Extracorporeal life support (ECLS) components designed to reduce clot formation have been developed, but thrombosis remains a significant challenge, requiring systemic anticoagulation. To address this, we evaluated a nitric oxide (NO) releasing extracorporeal circuit (ECC) in a 5 day ovine venovenous ECLS model without systemic anticoagulation. Fifteen sheep, weighing 40-52 kg, were instrumented and assigned to three groups (n = 5 each): 1) Control- "naïve" ECC without anticoagulation; 2) Sham-CarboSil-coated ECC; and 3) NOSA-NO-releasing ECC+100 ppm NO in the sweep gas. Animals were monitored until meeting two of the three end-point criteria defined by device resistance five times baseline, greater than 50% decrease in blood flow after RPM adjustments, or post-oxygenator SO2 less than 95%. Extracorporeal Life Support flow was adjusted from 1 L/min (0-24 h), 0.75 L/min (25-48 h) to 0.5 L/min (49 h to study end). Data collected included hemodynamics, ECC performance, coagulation markers, cellular activity, and NO toxicity. The NO-releasing ECC prolonged survival to ~120 h with lower resistance and plasma-free hemoglobin, stable coagulation, and final MetHb levels less than 5%. Activated clotting times (ACTs) were less than 200 s in all groups. These findings suggest that NO-releasing ECCs in a highly translational preclinical ovine model may improve ECLS safety while reducing reliance on systemic anticoagulation.

ABSTRACT Catheter-associated urinary tract infections (CAUTIs) are the most prevalent nosocomial infections. These infections impose a significant financial burden on the healthcare system and are responsible for approximately 15,000 deaths each year in the US. CAUTIs are caused by free-floating bacteria that adhere to the surface of Foley catheters and the patient’s uroepithelium, forming protective biofilms that enhance bacterial survival and resistance. In this study, a nitric oxide (NO)-releasing liquid formulation is used to inflate the Foley catheter balloon, aiming to inhibit biofilm formation and reduce the risk of CAUTIs. The solution of S-nitrosoglutathione (GSNO), an endogenous NO donor molecule, was used as the catheter inflation fluid. To assess the NO-release profile, the GSNO was dissolved in various pH-adjusted (pH 6.5) aqueous media (deionized water, phosphate-buffered saline, and 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid [HEPES] buffers). The GSNO/HEPES formulation sustained NO fluxes through the inflated balloon wall above 0.5 × 10⁻¹⁰ mol·min⁻¹·cm⁻² for up to 10 days, as well as significantly reduced the planktonic bacteria counts and surface colonization against Escherichia coli . The safety of the optimized GSNO formulation was evaluated in vivo in a 7-day preclinical porcine model. The formulation showed no signs of NO-related toxicity. Overall, this study presents a promising, translatable approach to substantially reducing the risk of CAUTIs in healthcare settings. IMPORTANCE Catheter-associated urinary tract infections (CAUTIs) are the most frequent hospital-acquired infections and pose serious risks for patients with Foley catheters in hospitals, nursing homes, and other care facilities. These infections lead to significant mortality and healthcare expenses and are especially difficult to prevent due to bacterial biofilms on catheter surfaces. Our study introduces a novel solution, a nitric oxide (NO)-releasing balloon inflation fluid using S-nitrosoglutathione (GSNO), that continuously releases antimicrobial NO for up to 10 days. This approach significantly reduces bacterial counts and surface colonization by Escherichia coli , a main cause of CAUTIs, while demonstrating safety in preclinical animal testing. Our findings suggest that this strategy could reduce infection risk and improve outcomes for catheterized patients across diverse healthcare settings.

S-Nitrosoglutathione (GSNO) is an endogenous nitric oxide (NO) donor molecule. Photolysis of solid GSNO may be utilized effectively as an NO source for inhaled nitric oxide (iNO) therapy. In this work, solid GSNO was photolyzed using narrow-band LED lights as a function of wavelength and intensity of incident light. The photolytic NO release efficiency, the presence of any decomposition products, as well as the morphology and the photolytic pathways for GSNO photo-decomposition were examined using NMR, UV, FTIR, ESR, TGA, DSC, SEM and UPLC-MS. The initial photochemical yield for NO release was 0.37 ± 0.03%, 0.10 ± 0.01%, and 0.02 ± 0.01% (n = 3 independent experiments) respectively using 340 nm, 385 nm, and 470 nm of LED light sources. The photolyzed solid GSNO reacted with oxygen in ambient air and immediately produced an oxygenated glutathione-derived species with m/z ratio (∼354) suggesting the possible formation of sulfonyl peroxyl radical GS(O)2O• without a measurable stable thiyl radical formation. However, the photolyzed product did not recombine with NO to reform the GSNO, strongly suggesting that the photolysis of GSNO is irreversible under our conditions. Based on PXRD and SEM analysis, after photolysis and NO generation the GSNO lost its crystallinity. Hence, these data may prove useful for development of GSNO as a solid-phase NO source for iNO therapy.

