In SITU nitric oxide generating materials and methods for making and using the same

Abstract

In accordance with the purpose(s) of the present disclosure, as embodies and broadly described herein, the disclosure, in one aspect, relates to materials and articles that generate nitric oxide in situ in a subject once the material or article has been introduced into the subject. The articles possess numerous advantages including, but not limited to, reducing or preventing the growth of microbes on the article as well as reduce or prevent inflammation and the adhesion of platelets on the article when introduced into the subject.

Description

T|H Docket: 222105-2380 IN SITU NITRIC OXIDE GENERATING MATERIALS AND METHODS FOR MAKING AND USING THE SAME STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under grant number R01HL157587 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 / 694,981, filed on September 16, 2024, the contents of which are incorporated by reference herein in their entireties. BACKGROUND

[0003] Silicone-based (-[O-Si(CH3)2]n-) biomaterials are the most commonly employed biomaterials for implantable device applications owing to their bio-inertness, anti- adhesiveness, and low cytotoxicity. This has led to the use of silicone-based devices for use in long-term (months to years) applications such as defect fillers in lumpectomy or muscle atrophy conditions for augmentation or restoration of the defects, and as tracheobronchial or vascular stents. Silicone-based devices have also been used for short-term applications (weeks to months) such as in catheters and totally implanted venous access ports (TIVADs). Though being a versatile biomaterial, silicone-based devices are still challenged by complications relating to implant-associated infections and fibrous capsule formation. Chronic infections associated with such implantable devices occur at an incidence rate of ~ 5%, but the impending risk factors relating to antimicrobial resistance and the vulnerability to cause serious complications in immunocompromised and elderly patients, exacerbate this low- frequency number.[1]Similarly, a 10.6% incidence rate of fibrous capsule formation is noticed in silicone implants used for augmentative or restoration procedures leading to pain, discomfort at the implant site, and ultimately device failure.[2]

[0004] As with any foreign device implanted in the body, it evokes a foreign body response which is a sequential cascade of biomolecular and cellular events that decides the fate of the implanted biomaterial (Figure 1). Soon after implantation, the biomaterial surface is crowded by the blood proteins, followed by the immune cells engaging with the surface. If the materialT|H Docket: 222105-2380 surface is not conducive, it triggers the activation of the chronic inflammatory phase which involves the activation of macrophages, the formation of foreign body giant cells and myofibroblast phenotype conversion leading to fibrous encapsulation. Pertinent to note here, the fibroblast activation and its phenotype control is dependent on the inflammatory environment which is closely regulated by macrophage-fibroblast crosstalk.[3]The presence of implant-associated opportunistic microbes can further worsen the chronic inflammation phase divulging out as infection at the implant site. Pharmacological interventions involve the systemic administration of immunomodulatory drugs or antibiotics to curtail these complications. Use of broad-spectrum anti-inflammatory drugs help in long-term maintenance while short-term administration of steroids or anti-fibrotic drugs aid in transiently inhibiting initial immune cell engagement.[2a, 4]However, the use of these anti-inflammatory drugs systemically or locally has multiple and can lead to differential non-specific effects invivo and associated toxicity.[5]the widespread use of antibiotics to prevent implant- associated infections has raised concerns relating to multidrug-resistance and the evolution of superbugs.[6]SUMMARY

[0005] In accordance with the purpose(s) of the present disclosure, as embodies and broadly described herein, the disclosure, in one aspect, relates to materials and articles that generate nitric oxide in situ in a subject once the material or article has been introduced into the subject. The articles possess numerous advantages including, but not limited to, reducing or preventing the growth of microbes on the article as well as reduce or prevent inflammation and the adhesion of platelets on the article when introduced into the subject.

[0006] Other systems, methods, features, and advantages of the present disclosure 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 systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.T|H Docket: 222105-2380 BRIEF DESCRIPTION OF THE DRAWINGS

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

[0008] Figure 1 shows the sequence of events noticed with a silicone biomaterial associated with fibrosis at the implant site (with timeline in parenthesis). A silicone implant used as defect filler in a lumpectomy is illustrated here: (i) after implantation, the implant surface is crowded by blood proteins from the microvasculature (minutes to hours); (ii) acute inflammation is noticed when immune cells such as neutrophils, macrophages and lymphocytes engage with the implant (days), (iii) chronic inflammation is triggered which initiates fibroblast infiltration and foreign body cell formation (days to weeks); (iv) myofibroblast activation and fibrous encapsulation of implant (weeks to months) leads to implant rejection and need for replacement.

[0009] Figures 2A-2C show the synthesis and characterization of mesoporous nanobioactive glass nitric oxide generating particles. A) hexadecyltrimethylammonium bromide (CTAB) assisted sacrificial templating synthesis of nanobioactive glass by modified Stöber’s method; B) Fourier transform infrared spectra of synthesized nano-bioactive glass (nBG), copper doped BG (nBG-Cu), strontium doped BG (nBG-Sr); C) Transmission electron microscopy (TEM) analysis of synthesized nanoparticles showing dendrimer-like nature of glass network; selected area energy diffraction (SAED) showing amorphous nature of the nano-bioactive glasses and energy dispersal spectral (EDS) mapping of particles confirming stoichiometry of the glass composition (scale bars represented are 100 nm).

[0010] Figures 3A-3H show the facile modification of silicone rubber (SR) substrates by dip coating method to develop a nitric oxide generating (NOgen) interface. A) dip coating of SR substrates with different concentration of nanobioactive glasses with room temperature curing silicone (RTV) and B) the resultant gross morphological appearance of coated substrates; C) chemiluminescence nitric oxide analysis of coated substrates (total moles of NO generated per 1 µM GSNO / 30 µM GSH injection) and D) its representative NO generation curve for different concentrations of i) nBG-Cu, ii) nBG-Sr, and iii) nBG; E) Reaction scheme summarizing the mechanism and regeneration of metal ion assisted nitric oxide generation from the NOgen interface; F) Understanding the mechanism of decomposition of GSNO by metal ion (+1 oxidation state) by arresting it with a ligand (1,10- phenanthroline); G) chemiluminescence nitric oxide analysis of decomposition of 1 µM GSNO (with 30 µM GSH) in i) presence of copper or ii) strontium salts and chelating agents ethylenediaminetetraaceticT|H Docket: 222105-2380 acid (EDTA) or 1,10 phenanthroline; and with NOgen films: iii) nBG-Cu 50, iv) nBG-Sr 50; H) color change post incubation with ligand (1,10 phenanthroline) indicative of metal (+1 oxidation state)-ligand complexation formation, and the arrest of NO generation. [Data are presented as means ± S.D; (n ≥ 4) for nitric oxide analysis); Parametric ANOVA, Tukey’s post hoc test was performed; * indicates significance at p ≤ 0.05].

[0011] Figures 4A-4C show the multifunctional NOgen interface on silicone rubber (SR) substrates bestow antibacterial and antithrombotic attributes. A) antibacterial activity of NOgen and SR control substrates assessed by challenging against planktonic cultures of two commonly noted bacterial strains associated with implant infections: i) Gram-negative, Escherichia coli and ii) Gram-positive Staphylococcus aureus; in presence of 1 µM GSNO / 30 µM GSH supplementation every hour for 4 h; B) antithrombotic effect of NOgen substrates tested by exposing porcine derived platelets to these substrates for 90 min in presence of 1 µM GSNO / 30 µM GSH supplementation; C) illustration depicting the metal ion assisted NO generation from NOgen interface holding promise in reducing implant associated infection and reducing platelet activation (thrombus formation). [Data are presented as means ± S.D; ((n ≥ 4) for antibacterial study; (n ≥ 6) for platelet adhesion study); Parametric ANOVA, Tukey’s post hoc test was performed; same letters indicate non-significance, whereas different letters indicate significance at p ≤ 0.05]

[0012] Figures 5A-5C show the cytocompatibility and in vitro fibrosis assay on NOgen interface and its leachates. A) Cytocompatibility assessment using human fibroblasts by following ISO standard (ISO10993-5: 2009) protocol by (i) collecting leachates and (ii) through indirect contact method. Ascorbic acid- induced upregulation of fibroblast activity and the effect of leachate on its downregulation (by nBG-Sr 50) tested by: B) i) collagen estimation by Sirius red assay and ii) representative well images showing red coloration of collagen deposits; C) i) fibroblast migration assessed by in vitro wound closure and ii) representative phase-contrast micrographs. [Data are presented as means ± S.D; (n ≥ 4); Parametric ANOVA, Tukey’s post hoc test was performed; same letters indicate non-significance, whereas different letters or * indicate significance at p ≤ 0.05].

[0013] Figures 6A-6C show the effect of copper and strontium from NOgen interface leachates on macrophages. A) Flow cytometric analysis of naïve macrophages treated with 14-day leachates for 72 h to polarize them in vitro and representative dot plots for different groups are shown: i) cell control, ii) SR control, iii) nBG 50, iv) nBG-Cu 50 and v) nBG-Sr 50; and the B) M2 / M1 macrophage index calculated from the populations. C) Qualitative representation of the immunostained population of macrophages treated with the leachatesT|H Docket: 222105-2380 by fluorescent microscopy. [Data are presented as means ± S.D; (n ≥ 3); Parametric ANOVA, Tukey’s post hoc test was performed; same letters indicate non-significance, whereas different letters indicate significance at p ≤ 0.05].

[0014] Figures 7A-7E show the effect of NOgen interface on macrophages. Immunomodulatory effect of NO generated from NOgen interface on macrophages over a period of 10 days, A) using the strategy depicted in the scheme; M2 / M1 macrophage bias index calculated from the flow cytometric analysis of macrophage population retrieved at B) Day-3 and C) Day-10; Qualitative representation of the immunostained population of macrophages retrieved at D) Day-3 and E) Day-10 by fluorescent microscopy. [Data are presented as means ± S.D; (n ≥ 3); Parametric ANOVA, Tukey’s post hoc test was performed; same letters indicate non-significance, whereas different letters indicate significance at p ≤ 0.05].

[0015] Figures 8A-8B show the proangiogenic effect of leachates and NOgen interface. A) In vitro collagen tube formation performed using human umbilical vein endothelial cells on i) custom microscope molds, following the ii) method illustrated in the scheme and iii) assessing the number of nodes and tube length of matured endothelial cell network after staining. B) i) Fluorescent images (representative), ii) tube length measured and iii) number of tubes and nodes noted per frame from the images for different groups. [Data are presented as means ± S.D; (n ≥ 3); Parametric ANOVA, Tukey’s post hoc test was performed; same letters indicate non-significance, whereas different letters indicate significance at p ≤ 0.05]

[0016] Figure 9 shows the Energy Dispersal Spectra (EDS) for the bioactive glass particles used in the study.

[0017] Figures 10A-10D show dynamic light scattering analysis on synthesized nanobioactive glass for A) nBG, B) nBG-Cu, C) nBG-Sr; D) gross appearance of nBG-Cu and nBG-Sr (50 mg / mL) suspension stable up to 4h in ethanol at ambient conditions.

