Mimicking the Endothelium: Dual Action Heparinized Nitric Oxide Releasing Surface.

The management of thrombosis and bacterial infection is critical to ensure the functionality of medical devices. While administration of anticoagulants is the current antithrombotic clinical practice, a variety of complications, such as uncontrolled hemorrhages or heparin-induced thrombocytopenia, can occur. Additionally, infection rates remain a costly and deadly complication associated with use of these medical devices. It has been hypothesized that if a synthetic surface could mimic the biochemical mechanisms of the endothelium of blood vessels, thrombosis could be reduced, anticoagulant use could be avoided, and infection could be prevented. Herein, the interfacial biochemical effects of the endothelium were mimicked by altering the surface of medical grade silicone rubber (SR). Surface modification was accomplished via heparin surface immobilization (Hep) and the inclusion of a nitric oxide (NO) donor into the SR polymeric matrix to achieve synergistic effects (Hep-NO-SR). An in vitro bacteria adhesion study revealed that Hep-NO-SR exhibited a 99.46 ± 0.17% reduction in viable bacteria adhesion compared to SR. An in vitro platelet study revealed Hep-NO-SR reduced platelet adhesion by 84.12 ± 6.19% compared to SR, while not generating a cytotoxic response against fibroblast cells. In a 4 h extracorporeal circuit model without systemic anticoagulation, all Hep-NO-SR samples were able to maintain baseline platelet count and device patency; whereas 66% of SR samples clotted within the first 2 h of study. Results indicate that Hep-NO-SR creates a more hemocompatible and antibacterial surface by mimicking two key biochemical functions of the native endothelium.

Nitric oxide affects cisplatin cytotoxicity oppositely in A2780 and A2780-CDDP cells via the connexin32/gap junction.

Chemoresistance is a main obstacle in ovarian cancer therapy and new treatment strategies and further information regarding the mechanism of the medication cisplatin are urgently needed. Nitric oxide has a critical role in modulating the activity of chemotherapeutic drugs. Our previous work showed that connexin32 contributed to cisplatin resistance. However, whether nitric oxide is involved in connexin32-mediated cisplatin resistance remains unknown. In this study, using A2780 and A2780 cisplatin-resistant cells, we found that S-nitroso-N-acetyl-penicillamine, a nitric oxide donor, attenuated cisplatin toxicity by decreasing gap junctions in A2780 cells. Enhancement of gap junctions using retinoic acid reversed the effects of S-nitroso-N-acetyl-penicillamine on cisplatin toxicity. In A2780 cisplatin-resistant cells, however, S-nitroso-N-acetyl-penicillamine enhanced cisplatin toxicity by decreasing connexin32 expression. Downregulation of connexin32 expression by small interfering RNA exacerbated the effects of S-nitroso-N-acetyl-penicillamine on cisplatin cytotoxicity and upregulation of connexin32 expression by pcDNA transfection reversed the effects of S-nitroso-N-acetyl-penicillamine on cisplatin cytotoxicity. Our study suggests for the first time that combining cisplatin with nitric oxide in clinical therapies for ovarian cancer should be avoided before cisplatin resistance emerges. The present study provides a productive area of further study for increasing the efficacy of cisplatin by combining cisplatin with the specific inhibitors or enhancers of nitric oxide in clinical treatment.

Semiconducting polymer nanotheranostics for NIR-II/Photoacoustic imaging-guided photothermal initiated nitric oxide/photothermal therapy.