In order to overcome the high costs and logistical issues associated with nitric oxide (NO) storage tanks, a portable tankless prototype device has been developed for precise controlled release of inhaled nitric oxide (iNO) gas from a solid NO donor, S-nitroso-N-acetyl penicillamine (SNAP) stored within "coffee pod" style replaceable cartridges. The device utilizes LED light to trigger the release of NO from SNAP, and the concentration of the delivered NO gas is continuously monitored with amperometric sensors and regulated by a feedback control system via modulating the light intensity. The system enables facile adjustment of NO concentrations, allowing for the reliable delivery of low-dose NO (up to 10 ppm, as demonstrated here) across clinically relevant air flow rates, with the prototype specifically designed for air flow up to 4 L/min. Importantly, NO generation is achieved without the need for nitrogen (N₂) as a carrier gas, enabling safe and simple operation using ambient air even in home settings. To ensure the safety of this device, the key factors affecting the formation of the major undesired impurity, nitrogen dioxide (NO2), during NO generation were carefully considered and minimized to levels suitable for NO inhalation therapy (<1 ppm). The gas phase NO generated by this device significantly reduced the biofilm density as measured by crystal violet staining and the viable bacterial counts in the mucoid P. aeruginosa biofilm established on a plastic surface in vitro and in sputum isolated from cystic fibrosis (CF) patients ex vivo. With the capacity to maintain 10 ppm NO at up to 4 L/min air flow for over five hours, this portable system demonstrates promise for safe, low-cost, in-home iNO delivery as an adjunctive therapy to reduce biofilm-associated bacterial burden in the lungs of cystic fibrosis patients.

This study presents the utilization of a novel, highly lipophilic nitric oxide (NO) donor molecule, S-nitroso-1-adamantanethiol (SNAT), for developing an NO-emitting polymer surface aimed at preventing thrombus formation and bacterial infection in extracorporeal circuits (ECCs). S-nitroso-1-adamantanethiol, a tertiary nitrosothiol-bearing adamantane species, was synthesized, characterized, and used to impregnate polyvinyl chloride (PVC) tubing for subsequent in vivo evaluation. The impregnation process with SNAT preserved the original mechanical strength of the PVC. In vitro assessments revealed sustained NO release from the SNAT-impregnated PVC tubing (iSNAT), surpassing or matching endothelial NO release levels for up to 42 days. The initial NO release remained stable even after 1 year of storage at −20°C. The compatibility of iSNAT with various sterilization techniques (OPA Plus, hydrogen peroxide, EtO) was tested. Acute in vivo experiments in a rabbit model demonstrated significantly reduced thrombus formation in iSNAT ECCs compared with controls, indicating the feasibility of iSNAT to mitigate coagulation system activation and potentially eliminate the need for systemic anticoagulation. Moreover, iSNAT showed substantial inhibition of microbial biofilm formation, highlighting its dual functionality. These findings underscore the promising utility of iSNAT for long-term ECC applications, offering a multifaceted approach to enhancing biocompatibility and minimizing complications.