[0018] Figure 11 shows Chemiluminescence-based nitric oxide analysis of coated substrates representing total moles of NO generated at 37 ºC, in phosphate buffered saline PBS, pH 7.4 supplemented with A) 0.3 µM and 9 µM GSNO and reduced glutathione and B) 0.09 µM and 2.7 µM GSNO and reduced glutathione. The GSH concentrations were also reduced in a ratio corresponding to the GSNO concentration. [Data are presented as means ± S.D; (n ≥ 4) for nitric oxide analysis); Parametric ANOVA, Tukey’s post hoc test was performed; same letters indicate non-significance, different letter indicates significance at p ≤ 0.05].T|H Docket: 222105-2380

[0019] Figures 12A-12B show UV-visible spectroscopic analysis of metal and 100 µM ligand (1,10 phenanthroline) interaction for different concentration of A) copper chloride and B) strontium chloride in i) the absence and ii) the presence of 1 µM GSNO and 30 µM GSH.

[0020] Figure 13 shows static water contact angle measurements on the silicone rubber (SR) substrates and the NOgen substrates. [Data are presented as means ± S.D; (n ≥ 3)]

[0021] Figures 14A-14C show elemental ion concentration quantified using inductively coupled plasma optical emission spectrophotometer (ICP-OES); A) predominant network former SiO2in leachate estimated by measuring Si ion concentration in nBG 50, nBG-Cu 50, and nBG-Sr 50; network modifiers (CuO or SrO) estimated in leachate in doped systems measured by B) quantifying Cu ion concentration (in nBG 50 and nBG-Sr leachates at observed timepoints, the Cu concentrations were below the limit of quantitation (< 0.04 μg / g of sample); by C) quantifying Sr ion concentration (in nBG 50 and nBG-Cu leachates at observed timepoints, the Sr concentrations were below the limit of quantitation (< 0.02 μg / g of sample). [Cumulative ion concentration presented as means ± S.D; (n ≥ 3)].

[0022] Figures 15A-15B show chemiluminescence-based nitric oxide analysis of coated substrates representing A) total moles of NO generated at 37 ºC, in phosphate buffered saline PBS, pH 7.4 supplemented with 1 µM GSNO and 30 µM reduced glutathione monitored over a period of 21 days and B) the corresponding flux values. [Data are presented as means ± S.D; (n ≥ 4)); Parametric ANOVA, Tukey’s post hoc test was performed; same letters indicate non-significance, different letter indicates significance at p ≤ 0.05]. The dotted red line indicates the threshold in which a differential behavior of macrophages where noticed, below the dotted line M2polarization was observed.

[0023] Figure 16 shows workflow for polarizing naïve macrophages to M1and M2macrophages in vitro with known cytokine stimulants and using them as reference populations to assess polarized macrophage population resulting from subsequent leachate treatment or NOgen exposure.

[0024] Figure 17 shows flow cytometric and immunostaining of M1and M2macrophages and their M2 / M1 polarization index calculated from flow-cytometric analysis.

[0025] Figure 18 shows flow cytometric analysis for NOgen substrates for macrophages retrieved at day 3.

[0026] Figure 19 shows flow cytometric analysis for NOgen substrates for macrophages retrieved at day 10.

[0027] Figure 20 shows monitoring the stability of 1μM GSNO and 30 μM GSH stability in phosphate buffered saline (pH 7.4) at 37 ºC at different time points. GSNO has an absorptionT|H Docket: 222105-2380 maximum at 335 nm, and the tested concentration (which was physiologically relevant) got reduced by 70 % at 1 h. This led us in supplementing GSNO / GSH every hour for bacterial studies.

[0028] The drawings illustrate only example embodiments and are therefore not to be considered limiting of the scope described herein, as other equally effective embodiments are within the scope and spirit of this disclosure. The elements and features shown in the drawings are not necessarily drawn to scale, emphasis instead being placed upon clearly illustrating the principles of the embodiments. Additionally, certain dimensions may be exaggerated to help visually convey certain principles. In the drawings, similar reference numerals between figures designate like or corresponding, but not necessarily the same, elements. DETAILED DESCRIPTION

[0029] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific compounds, synthetic methods, or uses as such may, of course, vary. 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.

[0030] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0031] 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.

[0032] 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.

[0033] 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.T|H Docket: 222105-2380 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.

[0034] 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.

[0035] 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 methods 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.

[0036] 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

[0037] In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below.

[0038] 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.T|H Docket: 222105-2380

[0039] 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 solvent” includes, but are not limited to, mixtures or combinations of two or more such solvents, and the like.

[0040] It should be noted that ratios, concentrations, amounts, rates, 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 and “about 5 to about 15” is also disclosed.

[0041] 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 the 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’”.

[0042] 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-rangesT|H Docket: 222105-2380 (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.

[0043] 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.

[0044] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated 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; and the number or type of embodiments described in the specification.

[0045] Disclosed are the components to be used to prepare the compositions disclosed herein as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed,T|H Docket: 222105-2380 specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B- D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

[0046] It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.

[0047] 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.

[0048] The term “pharmaceutically acceptable salt”, as used herein, means salts of the active principal agents which are prepared with acids or bases that are tolerated by a biological system or tolerated by a subject or tolerated by a biological system and tolerated by a subject when administered in a therapeutically effective amount. When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include, but are not limited to; sodium, potassium, calcium, ammonium, organic amino, magnesium salt, lithium salt, strontium salt or a similar salt. When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include, but are not limited to; those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,T|H Docket: 222105-2380 monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like.

[0049] 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.

[0050] 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 or microbes (e.g., bacteria, fungi, virus, 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.

[0051] 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,T|H Docket: 222105-2380 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,T|H Docket: 222105-2380 as well as other Enterobacteria, Brucella abortus and other Brucella species, Burkholderia cepacia, Burkholderia pseudomallei, Francisella tularensis, Bacteroides fragilis, 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).

[0052] The terms “fungus” or “fungi” include, but are not limited to yeasts such as, for example, Candida albicans or other Candida spp. including C. glabrata, C. rugosa, C. parapsilosis, C. tropicalis, or C. dubliniensis Fungi can also include dermatophytes such as, for example, Trichophyton spp. and Microsporum spp. (e.g., T. rubrum, T. interdigitale, T. tonsurans, T. violaceum, T. concentricum, T. schoenleinii, T. soudanense, T. mentagrophytes, T. equinum, T. erinacei, T. verrucosum, M. audouinii, M. ferrugineum, M. canis, M. gypseum, M. nanum, and / or M. cookie).

[0053] 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.

[0054] 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; and domesticated animals particularly petsT|H Docket: 222105-2380 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.

[0055] 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., not 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.

[0056] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0057] As used herein, the term “reduce” or “reducing” refers to deceasing the degree or amount of an event. For example, reducing biofilm formation can mean lower the amount of biofilm formation when using the compositions described herein when compared to the amount of biofilm formation when the compositions described herein are not used. Articles and Methods for Producing Nitric Oxide In Situ in a Subject

[0058] In accordance with the purpose(s) of the present disclosure, as embodies and broadly described herein, the disclosure, in one aspect, relates to materials and articles that generate nitric oxide in situ in a subject once the material or article has been introduced into the subject.

[0059] Nitric oxide (NO) is a potent gasotransmitter that exhibits a pleiotropic effect in regulating homeostasis and pathophysiology. Though it is a versatile biomaterial, silicone- based devices are still challenged by implant associated infections and fibrous capsule formation complications. Described herein are NO generating (NOgen) materials and articles that facilitate metal-ion mediated catalysis of endogenous S-nitrosothiols (NO donors). The NOgen articles and interface can generate physiologically relevant levels of NO having bactericidal and anti-thrombotic effects to combat implant-associated early onsite infection and thrombosis.

[0060] In one aspect, the nitric oxide generating material is a bioactive glass composed of silica particles doped with one or more monovalent cationic metals, divalent cationic metals, trivalent cationic metals, or any combination thereof. The selection of the silica used toT|H Docket: 222105-2380 produce the bioactive glass particles described herein can vary. In one aspect, the silica is amorphous silica, a fumed silica, a nanocrystalline silica, ceramic silica, colloidal silica, a silica coating, a silica film, organically modified silica, silica gel, a bioactive glass. In another aspect, the silica is mesoporous silica. In one aspect, the mesoporous silica includes pores in the range of between about 20 angstroms to about 500 angstroms.

[0061] In one aspect, the monovalent cations present in the bioactive glass particles described herein include sodium, potassium, lithium, or any combination thereof. In another aspect, the divalent cations present in the bioactive glass particles described herein includes Cu, Sr, Zn, Ca, Mg, Ni, Co, Mn, Cr, Pd, Pt, or any combination thereof. In another aspect, the trivalent cations present in the bioactive glass particles described herein includes Fe, Au, V, and any combination thereof.

[0062] In one aspect, the bioactive glass particles described herein include CaO, Na2O, P2O5, and any combination thereof. In another aspect, the bioactive glass particles described herein include CaO and P2O5. In another aspect, the doped silica particles include CaO, P2O5, and SrO or CuO.

[0063] In one aspect, the bioactive glass particles described herein have the formula I (SiO2)-(CaO)-(P2O5)-X, wherein X is a monovalent cationic metal, divalent cationic metal, or any combination thereof, SiO2is from about 40 molar percent to about 80 molar percent, CaO is from about 10 molar percent to about 30 molar percent, P2O5is from about 0.1 molar percent to about 10 molar percent, and X is greater than 0 molar percent to about 5 molar percent, wherein the sum is 100 molar percent.

[0064] In one aspect, SiO2in formula I is 40 molar percent, 45 molar percent, 50 molar percent, 55 molar percent, 60 molar percent, 65 molar percent, 70 molar percent, 75 molar percent, or 80 molar percent, where any value can be a lower and upper endpoint of a range (e.g., 45 molar percent to 75 molar percent).

[0065] In one aspect, CaO in formula I is 10 molar percent, 15 molar percent, 20 molar percent, 25 molar percent, or 30 molar percent, where any value can be a lower and upper endpoint of a range (e.g., 15 molar percent to 25 molar percent).

[0066] In one aspect, P2O5in formula I is 0.1 molar percent, 1 molar percent, 2 molar percent, 3 molar percent, 4 molar percent, 5 molar percent, 6 molar percent, 7 molar percent, 8 molar percent, 9 molar percent, or 10 molar percent, where any value can be a lower and upper endpoint of a range (e.g., 2 molar percent to 7 molar percent).

[0067] In one aspect, X in formula I is SrO or CuO in the amount of 0.1 molar percent, 1 molar percent, 2 molar percent, 3 molar percent, 4 molar percent, or 5 molar percent, whereT|H Docket: 222105-2380 any value can be a lower and upper endpoint of a range (e.g., 2 molar percent to 5 molar percent).

