NO) from polymers has been highly effective toward improving the thromboresistivity of blood-contacting devices (Smith et al., 1996; Hanson et al., 1995; Mowery et al., 2000; Schoenfisch et al., 2000; Espadas-Torre et al., 1997) and reducing bacterial adhesion and biofilm formation in vitro (Nablo et al., 2001). Micropatterning techniques offer a novel approach for designing surfaces that release NO while retaining functionality for other applications (e.g., sensors). Herein, the fabrication and characterization of micropatterned NO-releasing materials based on aminosilane-containing sol- gels is described. The stability of sol-gel micropatterns under both solution and high-pressure conditions is assessed using atomic force microscopy (AFM). Heterogeneously modified substrates are further characterized both with respect to NO release kinetics and in vitro platelet adhesion. In vitro studies indicate that micropatterned sol-gels are capable of releasing NO from distinct regions on a substrate and effectively reducing biofouling L Definitions

Following long-standing patent law convention, the terms "a" and "an" refer to "one or more" when used in this application, including the claims.

The term "controlled release profile", as used herein, refers to administration of an active agent such as NO in a pre-selected manner, wherein the temporal features and dose of agent release are predictable. In the case of the use of a coated implant during surgery, a short continual controlled release profile can be provided. In the case of reducing an initial biofouling response, a controlled release profile characterized by an initial surge in release followed by a trailing off of the release can be provided. In the case of a long-term implantation, a controlled release profile characterized by long-term continual controlled release of NO can be provided.

The term "subject", as used herein, refers to any proposed recipient of an implant. Contemplated subjects include warm-blooded vertebrates, including mammals such as humans, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economic importance (animals raised on farms for consumption by humans), and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses. Also contemplated is the treatment of birds, including the treatment of those kinds of birds that are endangered and/or kept in zoos, as well as fowl, and more particularly domesticated fowl, for example, poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Thus, contemplated is the treatment of livestock, including, but not limited to domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like. The term "biocompatible" is used herein to refer to a material or implant that is compatible with a biological system, such as that of a subject as defined herein. The term "biocompatible" also refers to a material or substrate that can be implanted internally in a subject, wherein the material or substrate has been adapted to resist biofouling as defined herein, such as by treatment with an anti-biofouling agent. The term "biofouling" refers to undesirable contamination of a substrate (e.g., an implantable substrate) by a biological agent. The presently claimed subject matter can encompass biofouling of any substrate by any biological agent (in vivo and in vitro). Representative forms of biofouling include, but are not limited to protein adhesion, platelet adhesion, and microbial adhesion. Representative microbial species include any commonly associated with implant biofouling, such as, but not limited to, Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus mutans, Bacillus cereus, Rhodobacter sphaeroides, and Pseudomonas aeruginosa. The term "thrombosis" is used herein to refer to the aggregation of platelets to form a dense network of cells or a thrombus (blood clot).

The term "thromboresistant" is used herein to refer to a material or implant that is resistant to biofouling caused by platelet adhesion and subsequent thrombus formation in vitro and/or in vivo. As used herein, the terms "micro", "microscopic", "micrometer-sized",

"microstructured", "microscale", "micro-complexes", "microparticles", and grammatical derivatives thereof are used synonymously and interchangeably and refer to a structure that has a longest dimension of about 1000 μm or less. Thus, microstructures can be structures ranging in size from about 1 to 1000 μm, including 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 50, 100, 500 and 750 μm, as well as intermediate values. A microparticle can have any diameter less than or equal to 1000 μ , including 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 50, 100, 500 and 750 μm. In one embodiment, a microstructure comprising a nitric oxide donor moiety is provided in accordance with the presently claimed subject matter.

I General Considerations

Nitric oxide (NO), a diatomic free radical naturally synthesized in subjects when L-arginine is converted to L-citrulline (Moncada et al., 1993), serves multiple bioregulatory processes in the cardiovascular, respiratory, gastrointestinal, genitourinary, and central and peripheral nervous systems (Marietta et al., 1990). The specific function of NO is regulated by both the location of release and the local amounts generated. Although it is not the inventors' desire to be bound by a particular theory of operation, the properties of NO that are believed to be beneficial in developing more biocompatible implants include its involvement in the regulation of platelet activation and aggregation and macrophage-induced phagocytosis. It is established that at a site of injury, endothelial cells lining the vascular walls release low levels of NO to regulate the activity of platelets, such that excessive adhesion and thrombus formation do not occur (Jurasz et al., 2000). Furthermore, it is known that both monocytes and macrophages synthesize NO when stimulated by bacteria and other pathogens (Albina & Reichner, 1998; Lopez-Farre et al., 1998). Although the mechanism remains unclear, NO induces a series of morphological changes in the pathogenic cell, ultimately resulting in death (Lopez-Farre et al., 1998). NO release from diazemiumdiolate-containing materials can reduce platelet adhesion by mimicking the endothelium NO-release of healthy blood vessels (Diodati et al, 1993; Radomski et al., 1992; Hanson et al., 1995; Smith et al., 1996). Recently, the use of (Z)-1 -[Λ/-methyl-Λ/-[6-(/V- methylammoniohexyl)-amino]]diazen-1-ium-1 ,2,-diolate (MAHMA/N₂O₂) for improving the thromboresistivity (ability to inhibit the formation of a surface thrombosis) of hydrophobic polymers for use as coatings for blood- contacting intravascular sensors and extracorporeal circuits was reported (Espadas-Torre et al., 1997; Mowery et al., 1998; Mowery & Meyerhoff, 1999a; Mowery& Meyerhoff, 1999b; Mowery et al., 2000; Schoenfisch et al., 2000). The presently claimed subject matter pertains in part to the use of NO releasing coatings with improved anti-biofouling characteristics for developing implants that function more effectively in vivo. 111. Anti-Biofoulinq Agent