Background: Children with end-stage lung disease are commonly managed with extracorporeal life support (ECLS) as a bridge to lung transplantation. A pumpless artificial lung (MLung) is a portable alternative to ECLS and it allows for ambulation. Both ECLS and pumpless artificial lungs require systemic anticoagulation which is associated with hemorrhagic complications. We tested the MLung with a novel Nitric Oxide (NO) Surface Anticoagulation (NOSA) system, to provide local anticoagulation for 72 h of support in a pediatric-size ovine model. Methods: Four mini sheep underwent thoracotomy and cannulation of the pulmonary artery (inflow) and left atrium (outflow), recovered and were monitored for 72hr. The circuit tubing and connectors were coated with the combination of an NO donor (diazeniumdiolated dibutylhexanediamine; DBHDN2O2) and argatroban. The animals were connected to the MLung and 100 ppm of NO was added to the sweep gas. Systemic hemodynamics, blood chemistry, blood gases, and methemoglobin were collected. Results: Mean device flow was 836 +/- 121 mL/min. Device outlet saturation was 97 +/- 4%. Pressure drop across the lung was 3.5 +/- 1.5 mmHg and resistance was 4.3 +/- 1.7 mmHg/L/min. Activated clotting time averaged 170 +/- 45s. Methemoglobin was 2.9 +/- 0.8%. Platelets declined from 590 +/- 101 at baseline to 160 +/- 90 at 72 h. NO flux (x10-10 mol/min/cm2) of the NOSA circuit averaged 2.8 +/- 0.6 (before study) and 1.9 +/- 0.1 (72 h) and across the MLung 18 +/- 3 NO flux was delivered. Conclusion: The MLung is a more portable form of ECLS that demonstrates effective gas exchange for 72 h without hemodynamic changes. Additionally, the NOSA system successfully maintained local anticoagulation without evidence of systemic effects. (c) 2023 Elsevier Inc. All rights reserved.

Introduction: Data has shown that coating or full impregnation of extracorporeal circuits (ECC) with nitric oxide (NO) donors can provide efficient local anticoagulation by mimicking the natural anticoagulation mechanism of the healthy endothelium. However, these modifications can negatively affect the mechanical properties of the ECC components. This study aimed to develop and optimize a novel NO-releasing surface modification technique, that modifies only the inner surface of the ECC tubing via solvent swelling (“semi-impregnation”) with a strongly lipophilic NO donor, S-nitroso-adamantanethiol (SNAT), providing effective local anticoagulation by continuous NO release, meanwhile preserving the original mechanical properties of the ECC. Methods: The ECCs were semi-impregnated with 1g/mL SNAT dissolved in two different ratios of solvents [acetone(Ac):plasticizer(P):methanol(MeOH)] (2:1:2 and 1:3:1). These ECCs were then tested and compared to naïve ECC in vitro for NO donor loading, NO release profile (ozone chemiluminescence), and tensile strength test (texture analyzer) to find the optimal semi-impregnation conditions. The antibacterial properties of the new ECCs were assessed for 7 days using Gram+ and Gram- bacteria strains. The anticoagulation properties of the optimized, semi-swelled SNAT circuit (n=3) were tested in vivo, in an acute rabbit model and compared to naïve, control ECC (n=9). Results: By the “semi-impregnation” of the ECC, 50% less NO donor was consumed compared to full impregnation, making the fabrication process more cost-efficient. The 2:1:2 solvent combination yielded significantly higher loading vs. the 1:3:1 group (p≤0.05, Table 1). The tensile strength of the 1:3:1 group was not significantly different from the naïve control (p≥0.05, Table 1) and maintained the original properties better vs. 2:1:2 group (Table 1). Although, the 2:1:2 group had overall higher NO-releasing capacity when comparing values on day 21 of NO release, the 1:3:1 group still showed sufficient NO flux (Table 1). The biofilm study also showed adequate antibacterial properties in both groups with ca.2 log reduction in bacterial colonies (Table 1). Finally, when the optimized SNAT circuit was tested and compared to control ECC in the 4 hr long rabbit model during the evaluation of anticoagulation properties less thrombus area: SNAT 0.5±0.4cm2 vs. control 9.8±0.8 cm2; and higher preservation of platelets 108 % vs. 74 % of baseline, respectively. Conclusion: These data suggest that the optimized SNAT-based surface anticoagulation with “semi-impregnation” provides a more cost-efficient way of surface modification with adequate NO flux and with preserving the original mechanical characteristics of the ECC. It also has excellent antithrombogenic properties without the need of systemic anticoagulation in vivo. Future studies include prolonged preclinical studies of the SNAT ECC.