[0068] In one aspect, the bioactive glass particles described herein include CaO, Na2O, and P2O5. In another aspect, the bioactive glass particles described herein include CaO, Na2O, P2O5, and SrO or CuO

[0069] In one aspect, the bioactive glass particles described herein have formula II (SiO2)-(CaO)-(Na2O)-(P2O5)-X, wherein X is a monovalent cationic metal, divalent cationic metal, or any combination thereof, SiO2is from about 40 molar percent to about 80, CaO is from about 10 molar percent to about 30 molar percent, P2O5is from about 0.1 molar percent to about 10 molar percent, X is greater than 0 molar percent to about 5 molar percent, and Na2O is from about 1 molar percent to about 30 molar percent, wherein the sum is 100 molar percent.

[0070] In one aspect, SiO2in formula II is 40 molar percent, 45 molar percent, 50 molar percent, 55 molar percent, 60 molar percent, 65 molar percent, 70 molar percent, 75 molar percent, or 80 molar percent, where any value can be a lower and upper endpoint of a range (e.g., 45 molar percent to 75 molar percent).

[0071] In one aspect, CaO in formula II is 10 molar percent, 15 molar percent, 20 molar percent, 25 molar percent, or 30 molar percent, where any value can be a lower and upper endpoint of a range (e.g., 15 molar percent to 25 molar percent).

[0072] In one aspect, P2O5in formula II is 0.1 molar percent, 1 molar percent, 2 molar percent, 3 molar percent, 4 molar percent, 5 molar percent, 6 molar percent, 7 molar percent, 8 molar percent, 9 molar percent, or 10 molar percent, where any value can be a lower and upper endpoint of a range (e.g., 2 molar percent to 7 molar percent).

[0073] In one aspect, Na2O in formula II is 1 molar percent, 5 molar percent, 10 molar percent, 15 molar percent, 20 molar percent, 25 molar percent, or 30 molar percent, where any value can be a lower and upper endpoint of a range (e.g., 15 molar percent to 25 molar percent).

[0074] In one aspect, X in formula II is SrO or CuO in the amount of 0.1 molar percent, 1 molar percent, 2 molar percent, 3 molar percent, 4 molar percent, or 5 molar percent, where any value can be a lower and upper endpoint of a range (e.g., 2 molar percent to 5 molar percent).

[0075] The bioactive glass particles described herein can be produced using a sol-gel process. The Examples provide non-limiting procedures for making the bioactive glass particles described herein. The bioactive glass particles described herein produce nitric oxideT|H Docket: 222105-2380 in situ; thus, the inclusion or use of nitric oxide compounds with the doped silica particles is not necessary.

[0076] In one aspect, the bioactive glass particles described herein are used as nanoparticles. In one aspect, the bioactive glass particles described herein have an average diameter of about 100 nm to about 500 nm. Methods for determining the average particle size of the bioactive glass particles described herein are provided in the Examples.

[0077] In another aspect, the articles described herein include strontium ions as the in situ nitric oxide generating material, wherein the strontium ions generate nitric oxide in situ when introduced into a subject. In one aspect, the strontium ions are derived from strontium oxide. In another aspect, strontium ions are derived from a pharmaceutically acceptable salt of strontium. Examples of pharmaceutically acceptable salts of strontium include, but are not limited to, chloride, bromide, nitrate, carbonate, acetate, propionate, isobutyrate, maleate, malonate, benzoate, succinate, suberate, fumarate, lactate, mandelate, phthalate, benzenesulfonate, p-tolylsulfonate, citrate, tartrate, and methanesulfonate. Applications of In Situ Nitric Oxide Generating Materials and Articles

[0078] The in situ nitric oxide generating materials and articles described herein possess unique properties that make them suitable in a variety of different applications. The in situ nitric oxide generating materials and articles described herein are biocompatible, which make them useful in biomedical applications. Nitric oxide generated in situ in tandem with the low therapeutic release of metal ions from the in situ nitric oxide generating materials and articles can regulate numerous biological mechanisms including, but not limited to, cellular fate pertaining to fibroblasts, macrophages, and endothelial cells.

[0079] The in situ nitric oxide generating materials described herein can be applied to an article using techniques known in the art for applying solid particles to a surface. For example, the in situ nitric oxide generating materials can be applied by electro-spraying or other spray techniques. In another aspect, the in situ nitric oxide generating materials can be formulated with a coating composition prior to being applied to the article. In one aspect, the in situ nitric oxide generating materials can be formulated with a silicone coating such as, for example, a room temperature vulcanizing (RTV) silicone then subsequently applied to the surface of the article. In one aspect, the article to be coated is composed of a polysiloxane such as, for example, polydimethylsiloxane.

[0080] In another aspect, the in situ nitric oxide generating materials can be integrated or dispersed throughout the article. In one aspect, the article is composed of a polymeric materal such as polysiloxane (e.g., polydimethylsiloxane). Polysiloxane can be admixed with the inT|H Docket: 222105-2380 situ nitric oxide generating materials, and the resulting composition can be added to a mold to produce an article with a desired shape. In another aspect, the in situ nitric oxide generating material can be incorporated into a ceramic or other porous material.

[0081] In one aspect, an article or surface of an article can be coated with the in situ nitric oxide generating materials described herein where it is desirable to reduce or prevent biofouling (e.g. growth of bacteria, adhesion of platelets, adhesion of fibrinogen). Biofilm and thrombus formation on surfaces results in significant morbidity and mortality worldwide, which highlights the importance of the development of efficacious fouling-prevention approaches.

[0082] In one aspect, the in situ nitric oxide generating materials described herein are useful in applications where it is desirable to reduce or prevent biofouling. Implantable medical devices are a leading cause of infection such as nosocomial infections. Implantable devices coated with or constructed of the bioactive glass particles described herein can reduce or prevent biofouling in a subject when the device is introduced into the subject. In one aspect, the in situ nitric oxide generating materials described herein can reduce or prevent bacterial growth on a surface of an implantable device. In another aspect, the compositions described herein can reduce or prevent biofilm formation on a surface of an implantable device.

[0083] In one aspect, the implantable device is 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. The in situ nitric oxide generating materials described herein are biocompatible (e.g., with fibroblast cells), which makes them useful in implantable medical devices. In one aspect, the device is composed of PDMS, PCL, or Elasteon.

[0084] In one aspect, the in situ nitric oxide generating materials described herein can prevent the growth of bacteria on an article, in which the method includes applying the in situ nitric oxide generating materials as above to at least one surface of the article. The coated article can prevent about at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the growth of bacteria when compared to an uncoated article.

[0085] In one aspect, the in situ nitric oxide generating materials described herein can prevent the adhesion of platelets on an article, in which the method includes applying the in situ nitric oxide generating materials described herein as above to at least one surface of the article. The coated article can prevent about at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the adhesion of platelets when compared to an uncoated article.

[0086] In another aspect, the in situ nitric oxide generating materials described herein can reduce or prevent inflammation when introduced into a subject. Not wishing to be bound byT|H Docket: 222105-2380 theory, one or more metal cations leached from the in situ nitric oxide generating materials described herein can suppress the collagen expression and migration of activated fibroblasts, while favouring M2phenotype bias in macrophages. Differential NO flux observed over time from NOgen article helps switch macrophages from proinflammatory M1phenotype to M2anti- inflammatory phenotype. Moreover, the synergistic effect of leachate and NO generated by the bioactive glass particles described herein demonstrates a proangiogenic effect by aiding endothelial network maturation in vitro. Aspects

[0087] Aspect 1. An article comprising at least one surface coated with silica particles doped with one or more monovalent cationic metals, divalent cationic metals, trivalent cationic metals, or any combination thereof.

[0088] Aspect 2. An article comprising silica particles doped with one or more monovalent cationic metals, divalent cationic metals, trivalent cationic metals, or any combination thereof dispersed throughout the article.

[0089] Aspect 3. The article of Aspect 1 or 2, wherein the silica comprises amorphous silica, a fumed silica, a nanocrystalline silica, ceramic silica, colloidal silica, a silica coating, a silica film, organically modified silica, mesoporous silica, silica gel, a bioactive glass.

[0090] Aspect 4. The article of Aspect 1 or 2, wherein the silica comprises mesoporous silica.

[0091] Aspect 5. The article of any one of Aspects 1-4, wherein the monovalent cations comprise sodium, potassium, lithium, or any combination thereof.

[0092] Aspect 6. The article of any one of Aspects 1-4, wherein the divalent cations comprise Cu, Sr, Zn, Ca, Mg, Ni, Co, Mn, Cr, Pd, Pt, or any combination thereof.

[0093] Aspect 7. The article of any one of Aspects 1-4, wherein the trivalent cations comprise Fe, Au, V, and any combination thereof.

[0094] Aspect 8. The article of any one of Aspects 1-7, wherein the silica particles comprise CaO, Na2O, P2O5, and any combination thereof.

[0095] Aspect 9. The article of any one of Aspects 1-7, wherein the silica particles comprise CaO and P2O5.

[0096] Aspect 10. The article of any one of Aspects 1-7, wherein the silica particles comprise CaO, P2O5, and SrO or CuO.

[0097] Aspect 11. The article of any one of Aspects 1-7, wherein the silica particles comprise (SiO2)-(CaO)-(P2O5)-X, wherein X is a monovalent cationic metal, divalent cationic metal, or a combination thereof, SiO2is from about 40 molar percent to about 80 molarT|H Docket: 222105-2380 percent, CaO is from about 10 molar percent to about 30 molar percent, P2O5is from about 0.1 molar percent to about 10 molar percent, and X is greater than 0 molar percent to about 5 molar percent, wherein the sum is 100 molar percent.

[0098] Aspect 12. The article of Aspect 11, wherein X is SrO or CuO.

[0099] Aspect 13. The article of any one of Aspects 1-7, wherein the silica particles comprise CaO, Na2O, and P2O5.

[0100] Aspect 14. The article of any one of Aspects 1-7, wherein the silica particles comprise CaO, Na2O, P2O5, and SrO or CuO

[0101] Aspect 15. The article of any one of Aspects 1-7, wherein the silica particles comprise (SiO2)-(CaO)-(Na2O)-(P2O5)-X, wherein X is a monovalent cationic metal, divalent cationic metal, or any combination thereof, SiO2is from about 40 molar percent to about 80, CaO is from about 10 molar percent to about 30 molar percent, P2O5is from about 0.1 molar percent to about 10 molar percent, X is greater than 0 molar percent to about 5 molar percent, and Na2O is from about 1 molar percent to about 30 molar percent, wherein the sum is 100 molar percent.

[0102] Aspect 16. The article of Aspect 15, wherein X is SrO or CuO.

[0103] Aspect 17. The article of any one of Aspects 1-16, wherein the silica particles do not include a nitric oxide releasing compound.

[0104] Aspect 18. The article of any one of Aspects 1-17, wherein the coating of silica particles further comprises a room temperature vulcanizing silicone.

[0105] Aspect 19. An article comprising at least one surface coated with strontium ions.

[0106] Aspect 20. An article comprising strontium ions dispersed throughout the article.

[0107] Aspect 21. The article of Aspects 19 or 20, wherein the strontium ions are derived from strontium oxide or a pharmaceutically acceptable salt of strontium.