In one embodiment an anti-biofouling agent comprises: (a) a nitric oxide (NO) donor moiety; and (b) a matrix adapted to release NO in a controlled release profile. A matrix composition provides for the controlled release of NO in an in vivo or other desired setting. A representative NO donor moiety comprises a diazeniumdiolate moiety (Figure 3, Scheme 2)

Diazeniumdiolates are stable at low temperatures in the absence of hydrogen donors (e.g., water). While stable under ambient conditions, diazeniumdiolates undergo spontaneous (non-enzymatic) generation of NO in aqueous solution as shown in Figure 3, Scheme 3. The rate of this type of NO release is tunable by varying the pH, temperature, environment, and/or amine precursor structure. Depending on the particular structure of the amine precursor, the aqueous solution half-lives of diazeniumdiolates at pH 7.4 and 37°C range from less than one minute to several days. Background research efforts concerning diazeniumdiolates are disclosed in Maragos et al., 1991 ; Hrabie et al., 1993; and Hrabie & Keefer, 2002.

To address toxicity concerns, an aspect of the presently claimed subject matter pertains to the synthesis and characterization of NO-releasing matrix materials wherein the NO donors are covalently bound to a silane backbone. Therefore, undesirable leaching of residual amines into the surrounding aqueous environment is avoided because the NO donor remains covalently linked to the silane. In a representative embodiment, a matrix composition comprises a sol-gel. Sol-gels are materials that can be made at low-temperatures from alkoxide precursors and water. Sol-gel preparation processes can generally be divided into the following four categories: mixing (to form a solution), gelation, aging, and drying. In a typical procedure, an alkoxide precursor is mixed with water, a catalyst (acid or base), and a mutual solvent such as methanol or ethanol, to form a solution (the sol). Hydrolysis results in the formation of silanol groups (Si-OH; Figure 3, Scheme 1), which cross-react further with either silanol groups or ester groups to form siloxane polymers where R is an alkoxy group (e.g., methoxy or ethoxy group), for example, and R' can be an amino moiety, for example (Figure 3, Scheme 2). Polycondensation reactions then lead to the formation of polymeric gel. During aging, polycondensation reactions continue, thereby increasing the tensile strength and density of the gel. In the drying process, residual solvent is removed from the interconnected pore network. The type of silane, reaction rates, and the degree to which the reactions go to completion determine the physical porosity, rigidity, and wettability of the resulting sol- gel. By controlling a range of processing conditions including pH, solvent, water content, drying time, and the type and concentration of the silane precursor (or precursors), materials with an assortment of physical properties (e.g., wettability and micropore structures) can be prepared. A variety of sol-gel materials capable of generating NO atNariable rates and amounts (without concomitant leaching) is thus provided herein.

A range of diazeniumdiolate-containing sol-gels has been synthesized using various processing conditions, silicon alkoxide precursors, and silicon amino-alkoxides (also referred to herein as alkoxysilanes and aminosilanes respectively). Because aminosilanes (which facilitate diazeniumdiolate formation) hydrolyze considerably faster than alkoxysilanes (Charbouillot et al., 1988; Cao & Hunt, 1994; Badini et al., 1995; Zhmud & Sonnefeld, 1996), sol-gels were prepared in a 2-step synthesis involving the pre-hydrolysis of the alkoxysilane (by combining appropriate amounts of the alkoxysilane, water, co-solvent, and catalyst), followed by the addition of aminosilane. The amine functional groups in the dried sol-gel were converted to diazeniumdiolates by exposure to high pressures of NO (about 5 atmospheres (atm) for 60-72 hours; Keefer et al., 1996) as shown in Figure 3, Scheme 2. Diazeniumdiolate formation was confirmed by the presence of a characteristic peak at 235-250 nm in the UV-Vis spectra of sol-gels spin- coated onto quartz slides.

Thus, in one example, the sol-gel comprises an aminosilane. In another example, the aminosilane can be selected from the group consisting of a monoalkoxysilane, a dialkoxysilane, a trialkoxysilane, and combinations thereof. The incorporation of an aminosilane into a silicon-based sol-gel matrix allows for modification of the material to controllably release NO. Exposing diamine groups to high pressures of NO under anaerobic conditions results in the formation of diazeniumdiolates X[N(O)NO]^" as shown in Figure 3.

To enhance the stability of the sol-gel, aminosilanes can also be combined with a suitable cross-linking agent such as an organosilane (e.g., methyltrimethoxysilane (MTMOS)), ethanol, and water. Representative sol- gel components thus include (aminoethylaminomethyl)-phenylethyl- trimethoxysilane (AEMP3) in methyltrimethoxysilane (MTMOS), which are employed in Examples 1-3. Other representative components include: N-(6-aminohexyl)aminopropyltrimethoxysilane (AHAP3), (3-trimethoxysilylpropyl)diethylenetriamine (DET3), isobutyltrimethoxysilane (BTMOS), Λ/-(2-aminoethyl)-3-aminopropyltrimethoxysilane,

Λ/-(2-aminoethyl)-3-aminopropylmethyldimethoxysilane, bis[(3-trimethoxysilyl)propyl]-ethylenediamine, ethyltrimethoxysilane, hexyltrimethoxysilane, and octyltrimethoxysilane. The aminosilane component of a sol-gel of the presently claimed subject matter is present in an amount up to 100% of the total silane. Particular percentages are chosen for a variety of reasons, including increasing or decreasing viscosity of the sol-gel material and/or providing for more or less NO donor moiety, depending on the desired controlled release profile.