Patients with chronic rhinosinusitis (CRS) often show persistent colonization by bacteria in the form of biofilms which are resistant to antibiotic treatment. One of the most commonly isolated bacteria in CRS is Staphylococcus aureus (S. aureus). Nitric oxide (NO) is a potent antimicrobial agent and disperses biofilms efficiently. We hypothesized that S-nitrosoglutathione (GSNO), an endogenous NO carrier/donor, synergizes with gentamicin to disperse and reduce the bacterial biofilm density. We prepared GSNO formulations which are stable up to 12 months at room temperature and show the maximum amount of NO release within 1 h. We examined the effects of this GSNO formulation on the S. aureus biofilm established on the apical surface of the mucociliary-differentiated airway epithelial cell cultures regenerated from airway basal (stem) cells from cystic fibrosis (CF) and CRS patients. We demonstrate that for CF cells, which are defective in producing NO, treatment with GSNO at 100 μM increased the NO levels on the apical surface and reduced the biofilm bacterial density by 2 log units without stimulating pro-inflammatory effects or inducing epithelial cell death. In combination with gentamicin, GSNO further enhanced the killing of biofilm bacteria. Compared to placebo, GSNO significantly increased the ciliary beat frequency (CBF) in both infected and uninfected CF cell cultures. The combination of GSNO and gentamicin also reduced the bacterial density of biofilms grown on sinonasal epithelial cells from CRS patients and improved the CBF. These findings demonstrate that GSNO in combination with gentamicin may effectively reduce the density of biofilm bacteria in CRS patients. GSNO treatment may also enhance the mucociliary clearance by improving the CBF.

Electrocatalytic nitric oxide (NO) generation from nitrite (NO2-) within a single lumen of a dual-lumen catheter using CuII-ligand (CuII-L) mediators have been successful at demonstrating NO's potent antimicrobial and antithrombotic properties to reduce bacterial counts and mitigate clotting under low oxygen conditions (e.g., venous blood). Under more aerobic conditions, the O2 sensitivity of the Cu(II)-ligand catalysts and the reaction of O2 (highly soluble in the catheter material) with the NO diffusing through the outer walls of the catheters results in a large decreases in NO fluxes from the surfaces of the catheters, reducing the utility of this approach. Herein, we describe a new more O2-tolerant CuII-L catalyst, [Cu(BEPA-EtSO3)(OTf)], as well as a potentially useful immobilized glucose oxidase enzyme-coating approach that greatly reduces the NO reactivity with oxygen as the NO partitions and diffuses through the catheter material. Results from this work demonstrate that very effective NO fluxes (>1*10-10 mol min-1 cm-2) from a single-lumen silicone rubber catheter can be achieved in the presence of up to 10% O2 saturated solutions.

Due to recent legalization of marijuana across many states in the U.S., there is an increased concern of users driving while impaired/intoxicated with delta(9)-tetrahydrocannabinol (delta(9)-THC), the principal psychoactive constituent of cannabis/marijuana. Hence, there is a need for a rapid roadside detection of this drug that can be used to accurately screen drivers. Current field sobriety tests rely on a series of physical and mental exercises administered during DUI investigations to help determine a driver's level of impairment. Due to their portability and effectiveness, screen printed carbon electrodes (SPCEs) are ideal to work with when it comes to devising a low-cost screening device for roadside testing. SPCE's can potentially detect low levels of delta(9)-THC in an individual's saliva via electrochemical oxidation of delta(9)-THC. Herein we report a fast, cheap, and accurate approach to electrochemically detect 1-20 mu M delta(9)-THC in a 1 mL sample of artificial oral fluid (AF-OF) diluted to 50 % with a buffer/electrolyte solution using differential pulse voltammetry (DPV) at the surface of a small SPCE. Implications for the use of this method to screen intoxicated drivers are discussed.

S-Nitroso-N-acetylpenicillamine (SNAP) is among the most common nitric oxide (NO)-donor molecules and its solid-state photolytic decomposition has potential for inhaled nitric oxide (iNO) therapy. The photochemical NO release kinetics and mechanism were investigated by exposing solid-state SNAP to a narrow-band LED as a function of nominal wavelength and intensity of incident light. The photolytic efficiency, decomposition products, and the photolytic pathways of the SNAP were examined. The maximum light penetration depth through the solid layer of SNAP was determined by an optical microscope and found to be within 100-200 μm, depending on the wavelength of light. The photolysis of solid-state SNAP to generate NO along with the stable thiyl (RS·) radical was confirmed using Electron Spin Resonance (ESR) spectroscopy. The fate of the RS· radical in the solid phase was studied both in the presence and absence of O2 using NMR, IR, ESR, and UPLC-MS. The changes in the morphology of SNAP due to its photolysis were examined using PXRD and SEM. The stable thiyl radical formed from the photolysis of solid SNAP was found to be reactive with another adjacent thiyl radical to form a disulfide (RSSR) or with oxygen to form various sulfonyl and sulfonyl peroxyl radicals {RS(O)xO·, x = 0 to 7}. However, the thiyl radical did not recombine with NO to reform the SNAP. From the PXRD data, it was found that the SNAP loses its crystallinity by generating the NO after photolysis. The initial release of NO during photolysis was increased with increased intensity of light, whereas the maximum light penetration depth was unaffected by light intensity. The knowledge gained about the photochemical reactions of SNAP may provide important insight in designing portable photoinduced NO-releasing devices for iNO therapy.