[0108] Aspect 22. The article of any one of Aspects 19-21, wherein the strontium ions are incorporated into a polymer matrix, a gel, a glass, or a ceramic material.

[0109] Aspect 23. The article of any one of Aspects 1-22, wherein the article comprises a polymeric grade material, a medical device, a surface or article in a hospital or medical facility, a wound dressing.

[0110] Aspect 24. The article of any one of Aspects 1-23, wherein the surface comprises a polysiloxane.

[0111] Aspect 25. The article of any one of Aspects 1-24, wherein the article comprises an implantable medical device.T|H Docket: 222105-2380

[0112] Aspect 26. The article of Aspect 25, wherein the implantable medical device comprises a urinary catheter, artificial heart valve, a vascular catheter, a graft, or a stent.

[0113] Aspect 27. The article of any one of Aspects 1-26, wherein the article generates nitric oxide when introduced into a subject.

[0114] Aspect 28. The article of any one of Aspects 1-26, wherein the article reduces or prevents the growth of microbes on the article when introduced into a subject.

[0115] Aspect 29. The article of any one of Aspects 1-26, wherein the article reduces or prevents the adhesion of platelets on the article when introduced into a subject.

[0116] Aspect 30. The article of any one of Aspects 1-26, wherein the article reduces or prevents inflammation when introduced into a subject.

[0117] Aspect 31. A method of generating nitric oxide in situ in a subject, the method comprising introducing the article in any one of Aspects 1-30 into the subject. EXAMPLES

[0118] Now having described the embodiments of the disclosure, in general, the examples describe some additional embodiments. While embodiments of the present disclosure are described in connection with the example and the corresponding text and figures, there is no intent to limit embodiments of the disclosure to these descriptions. 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.

[0119] MATERIALS AND METHODS

[0120] Chemical reagents conforming to ACS reagent grade (purity ≥ 98%) such as hexadecyltrimethylammonium bromide (CTAB), Tris(hydroxymethyl)aminomethane (Tris base), tetraethylorthosilicate (TEOS), calcium nitrate tetrahydrate, triethylphosphaste (TEP), copper (II) chloride dihydrate, strontium chloride, hexahydrate, sodium nitrite, L-ascorbic acid, Direct red 80, triton-X 100, tween-20 and ethylenediaminetetraacetic acid (EDTA) were obtained from Millipore-Sigma (St. Louis, MO, USA); 1,10-phenanthroline was obtained from Fisher Scientific (Fair Lawn, NJ, USA). All solvents, bases, acids, and biological reagents were procured from Fisher Scientific (Fair Lawn, NJ, USA), unless otherwise mentioned differently in the subsequent text.

[0121] Synthesis of mesoporous bioactive glass nanoparticles: A modified Stöber’s method was adopted for synthesis of mesoporous bioactive glass belonging to the glass composition: 70SiO2.25CaO.5P2O5using a sacrificial templating technique.[23, 28]CTAB was chosen as the surfactant to form a micellar template acting as caping agent for the dendrimericT|H Docket: 222105-2380 silica network formation using TEOS as the SiO2(network formers) precursor to which other non-bridging oxide precursors (network modifiers) were sequentially added. Briefly, 4 mM CTAB in Tris-HCl buffer (10mM, pH 8.0) was used to hydrolyse TEOS for 1 h at ambient conditions (25 ± 2 ºC) under continuous stirring. The CaO and P2O5precursors in the form of calcium nitrate tetrahydrate and TEP were subsequently added after 1 h intervals in succession to each precursor addition. The molar ratio of the constituents was maintained at 70:25:5, TEOS: calcium nitrate tetrahydrate: TEP. The reaction was allowed to continue for 48 h under stirring conditions, after which the nanobioactive glass (nBG) precipitates were collected by centrifugation. For the synthesis of doped bioactive glass nanoparticles, either copper doped nanobioactive glass (nBG-Cu) or strontium doped bioactive glass (nBG-Sr), copper (II) chloride dihydrate or strontium chloride was added after CaO precursor addition step as noted earlier for nBG synthesis, followed by same processing steps. The molar ratio of the constituents was maintained at 70:20:5:5, TEOS: calcium nitrate tetrahydrate: copper (II) chloride dihydrate: TEP for nBG-Cu, while 70:20:5:5, TEOS: calcium nitrate tetrahydrate: strontium chloride: TEP for nBG-Sr, respectively.

[0122] Dip coating of silicone substrates to obtain NOgen interfaces: Medical grade silicone rubber (SR) sheets – thickness 1 mm (McMaster-Carr, Douglasville, GA, USA) were used as substrates for developing the NOgen interfaces. A dip coating strategy was adopted wherein room temperature vulcanizing (RTV) silicone (Dowsil™ RTV 3140, Midland, MI, USA) was chosen as the polymer so it can bond well with SR substrates without delamination and cured at ambient conditions. Depending on the concentration of nanoparticle in suspension, the experimental groups were denoted as: nBG-Cu 5, nBG-Cu 15, nBG-Cu 25 and nBG-Cu 50 for concentrations 5, 15, 25, and 50 mg / mL respectively (likewise for other sample types also). Control substrates (SR control) for biological experiments were made from SR (6 mm punchouts), dip coated with 200 mg / mL RTV (thrice) and similar procedure followed for curing and removal of any residual solvent.

[0123] Synthesis of S-nitrosoglutathione (NO donor) for NOgen studies

[0124] GSNO was synthesized based on our previously published method

[0031] from reduced glutathione (GSH) (Alfa Aesar, Waltham, MA, USA). Briefly, 16 mM GSH was dissolved in 0.48 M HCl, and this mixture was cooled down in ice bath. Excess sodium nitrate (17 mM) was added to this cooled mixture and allowed to react for 40 min. Cold acetone was added to crash out GSNO as pink precipitate which was collected by vacuum filtration. The precipitate was washed with cold acetone, followed with deionised water to remove any unreacted reagents, and dried under vacuum overnight. The dried GSNO product was groundT|H Docket: 222105-2380 using mortar and pestle and stored in -20 ºC until further use. The purity of the synthesized GSNO was tested using a Nitric Oxide Analyzer (NOA) and was found to be >95%.

[0125] Physicochemical characterizations

[0126] The synthesized nanoparticles were characterized for their size, morphology and functional composition. Hydrodynamic radius, polydispersivity index and surface charge was analysed by dynamic light scattering studies by Zetasizer Nano S90 (Malvern Panalytical, UK) equipped with Zeta potential measurement. Surface microstructure and compositional analysis of the synthesized nanoparticles was analysed by high resolution scanning transmission electron microscope (HR-STEM, Hitachi SU9000EA, Japan) equipped with Oxford large area windowless - energy dispersive spectroscopy (EDS) and micro-electron diffraction to gather selected area energy diffraction (SAED) detectors. The functional composition of fingerprint regions of synthesized nanoparticles was analysed using a Fourier transform infrared spectrometer (Spectrum Two, PerkinElmer, USA).

[0127] The static water contact angle (droplet volume = 5 µL) of dip coated NOgen substrates (n ≥ 5), was measured using contact angle goniometer (Ossila Ltd., UK). The metal ion leachate from the dip coated substrates at different time points were quantified using inductively coupled plasma optical emission spectrophotometer (ICP-OES) Perkin Elmer 8300 (PerkinElmer, USA). The nitric oxide generated from NOgen substrates was measured using Sievers chemiluminescence nitric oxide analyser (Zysense NOA 280i, G.E. Analytical, USA).

[0128] Antibacterial studies

[0129] The antibacterial efficacy of the NO from NOgen substrates (n ≥ 4) was challenged against planktonic cultures of two commonly noted bacterial strains associated with implant infections: Gram-negative, Escherichia coli (ATCC 25922) and Gram-positive Staphylococcus aureus (ATCC 6538), which were procured from American Type Culture Collection (ATCC, VA, USA). To assess the reduction of planktonic bacteria population bacterial suspension was serially diluted and plated in LB agar plates using a spiral plater (Eddy Jet 2, IUL Instruments, Spain). Colonies were counted using an automated colony counter (Sphere Flash, IUL Instruments, Spain).

[0130] Platelet adhesion study

[0131] The protocols for the ethical use of freshly drawn whole porcine blood and its platelet-rich (PRP) and platelet-poor (PPP) plasma were approved by the Institutional Animal Care and Use Committee. Platelet solution in the density of 108platelets / mL was supplemented with 1 µM GSNO and 30 µM GSH (final concentration) and incubated for 90 min at 37 ºC under mild rocking conditions with the substrates. The substrates were retrievedT|H Docket: 222105-2380 and rinsed in PBS to remove loosely adhered platelet, and the adhered platelets to substrates was detected by lactate dehydrogenase (LDH) released was quantified using Roche LDH detection kit (Millipore-Sigma, St. Louis, MO, USA).

[0132] In vitro biological studies

[0133] The cell instructive facets of the NOgen substrates were tested on human fibroblasts, endothelial cells and macrophages. For these the following cell lines were obtained: human fibroblasts (BJ, CRL-2522, ATCC, USA), human umbilical vein endothelial cells (HUVECs) (PCS-100-010, ATCC, USA) and human monocytic cell line (THP-1) (TIB- 202, ATCC, USA) and maintained in their respective culture media. All consumables and biological reagents were procured from Thermo Fisher Scientific (Waltham, MA, USA).

[0134] Cytocompatibility assessment: The cytocompatibility assessment was done following ISO standard (ISO10993-5: 2009) protocol by (i) collecting leachates and (ii) through indirect contact method. NOgen and SR control substrates (n = 4) (6 mm diameter, ~ 1 mm thick) were UV sterilized prior to experimentation. Fibroblasts were seeded in 96 well plates (8000 cells / well) and after 24 h post-seeding, 100 µL of collected leachates were added as treatment (for 24 h). The relative cell viability percentage of the treated and non-treated (cell control group) were assessed using 0.5 mg / mL MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) reagent (Millipore-Sigma, St. Louis, MO, USA). Similarly, for indirect contact method 25,000 cells were seeded per well in a 24 well plate and after 24 h post- seeding sterilized specimens were exposed to cells via a transwell insert (6.5 mm diameter polycarbonate 3 µm pore size, Millipore-Sigma, St. Louis, MO, USA). After 24 h of exposure, the relative cell viability percentage of the treated and non-treated (cell control group) were assessed using MTT assay.

[0135] In vitro fibrosis assay: The effect of 14 days leachate on fibroblasts was further studied by setting up an in vitro fibrosis assay and quantifying the reduction of fibroblast activity in presence of a known stimulant ascorbic acid.

[0046] Fibroblasts were seeded on 12 well plates at a seeding density of 105cells / well, and culture media were supplemented with 100 µM ascorbic acid to stimulate collagen synthesis for 24 h. The collagen secreted by cells were quantified by Sirius Red assay using type-1 rat tail collagen (Corning, Millipore-Sigma, St. Louis, MO, USA) as standards and was read at 550 nm using a multiplate reader (Cytation 5, BioTek, Agilent, USA).