Gasotransmitters with their cytotoxicity in high concentration have become the focus of attention. For such concentration depended therapy, how to effectively deliver gases and precisely control gases release to the lesion as well as combine them with other therapy to achieve precise therapeutics is still a big challenge. Herein, we realize single near-infrared (NIR) laser-initiated nitric oxide (NO) therapy/photothermal therapy (PTT) using semiconducting polymer nanoparticles (SPNs, PFTDPP) combing s-nitrosothiol groups (the NO donor, SNAP). By the good photothermal conversion effect of SPNs, NIR laser energy can be spatio-temporally controlled to convert into heat to decompose s-nitrosothiol. Meanwhile, considering the accompanied PTT produced by photothermal, we can easily and precisely conduct a dual therapy (NO therapy/PTT) under single NIR laser irradiation. Additionally, semiconducting polymer with its structural modifiability and spectral adjustability can provide a second NIR window & photoacoustic (NIR II/PA) imaging for guiding photothermal initiated NO/photothermal therapy. PFTDPP showed a high photothermal conversion efficiency of 48% and good dual-mode imaging signals (NIR-II/photoacoustic). Cellular test illustrated that NO combined photothermal presented more prominent cytotoxicity than any one of them individually. As the tumor pinpointed in vivo by dual-mode imaging (NIR II/PA), this nanotheranostics provided a tumor inhibition of 77%. Consequently, such phototheranostics produced a new design thought for effectively deliver and precisely controlled release of drugs for oncology. And also, it expanded the application range of gasotransmitters combined therapy that shall have a promising application foreground.

Effects of nitric oxide donor on the lung functions in a saline lavage-induced model of ARDS.

Acute respiratory distress syndrome (ARDS) is characterized by acute hypoxemia, neutrophil-mediated inflammation, and lung edema formation. Whereas lung damage might be alleviated by nitric oxide (NO), goal of this study was to evaluate if intratracheal NO donor S-nitroso-N-acetylpenicillamine (SNAP) can positively influence the lung functions in experimental model of ARDS. New Zealand rabbits with respiratory failure induced by saline lavage (30 ml/kg, 9+/-3 times) were divided into: ARDS group without therapy, ARDS group treated with SNAP (7 mg/kg i.t.), and healthy Control group. During 5 h of ventilation, respiratory parameters (blood gases, ventilatory pressures) were estimated. After anesthetics overdosing, left lung was saline-lavaged and cell count, cell viability and protein content in bronchoalveolar lavage fluid (BALF) were measured. Right lung tissue was used for estimation of wet/dry weight ratio, concentration of NO metabolites, and histomorphological investigation. Repetitive lung lavage induced lung injury, worsened gas exchange, and damaged alveolar-capillary membrane. Administration of SNAP reduced cell count in BALF, lung edema formation, NO metabolites, and histopathological signs of injury, and improved respiratory parameters. Treatment with intratracheal SNAP alleviated lung injury and edema and improved lung functions in a saline-lavaged model of ARDS suggesting a potential of NO donors also for patients with ARDS.

In vivo and in silico evaluation of a new nitric oxide donor, S,S'-dinitrosobucillamine.

PURPOSE: In a previous work, we have synthetized a new dinitrosothiol, i.e. S,S'-dinitrosobucillamine BUC(NO)2 combining S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-N-acetylcysteine (NACNO) in its structure. When exposed to isolated aorta, we observed a 1.5-fold increase of •NO content and a more potent vasorelaxation (1 log higher pD2) compared to NACNO and SNAP alone or combined (Dahboul et al., 2014). In the present study, we analyzed the thermodynamics and kinetics for the release of •NO through computational modeling techniques and correlated it to plasma assays. Then BUC(NO)2 was administered in vivo to rats, assuming it will induce higher and/or longer hypotensive effects than its two constitutive S-mononitrosothiols. METHODS: Free energies for the release of •NO entities have been computed at the density functional theory level assuming an implicit model for the aqueous environment. Degradation products of BUC(NO)2 were evaluated in vitro under heating and oxidizing conditions using HPLC coupled with tandem mass spectrometry (MS/MS). Plasma from rats were spiked with RSNO and kinetics of RSNO degradation was measured using the classical Griess-Saville method. Blood pressure was measured in awake male Wistar rats using telemetry (n = 5, each as its own control, 48 h wash-out periods between subcutaneous injections under transient isoflurane anesthesia, random order: 7 mL/kg vehicle, 3.5, 7, 14 µmol/kg SNAP, NACNO, BUC(NO)2 and an equimolar mixture of SNAP + NACNO in order to mimic the number of •NO contained in BUC(NO)2). Variations of mean (ΔMAP, reflecting arterial dilation) and pulse arterial pressures (ΔPAP, indirectly reflecting venodilation, used to determine effect duration) vs. baseline were recorded for 4 h. RESULTS: Computational modeling highlights the fact that the release of the first •NO radical in BUC(NO)2 requires a free energy which is intermediate between the values obtained for SNAP and NACNO. However, the release of the second •NO radical is significantly favored by the concerted formation of an intramolecular disulfide bond. The corresponding oxidized compound was also characterized as related substance obtained under degradation conditions. The in vitro degradation rate of BUC(NO)2 was significantly greater than for the other RSNO. For equivalent low and medium •NO-load, BUC(NO)2 produced a hypotension identical to NACNO, SNAP and the equimolar mixture of SNAP + NACNO, but its effect was greater at higher doses (-62 ± 8 and -47 ± 14 mmHg, maximum ΔMAP for BUC(NO)2 and SNAP + NACNO, respectively). Its duration of effect on PAP (-50%) lasted from 35 to 95 min, i.e. shorter than for the other RSNO (from 90 to 135 min for the mixture SNAP + NACNO). CONCLUSION: A faster metabolism explains the abilities of BUC(NO)2 to release higher amounts of •NO and to induce larger hypotension but shorter-lasting effects than those induced by the SNAP + NACNO mixture, despite an equivalent •NO-load.

