Multifunctional S-Nitroso-N-acetylpenicillamine-Incorporated Medical-Grade Polymer with Selenium Interface for Biomedical Applications.

Modern crises in implantable or indwelling blood-contacting medical devices are mainly due to the dual problems of infection and thrombogenicity. There is a paucity of biomaterials that can address both problems simultaneously through a singular platform. Taking cues from the body's own defense mechanism against infection and blood clotting (thrombosis) via the endogenous gasotransmitter nitric oxide (NO), both of these issues are addressed through the development of a layered S-nitroso-N-acetylpenicillamine (SNAP)-doped polymer with a blended selenium (Se)-polymer interface. The unique capability of the SNAP-Se-1 polymer composites to explicitly release NO from the SNAP reservoir as well as generate NO via the incorporated Se is reported for the first time. The NO release from the SNAP-doped polymer increased substantially in the presence of the Se interface. The Se interface was able to generate NO in the presence of S-nitrosoglutathione (GSNO) and glutathione (GSH), demonstrating the capability of generating NO from endogenous S-nitrosothiols (RSNO). Scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) traced distribution of elemental Se nanoparticles on the interface and the surface properties were evaluated by surface wettability and roughness. The SNAP-Se-1 efficiently inhibited the growth of bacteria and reduced platelet adhesion while showing minimal cytotoxicity, thus potentially eliminating the risks of systemic antibiotic and blood coagulation therapy. The SNAP-Se-1 exhibited antibacterial activity of ∼2.39 and ∼2.25 log reductions in the growth of clinically challenging adhered Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. SNAP-Se-1 also significantly reduced platelet adhesion by 85.5% compared to corresponding controls. A WST-8-based cell viability test performed on NIH 3T3 mouse fibroblast cells provided supporting evidence for the potential biocompatibility of the material in vitro. These results highlight the prospective utility of SNAP-Se-1 as a blood-contacting infection-resistant biomaterial in vitro which can be further tuned by application specificity.

72-Hour in vivo evaluation of nitric oxide generating artificial lung gas exchange fibers in sheep.

The large, densely packed artificial surface area of artificial lungs results in rapid clotting and device failure. Surface generated nitric oxide (NO) can be used to reduce platelet activation and coagulation on gas exchange fibers, while not inducing patient bleeding due to its short half-life in blood. To generate NO, artificial lungs can be manufactured with PDMS hollow fibers embedded with copper nanoparticles (Cu NP) and supplied with an infusion of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP). The SNAP reacts with Cu NP to generate NO. This study investigates clot formation and gas exchange performance of artificial lungs with either NO-generating Cu-PDMS or standard polymethylpentene (PMP) fibers. One miniature artificial lung (MAL) made with 10 wt% Cu-PDMS hollow fibers and one PMP control MAL were attached to sheep in parallel in a veno-venous extracorporeal membrane oxygenation circuit (n = 8). Blood flow through each device was set at 300 mL/min, and each device received a SNAP infusion of 0.12 µmol/min. The ACT was between 110 and 180 s in all cases. Blood flow resistance was calculated as a measure of clot formation on the fiber bundle. Gas exchange experiments comparing the two groups were conducted every 24 h at blood flow rates of 300 and 600 mL/min. Devices were removed once the resistance reached 3x baseline (failure) or following 72 h. All devices were imaged using scanning electron microscopy (SEM) at the inlet, outlet, and middle of the fiber bundle. The Cu-PDMS NO generating MALs had a significantly smaller increase in resistance compared to the control devices. Resistance rose from 26 ±â€¯8 and 23 ±â€¯5 in the control and Cu-PDMS devices, respectively, to 35 ±â€¯8 mmHg/(mL/min) and 72 ±â€¯23 mmHg/(mL/min) at the end of each experiment. The resistance and SEM imaging of fiber surfaces demonstrate lower clot formation on Cu-PDMS fibers. Although not statistically significant, oxygen transfer for the Cu-PDMS MALs was 13.3% less than the control at 600 mL/min blood flow rate. Future in vivo studies with larger Cu-PDMS devices are needed to define gas exchange capabilities and anticoagulant activity over a long-term study at clinically relevant ACTs. STATEMENT OF SIGNIFICANCE: In artificial lungs, the large, densely-packed blood contacting surface area of the hollow fiber bundle is critical for gas exchange but also creates rapid, surface-generated clot requiring significant anticoagulation. Monitoring of anticoagulation, thrombosis, and resultant complications has kept permanent respiratory support from becoming a clinical reality. In this study, we use a hollow fiber material that generates nitric oxide (NO) to prevent platelet activation at the blood contacting surface. This material is tested in vivo in a miniature artificial lung and compared against the clinical standard. Results indicated significantly reduced clot formation. Surface-focused anticoagulation like this should reduce complication rates and allow for permanent respiratory support by extending the functional lifespan of artificial lungs and can further be applied to other medical devices.

Nitric oxide releasing vascular catheters for eradicating bacterial infection.