Currently, the controlled release of nitric oxide (NO) plays a crucial role in various biomedical applications. However, injectable NO-releasing materials remain an underexplored research field to date. In this study, via the incorporation of S-nitroso-N-acetyl-penicillamine (SNAP) as an NO donor, a family of NO-releasing injectable hydrogels was synthesized through the in situ cross-linking between sodium alginate and calcium ion induced by D-(+)-gluconate δ-lactone as an initiator. Initially, the organic functional groups and the corresponding morphologies of the resulting injectable hydrogels were characterized by IR and SEM spectroscopies, respectively. The NO release times of hydrogels with different SNAP loading amounts could reach up to 36-47 h. Due to the release of NO, the highest antibacterial rates of these injectable hydrogels against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were up to 95 %, respectively. Furthermore, the matrix of these hydrogels demonstrated great water absorption ability, swelling behavior, and degradation performance. Finally, we expect that these NO-releasing injectable hydrogels could have great potential applications various biomedical material fields.

Clinical translation of the extracorporeal artificial placenta (AP) is impeded by the high risk for intracranial hemorrhage in extremely premature newborns. The Nitric Oxide Surface Anticoagulation (NOSA) system is a novel non-thrombogenic extracorporeal circuit. This study aims to test the NOSA system in the AP without systemic anticoagulation. Ten extremely premature lambs were delivered and connected to the AP. For the NOSA group, the circuit was coated with DBHD-N2O2/argatroban, 100 ppm nitric oxide was blended into the sweep gas, and no systemic anticoagulation was given. For the Heparin control group, a non-coated circuit was used and systemic anticoagulation was administered. Animals survived 6.8 ± 0.6 days with normal hemodynamics and gas exchange. Neither group had any hemorrhagic or thrombotic complications. ACT (194 ± 53 vs. 261 ± 86 s; p < 0.001) and aPTT (39 ± 7 vs. 69 ± 23 s; p < 0.001) were significantly lower in the NOSA group than the Heparin group. Platelet and leukocyte activation did not differ significantly from baseline in the NOSA group. Methemoglobin was 3.2 ± 1.1% in the NOSA group compared to 1.6 ± 0.6% in the Heparin group (p < 0.001). The AP with the NOSA system successfully supported extremely premature lambs for 7 days without significant bleeding or thrombosis.

Although there are many techniques to detect pathogenic bacteria, most of them are only suited for in vitro diagnostics. We report a urinary catheter-based colorimetric sensor for potential on-body detection of E. coli, the most prevalent bacterial species in urinary tract infections associated with the use of urinary catheters. In urine, indole is secreted by E. coli and reacts with a nitrosating agent incorporated in a silicone catheter. A red dimeric product, indoxyl red, is generated within silicone rubber to allow for color-based indole sensing with high sensitivity, linearity, and specificity. This reaction is initiated by the nitrosation reaction of indole at its C-3 position via reagents like sodium nitrite or S-nitroso-N-acetyl-penicillamine under aerobic conditions. The generated 3-nitrosoindole undergoes tautomerization, dimerization, and deoximation to form indoxyl red with high absorbance at 537 nm. In contrast to other indole sensors, the presented method can be applied in real catheters to detect indole and E. coli in biofluids such as urine. The is because (1) S-nitroso-N-acetyl-penicillamine, the nitrosating agent, can be impregnated into silicone elastomers, (2) indole from urine is extracted into silicone due to its hydrophobicity, and (3) the high acidity and oxygen solubility of silicone facilitates the sensing reaction within the silicone matrix. This silicone-based colorimetric sensor clearly differentiates E. coli below and above 105 CFU/mL, which is the threshold concentration of bacteriuria. We expect that early diagnosis of urinary tract infections using the naked eye is possible by functionalizing an exposed section of urinary catheters with the proposed molecular probe.