In one embodiment a sol-gel comprises a composition having the general formula (I):

(R¹)_n-Si-(R²)_4-n, where R¹ and R² can be the same or different and can comprise hydrogen, amino, alkyl, alkylamino (including primary, secondary and tertiary amines), alkoxy, alkenyl, alkenylamino (including primary, secondary and tertiary amines), alkenoxy, alkynyl, alkynlamino (including primary, secondary and tertiary amines), alkynoxy, aryl, arylamino (including primary, secondary and tertiary amines), or aryloxy groups as defined herein below, and combinations thereof, and where n is 1 , 2, 3, or 4. The composition can be referred to as an "organosilane". When an amino group is present, whether as a substituent on the longest carbon chain or as a constituent of the longest carbon chain, the compound is also referred to herein as an "aminosilane", as noted above.

"Alkyl" refers to an aliphatic hydrocarbon group whϊcTTcari be straight or branched having 1 to about 60 carbon atoms in the chain. The alkyl group can be optionally substituted with one or more alkyl group substituents, which can be the same or different, where "alkyl group substituent" includes halo, amino, aryl, hydroxy, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkoxycarbonyl, oxo, cycloalkyl, and sila (Si). There can be optionally inserted along the alkyl chain one or more oxygen, silicon, sulphur, or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is lower alkyl. When an amino group atom is present the "alkyl" can also be described as an "alkylamino". "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, serf-butyl, teπf-butyl, n-pentyl, heptyl, octyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl, and hexadecyl. Exemplary alkyl groups include, but are not limited to branched or straight chain alkyl groups of 6 to 50 carbons, and also include the lower alkyl groups of 1 to about 4 carbons and the higher alkyl groups of about 12 to about 16 carbons.

"Alkenyl" refers to an alkyl group containing at least one carbon- carbon double bond. The alkenyl group can be optionally substituted with one or more "alkyl group substituents". Exemplary alkenyl groups include vinyl, allyl, n-pentenyl, decenyl, dodecenyl, tetradecadienyl, heptadec-8-en- 1-yl, and heptadec-8,11-dien-1-yl. Alkenoxy forms of alkenyls as defined herein are also provided. When an amino group is present the "alkenyl" or "alkenoxy" can also be described as an "alkenylamino" or "alkenoxyamino". "Alkynyl" refers to an alkyl group containing a carbon-carbon triple bond. The alkynyl group can be optionally substituted with one or more "alkyl group substituents". Exemplary alkynyl groups include ethynyl, propynyl, n- pentynyl, decynyl, and dodecynyl. Exemplary alkynyl groups include the lower alkynyl groups. Alkynoxy forms of alkynyls as defined herein also provided. When an amino group is present the "alkynyl" or "alkynoxy" can also be described as ^"an^""alkyήylarriiriό" or "alkynδxyarriiriό";

"Cycloalkyl" refers to a non-aromatic mono- or multicyclic ring system of about 4 to about 10 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group can be also optionally substituted with an aryl group substituent, oxo, and/or alkylene. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl, and cycloheptyl. Exemplary multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.

"Aryl" refers to an aromatic carbocyclic radical containing about 6 to about 10 carbon atoms. The aryl group can be optionally substituted with one or more aryl group substituents which can be the same or different, where "aryl group substituent" includes alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxy, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and --NRR', where R and R' are each independently hydrogen, alkyl, aryl, and aralkyl. Exemplary aryl groups include substituted or unsubstituted phenyl and substituted or unsubstituted naphthyl. When an amino group is present the "aryl" can also be described as an "arylamino".

"Acyl" refers to an alkyl-CO- group wherein alkyl is as previously described. Exemplary acyl groups comprise alkyl of 1 to about 30 carbon atoms. Exemplary acyl groups also include acetyl, propanoyl, 2- methylpropanoyl, butanoyl, and palmitoyl. "Aroyl" refers to an aryl-CO- group wherein aryl is as previously described. Exemplary aroyl groups include benzoyl and 1- and 2-naphthoyl.

"Alkoxy" refers to an alkyl-O-- group wherein alkyl is as previously described. Exemplary alkoxy groups include methoxy, ethoxy, n-propoxy, i- propoxy, n-butoxy, sec-butoxy, terf-butoxy, and heptoxy. The alkoxy group can be optionally substituted with one or more "alkyl group substituents". When an amino i group is Tpreserit the '"alkoxy" cafTaϊso be described as^" an "alkoxyamino".

"Aryloxy" refers to an aryl-O- group wherein the aryl group is as previously described. Exemplary aryloxy groups include phenoxy and naphthoxy. The aryloxy group can be optionally substituted with one or more "aryl group substituents". When an amino group is present the "aryloxy" can also be described as an "aryloxyamino".

"Alkylthio" refers to an alkyl-S- group wherein alkyl is as previously described. Exemplary alkylthio groups include methylthio, ethylthio, i- propylthio, and heptylthio. "Arylthio" refers to an aryl-S-- group wherein the aryl group is as previously described. Exemplary arylthio groups include phenylthio and naphthylthio.

"Aralkyl" refers to an aryl-alkyl- group wherein aryl and alkyl are as previously described. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.

"Aralkyloxy" refers to an aralkyl-O-- group wherein the aralkyl group is as previously described. An exemplary aralkyloxy group is benzyloxy.

"Aralkylthio" refers to an aralkyl-S-- group wherein the aralkyl group is as previously described. An exemplary aralkylthio group is benzylthio.

