Kinetics of <i>S</i>-Nitrosation Processes in Aqueous Polymer Solution for Controlled Nitric Oxide Loading: Toward Tunable Biomaterials

An understanding of the nitrosation processes that dictate <i>S</i>-nitrosothiol formation in the presence of a polymer is crucial toward the controlled synthesis of nitric oxide (NO)-releasing materials, an important class of biomaterials that mimic the natural function of cells. Herein, the kinetics of <i>S</i>-nitrosoglutathione (GSNO) formation in the presence of dextran under a variety of nitrosation conditions, including the nitrosating agent and the dextran concentration, are reported. When comparing nitrous acid and <i>t</i>-butyl nitrite as the nitrosating agent, the use of nitrous acid results in 100% nitrosation of the thiol sites within less than a minute and <i>t</i>-butyl nitrite requires more than 5 min to reach completion. This trend establishes nitrous acid as a highly efficient nitrosating agent. In the presence of increasing dextran concentration from 0 w/v% to 10 w/v%, the extent of nitrosation decreases by approximately 5% and 30% using nitrous acid and <i>t</i>-butyl nitrite, respectively. With sufficient reaction time, either reagent leads to 100% nitrosation. This indicates that <i>t</i>-butyl nitrite is the preferred reagent for fine-tuned NO loading of thiol sites as the extent of reaction is greatly impacted by the polymer concentration. Taken together, these studies provide valuable insights regarding the ability to tailor NO storage within biomaterials for use in a wide range of clinical applications

Metal Organic Frameworks as Nitric Oxide Catalysts

The use of metal organic frameworks (MOFs) for the catalytic production of nitric oxide (NO) is reported. In this account we demonstrate the use of Cu₃(BTC)₂ as a catalyst for the generation of NO from the biologically occurring substrate, S-nitrosocysteine (CysNO). The MOF catalyst was evaluated as an NO generator by monitoring the evolution of NO in real time via chemiluminescence. The addition of 2, 10, and 15-fold excess CysNO to MOF-Cu^II sites and cysteine (CysH) resulted in catalytic turnover of the active sites and nearly 100% theoretical yield of the NO product. Control experiments without the MOF present did not yield appreciable NO generation. In separate studies the MOF was found to be reusable over successive iterations of CysNO additions without loss of activity. Subsequently, the MOF catalyst was confirmed to remain structurally intact by pXRD and ATR-IR following reaction with CysNO and CysH

Accurate Nitric Oxide Measurements from Donors in Cell Media: Identification of Scavenging Agents

Nitric oxide (NO) is an essential messenger in human physiology, mediating cellular processes ranging from proliferation to apoptosis. The effects of NO are concentration dependent, and control over the instantaneous amount of NO available to cells is essential for determining the therapeutic NO dosages for various applications. As such, the development of NO therapeutic materials relies on accurate quantitative NO measurements that provide both total NO release from the NO donor as well as instantaneous NO concentrations. On the basis of the complexity of the cell media environment, inaccurate NO reporting often occurs for <i>in vitro</i> studies. These inaccuracies result from using inert media such as phosphate buffer saline (PBS), failing to account for the reactivity of media components. In this work, we describe a method for directly quantifying the instantaneous and total amounts of NO from commonly used NO donors in commercially available cell media routinely used for endothelial and neural cell lines. A riboflavin–tryptophan complex found in the media was identified as the major scavenger of NO in the cell media and likely reacts with NO via a radical–radical reaction. This finding significantly impacts the amount of available NO. The scavenging effects are concentration dependent on the riboflavin–tryptophan complex and the NO release rate from the NO donor. The results of this study provide insights on the exogenous amounts of NO that are present in cell media and may provide an explanation for differences in NO dosages between buffer experiments and <i>in vitro</i> and <i>in vivo</i> studies

Functionalization of Metal–Organic Frameworks To Achieve Controllable Wettability