[0136] The effect of leachate was also investigated on the migration ability of ascorbic acid treated fibroblasts. For this purpose, two-well silicone inserts (ibidi USA, Inc., Fitchburg, WS, USA) with a gap size ~ 500 µm were used in a 24 well plate. The rate of cell migration toT|H Docket: 222105-2380 close the gap was assessed by taking phase contrast images using a phase contrast microscope (Advanced Microscopy Group’s AMG EVOS microscope, USA) at regular time intervals. The percentage closure (n = 4) was estimated from the phase contrast images using Image-J (National Institutes of Health, USA) software.

[0137] Macrophage polarization studies: Human monocyte cell line (THP-1) was cultured as suspensions in RPMI-1640 media supplemented with 10 % (v / v) FBS, 0.05 mM mercaptoethanol and penicillin-streptomycin (10 U / mL and 10 μg / mL, respectively). Monocytes in suspension were inducted into adherent naïve macrophages (M0) by phorbol 12-myristate 13-acetate (PMA). The naïve macrophages were polarised into either pro- inflammatory (M1) or anti-inflammatory (M2) phenotype by treating them with either 20 ng / mL interferon-γ (IFN-γ) or interleukin-4 (IL-4) (Peprotech, Thermo Fisher Scientific) respectively, for 72 h. This served as the workflow for control cell experiments. For testing the effect of leachates (14-day) and the NOgen films (n ≥ 3), THP-1 monocytes were seeded at a density of 105cells per well in 24 well plates and induced to M0 macrophages by PMA treatment. Meanwhile for testing the effect of NO from NOgen substrates, UV-sterilized substrates (6mm diameter, ~1 mm thickness) were exposed to M0macrophages using transwell inserts (6.5 mm polycarbonate 3 µm pore size) in 1mL of monocyte culture media with 1 µM GSNO and 30 µM GSH injections supplemented every 5 h for 72 h (13 injections in total). The macrophages obtained at day-3 and day-10 were subsequently analysed.

[0138] The polarized macrophages were trypsinized and characterized for the population distribution using flow cytometry by staining for mouse monoclonal against human CD 68 (pan macrophage marker) and rabbit polyclonal against human CCR7 (pro-inflammatory marker M1) and rabbit polyclonal against human CD 206 (anti-inflammatory marker M2). The cells stained with secondary antibody cocktail consisting of Alexa Fluor™ 488 conjugated goat-anti mouse Ig and Alexa Fluor™ 647 conjugated- donkey-anti rabbit Ig and was analysed in a flow cytometer (Cytek® Aurora, Cytek® Biosciences, USA). For qualitative assessment of polarized macrophages, immunocytochemistry using the same antibodies were performed. The stained cells were washed with PBS (pH 7.4) with 0.1% (v / v) tween-20 and imaged using a fluorescence microscope (AMG EVOS-FL, USA).

[0139] In vitro angiogenesis assay: HUVECs were maintained in endothelial cell growth media (Lonza EGM-2™) supplemented with its bullet kit components (Lonza Lifescience, Walkersville, MD, USA). Type-1 rat tail collagen was used as the basement substrate to encapsulate HUVECs and the extent of sprouting (node formation) and tube extension within the collagen gel was studied by staining the F-actin cytoskeleton. The stained endothelial tubeT|H Docket: 222105-2380 network was imaged using a fluorescence microscope (AMG EVOS-FL, USA). The fluorescent images were analysed using Image-J software to estimate the number of endothelial nodes and the tube length.

[0140] Statistical analysis

[0141] All the experiments were performed in biological or technical replicates (n ≥ 3), unless otherwise noted in the respective methods section and the results are represented as mean ± standard deviation (mean ± SD). One-way analysis of variance (ANOVA) via Tukey’s test was performed on parametric data sets using Origin 2018b graphing and analysis software (OriginLabs, USA) to analyse the statistical significance and the results between two test groups were considered statistically significant if the p-value was found to be less than 0.5.

[0142] RESULTS AND DISCUSSION

[0143] As the first steps in materializing an in situ NOgen interface, we synthesized the building blocks in the form of sol-gel derived nano-bioactive glass particles. Bioactive glasses have long been used as resorbable defect fillers in bone and dental application. In addition to this, bioactive glasses have gained traction for use in implantable device applications such as neural, angiogenesis, gastro-intestinal and wound regeneration.

[0024] A 70S bioactive glass ternary glass network was chosen here owing to its better bioactivity, higher density of Si-OH exposed groups (ideal for bonding when coating), and enhanced glass dissolution traits

[0025] (in comparison to well characterized 45S5 melt-derived bioactive glasses), making it a resorbable bioceramic without eliciting any adverse immune response while being cytocompatible.

[0144] Utilizing mesoporous doped nano-bioactive glass as building blocks for nitric oxide generation

[0145] For obtaining the sol-gel derived nano-bioactive glass particles we resorted to use a modified Stöber’s method with the help of a sacrificial template - hexadecyltrimethylammonium bromide (CTAB). This surfactant here helped in templating and served as a capping agent to control the size of the synthesized particles. Moreover, when the templating agent is removed it results in obtaining a mesoporous network (Figure 2A), which has shown immense potential in drug delivery applications owing to its large specific area, low density, and good penetration capability.

[0026] These properties are desired in a nanoparticle system which is intended to be used as an in situ nitric oxide nano-generator, where NO donor should be able to access the catalytic site and decompose easily. For the catalytic sites, we investigated the potency of two metal ions: copper and strontium in the current work. These metals were introduced as oxides which acted as network modifier within the tetrahedraT|H Docket: 222105-2380 silicate network, connected by –Si–O–Si– bridging oxygen bonds. Thus, the bioactive glass particles obtained from 70S bioactive glass: 70SiO2.25CaO.5P2O5were termed nBG, copper doped network: 70SiO2.20CaO.5CuO.5P2O5as nBG-Cu, or strontium doped network: 70SiO2.20CaO.5SrO.5P2O5as nBG-Sr. Both copper and strontium are bivalent cations that exhibit +1 and +2 oxidation states and are predominantly present in their +2-oxidation state within the glass network in its oxide form. Though copper has been well validated in its role to catalyse S-nitrosothiols,

[0018] this is the first report investigating the catalytic potency of strontium. Moreover, strontium has been reported to possess other ancillary benefits in regulating cellular fate involving neuronal plasticity, angiogenesis and osteoclast activity.

[0027] Infrared spectra of the synthesized nBG, nBG-Cu and nBG-Sr confirmed the fingerprint regions of the bioactive glass network (Figure 2B). The stretching vibration of Si-O-Si at 1093 cm-1(as a shoulder peak) and stretching vibration of O-Si-O at 800 cm-1from the SiO2network formers; the P-O bending vibration at 962 cm-1from P2O5network modifiers

[0023] were noticed in all the sample types.

[0146] Previously, it has been reported that the concentration of the surfactant dictates the templating characteristics and order of the mesoporous network. For instance, when 0.4 mM CTAB was used, it resulted in particle size of ~ 150 nm, whereas 10 mM resulted in ~ 100 nm particles.

[0028] Similarly, anything below 3 mM CTAB had a specific area of ~ 444 m2 / g while above 6 mM had a specific area of ~ 970 m2 / g. For our system, we wanted to use an optimal concentration that had a balance of the right particle size and the specific area, and the 4 mM CTAB concentration was investigated here which yielded us size of the order ~ 100 nm (Figure 2C). Monodispersed spherical nano-bioactive glass particles with dendrimer-like projections arising from maturation of the bioactive glass network were obtained (as observed from the transmission electron micrographs) while no significant difference in size was noted between the groups (nBG, nBG-Cu and nBG-Sr). Moreover, we opted for warm ethanol refluxing

[0023] to remove the templating agent to create the mesopores (instead of calcination) which resulted in nanoparticles having an amorphous nature as observed by the diffused rings from selected area energy diffraction (SAED) patterns (Figure 2C and Figure 9). The energy dispersal spectral (EDS) confirmed the stochiometric composition of the synthesized glass network where the incorporation of Cu (in nBG-Cu), Sr (in nBG-Sr), and calcium (Ca), phosphate (P) as network modifiers were mapped and found to be distributed throughout the network former Si (in SiO2) (Figure 2C). The hydrodynamic radii of nanoparticles were analysed (dispersant: ethanol, viscosity = 1.0740 cP and refractive index = 1.361) (Figures 10A-C) and the average diameter (polydispersity index - PDI, Zeta potential – ζ, inT|H Docket: 222105-2380 parenthesis) in suspension for nBG was 358 ± 181 nm (PDI = 0.181; ζ = 13.6 mV), for nBG- Cu was 222 ± 77 nm (PDI = 0.295; ζ = 12.8 mV), and for nBG-Sr was 280 ± 107 nm (PDI = 0.221; ζ = 13.2 mV). No statistical significance was noted between the hydrodynamic radii and the zeta potential of the analysed samples and the values were in accordance with nano- bioactive glass systems previously reported.

[0023] The network modifiers used here did not significantly impact the microstructure or its property in suspension. The suspensions were stable in ethanol after 4h (Figure 10D), which is indicative that they are suitable for dip coating application when incorporated in a polymer solution.

[0147] Facile modification of silicone substrates to obtain NOgen interface

[0148] NO is one of the classical gasotransmitters (the other two being hydrogen sulfide and carbon monoxide) which regulate key cellular signalling cascades of vascular, neural, and immune homeostasis. NO is generated by endothelium with an estimated flux 0.5 to 4 x 10-10mol cm-2min-1

[0029] as a product of oxidation of L-arginine into L-citruline mediated by enzyme (endothelial nitric oxide synthase – eNOS) in the presence of molecular oxygen and reduced NADPH (nicotinamide adenine dinucleotide phosphate) as co-substrates. In addition to eNOS, neurnonal NOS (nNOS) and inducible NOS (iNOS) expressed by cells of neuronal lineage and immune cells (respectively) can also facilitate the production of NO in vivo. NO is short- lived and it exerts its cellular signalling by activating a key secondary signalling messenger - soluble guanylate cyclase (sGC) which activates the NO / sGC / cGMP (cyclic guanosine monophosphate) signalling axis.

[0030] NO generated by these cells also exerts another important role which is S-nitrosylation of thiol groups of L-cysteine of proteins generating S-nitrosothiols (such as S-nitrosoalbumin or S-nitrosoglutathione - GSNO) which are generally present in the human body and are the major sources of NO donors that can be harnessed to generate NO in an enzyme and cell-independent fashion.