"Dialkylamino" refers to an --NRR' group wherein each of R and R' is independently an alkyl group as previously described. Exemplary dialkylamino groups include ethylmethylamino, dimethylamino, and diethylamino. "Alkoxycarbonyl" refers to an alkyl-O-CO- group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.

"Aryloxycarbonyl" refers to an aryl-O-CO- group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl. "Aralkoxycarbonyl" refers to an aralkyl-O--CO-- group. An exemplary aralkoxycarbonyT group is benzyloxycarbonyl.

"Carbamoyl" refers to an H2N-CO- group.

"Alkylcarbamoyl" refers to a R'RN-CO- group wherein one of R and R' is hydrogen and the other of R and R' is alkyl as previously described. "Dialkylcarbamoyl" refers to R'RN~CO- group wherein each of R and

R' is independently alkyl as previously described.

"Acyloxy" refers to an acyl-O- group wherein acyl is as previously described.

"Acylamino" refers to an acyl-NH- group wherein acyl is as previously described. "Aroylamino" refers to an aroyl-NH- group wherein aroyl is as previously described.

"Halo" or "halide" refers to fluoride, chloride, bromide, or iodide.

The versatility of the sol-gel process in terms of tuning NO-release properties by varying both the type and amount of aminosilane, combined with the variety of pattern geometries that can be created using micropatterning techniques, provides for numerous types of heterogeneous NO-releasing surfaces with a broad range of applications as disclosed herein. IV. Crafting a Patterned Surface

Disclosed herein is a method for preparing a substrate that can resist biofouling. In a representative embodiment, the method comprises applying an anti-biofouling agent to a substrate in a pre-determined pattern. Optionally, the anti-biofouling agent comprises: (a) a nitric oxide (NO) donor moiety; and (b) a matrix adapted to release NO in a controlled release profile. Optionally, the substrate is an implantable substrate. The terms "implantable substrate" and "implant" are used interchangeably herein.

In another embodiment, the substrate can be used for in situ applications, for example, "lab on a chip" applications. By way of particular example a substrate comprising wells, channels, etc. that resist biofouling can be prepared in accordance with the method disclosed herein for experiments dealing with blood or other common biological samples wherein resistance to biofouling is appropriate.

The pre-determined pattern can comprise any suitable pattern, and the pattern in typically chosen based an envisioned end use for the substrate. The term "pre-determined pattern" encompasses a pattern that facilitates proper function while enhancing biocompatibility. For a number of biomedical devices, a coating can influence its ability to function properly. For example, in a "lab on a chip" setting, the predetermined pattern can comprise a microarray of wells, channels, etc. for receiving analytes, reagents, etc. As an additional example, modifying a biosensor with a NO- releasing membrane can result in improved biocompatibility at the expense of analytical sensitivity due to reduced analyte diffusion (to the electrode). Thus, a majority of the surface of the biosensor substrate remains unmodified, and the substrate is protected from biofouling by the heterogeneous release of NO from strategically positioned microstructures that are patterned in a microarray. While the following discussion focuses on patterning via placement of microarrays, patterning of the surface of the substrate in a macro approach, that is coating only a portion of the surface, is also encompassed.

Background research efforts for patterning polymers onto surfaces have recently been described. For example, Clark and coworkers employed microfluidic networks to construct arrays (lines) of polymer-encapsulated protein patterns onto glass substrates (Kim et al., 2001). Whitesides et al. have reported on an alternate strategy that involves the application of pressure to a polymeric template in order to produce microarrays of dots and lines on a substrate (Yang et al., 1998; Jackman et al., 1999; James et al., 1998). Heretofore, no methods for patterning a matrix on a substrate to impart resistance to biofouling have been disclosed in the art. An aspect of the presently claimed subject matter pertains to a method for modifying surfaces of substrates with arrays of NO-releasing sol- gel microstructures. Sol-gel micropatterned surfaces can be prepared by both capillary flow and applied pressure methods using elastomeric templates, as shown schematically in Figures 1A-1C. In one embodiment, silicon masters are fabricated as follows: 1 ) silicon (Si) wafers are spin- coated with a photoresist, (such as a NFR55cp negative photoresist); 2) a photomask and ultraviolet (UV) light is then used to create a pattern in the resist; and 3) the photo-transferred pattern is developed. The length and width of features on the master are varied by changing the physical size of the chrome-plated features on the photomask used in step (2). Aluminum (Al) metal is then blanket deposited onto the wafers via electron beam (e- beam) evaporation. This metal is patterned using a tape lift-off process to remove any Al covering the resist. The metal serves as a. mask to etch the pattern into the Si with straight sidewalls using magnetically enhanced reactive ion etching. Feature heights are altered by varying the etching time. After etching to the desired depth, the Al mask is stripped with a chemically selective wet etch to yield a durable, reusable, dimensionally accurate, and chemically stable mold.

In one embodiment, the molds are then fluorinated with a suitable fluorination source (e.g., (heptadecafluoro-1 ,1 ,2,2-tetra-hydrodecyl)- trichlorosilane) which is optionally applied as a vapor under a nitrogen environment for a suitable time (e.g., 35 minutes) and which acts to prevent bonding of polydimethylsiloxane (PDMS) with the silicon wafer during the curing process (Espadas-Torre & Meyerhoff, 1995). The fluorinated master is then coated with PDMS, which is allowed to cure, to yield an elastomeric template.

Fabrication of surfaces by capillary flow involves placing the template on a substrate to create a series of microchannels. As shown in Figure 1B, sol solution is placed at one end of the array and drawn into the channels by capillary flow. After curing of the sol-gel, the template is removed to yield a micropatterned surface. Micropatterned arrays of sol-gel lines with dimensions including but not limited to 1-15 μm have been constructed in this embodiment.