The overall versatility of a material can be immensely expanded by the ability to controllably tune its hydrophobicity. Herein we took advantage of steric bias to demonstrate that tricarboxylate metal–organic frameworks (MOFs) can undergo covalent postsynthetic modification to confer various degrees of hydrophobicity. MOF copper 2-aminobenzene-1,3,5-tricarboxylate was modified with varying-length aliphatic carbon chains. Unmodified Cu₃(NH₂BTC)₂ degrades in minutes upon contact with water, whereas modification as low as 14% results in powders that show significantly enhanced hydrophobic character with contact angles up to 147°. The modified material is capable of withstanding direct contact with water for 30 min with no visual evidence of altered surface characteristics. A linear relationship was observed between the length of the tethered chain and the water contact angle. These results reveal a predictable method for achieving a range of desirable sorption rates and highly controllable hydrophobic character. This work thereby expands the possibilities of rationally modifying MOFs for a plethora of target-specific applications

Nitric Oxide Releasing Tygon Materials: Studies in Donor Leaching and Localized Nitric Oxide Release at a Polymer-Buffer Interface

Tygon is a proprietary plasticized poly­(vinyl chloride) polymer that is used widely in bioapplications, specifically as extracorporeal circuits. To overcome issues with blood clot formation and infection associated with the failure of these medical devices upon blood contact, we consider a Tygon coating with the ability to release the natural anticlotting and antibiotic agent, nitric oxide (NO), under simulated physiological conditions. These coatings are prepared by incorporating 20 w/w% <i>S</i>-nitrosoglutathione (GSNO) donor into a Tygon matrix. These films release NO on the order of 0.64 ± 0.5 × 10^–10 mol NO cm^–2 min^–1, which mimics the lower end of natural endothelium NO flux. We use a combination of assays to quantify the amount of GSNO that is found intact at different time points throughout the film soak, as well as monitor the total thiol content in the soaking solution due to any analyte that has leached from the polymer film. We find that a burst of GSNO is released from the material surface within 5 min to 1 h of soaking, which only represents 0.25% of the total GSNO contained in the film. After 1 h of film soak, no additional GSNO is detected in the soaking solution. By further considering the total thiol content in solution relative to the intact GSNO, we demonstrate that the amount of GSNO leached from the material into the buffer soaking solution does not contribute significantly to the total NO released from the GSNO-incorporated Tygon film (<10% total NO). Further surface analysis using SEM-EDS traces the elemental S on the material surface, demonstrating that within 5 min −1 h soaking time, 90% of the surface S is removed from the material. Surface wettability and roughness measurements indicate no changes between the GSNO-incorporated films pre- to postsoak that will be significant toward the adsorption of biological components, such as proteins, relative to the presoaked donor-incorporated film. Overall, we demonstrate that, for a 20 w/w% GSNO-incorporated Tygon film, relatively minimal GSNO leaching is experienced, and the lost GSNO is from the material surface. Varying the donor concentration from 5 to 30 w/w% GSNO within the film does not result in significantly different NO release profiles. Additionally, the steady NO flux associated with the system is predominantly due to localized release from the material, and not donor lost to soaking solution. The surface properties of these materials generally imply that they are useful for blood-contacting applications

Immobilization of Metal–Organic Framework Copper(II) Benzene-1,3,5-tricarboxylate (CuBTC) onto Cotton Fabric as a Nitric Oxide Release Catalyst

Immobilization of metal–organic frameworks (MOFs) onto flexible polymeric substrates as secondary supports expands the versatility of MOFs for surface coatings for the development of functional materials. In this work, we demonstrate the deposition of copper­(II) benzene-1,3,5-tricarboxylate (CuBTC) crystals directly onto the surface of carboxyl-functionalized cotton capable of generating the therapeutic bioagent nitric oxide (NO) from endogenous sources. Characterization of the CuBTC-cotton material by XRD, ATR-IR, and UV–vis indicate that CuBTC is successfully immobilized on the cotton fabric. In addition, SEM imaging reveals excellent surface coverage with well-defined CuBTC crystals. Subsequently, the CuBTC-cotton material was evaluated as a supported heterogeneous catalyst for the generation of NO using <i>S</i>-nitrosocysteamine as the substrate. The resulting reactivity is consistent with the activity observed for unsupported CuBTC particles. Overall, this work demonstrates deposition of MOFs onto a flexible polymeric material with excellent coverage as well as catalytic NO release from <i>S</i>-nitrosocysteamine at therapeutic levels

Surface-Anchored Metal–Organic Framework–Cotton Material for Tunable Antibacterial Copper Delivery