[0149] To produce a localized NO flux at a biomaterial implant site to harness the cell instructive facets of NO, a nitric oxide generating (NOgen) interface needs to be developed on the biomaterial. The NOgen interface can utilize the NO donors present in the vicinity to catalyse its decomposition to generate NO in situ. We intended to achieve this in a facile manner by dip coating silicone rubber (SR) substrates with different concentration (5, 15, 25, 50 mg / mL suspensions) of synthesized nanobioactive glasses in RTV silicone elastomer polymer to obtain conformal coatings which are cured at ambient temperatures (Figure 3A- B). Anything above 50 mg / mL concentration led in delamination of the coating and concentrations in 5 and 15 mg / mL resulted in non-homogenous coatings. The modified substrates were analysed for the NO generation capability (Figure 3C) using aT|H Docket: 222105-2380 chemiluminescence-based real-time NO analyser to determine the flux values in the monitored 30 min duration (Figure 3D, at 37 ºC, in phosphate buffered saline PBS, pH 7.4 supplemented with 1 µM and 30 µM GSNO and reduced glutathione - GSH respectively, akin to the physiological levels of GSNO and GSH in blood[18, 31]reported earlier). S-nitrosothiols (like GSNO) can be decomposed to release NO through photolytic, thermal and metal-ion assisted mechanisms. Among these, metal-ion-mediated catalysis was of interest to us here since copper (Cu2+ / Cu+1) has long been proven and extensively used as an effective reagent for GSNO catalysis.

[0032]

[0150] A gamut of bivalent metal ions such as Zn, Ca, Mg, Ni, Co, Mn, Cr, Pd, Pt and trivalent Fe, Au, V have been investigated for their potency to catalyse GSNO decomposition.[21, 33]Among which, Cu2+, Au3+, Pd2+, Pt2+and V3+were demonstrated to be the most potent metal-ion catalysts. Copper has been an important biological metal ion used as a cofactor in several metalloenzymes, we wanted to probe the efficacy of another important bivalent metal ion strontium (Sr) as a catalyst. This is the reason why we chose to dope the bioactive glass network with oxides of Cu (a known metal ion catalyst) and Sr which has several ancillary cell instructive benefits, to investigate the doped-nanobioactive glass systems to catalyse GSNO decomposition. A concentration-dependant increase in total moles generated from nBG-Cu and nBG-Sr groups were observed with a saturation reaching at 50 mg / mL concentrations dip coats (Figure 3C); total moles released from nBG-Cu 50 was 3.6 ± 1.12 nmol, nBG-Cu 25 was 3.58 ± 0.52 nmol; from nBG-Sr 50 was 2.96 ± 0.85 nmol, nBG-Sr 25 was 2.45 ± 0.48 nmol. Interestingly, the control nBG group did not aid in GSNO catalysis as the molecular constituents Si or P or Ca have previously been shown not to participate in NO generation

[0033] (Figure 3D iii). We also tested the potency of these doped nano-bioactive glass NOgen interface’s efficacy to generate NO at reduced GSNO and GSH concentration noted in other tissue space such as in human broncho alveolar lavage fluid (300 nM – 4 μM )

[0019] and in subcutaneous tissue level (~ 0.09 μM).

[0020] We found that the total moles of NO also reduced corresponding to the GSNO supplemented: (i) with 0.3 µM GSNO, total moles released for nBG-Cu 50 was 2.1 ± 0.41 nmol, nBG-Cu 25 was 1.22 ± 0.73 nmol; for nBG-Sr 50 was 1.0 ± 0.66 nmol, nBG-Sr 25 was 0.58 ± 0.08 nmol; (ii) with 0.09 µM GSNO, total moles released from nBG-Cu 50 was 0.48 ± 0.05 nmol, nBG-Cu 25 was 0.16 ± 0.07 nmol; from nBG-Sr 50 was 0.09 ± 0.006 nmol, nBG-Sr 25 was 0.05 ± 0.009 nmol (Figure 11).

[0151] The mechanism for the metal ion mediated catalysis was further studied by performing the decomposition of 1 µM GSNO (with 30 µM GSH) in presence of two types of metal chelators: (i) ethylenediaminetetraacetic acid (EDTA) which has specificity for +2T|H Docket: 222105-2380 valency state of metal ions and (ii) 1,10 phenanthroline which has specificity for +1 valency state of metal ions of copper and strontium.[33-34]It is regarded that the metal (M) ion used here exists in its +2 valency (M2+), which gets reduced by a reducing agent like glutathione (GSH), to yield a reduced form of M+ion which catalyses the GSNO decomposition to release NO and thereafter regenerating the M2+ion which participates in the catalysis again

[0033] (Figure 3E). Computational modelling has revealed that GSNO coordinates with the M+metal ion centre via its sulphur and once NO is released glutathione disassociates from the complex.

[0032] To test this hypothesis and whether it holds true for a bivalent cation such as Sr we wanted to arrest the active catalytic centre M+with 1,10 phenanthroline ligand (Figure 3F). Chemiluminescence NO analysis corroborated this, where in the presence of either 100 µM of metal ion salts: copper chloride (Figure 3Gi) or strontium chloride (Figure 3Gii) the NO catalysis which occurred typically got arrested instantaneously in the presence of EDTA while NO catalysis was retarded to a significant extent in the presence of 1,10-phenanthroline. UV- visible spectroscopic analysis of metal ion and 100 µM ligand (1,10 phenanthroline) interaction revealed that in the absence of a reducing agent (GSH), +1 valency state was not achieved; whereas in the presence of reducing agent +1 valency state was obtained and (metal-ligand)+1complex (having an absorption peak at λ = 450 nm) was formed (Figure 12A-12B). Whenmaxwe tested the NOgen efficacy of nBG-Cu 50 and nBG-Sr films in the presence of the 1,10 phenanthroline ligand, the NO catalysis was arrested significantly (Figure 3G iii-iv). It was interesting to note that when the films (NOgen substrates) were retrieved from the NO analysis chamber, there was a colour change (Figure 3H); white strontium films turned reddish brown whereas copper films changed color from teal to green. This is indicative of the (metal-ligand)+1complex having a reddish brown colored product formation having an absorption peak at λmax= 450 nm.

[0033]

[0152] Kinetic analysis on rate forms of metal ion-mediated GSNO decomposition has shown to have first-order dependencies (upon both concentration levels of GSNO and [32, 35]copper). The reaction rate constants (k) can be determined from NO flux profiles (Figure 3D i-ii) by plotting time (t) versus ln ([GSNO] / [GSNO] ), yielding linear equation (y = mx),t t=0where slope gives the rate constant (k = = - ; for equation 2 in Figure 3E). The rate constant for nBG-Cu 50 was found to be-1 -3 -1s and 3.67 x 10 s in presence of 1 µM GSNO (as NO donor) i.e. k of Cu mediated > k of Sr mediated ; whichexplains why Cu is a more potent catalyst in to Sr. The rate nBG-5 -150 films (Figure 3D iii) was found to be 2 x 10 s which served as the control group whereT|H Docket: 222105-2380 no metal-assisted catalysis of NO donor took place. The average flux values generated for 1 µM GSNO supplementation from nBG-Cu 50 was 1.72 x 10-10mol cm-2min-1and from nBG- Sr 50 was 1.12 ^ 10-10mol cm-2min-1which was in the physiologically relevant levels as seen in the endothelium.

[0029]

[0153] Multifunctional NOgen interface exhibit antibacterial and antithrombogenic properties

[0154] Several active and passive surfaces or interfaces have been developed on silicone rubber (SR) substrates to combat implant-associated thrombosis (occurs within minutes to hours) and early onsite infection (initiated within hours to days). Of particular interest are the interfaces that modulate the wettability of the surfaces to obtain either super-hydrophilic or super-hydrophobic surfaces. These strategies are passive strategies that prevent protein- fouling and cell (bacterial and mammalian) adhesion (Figure 1). However, these passive methods are generally regarded advantageous for short term applications as these coatings are not that durable in vivo and require use of complex chemistry. Liquid or lubricant infusion to make SR substrates slippery also face drawbacks due to the leaching or deswelling of these infused liquids which renders these antifouling surfaces ineffective. Pertinent to note here, our approach of introducing nano-bioactive glass by dip coating the SR substrates did not alter the surface wettability much as a modest increase in static water contact angle was seen with increasing nanoparticle concentration in comparison to SR control (Figure 13). Similarly, the water uptake percentage of SR control was found to be 0.5 ± 0.24 wt %, which was not significantly different for the modified substrates (nBG-50, nBG-Cu and nBG-Sr was found to be 0.49 ± 0.14 wt %, 0.53 ± 0.2 wt % and 0.51 ± 0.16 wt % respectively). NO-releasing materials have been very effective in curtailing microbial growth and colonization on NO donor- incorporated silicone-based.[15, 37]Here we tested the efficacy of physiologically relevant levels of NO generated from NOgen substrates when challenged against planktonic cultures of two commonly noted bacterial strains associated with implant infections: Gram-negative, Escherichia coli and Gram-positive Staphylococcus aureus (Figure 4A). The NOgen substrates were effective in reducing the viability of S. aureus by ~ 1.1 log reduction for nBG- Cu 50 (~ 92 % reduction), ~ 0.4 log reduction for nBG-Cu 25 (~70 % reduction); while ~ 1 log reduction for nBG-Sr 50 (~ 90 % reduction), and only ~ 0.2 log reduction for nBG-Sr 25 (~55 % reduction) in comparison to SR controls and nBG groups. In case of E. coli, ~ 1.4 log reduction for nBG-Cu 50 (~ 93.3 % reduction) and ~1.2 log reduction (~ 93 % reduction) for nBG-Sr 50 was observed. Since the NO levels generated here were in the physiological range, they are regarded to have a bactericidal effect while not imposing any adverse nitrosative orT|H Docket: 222105-2380 oxidative stress on mammalian cells.

[0038] Furthermore, we tested the hallmark anti-thrombotic effect of NO (for only 50 mg / mL groups, since it had better anti-bacterial efficacy) by checking the extent of activated platelets adhered on to SR control and NOgen substrates (Figure 4B). Both the NOgen surfaces exhibited a drastic reduction in adhered platelets: 81.5 ± 3.6 % reduction for nBG-Cu 50, and 77.5± 2.4 % reduction for nBG-Sr 50 group in comparison to SR control, showcasing the NOgen’s potency to inhibit activated platelet adhesion. The developed in situ NOgen interface (Figure 4C) which could generate NO from endogenous RSNOs, can be helpful in mitigating implant associated early onsite infection in medical devices and can also be effective in preventing implant-associated thrombosis for direct-blood contacting devices. The mechanism of action of NO to elicit antibacterial action relies on the nitrosation of amines and thiols of bacterial proteins, lipid peroxidation, and chemical damage to nucleic acids

[0017] ; while inhibition of platelet activation relies on activation of NO / sGC / cGMP signalling axis which downregulates the activity of a key cell-adhesion receptor (αIIbβ3integrin) present in platelets.[29-30]

[0155] Active strategies that rely on the release of an antimicrobial agent or anti-fibrotic agent or anti-thrombotic agent or even dual / multi- release systems hold promise. However, the bioactivity and undesired action of released cargo always poses a risk in regulatory clearance for clinical translation prospects. The 70S bioactive glass used here belongs to an amorphous resorbable glass network where the glass dissolution occurs at physiological conditions. We quantified the metal ions leached out from the SR control and modified NOgen substrates at different time points by incubating the films in phosphate buffered saline (pH 7.4) at 37 ºC (Figure 14A-C). The network formers (SiO2) in the form of Si ion concentration were found to be in the range of 80 to 150 μg / g of sample (cumulative release at day 14), which is in the cytocompatible range of 160 ppm

[0039] to regulate cellular fate monocyte-macrophage cell line, J774.2 (in vitro) and below the lethal dosage (LD50= 2000 mg / kg) for colloidal silica in rat models.