Patterned surfaces have also been prepared using the method of applied pressure (Figure 1C) where sol solution is placed directly on a substrate and covered with the elastomeric template. Pressure is then applied such that contact is made between the template and the underlying substrate. After formation and drying of the sol-gel features, the template is removed. Microarrays consisting of a variety of geometric constituents including, but not limited to sol-gel lines and spheres (1-5 μm), have been fabricated using this technique. V. Substrates

A substrate is also provided herein. The substrate comprises an anti- biofouling agent disposed on a surface of the substrate, wherein the anti- biofouling agent comprises: (a) a nitric oxide (NO) donor moiety; and (b) a matrix adapted to release the NO in a pre-determined profile. Optionally, the substrate is an implantable substrate. The terms "implantable substrate" and "implant" are used interchangeably herein.

In another embodiment the substrate can be used for in situ applications, for example, "lab on a chip" applications. By way of particular example a substrate comprising wells, channels, etc. that resist biofouling can be prepared in accordance with the method disclosed herein for experiments dealing with blood or other common biological samples wherein resistance to biofouling is appropriate. In one embodiment, the anti-biofouling agent is disposed on the substrate in a pre-determined pattern. In another embodiment, the predetermined pattern is a microarrayed pattern as disclosed herein above.

Representative implants include both intravascular and subcutaneous sensors as well as structural implants and in vitro microfluidic devices. Thus, additional representative implants include biosensors, catheters, orthopedic implants, sutures, staples, stents, breast implants, penile implants, other anatomical implants, screws, nails, plates, rods, and prostheses. Indeed, any implant as would be apparent to one of ordinary skill in the art upon review of the present disclosure falls within the scope of the presently claimed subject matter.

The presently claimed subject matter also provides a biosensor comprising an anti-biofouling agent disposed on a surface thereof in a predetermined pattern, wherein the anti-biofouling agent comprises: (a) a nitric oxide (NO) donor moiety; and (b) a matrix adapted to release the NO in a pre-determined profile. In another embodiment, the predetermined pattern is a microarrayed pattern as disclosed herein above.

Implant devices can be modified with various sol-gel patterning architectures to release NO while retaining the analytical response characteristics of an unmodified device (e.g., biosensor), since only specific regions on the substrate are functionalized with sol-gel. In this manner, the release of NO from the sol-gel patterns prevents biofouling without compromising the functionality of the implant. Microarray geometry, dimensions, and spacing between NO-releasing microstructures can be varied to most effectively inhibit cellular (platelets and bacteria) attachment while minimizing the surface area coated with sol-gel as shown schematically in Figure 6.

Examples The following Examples have been included to illustrate aspects of the presently claimed subject matter. Certain aspects of the following Examples are described in terms of techniques and procedures found or contemplated by the present inventors to work well in the practice of the presently claimed subject matter. These Examples are exemplified through the use of standard laboratory practices of the inventors. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the spirit and scope of the presently claimed subject matter.

Example 1 Surface Topography of Micropatterned Sol-Gel Substrates

The surface topography of micropatterned sol-gel substrates was characterized using a PICOSPM™ (Molecular Imaging of Phoenix, Arizona, United States of America) atomic force microscope (AFM) with Si₃N₄ tips (spring constant circa 0.12 N/m). Representative contact mode AFM images of 20% (aminoethylaminomethyl)-phenylethyl-trimethoxysilane (AEMP3) in methyltrimethoxysilane (MTMOS) sol-gel patterns on glass substrates are shown in Figures 2A-2D. These images illustrate the diversity both in terms of feature geometry and spacing between sol-gel regions that can be achieved with micropatterning methods. An array of 1 μm sol-gel dots separated by distances of 10 μm and arrays of sol-gel lines ranging from 1-5 μm separated by spaces ranging from 1-6 μ are included as examples of the variety of surface architectures provided herein.

Example 2 Conversion of Sol-Gels to NO-Releasing Materials The incorporation of an aminosilane such as AEMP3 into a silicon- based sol-gel matrix allows for modification of the material to controllably release NO. Exposing diamine groups to high pressures of NO under anaerobic conditions results in the formation of diazeniumdiolates X[N(O)NO]^" as shown in Figure 3, Scheme 3. Diazeniumdiolates are stable at low temperatures in the absence of hydrogen donors (e.g., water). To enhance the stability of the sol-gel and provide a matrix for controlling the NO-release profiles of the sol-gel, aminosilanes are combined with a suitable cross-linking agent such as MTMOS, ethanol, and water. Aminosilane-containing sol-gels are then converted to NO-releasing materials by exposure to 5 atm NO for 72 hours. Real-time NO release from micropatterned surfaces is assessed using a chemiluminescence nitric oxide analyzer (Model NOA™ 280, available from Sievers Instruments of Boulder, Colorado, United States of America). A representative NO release curve (i.e., a predetermined profile or a controlled release profile) for a 20% AEMP3/MTMOS micropattem (array of lines) is provided in Figure 4. Notably, the rate of NO release is consistent for the duration of the experiment.

Example 3 Evaluating Sol-Gel Micropattem Stability The utility of NO-releasing sol-gel micropatterns for biomedical implant applications is highly dependent on the stability of the patterned features under both high pressure and solution conditions. Micropattem stability was evaluated using contact mode AFM. Representative images of an array comprising 3 μm sol-gel lines a) before and b) after pressurizing to 5 atm NO for 60 hours are shown in Figures 5A and 5B, respectively. The integrity of the micropatterned array was maintained, indicating that the charging process used to convert aminosilanes to diazeniumdiolates is not destructive to the pattern.