In the present study, a new copper metal-organic framework (MOF)–cotton material was strategically fabricated to exploit its antibacterial properties for postsynthetic modification (PSM) to introduce a free amine to tune the physicochemical properties of the material. A modified methodology for carboxymethylation of natural cotton was utilized to enhance the number of nucleation sites for the MOF growth. Subsequently, MOF Cu₃(NH₂BTC)₂ was synthesized into a homogenous surface-supported film via a layer-by-layer dip-coating process. The resultant materials contained uniformly distributed 1 μm × 1 μm octahedral MOF crystals around each carboxymethylated fiber. Importantly, the accessible free amine of the MOF ligand allowed for the PSM of the MOF–cotton surface with valeric anhydride, yielding 23.5 ± 2.2% modified. The Cu²⁺ ion-releasing performance of the materials was probed under biological conditions per submersion in complex media at 37 °C. Indeed, PSM induces a change in the copper flux of the material over the first 6 h. The materials continue to slowly release Cu²⁺ ions beyond 24 h tested at a flux of 0.22 ± 0.003 μmol·cm^–2·h^–1 with the unmodified MOF–cotton and at 0.25 ± 0.004 μmol·cm^–2·h^–1 with the modified MOF–cotton. The antibacterial activity of the material was explored using Escherichia coli by testing the planktonic and attached bacteria under a variety of conditions. MOF–cotton materials elicit antibacterial effects, yielding a 4-log reduction or greater, after 24 h of exposure. Additionally, the MOF–cotton materials inhibit the attachment of bacteria, under both dry and wet conditions. A material of this type would be ideal for clothing, bandages, and other textile applications. As such, this work serves as a precedence toward developing uniform, tunable MOF–composite textile materials that can kill bacteria and prevent the attachment of bacteria to the surface

Sustained Nitric Oxide Release from a Tertiary <i>S</i>‑Nitrosothiol-based Polyphosphazene Coating

Nitric oxide (NO) occurs naturally in mammalian biochemistry as a critical signaling molecule and exhibits antithrombotic, antibacterial, and wound-healing properties. NO-forming biodegradable polymers have been utilized in the development of antithrombotic or antibacterial materials for biointerfacial applications, including tissue engineering and the fabrication of erodible coatings for medical devices such as stents. Use of such NO-forming polymers has frequently been constrained by short-term release or limited NO storage capacity and has led to the pursuit of new materials with improved NO release function. Herein, we report the development of an NO-releasing bioerodible coating prepared from poly­[bis­(3-mercapto-3-methylbut-1-yl glycinyl)­phosphazene] (POP-Gly-MMB), a polyphosphazene based on glycine and the naturally occurring tertiary thiol 3-mercapto-3-methylbutan-1-ol (MMB). To evaluate the NO release properties of this material, the thiolated polymer POP-Gly-MMB-SH was applied as a coating to glass substrates and subsequently converted to the NO-forming S-nitrosothiol (RSNO) derivative (POP-Gly-MMB-NO) by immersion in a mixture of <i>tert</i>-butyl nitrite (<i>t</i>-BuONO) and pentane. NO release flux from the coated substrates was determined by chemiluminescence-based NO measurement and was found to remain in a physiologically relevant range for up to 2 weeks (6.5–0.090 nmol of NO·min^–1·cm^–2) when immersed in pH 7.4 phosphate-buffered saline (PBS) at 37 °C. Furthermore, the coating exhibited an overall NO storage capacity of 0.89 ± 0.09 mmol·g^–1 (4.3 ± 0.6 μmol·cm^–2). Erosion of POP-Gly-MMB-NO in PBS at 37 °C over 6 weeks results in 14% mass loss, and time-of-flight mass spectrometry (TOF-MS) was used to characterize the organic products of hydrolytic degradation as glycine, MMB, and several related esters. The comparatively long-term NO release and high storage capacity of POP-Gly-MMB-NO coatings suggest potential as a source of therapeutic NO for biomedical applications

Nitric Oxide Generation from Endogenous Substrates Using Metal–Organic Frameworks: Inclusion within Poly(vinyl alcohol) Membranes To Investigate Reactivity and Therapeutic Potential