[0040] The Cu ion concentration leaching noted in nBG-Cu 50 at day-14 was ~ 66 μg / g of sample, which was also on par with the tolerance levels above which the initial hepatotoxic effects are noted in rats.

[0041] The Sr ion concentration in leachates from nBG-Sr at day-14 was ~ 40 μg / g of sample and is within the therapeutic dosage < 300 mg / kg administered to have beneficial effects in osteoporotic rodents.

[0042] Therefore, the current system was not only successfully able to generate and relatively sustain the NO generation at physiologically relevant levels for 21 days (Figure 15A-15B), and was also capable of releasing therapeutic ions (such as Cu or Sr) at levels which could have cell-instructive effects at the implant site. Calcium sensing receptors (CaSRs) and other G-protein coupled receptors (GPCR 6A) haveT|H Docket: 222105-2380 affinity for such bivalent cations, which upon activation by these cations mediate the downstream activity. These CaSR are constitutively expressed in fibroblasts and macrophages and they can impact their cellular fate.

[0043]

[0156] NOgen interface and the cell-instructive effect of strontium on fibroblasts

[0157] The foreign body response is a multi-staged process involving multiple cells (macrophages, neutrophils, and lymphocytes) in the implant vicinity (Figure 1). Macrophage trafficking and fibroblast activation are two crucial events that determine the extent of inflammation and fibrous encapsulation, thereby dictating the fate of the implanted biomaterial.

[0010] To prove the coatings used here to develop the NOgen interface are cytocompatible, we performed the cytocompatibility assessment using human fibroblasts following ISO standard (ISO10993-5: 2009) protocol by (i) exposing the leachates on fibroblasts and (ii) through contact method using transwell inserts which are followed for testing biomedical devices safety in vitro for implantable device (Figure 5A). All the tested groups (nBG, nBG-Cu and nBG-Sr; 25 and 50 mg / mL dipcoat variants) exhibited a percentage cellular viability index greater than the 70% tolerance threshold regarded as safe for use in implantable silicone-based medical devices.

[0044] Though no discernible difference between test groups and cell control was noted in contact method (Figure 5Aii), the leachates (Day-7 and Day-14) treated cells showed a decrease in viability index. The assay used relies on the reduction of MTT (3-[4,5- dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) by mitochondrial dehydrogenase enzyme to form purple formazan crystals which correlates to the viability. However, when cells are exposed to certain compounds or cargoes, the bioenergetics could be altered; the cells remain viable, but the mitochondrial activity is slightly reduced.

[0045]

[0158] We wanted to investigate this hypothesis further and hence used the leachates (day-14) to test its efficacy against activated fibroblasts. One of the pivotal events in implant- associated fibrosis is the recruitment and activation of fibroblasts in response to inflammatory triggers. This alongside with other signalling mechanisms differentiate into myofibroblasts, which secrete excessive collagenous matrix.

[0010] To activate the fibroblasts, we used a known stimulant, ascorbic acid (AA) that enters the cells via sodium-dependent vitamin C (SVCT 1 or 2) transporters and upregulates the collagen gene expression levels.

[0046] This is evident with the ~ 5-fold increase in collagen secretion noted in AA-treated fibroblasts compared to untreated control (Figure 5B). To these activated fibroblasts, the nBG-Cu 50 and nBG-Sr 50 leachate treatment had a considerable reduction in collagen secretion (~ 1.5-fold reduction for nBG-Cu and ~ 5-fold reduction in comparison to AA treated controls). We also assessed the migration potency of activated fibroblasts and the effect of leachates by an in vitro woundT|H Docket: 222105-2380 closure assay (Figure 5C). Overlapping with excessive extracellular matrix (ECM) production is the migration of protofibroblasts into these provisional ECM, which later get activated into myofibroblasts.[3]Hence, the recruitment of fibroblasts at the implant site becomes an important step in peri-implant fibrosis and if any active surface can retard this it could be beneficial. The nBG-Sr 50 leachate treated group significantly reduced the migration of these activated fibroblasts (~ 1.66-fold reduction in comparison to AA-treated cell control).

[0159] One of the key regulators of collagen gene expression is the speckled protein (Sp) family, where Sp 1 and Sp 3 transcription factors bind upstream to collagen-α1 gene and upregulate its expression.

[0047] The molecular mechanism for increased collagen expression in AA treated fibroblasts is due to the increased levels of malondialdehyde noticed in the cytosol which positively influences the expression of Sp1 and Sp3 proteins, thereby upregulating collagen expression.

[0046] Strategies that involve modifying biomedical surfaces, such as Ti surfaces with flavonoids (anti-inflammatory: quercetin or taxifolin)

[0048] drastically reduce the fibroblast activity. This is because, anti-inflammatory the inflammatory(particularly cyclooxygenase-2 COX-2 dependant) cascade which share a common nexus with Sp 1 and Sp 3 expression.

[0049] In our present investigation, 14-day leachates which had Sr concentration (~ 16 μM) therapeutic outcome downregulating collagen expression andretarding the fibroblast migration. Low doses of trace metal ions such as Mg, Se, and Sr have been found to downregulate COX-2 activity thereby having anti-inflammatory potential.

[0027] Moreover, strontium ranelate (Sr2+) has also been reported to downregulate the transforming growth factor TGF-β1 / NFκB (nuclear factor κ B) signalling axis

[0050] which is one of the predominant pathways targeted by multiple antifibrotic drug releasing platforms to combat capsular contracture[2a]and treating fibrosis.

[0051]

[0160] Effect of copper andfrom leachates of NOgen interface on macrophages

[0161] To test the anti-inflammatory effect of Sr, we treated these leachates on naïve human macrophages and assessed the polarization fate of macrophages (Figure 6). For this, as a reference population, we had inflammatory cytokine (interferon INF-γ) treated macrophages (now polarized into M1phenotype), and similarly anti-inflammatory cytokine (interleukin IL-4) treated macrophages (now polarised into M2phenotype) (Figure 16). These M1and M2populations had distinct morphological appearances: M0macrophages were more rounded, M1macrophages were filamentous and star-shaped, and M2had a more spread-out nature (Figure 16). The population was characterized quantitatively by flow-cytometry and qualitatively visualized using immunostaining by probing for cell surface markers: CD 68 (panT|H Docket: 222105-2380 macrophage marker), CCR7 (inflammatory marker present in M1) and CD 206 (anti- inflammatory marker M2macrophages) (Figure 17). As expected, INF-γ treated naïve macrophages predominantly expressed CCR7 while IL-4 treated naïve macrophages predominantly expressed CD 206; whereby we established that INF-γ inducted them into M1 phenotype (having low M2 / M1 index of ~ 0.01; < 1), and IL-4 inducted them into M2 phenotype (having high M2 / M1 index of ~ 5.1; > 1). Additionally, M0populations with basal CCR7 and CD206 expression levels were normalized to M2 / M1 index to 1 (from flowcytometric analysis).

[0162] When naïve macrophages were treated with leachates from SR control substrates and NOgen substrates, we noticed two distinct observations: (i) SR control, nBG 50, and nBG- Cu all favoured a pro-inflammatory bias, with M2 / M1 index: ~0.19, ~0.21 and ~0.41, respectively, while (ii) nBG-Sr favoured a regenerative or anti-inflammatory bias with M2 / M1 index of ~4.1 (Figure 6 A-B). Qualitative assessment from immunostaining also corroborated this observation with the nBG-Sr 50 leachate treated group having more M2macrophages expressing CD206, while other groups had M1macrophages expressing CCR7 (Figure 6C). This attests to the role of Sr as an active metal ion which when released at low therapeutic levels from NOgen interface can effectively modulate the local implant site. The immunomodulatory role of Sr has been established previously also in doped-bioceramic platforms used in dental or bone tissue engineering applications.

[0052] This is attributed to the role of Sr in decreasing the expression of COX-2

[0050] whereas Cu is known to increase the level of COX-2.[27a]

[0163] Immunomodulatory effect of NOgen interface on macrophages

[0164] The ancillary features of metal ion from leaching are now established, we wanted to investigate the effect of NO generated in situ from our NOgen interface on the fate of macrophages. The NO generation potency decreases over time (Figure 15A-15B) due to the leaching / dissolution of network modifiers (catalytic – Cu or Sr metal ions) and network formers from the amorphous bioactive glass network (Figure 14A-14C). Thus, the NO flux values also decreased over time which might affect the macrophages differently. Hence, we tested this hypothesis by performing a continuous study on the same NOgen films and their effect on polarizing at two different time points on day 3, for 72 h (exposure period of 72 h in situ generated NO) and on day 10 (exposure period of 72 h in situ generated NO) (Figure 7A).

[0165] Under in vitro conditions, S-nitrosothiols (such as GSNO) are unstable

[0033] and hence need to be supplemented frequently to maintain a steady supply of NO donors to generate nitric oxide. In generalized mathematical modelling, it took 300 – 400 min for a response to materialize in cells after ligand activation in receptor-mediated signal transduction,T|H Docket: 222105-2380 which holds true even for gasotransmitter-based signalling.

[0053] This was the reason that at every 5 h time point, the culture media was supplemented with endogenously relevant levels of 1 μM GSNO / 30 μM GSH for a 72 h exposure period to study the effect of NO generated on naïve macrophages. At day-3 (Figure 7B), all the groups favoured a pro-inflammatory response, with the nBG-Cu 50 and nBG-Sr (the catalytic groups) having M2 / M1 index of ~ 0.02 (<< 1) (potent inflammatory mediator) in comparison to SR control (M2 / M1 index: ~ 0.42, < 1) and nBG 50 (M2 / M1 index: ~ 0.21, < 1) (mild mediator). Interestingly, at day-10 (Figure 7C), SR control and GSNO control maintained the same trend as day-3 (mild pro-inflammatory mediator), while nBG 50 had an M2 / M1 index of ~ 0.88 (close to 1, neither pro nor anti- inflammatory bias). nBG-Cu 50 maintained its potent pro-inflammatory mediator M2 / M1 index at ~ 0.08 (<< 1), in contrast to nBG-Sr 50 which favoured a potent anti-inflammatory M2 / M1 index (~ 2.41 >> 1) bias.