An equally important consideration is the ability of micropatterns to withstand solution immersion. The charged array was submerged in buffer for 3 days and characterized in situ at distinct intervals using contact mode AFM (Figures 5A-5C). From these data it was determined that the integrity of microstructures is not compromised by solution, suggesting that sol-gel micropatterns provide a viable approach for heterogeneously releasing NO from a substrate for the purpose of enhancing implant biocompatibility.

Example 4 Evaluating the Thromboresistivity of NO-Releasing Sol-Gel Micropatterns The adhesion and activation of platelets in response to implantation results in the formation of a dense network of cells and polymeric fibrin known as a haemostatic plug or thrombus (Cazenave, 1986; Cazenave & Mulvihill, 1993).

Surface thrombosis frequently poses a significant risk to patients by rendering blood-contacting implant devices ineffective. The viability of such devices is thus highly dependent upon the ability of the material to resist this type of biofouling. Because the thromboresistivity of a material is directly related to its ability to prevent the adhesion of platelets, in vitro platelet adhesion studies are used in this Example to characterize the thromboresistivity of heterogeneous NO-releasing sol-gel microarrays.

Platelet rich plasma (PRP) was obtained from acid citrate dextrose (ACD)-anticoagulated porcine blood (1 part ACD to 9 parts whole blood) by centrifugation at 200 x g for 15 min at room temperature. Since calcium is a requirement for normal platelet activity, CaCI₂ was added to a total concentration of 0.25-0.50 mM Ca⁺² to maintain platelet activity. PRP was then incubated with sol-gel micropattem controls and identical microarrays capable of NO-release for 30 min at room temperature. The substrates were then rinsed with Tyrode's buffer to remove loosely adhered platelets. Adsorbed platelets were fixed with glutaraldehyde solution (1 %, Tyrode's buffer) to ensure preservation of cell morphology. Finally, the surfaces were rinsed with Tyrode's buffer and water before being chemically dried with ethanol and hexamethyldisilazane (HMDS) for scanning electron microscopy (SEM) analysis. Representative SEM images of platelet adhesion to a micropattem control consisting of 15 μm sol-gel lines separated by 10 μm of bare glass and the corresponding NO-releasing microarray are provided in Figures 7A and 7B. A significant reduction in the adhesion of platelets to the NO-releasing surface was observed. These data indicated that in vitro platelet adhesion was significantly inhibited by heterogeneous NO release from sol-gel microarrays.

References The references listed below as well as all references cited in the specification are incorporated herein by reference to the extent that they supplement, explain, provide a background for or teach methodology, techniques and/or compositions employed herein.

Albina JE & Reichner JS. Role of nitric oxide in mediation of macrophage cytotoxicity and apoptosis. Cane. Metas. Rev. 1998, 17, 19-53. Badini GE, Grattan KTV & Tseung ACC. Sol-Gels with Fiber-Optic Chemical Sensor Potential: Effects of Preparation, Aging, and Long-Term Storage. Rev Sci Instrum 1995, 66, 4034-4040. Banerjee R, Nageswari K & Puniyani RR. Hematological aspects of biocompatibility. Review article. J Biomat Appls 1997, 12, 57-76. Benmakroha Y, Zhang S & Rolfe P. Haemocompatibility of invasive sensors. Medical Biological Engineering Computing 1995, 33, 811-821. Boelens JJ, Zaat SAJ, Meeldijk J & Dankert J. Subcutaneous abscess formation around catheters induced by viable and nonviable staphylococcus epidermidis as well as by small amounts of bacterial cell wall components. J. Biomed. Mater. Res. 2000, 50, 546-556.

Brown MRW, Allison DG & Gilbert P. Resistance of bacterial biofilms to antibiotics: A growth rate-related effect? Antimicrob. Chemother.

1988, 22, 777-780. Cao W & Hunt AJ. Sol-Gel Processing Using Aminofunctional Silanes. Mat Res Soc Symp Proc 1994, 346, 631-636.

Cazenave JP. Interaction of platelets with surfaces. Blood-Surface

Interactions: Biological Principles Underlying Haemocompatibilitv with

Artificial Materials, Cazenave J, Davies J, Kazatchkine M & Van Aken

W, Editors. 1986, 89-105. Cazenave J & Mulvihill J. Platelet adhesion to surfaces. ]n The Role of

Platelets in Blood-Biomaterial Interactions, Missirilis Y & Wautier J-L,

Editors. 1993, 69-80. Chapman RG, Ostuni E, Liang MN, Meluleni G & Kim E. Polymeric thin films that resist the adsorption of proteins and the adhesion of bacteria. Langmuir 2001 , 17, 1225-1233.

Charbouillot Y, Ravaine D, Armand M & Poinsignon C. Aminosils: New Solid

State Protonic Materials by the Sol-Gel Process. J Non-Cryst Solids

1988, 103, 325-330. Chen J-H, Wei J, Chang C-Y, Laiw R-F & Lee Y-D. Studies on segmented polyetherurethane for biomedical application: effects of composition and hard-segment content on biocompatibility. Journal of Biomedical

Materials Research 1998, 41, 633-648. Costerton J, Stewart P & Greenberg E. Bacterial Biofilms: A Common Cause of Persistent Infections. Science 1999, 284, 1318-1322. Diodati JG, Quyyumi AA, Hussain N & Keefer LK. Complexes of nitric oxide with nucleophiles as agents for the controlled biological release of nitric oxide: antiplatelet effect. Thromb. Haemostas. 1993, 70, 654-

658. Dunne WM, Mason EO & Kaplan SL. Diffusion of rifampin and vancomycin through a staphylococcus epidermis biofilm. Antimicrob. Agents

Chemother. 1993, 37, 2522-2526. Elbert DL & Hubbell JA. Reduction of fibrous adhesion formation by a copolymer possessing an affinity for anionic surfaces. J. Biomed.