Improved hemocompatibility of silicone rubber extracorporeal tubing via solvent swelling-impregnation of S-nitroso-N-acetylpenicillamine (SNAP) and evaluation in rabbit thrombogenicity model.

UNLABELLED: Blood-contacting devices, including extracorporeal circulation (ECC) circuits, can suffer from complications due to platelet activation and thrombus formation. Development of nitric oxide (NO) releasing polymers is one method to improve hemocompatibility, taking advantage of the ability of low levels of NO to prevent platelet activation/adhesion. In this study a novel solvent swelling method is used to load the walls of silicone rubber tubing with the NO donor S-nitroso-N-acetylpenicillamine (SNAP). This SNAP-silicone rubber tubing exhibits an NO flux of ca. 1×10(-10)molcm(-2)min(-1), which mimics the range of NO release from the normal endothelium, which is stable for at least 4h. Images of the tubing before and after swelling, obtained via scanning electron microscopy, demonstrate that this swelling method has little effect on the surface properties of the tubing. The SNAP-loaded silicone rubber and silicone rubber control tubing are used to fabricate ECC circuits that are evaluated in a rabbit model of thrombogenicity. After 4h of blood flow, the SNAP-loaded silicone rubber circuits were able to preserve the blood platelet count at 64% of baseline (vs. 12% for silicone rubber control). A 67% reduction in the degree of thrombus formation within the thrombogenicity chamber was also observed. This study demonstrates the ability to improve the hemocompatibility of existing/commercial silicone rubber tubing via a simple solvent swelling-impregnation technique, which may also be applicable to other silicone-based blood-contacting devices. STATEMENT OF SIGNIFICANCE: Localized nitric oxide (NO) release can be achieved from biomedical grade polymers doped with S-nitroso-N-acetylpenicillamine (SNAP). Despite the promising in vitro and in vivo biocompatibility results reported for these NO releasing polymers, many of these materials may face challenges in being translated to clinical applications, especially in the areas of polymer processing and manufacturing. In this study, we report a solvent swelling-impregnation technique to incorporate SNAP into extracorporeal circuit (ECC) tubing. These NO-releasing ECCs were able to attenuate the activation of platelets and maintain their functionality, while significantly reducing the extent of thrombus formation during 4h blood flow in the rabbit model of thrombogenicity.

Nitric oxide suppresses LPS-induced inflammation in a mouse asthma model by attenuating the interaction of IKK and Hsp90.