The interaction of blood proteins with an implant surface is not only a fundamental phenomenon but is also key to several important medical complications. Plasma proteins binding on the surface of intravascular catheters can promote bacterial adhesion leading to the risk of local and systemic complications such as catheter-related blood infections (CRBIs). The incidences of CRBIs in the United States amount to more than 250,000 cases/year with an attributable mortality of up to 35% and an annual healthcare expenditure of $2.3 billion approximately. This demands the development of truly nonthrombogenic and antimicrobial catheters. In the present study, catheters were fabricated by incorporating a nitric oxide (NO) donor molecule, S-nitroso-N-acetyl-penicillamine (SNAP) in a hydrophobic medical grade polymer, Elasteon-E2As. NO offers antithrombotic and antibacterial attributes without promoting drug resistance and cytotoxicity. E2As-SNAP catheters were first coated with fibrinogen, a blood plasma protein plays a key role in clot formation and eventual bacterial adhesion to the implant surface. The suitability of the catheters for biomedical applications was tested in vitro for contact angle, NO release kinetics, inhibition of bacteria, and absence of cytotoxicity toward mammalian cells. The highly hydrophobic catheters released NO in the physiological range that inhibited >99% bacterial viability on fibrinogen-coated catheters in a 24 h study. No toxic response of E2As-SNAP catheters leachate was observed using a standard cytotoxicity assay with mouse fibroblast cells. Overall, the results showed that the E2As-SNAP catheters can inhibit viable bacteria even in the presence of blood proteins without causing a cytotoxic response. The fundamentals of this study are applicable to other blood-contacting medical devices as well. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2849-2857, 2018.

Nitric oxide-donor SNAP induces Xenopus eggs activation.

Nitric oxide (NO) is identified as a signaling molecule involved in many cellular or physiological functions including meiotic maturation and parthenogenetic activation of mammalian oocytes. We observed that nitric oxide donor SNAP was potent to induce parthenogenetic activation in Xenopus eggs. NO-scavenger CPTIO impaired the effects of SNAP, providing evidence for the effects of the latter to be specific upon NO release. In Xenopus eggs, SNAP treatment induced pigment rearrangement, pronucleus formation and exocytosis of cortical granules. At a biochemical level, SNAP exposure lead to MAPK and Rsk inactivation within 30 minutes whereas MPF remained active, in contrast to calcium ionophore control where MPF activity dropped rapidly. MAPK inactivation could be correlated to pronuclear envelope reformation observed. In SNAP-treated eggs, a strong increase in intracellular calcium level was observed. NO effects were impaired in calcium-free or calcium limited medium, suggesting that that parthenogenetic activation of Xenopus oocytes with a NO donor was mainly calcium-dependent.

Correlation of in vitro and in vivo kinetics of nitric oxide donors in ocular tissues.

In the eye, nitric oxide (NO) is involved in the regulation of intraocular pressure (IOP) and ocular blood flow. The main purpose of this study was to measure the kinetics of NO release from NO donors in ocular cells and tissues using in vivo and in vitro models and demonstrate the link between the kinetics of NO release with the functional effect, IOP. Nitric oxide release was measured in human ocular cells using a fluorescent dye, diaminofluorescein (DAF), following treatment with short-acting sodium nitroprusside (SNP) and longer-acting S-nitroso-N-acetylpenicillamine (SNAP) NO donors. Both SNP and SNAP were also administered topically to rabbits; IOP was measured and levels of NO and cGMP were assessed as biomarkers over a time course in the aqueous humor (AH) and iris/ciliary body (ICB). Time- and concentration-dependent increases in NO level were produced by SNP and SNAP in human ocular cells. Both NO and cGMP levels appeared to be elevated following treatment with the aforementioned NO donors in rabbit ocular tissues. Transient IOP lowering was accompanied with these biochemical estimations in rabbits, with time of maximal effect being shifted to the right for longer-acting SNAP as compared with short-acting SNP. In vitro and in vivo NO/cGMP assay results displayed a correlation between short- and longer-acting NO donors, discriminating their respective temporal actions in the eye. Due to their translatability, the in vitro DAF assay and in vivo NO fluorometric assay can therefore be potentially useful in screening novel NO donors with different temporal/kinetic profiles.

Effect of hypoxia on vasodilator responses to S-nitroso-N-acetylpenicillamine and levcromakalim in guinea pig basilar artery.

Ischaemic stroke is characterised by reduction of blood flow, tissue hypoxia, energy depletion and neuronal death. Drugs causing vasodilatation of cerebral arteries may potentially enhance blood supply to the ischaemic area and improve clinical outcome. However, vasodilators could also reduce cerebral blood flow in the ischaemic region by acting on blood vessels in non-ischaemic tissue, a phenomenon known as blood flow steal. To explore whether these drugs could act selectively on cerebral blood vessels in a hypoxic environment, we examined the effect of hypoxia on vasodilator responses to the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) and the ATP-dependent potassium channel (K(ATP)) opener levcromakalim in guinea-pig basilar arteries contracted by endothelin-1. Hypoxia considerably enhanced the vasodilator responses to SNAP, while those to levcromakalim were unaffected. In the presence of the NO synthase inhibitor N(G)-nitro-L-arginine, hypoxia no longer enhanced the vasodilator response to SNAP and suppressed responses to levcromakalim. The results show that the NO donor SNAP, but not the K(ATP) opener levcromakalim, is a more effective vasodilator of cerebral arteries contracted by endothelin-1 during hypoxia than under control conditions. Hypoxia-induced inhibition of basal NO synthesis could explain this enhancement of the vasodilator response to SNAP. Thus, NO donors may have a selective effect on blood vessels in ischaemic brain areas and therefore warrant further evaluation as therapeutic agents in cerebral ischaemia.