Mater. Res. 1998, 42, 55-65. Espadas-Torre C & Meyerhoff ME. Thrombogenic Properties of Untreated and Poly(ethylene oxide)-Modified Polymeric Matrixes Useful for

Preparing Intraarterial Ion-Selective Electrodes. Anal. Chem. 1995,

67, 3108-3114. Espadas-Torre C, Oklejas V, Mowery KA & Meyerhoff ME. Thromboresistant chemical sensors using combined nitric oxide release/ion sensing polymeric films. J. Am. Chem. Soc. 1997, 119, 2321-2322. Gottenbos B, van der Mei HC & Busscher HJ. Initial adhesion and surface growth of staphylococcus epidermidis and pseudomonas aeruginosa on biomedical polymers. J. Biomed. Mater. Res. 2000, 50, 208-214. Hanson SR, Hutsell TC, Keefer LK, Mooradian DL & Smith DJ. Nitric oxide donors: a continuing opportunity in drug design. Adv. Pharmacol.

1995, 34, 383-398. Hrabie JA & Keefer LK. Chemistry of the nitric oxide-releasing diazeniumdiolate ("nitrosohydroxylamine") functional group and its oxygen-substituted derivatives. Chem. Rev. 2002, 102, 1135-1154.

Hrabie JA, Klose J R, Wink DA & Keefer LK. New nitric oxide-releasing zwitterions derived from polyamines. J. Org. Chem. 1993, 58, 1472-

1476. Imazato S, Imai T, Russell RRB, Torii M & Ebisu S. Antibacterial activity of cured dental resin incorporating the antibacterial monomer MDPB and an adhesion-promoting monomer. J. Biomed. Mater. Res. 1998, 39,

511-515. Jackman RJ, Duffy DC, Cherniavskaya O & Whitesides GM. Using elastomeric membranes as dry resists for dry lift-off. Langmuir 1999, 15, 2973-2984.

James CD, Davis RC, Kam L, Craighead HG & Isaacson M. Patterned protein layers on solid substrates by thin microcontact printing.

Langmuir 1998, 14, 741-744. Jurasz P, Radomski A, Sawicki G, Mayers I & Radomski M. Nitric Oxide and Platelet Function. Nitric Oxide Biology and Pathology; Academic

Press: New York, 2000; pp 823-840 Keefer LK, Nims RW, Davies KM & Wink DA. "NONOates" (1 -substituted diazen-1-ium-1 ,2-diolates) as nitric oxide donors: convenient nitric oxide dosage forms. Meth Enzymol, 1996, 268, 281-293. Kim Y-D, Park CB & Clark DS. Stable sol-gel microstructured and microfluidic networks for protein patterning. Biotechnol. Bioeng. 2001,

73, 331-337. Kyrolainen M, Rigsby P, Eddy S & Vadgama P. Bio-/haemocompatibility: implications and outcomes for sensors? Ada Anaesth. Scand. 1995, 104, 55-60.

Lopez-Farre A, Rodriguez-Feo JA, Sanchez de Miguel L, Rico L, Casado S

Role of nitric oxide in the control of apoptosis in the microvasculature.

Intl J Biochem Cell Biol 1998, 30, 1095-1106. Maragos CM, Morley D, Wink DA, Dunams TM, Saavedra JE, Hoffman A, Bove AA, Isaac L, Hrabie JA & Keefer LK Complexes of .NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects. J. Med. Chem. 1991, 34, 3242-3247. Marietta MA, Tayeh MA & Hevel JM. Unraveling the biological significance of nitric oxide. BioFactors 1990, 2, 219-225. Marois Y, Chakfe N, Guidoin R & Duhamel RC. An albumin-coated polyester arterial graft: in vivo assessment of biocompatibility and healing characteristics. Biomate als 1996, 17, 3-14. Moncada S & Higgs A. The L-arginine-nitric oxide pathway. N. Engl. J. Med.

1993, 30, 2002-2011. Mowery KA, et al., Polym. Mater. Sci. Eng. 1998, 79, 33-34. Mowery KA & Meyerhoff ME. Polymer 1999a, 40, 6203-6207. Mowery KA & Meyerhoff, ME. Electroanalysis 1999b, 11, 681-686. Mowery KA, Schoenfisch MH, Saavedra JE, Keefer LK & Meyerhoff ME.

Preparation and characterization of hydrophobic polymeric films that are thromboresistant via nitric oxide release. Biomatehals 2000, 21, 9-

21. Nablo BJ, Chen TY, Schoenfisch MH. Sol-gel derived nitric oxide releasing materials that reduce bacterial adhesion. J. Am. Chem. Soc. 2001,

123, 9712-9713. O'Conner SM, Gehrke SH & Retzinger GS. Ordering of poly(ethylene oxide)/poly(propylene oxide) triblock copolymers in condensed films.

Langmuir 1999, 15, 2580-2585. Pasic M, Muller-Glauser W, von Segesser L, Odermatt B & Lachat M.

Endothelial cell seeding improves patency of synthetic vascular grafts: manual versus automatized method. European Journal of Cardio-

Thoracic Surgery 1996, 10, 372-379. Potera C. Microbiology: Forging a link between biofilms and disease.