A feature of allergic airway disease is the observed increase of nitric oxide (NO) in exhaled breath. Gram-negative bacterial infections have also been linked with asthma exacerbations. However, the role of NO in asthma exacerbations with gram-negative bacterial infections is still unclear. In this study, we examined the role of NO in lipopolysaccharide (LPS)-induced inflammation in an ovalbumin (OVA)-challenged mouse asthma model. To determine whether NO affected the LPS-induced response, a NO donor (S-nitroso-N-acetylpenicillamine, SNAP) or a selective inhibitor of NO synthase (1400W) was injected intraperitoneally into the mice before the LPS stimulation. Decreased levels of proinflammatory cytokines were demonstrated in the bronchoalveolar lavage fluid from mice treated with SNAP, whereas increased levels of cytokines were found in the 1400W-treated mice. To further explore the molecular mechanism of NO-mediated inhibition of proinflammatory responses in macrophages, RAW 264.7 cells were treated with 1400W or SNAP before LPS stimulation. LPS-induced inflammation in the cells was attenuated by the presence of NO. The LPS-induced IκB kinase (IKK) activation and the expression of IKK were reduced by NO through attenuation of the interaction between Hsp90 and IKK in the cells. The IKK decrease in the lung immunohistopathology was verified in SNAP-treated asthma mice, whereas IKK increased in the 1400W-treated group. We report for the first time that NO attenuates the interaction between Hsp90 and IKK, decreasing the stability of IKK and causing the down-regulation of the proinflammatory response. Furthermore, the results suggest that NO may repress LPS-stimulated innate immunity to promote pulmonary bacterial infection in asthma patients.

Nitric oxide effects on the function of aged cells ex vivo and in vivo.

BACKGROUND: Angiogenesis is impaired in most aged tissues. Accordingly, there is great interest in interventions that improve the ability of aged cells to undergo blood vessel formation and subsequent tissue repair. MATERIALS AND METHODS: Nitric oxide (NO), a mediator proposed to enhance angiogenesis, was administered (as the precursor SNAP, S-nitroso-N-acetylpenicillamine) to aortic ring explants from aged mice and to aged mice in two separate in vivo experiments: a PVA sponge implant model of angiogenesis and full thickness excisional dermal wounds. RESULTS: SNAP inhibited angiogenesis from the mouse aortic ring explants. However, there was a trend toward increased blood vessel formation in the sponges from the aged mice treated with SNAP. SNAP did not detectably enhance dermal wound healing or angiogenesis, but it significantly inhibited epidermal closure. CONCLUSION: These data underscore the complexity of using a single agent, even one with multiple mechanisms such as NO, to improve a clinical outcome such as angiogenesis or wound repair in aged animals.

Evidence for a ceiling of cardioprotection with a nitric oxide donor-induced delayed preconditioning in rabbits.

Although extensive attention has been devoted to the window of preconditioning, only few studies investigated the efficacy of preconditioning against ischemia with increasing durations. To date, a "ceiling of protection" has been demonstrated to occur with early preconditioning but nothing is known about delayed preconditioning. Accordingly, the efficacy of a nitric oxide (NO)-donor-induced delayed preconditioning was tested against ischemic insults of increasing duration. Accordingly, 65 rabbits received a 75-min intravenous infusion of either saline (control group), or an NO-donor (S-nitroso-N-acetylpenicillamine) at 3 microg/kg/min (SNAP-3 group) or 30 microg/kg/min (SNAP-30 group). Twenty-four hours later, rabbits randomly underwent either a 15-, 20-, or a 30-min coronary artery occlusion (CAO). Infarct size was assessed after 72-h reperfusion (triphenyltetrazolium chloride staining, percentage of the area at risk). After 15-min CAO, both SNAP-3 and SNAP-30 reduced infarct size compared with control (10 +/- 3, 5 +/- 1 versus 29 +/- 8%, respectively; p < 0.05). After 20-min CAO, significant cardioprotection was only observed with SNAP-30 (29 +/- 4, 21 +/- 6 versus 36 +/- 2% for SNAP-3, SNAP-30 versus control, respectively). After 30-min CAO, both SNAP-3 and SNAP-30 failed to reduce infarct size (48 +/- 2, 50 +/- 5 versus 50 +/- 4% for SNAP-3, SNAP-30 versus control, respectively). In conclusion, this study demonstrates a dose-related ceiling of protection with delayed preconditioning induced by an NO donor. It supports that delayed preconditioning might exert its maximal beneficial effect with early reperfusion and this finding supports the necessary use of different durations of ischemia when investigating cardioprotective strategies.