Effective prevention of pacing lead infection could reduce morbidity and save lives. It has been demonstrated that nitric oxide (NO) can inhibit bacterial adhesion and reduce biofilm formation. We tested a diazeniumdiolated dibutylhexanediamine (DBHD/N2O2, a potent NO donor) coated pacing lead to prevent its bacterial infection.

Background: NO is an endogenous molecule with several important biological functions. Besides its vasodilator action, it also inhibits platelet activation, adhesion, and stimulates disaggregation of preformed platelet aggregates. Healthy endothelial cells that line the inner walls of blood vessels emit NO at a flux of 0.5-4×10-10 mol/min/cm2 level. Extracorporeal Life Support (ECLS) circuits with surface anticoagulation properties could eliminate the need for systemic anticoagulation to mitigate the risk of intracranial hemorrhage. Currently, there is no commercially available NO releasing circuit. Technologies in the research phase involve use of diazeniumdiolate type NO donors, however, those are prone to form carcinogenic nitrosamines, posing long-term risks to the patients. Thus, there is a strong need for developing new and stable lipophilic NO donor molecules that can be used in ECLS circuits. Methods: We synthesized a novel, highly lipophilic (predicted logP = 3.439) S-nitrosothiol type NO donor molecule (SNAT). Solvent impregnation was optimized for PVC tubing (Tygon 3/8” ID) and was prepared with 1000 mg/mL SNAT. The surface characteristics of the ECLS circuit was analyzed with scanning electron microscopy (SEM). The in vitro NO release properties were tested with ozone chemiluminescence. Storage stability at different temperatures was also assessed. Antibacterial properties were tested for S. aureus, P. aeruginosa, and E. coli. The effect of different sterilization techniques (EtO, H2O2, ortho-phthalaldehyde solution) was also analyzed. The antithrombogenicity of the impregnated circuit was evaluated in an in vivo rabbit thrombogenicity model with arterial venous shunt for 4h. Results: The SEM images showed no difference in the surface characteristics of the impregnated vs. the untreated control circuits. The initial NO flux of the impregnated circuits was 37×10-10 mol/min/cm2 which decreased below endothelial levels after 35 days. The SNAT impregnated circuits exhibited fair storage stability up to one year at -20°C and 4°C (100% and >50%, respectively, based on first day NO release). The biofilm study showed 3 log reduction of bacteria CFU after 7 days, while the bacteria killing effect of the NO circuit revealed 6-7 log reduction. H2O2 and the liquid chemical sterilizations preserved the NO release (>75%), while EtO decreased it by 50%. After the 4-h long rabbit model the NO releasing circuit showed 0.001(+/-0.000) cm2 (n=3) blood clot area vs. control with 11.18 (+/0.350) cm2 (n=6). Conclusion: These data suggest that SNAT is a very promising novel lipophilic RSNO type NO donor with potential to be used in all forms of extracorporeal circuits to increase hemocompatibility and lower the risks of thrombotic complications without need for systemic anticoagulation. Acknowledgements: This work was supported by National Institutes of Health R21EB024038-02 and 1R01HL15510001A1, and by generous gift from the Reid Family Foundation.

Throughout his career, Mark Meyerhoff, the Philip J. Elving Professor of Chemistry at the University of Michigan, has been exploring chemical sensors for biomedical applications. Recent work has involved the development of novel nitric oxide (NO)-releasing implantable sensors or monitoring important analytes continuously in vivo. For his work Meyerhoff has been awarded the 2021 ANACHEM award, which is presented annually to an outstanding analytical chemist based on activities in teaching, research, administration or other activity which has advanced the art and science of the field. Meyerhoff spoke to us about this work, his career, and what being presented this award at this fall's SciX event means to him.

Over the past 30 years, the significance of nitric oxide (NO) has become increasingly apparent in mammalian physiology. It is biosynthesized by three isoforms of nitric oxide synthases (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Neuronal and eNOS both produce low levels of NO (nM) as a signaling agent and vasodilator, respectively. Inducible (iNOS) is present in activated macrophages at sites of infection to generate acutely toxic (μM) levels of NO as part of the mammalian immune defense mechanism. These discoveries have led to numerous animal and clinical studies to evaluate the potential therapeutic utility of NO in various medical operations/treatments, primarily using NO gas (via gas-cylinders) as the NO source. In this review, we focus specifically on recent advances in the electrochemical generation of NO (E-NOgen) as an alternative means to generate NO from cheap and inert sources, and the fabrication and testing of biomedical devices that utilize E-NOgen to controllably generate NO for medical applications.