Science 1999, 283, 1838-1839. Radomski MW, Rees DD, Dutra A. & Moncada S. S-nitroso-glutathione inhibits platelet activation in vitro and in vivo. Br. J. Pharmacol. 1992,

107, 745-749. Ratner BD, Molecular design strategies for biomaterials that heal.

Macromolecular Symposia 1998, 130, 327-335. Razatos A, Ong YL, Boulay F, Elbert DL & Hubbell JA. Force measurements between bacteria and poly(ethylene glycol)-coated surfaces. Langmuir 2000, 16, 9155-9158.

Schoenfisch MH, Mowery KA, Rader MV, Baliga N, Wahr JA. Improving the thromboresistivity of chemical sensors via nitric oxide release: fabrication and in vivo evaluation of NO-releasing oxygen-sensing catheters." Anal. Chem. 2000, 72, 1119-1126.

Smith DJ, Chakravarthy D, Pulfer S; Simmons ML Hrabie JA Nitric oxide- releasing polymers containing the [N(O)NO]^" group. J. Med. Chem. 1996, 39, 1148-1156.

Suh H. Recent advances in biomaterials. Yonsei Medical Journal 1998, 39, 87-96.

Vacheethasanee K & Marchant RE. Surfactant polymers designed to suppress bacterial (staphylococcus epidermis) adhesion on biomaterials. J. Biomed. Mater. Res. 2000, 50, 302-312.

Walpoth BH, Rogulenko R, Tikhvinskaia E, Gogolewski S & Schaffner T. Improvement of patency rate in heparin-coated small synthetic vascular grafts. Circulation 1998, 98, 11319-323; discussion II324.

Weerwind PW, van der Veen FH, Lindhout T, de Jong DS & Cahalan PT. Ex vivo testing of heparin-coated extracorporeal circuits: bovine experiments. International Journal of Artificial Organs 1998, 21, 291- 298.

Yang P, Deng T, Zhao D, Feng P & Pine D. Hierarchically ordered oxides. Science 1998, 282, 2244-2246.

Yoda R. Elastomers for biomedical applications. Journal of Biomatehals Science, Polymer Edition 1998, 9, 561-626. Zhmud BV & Sonnefeld J. Aminopolysiloxane Gels: Production and Properties. J Non-Cryst Solids 1996, 195, 16-27. It will be understood that various details of the presently claimed subject matter can be changed without departing from the scope of the presently claimed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMS What is claimed is:

1. A method for preparing a substrate, the method comprising applying an anti-biofouling agent to a substrate in a pre-determined pattern, wherein the anti-biofouling agent comprises:

(a) a nitric oxide (NO) donor moiety; and

(b) a matrix adapted to release the NO in a controlled release profile.

2. The method of claim 1, wherein the predetermined pattern is a microarrayed pattern.

3. The method of claim 1 , wherein the matrix comprises a sol-gel.

4. The method of claim 3, wherein the sol-gel comprises an aminosilane, an organosilane, or a combination thereof.

5. The method of claim 3, wherein the sol-gel comprises a composition having the general formula (R¹)_n-Si-(R²)_4-n, where R¹ and R² are the same or different and are selected from the group consisting of hydrogen, amino, alkyl, alkylamino, alkoxy, alkenyl, alkenylamino, alkenoxy, alkynyl, alkynlamino, alkynoxy, aryl, arylamino, aryloxy and combinations thereof, and where n is 1 , 2, 3, or 4.

6. The method of claim 4, wherein the aminosilane is present in an amount ranging from 1 % to 100% of total silane.

7. The method of claim 1 , further comprising placing the substrate at an implant site in a subject.

8. A substrate comprising an anti-biofouling agent disposed on a surface of the substrate in a pre-determined pattern, wherein the anti- biofouling agent comprises:

(a) a nitric oxide (NO) donor moiety; and

(b) a matrix adapted to release the NO in a controlled release profile.

9. The substrate of claim 8, wherein the predetermined pattern is a microarrayed pattern.

10. The substrate of claim 8, wherein the matrix comprises a sol- gel.

11. The substrate of claim 10, wherein the sol-gel comprises an aminosilane, an organosilane, or a combination thereof.

12. The substrate of claim 10, wherein the sol-gel comprises a composition having the general formula (R¹)_n-Si-(R²) _-n, where R¹ and R² are the same or different and are selected from the group consisting of hydrogen, amino, alkyl, alkylamino, alkoxy, alkenyl, alkenylamino, alkenoxy, alkynyl, alkynlamino, alkynoxy, aryl, arylamino, aryloxy and combinations thereof, and where n is 1 , 2, 3, or 4.

13. The substrate of claim 11 , wherein the aminosilane is present in an amount ranging from 1% to 100% of total silane.

14. The substrate of claim 8, wherein the substrate is adapted for implantation in a subject.

15. An anti-biofouling composition comprising:

(a) a nitric oxide (NO) donor moiety; and

(b) a sol-gel matrix adapted to release the NO in a pre-determined profile, the sol-gel matrix comprising an aminosilane, an organosilane, or a combination thereof.

16 The anti-biofouling composition of claim 15 wherein the sol-gel matrix comprises a composition having the general formula (R¹)n-Si-(R²)_4-n, where R¹ and R² are the same or different and are selected from the group consisting of hydrogen, amino, alkyl, alkylamino, alkoxy, alkenyl, alkenylamino, alkenoxy, alkynyl, alkynlamino, alkynoxy, aryl, arylamino, aryloxy and combinations thereof, and where n is 1 , 2, 3, or 4.

17. The anti-biofouling composition of claim 15, wherein the aminosilane is present in an amount ranging from 1 % to 100% of total silane.