Cu-BTTri (H₃BTTri = 1,3,5-tris­[1<i>H</i>-1,2,3-triazol-5-yl]­benzene) is a water-stable, copper-based metal–organic framework (MOF) that exhibits the ability to generate therapeutic nitric oxide (NO) from <i>S</i>-nitrosothiols (RSNOs) available within the bloodstream. Immobilization of Cu-BTTri within a polymeric membrane may allow for localized NO generation at the blood–material interface. This work demonstrates that Cu-BTTri can be incorporated within hydrophilic membranes prepared from poly­(vinyl alcohol) (PVA), a polymer that has been examined for numerous biomedical applications. Following immobilization, the ability of the MOF to produce NO from the endogenous RSNO <i>S</i>-nitrosoglutathione (GSNO) is not significantly inhibited. Poly­(vinyl alcohol) membranes containing dispersions of Cu-BTTri were tested for their ability to promote NO release from a 10 μM initial GSNO concentration at pH 7.4 and 37 °C, and NO production was observed at levels associated with antithrombotic therapeutic effects without significant copper leaching (<1%). Over 3.5 ± 0.4 h, 10 wt % Cu-BTTri/PVA membranes converted 97 ± 6% of GSNO into NO, with a maximum NO flux of 0.20 ± 0.02 nmol·cm^–2·min^–1. Furthermore, it was observed for the first time that Cu-BTTri is capable of inducing NO production from GSNO under aerobic conditions. At pH 6.0, the NO-forming reaction of 10 wt % Cu-BTTri/PVA membrane was accelerated by 22%, while an opposite effect was observed in the case of aqueous copper­(II) chloride. Reduced temperature (20 °C) and the presence of the thiol-blocking reagent <i>N</i>-ethylmaleimide (NEM) impair the NO-forming reaction of Cu-BTTri/PVA with GSNO, with both conditions resulting in a decreased NO yield of 16 ± 1% over 3.5 h. Collectively, these findings suggest that Cu-BTTri/PVA membranes may have therapeutic utility through their ability to generate NO from endogenous substrates. Moreover, this work provides a more comprehensive analysis of the parameters that influence Cu-BTTri efficacy, permitting optimization for potential medical applications

Metal–Organic Framework/Chitosan Hybrid Materials Promote Nitric Oxide Release from <i>S</i>‑Nitrosoglutathione in Aqueous Solution

It has been previously demonstrated that copper-based metal–organic frameworks (MOFs) accelerate formation of the therapeutically active molecule nitric oxide (NO) from <i>S</i>-nitrosothiols (RSNOs). Because RSNOs are naturally present in blood, this function is hypothesized to permit the controlled production of NO through use of MOF-based blood-contacting materials. The practical implementation of MOFs in this application typically requires incorporation within a polymer support, yet this immobilization has been shown to impair the ability of the MOF to interact with the NO-forming RSNO substrate. Here, the water-stable, copper-based MOF H₃[(Cu₄Cl)₃-(BTTri)₈] (H₃BTTri = 1,3,5-tris­(1<i>H</i>-1,2,3-triazol-5-yl)­benzene), or Cu-BTTri, was incorporated within the naturally derived polysaccharide chitosan to form membranes that were evaluated for their ability to enhance NO generation from the RSNO <i>S</i>-nitrosoglutathione (GSNO). This is the first report to evaluate MOF-induced NO release from GSNO, the most abundant small-molecule RSNO. At a 20 μM initial GSNO concentration (pH 7.4 phosphate buffered saline, 37 °C), chitosan/Cu-BTTri membranes induced the release of 97 ± 3% of theoretical NO within approximately 4 h, corresponding to a 65-fold increase over the baseline thermal decomposition of GSNO. Furthermore, incorporation of Cu-BTTri within hydrophilic chitosan did not impair the activity of the MOF, unlike earlier efforts using hydrophobic polyurethane or poly­(vinyl chloride). The reuse of the membranes continued to enhance NO production from GSNO in subsequent experiments, suggesting the potential for continued use. Additionally, the major organic product of Cu-BTTri-promoted GSNO decomposition was identified as oxidized glutathione via mass spectrometry, confirming prior hypotheses. Structural analysis by pXRD and assessment of copper leaching by ICP-AES indicated that Cu-BTTri retains crystallinity and exhibits no significant degradation following exposure to GSNO. Taken together, these findings provide insight into the function and utility of polymer/Cu-BTTri systems and may support the development of future MOF-based biomaterials