[0166] Qualitative assessment from immunostaining also corroborated this observation with nBG-Sr 50 and nBG-Cu NOgen groups having predominantly CCR7 expressing M1macrophages at day-3. Consequently, a bias switch was noted for nBG-Sr 50 NOgen group at day-10 where CD206 expressing M2 macrophages were present more here in comparison to other groups (Figure 7D-E). NO as a gasotransmitter plays several crucial roles in maintaining vascular and immune homoeostasis. Such a potent pleiotropically active signalling molecule regulates its diverse biological activity by virtue of its concentration, and duration on its target cells and their sensitivity to it. The controversial role of this double-edged molecule which has been extensively seen to be associated with pathological conditions, also has been proven to possess a protective effect (against ischemia and thrombosis).

[0054] In our current investigation we noticed that when the NOgen groups (nBG-Cu 50, nBG-Sr 50, at day- 3) produce NO concentration > 2.5 nM (Figure 15A-15B), observed at day 1, 3; corresponding to a flux value of > 1.5 ^ 10-10mol cm-2min-1), this concentration was favouring a M1macrophage polarization bias; whereas when the NO concentration dropped below 0.8 nM (for nBG-Sr 50, at day 7 (Figure 15A-15B), corresponding to flux value < 0.4 ^ 10-10mol cm-2min-1), favoured a M2macrophage polarization bias, showcasing a dose-dependent immunomodulation. At lower concentration, NO is known to activate its classical NO / sGC / cGMP (cyclic guanosine monophosphate) signalling axis

[0030] , however at relatively higher concentrations NO (> 60 nM) it activates phosphoinositide 3-kinase (PI3K)

[0055] and mediates the phosphorylation of protein kinase B (Akt) leading to enhanced nuclear binding of transcription factor NF-κB to its inflammatory modulator sequences

[0054] . For the success of any biomaterial, it should never favour only M1or only M2macrophage bias; as prolongedT|H Docket: 222105-2380 inflammation could lead to extensive fibrosis, while rapid M2 activation could result in delayed onset of fibrosis owing to inadequate remodelling and immune cell engagement.

[0056] A delicate balance is needed which was observed in nBG-Sr NOgen interface which favours pro- inflammatory bias initially and then aids in M2phenotype activation.the tandem effects of metal ion release (say after 14 days) would further reduce the fibroblast activation, leading to attenuation of fibrotic response.

[0167] Proangiogenic effect of NOgen interface

[0168] Neo-vascularization is critical for long-term implantable devices such as defect fillers, stents and sensors. The formation of new capillary blood vessels helps in remodeling the tissue space at the implant site better and reduces the extent of fibrous capsule formation. Strategies involving the release of growth factors (such as vascular endothelial growth factor VEGF) and anti-inflammatory drugs (like dexamethasone) from antifouling substrates have been shown to be effective in rat models

[0057] . Hence, we evaluated the proangiogenic potential of our NOgen interface by performing a collagen gel tube formation assay (Figure 8A). The collagen gel with embedded endothelial cells mimics the tissue space (Figure 1) and NOgen substrates exposed to them helped us to quantify the extent of nodal points and tube length of the endothelial cell network generated in response to the NO generated and leachate treatment.

[0169] An increased number of nodes / sprouts and tubes were observed in the Cu and Sr incorporated NOgen groups (with no significance noted between nBG-Cu 50 and nBG-Sr 50). Similarly, longer endothelial tubes were formed in nBG-Cu 50 ad nBG-Sr 50 groups, indicating the NO generated from these interfaces facilitated the maturation of the endothelial cells, and the leachate treatment also did not affect the viability of the endothelial cells (Figure 8B). This is in correlation to other reports where Cu-based metal-organic framework (MOF) modified Ti surfaces (for vascular stents) aided in situ NO generation promoting rapid reendothelialization and suppressing smooth muscle cell activity

[0058] . The NOgen surfaces were thus holistically investigated for their cell-instructive properties on platelets, fibroblasts, macrophages, and endothelial cells. It is obvious that NO has pleiotropic roles and its effect on these cells had a favourable outcome which attests to the prospects of our nBG-Sr based NOgen interface in preventing implant-associated fibrosis. Moreover, the physiologically relevant levels of NO generated had a modest bactericidal effect but the catalytic nBG-Sr or nBG-Cu NOgen interface’s antibacterial ability can always be improved by incorporating NO donors (diazeniumdiolate or S-nitroso-N-acetylpenicillamine) such as by blending them in the polymer and having another conformal topcoat on SR substrates. However, the NO flux that would beT|H Docket: 222105-2380 generated needs to be validated to regulate cellular fate. Though the current work carried out a thorough in vitro investigation, in vivo performance of the nBG-Cu or nBG-Sr based NOgen interfaces also needs to be validated in suitable animal models for long-term resorption kinetics which would be the basis for our future studies. Furthermore, the molecular mechanisms involving several shared key mediators of inflammation, fibrosis, and angiogenesis need to be studied through multiomics-based analysis which would give us more insights into NO’s regulatory roles.

[0170] CONCLUSION

[0171] In summary, herein to attain an in situ NO generation to harness the pleiotropic effects of NO, a catalytic interface was developed using mesoporous doped-nanobioactive glass particles. The role of copper and strontium in acting as metal catalysts to decompose endogenous reservoirs of S-nitrosothiols was investigated. The facile modification of silicone rubber substrates by dip coating them with these nanoparticles helped generate physiologically relevant levels of NO. NOgen interfaces exhibited bactericidal and antithrombotic properties. In addition to NO, these NOgen interfaces also served as an active surface capable of releasing low therapeutic doses of copper or strontium ions which had other ancillary cell-instructive benefits. The strontium ion from leachates suppressed the collagen expression and migration of activated fibroblasts. The NO generated from NOgen interfaces regulated the macrophages bestowing an immunomodulatory effect. Altogether, the multifunctional NOgen interfaces developed here serve as a valuable strategy in combating implant-associated infections and attenuating implant-associated fibrosis noted in silicone- based implantable devices.

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Claims

T|H Docket: 222105-2380 CLAIMS 1. An article comprising at least one surface coated with silica particles doped with one or more monovalent cationic metals, divalent cationic metals, trivalent cationic metals, or any combination thereof.

2. An article comprising silica particles doped with one or more monovalent cationic metals, divalent cationic metals, trivalent cationic metals, or any combination thereof dispersed throughout the article.

3. The article of claim 1 or 2, wherein the silica comprises amorphous silica, a fumed silica, a nanocrystalline silica, ceramic silica, colloidal silica, a silica coating, a silica film, organically modified silica, mesoporous silica, silica gel, a bioactive glass.

4. The article of claim 1 or 2, wherein the silica comprises mesoporous silica.

5. The article of claim 1 or 2, wherein the monovalent cations comprise sodium, potassium, lithium, or any combination thereof.

6. The article of claim 1 or 2, wherein the divalent cations comprise Cu, Sr, Zn, Ca, Mg, Ni, Co, Mn, Cr, Pd, Pt, or any combination thereof.

7. The article of claim 1 or 2, wherein the trivalent cations comprise Fe, Au, V, and any combination thereof.

8. The article of claim 1 or 2, wherein the silica particles comprise CaO, Na2O, P2O5, and any combination thereof.

9. The article of claim 1 or 2, wherein the silica particles comprise CaO and P2O5.

10. The article of claim 1 or 2, wherein the silica particles comprise CaO, P2O5, and SrO or CuO.

11. The article of claim 1 or 2, wherein the silica particles comprise (SiO2)-(CaO)-(P2O5)-X, wherein X is a monovalent cationic metal, divalent cationic metal, or a combination thereof, SiO2is from about 40 molar percent to about 80 molar percent, CaO is from about 10 molar percent to about 30 molar percent, P2O5is from about 0.1 molar percent to about 10 molar percent, and X is greater than 0 molar percent to about 5 molar percent, wherein the sum is 100 molar percent.

12. The article of claim 11, wherein X is SrO or CuO.

13. The article of claim 1 or 2, wherein the silica particles comprise CaO, Na2O, and P2O5.

14. The article of claim 1 or 2, wherein the silica particles comprise CaO, Na2O, P2O5, and SrO or CuO 15. The article of claim 1 or 2, wherein the silica particles comprise (SiO2)-(CaO)-(Na2O)-(P2O5)-X, wherein X is a monovalent cationic metal, divalent cationicT|H Docket: 222105-2380 metal, or any combination thereof, SiO2is from about 40 molar percent to about 80, CaO is from about 10 molar percent to about 30 molar percent, P2O5is from about 0.1 molar percent to about 10 molar percent, X is greater than 0 molar percent to about 5 molar percent, and Na2O is from about 1 molar percent to about 30 molar percent, wherein the sum is 100 molar percent.

16. The article of claim 15, wherein X is SrO or CuO.

17. The article of claim 1 or 2, wherein the silica particles do not include a nitric oxide releasing compound.

18. The article of claim 1 or 2, wherein the coating of silica particles further comprises a room temperature vulcanizing silicone.

19. The article of claim 1 or 2, wherein the article comprises a polymeric grade material, a medical device, a surface or article in a hospital or medical facility, a wound dressing.

20. The article of claim 1, wherein the surface comprises a polysiloxane.

21. The article of claim 1 or 2, wherein the article comprises an implantable medical device.

22. The article of claim 21, wherein the implantable medical device comprises a urinary catheter, artificial heart valve, a vascular catheter, a graft, or a stent.

23. The article of claim 1 or 2, wherein the article generates nitric oxide when introduced into a subject.

24. The article of claim 1 or 2, wherein the article reduces or prevents the growth of microbes on the article when introduced into a subject.

25. The article of claim 1 or 2, wherein the article reduces or prevents the adhesion of platelets on the article when introduced into a subject.

26. The article of claim 1 or 2, wherein the article reduces or prevents inflammation when introduced into a subject.

27. A method of generating nitric oxide in situ in a subject, the method comprising introducing the article of claim 1 or 2 into the subject.

28. An article comprising at least one surface coated with strontium ions.

29. An article comprising strontium ions dispersed throughout the article.

30. The article of claim 28 or 29, wherein the strontium ions are derived from strontium oxide or a pharmaceutically acceptable salt of strontium.

31. The article of claim 28 or 29, wherein the strontium ions are incorporated into a polymer matrix, a gel, a glass, or a ceramic material.

32. The article of claim 28 or 29, wherein the article comprises a polymeric grade material, a medical device, a surface or article in a hospital or medical facility, a wound dressing.T|H Docket: 222105-2380 33. The article of claim 28, wherein the surface comprises a polysiloxane.

34. The article of of claim 28 or 29, wherein the article comprises an implantable medical device.

35. The article of claim 34, wherein the implantable medical device comprises a urinary catheter, artificial heart valve, a vascular catheter, a graft, or a stent.

36. The article of claim 28 or 29, wherein the article generates nitric oxide when introduced into a subject.

37. The article of claim 28 or 29, wherein the article reduces or prevents the growth of microbes on the article when introduced into a subject.

38. The article of of claim 28 or 29, wherein the article reduces or prevents the adhesion of platelets on the article when introduced into a subject.

39. The article of claim 28 or 29, wherein the article reduces or prevents inflammation when introduced into a subject.

40. A method of generating nitric oxide in situ in a subject, the method comprising introducing the article of claim 28 or 29 into the subject.

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