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Constant or intense light degenerates the retina and retinal pigment epithelial cells. Light generates reactive oxygen species and nitric oxide leading to initial reactions of retinal degeneration. Apoptosis is the primary mechanism of abnormal death of photoreceptors, retinal ganglion cells, or retinal pigment epithelium (RPE) in degenerative retinal diseases, including diabetic retinopathy and age-related macular degeneration. The current study evaluated the function of erythropoietin (EPO) on angiogenesis and apoptosis in the retina and RPE under oxidative stress. We determined the pro-angiogenic and antiapoptotic mechanism of EPO under stress conditions using a conditional EPO knockdown model using siRNA, EPO addition, proteomics, immunocytochemistry, and bioinformatic analysis. Our studies verified that EPO protected retinal cells from light-, hypoxia-, hyperoxia-, and hydrogen peroxide-induced apoptosis through caspase inhibition, whereas up-regulated angiogenic reactions through vascular endothelial growth factor (VEGF) and angiotensin pathway. We demonstrated that the EPO expression in the retina and subsequent serine/threonine/tyrosine kinase phosphorylations might be linked to oxidative stress response tightly to determining angiogenesis and apoptosis. Neuroprotective roles of EPO may involve the balance between antiapoptotic and pro-angiogenic signaling molecules, including BCL-xL, c-FOS, caspase-3, nitric oxide, angiotensin, and VEGF receptor. Our data indicate a new therapeutic application of EPO toward retinal degeneration based on the dual roles in apoptosis and angiogenesis at the molecular level under oxidative stress.

Diabetic foot ulcers (DFU) are a major health problem associated with diabetes mellitus. Impaired nitric oxide (NO) production has been shown to be a major contributor to the dysregulation of healing in DFU. The level of impairment is not known primarily due to challenges with measuring NO. Herein, we report the actual level of NO produced by human dermal fibroblasts cultured under normal and high glucose conditions. Fibroblasts produce the extracellular matrix, which facilitate the migration of keratinocytes to close wounds. The results show that NO production was significantly higher in normal glucose compared to high glucose conditions. The real-time NO detected was compared to the nitrite present in the culture media and there was a direct correlation between real-time NO and nitrite in normal glucose conditions. However, real-time NO detection and nitrite measurement did not correlate under high glucose conditions. The inducible nitric oxide synthase (iNOS) enzyme responsible for NO production was upregulated in normal and high glucose conditions and the proliferation rate of fibroblasts was not statistically different in all the treatment groups. Relying only on nitrite to assess NO production is not an accurate determinant of the NO present in the wound bed in pathological states such as diabetes mellitus.

Microgels that can generate antipathogenic levels of hydrogen peroxide (H 2 O 2 ) through simple rehydration in solutions with physiological pH are described herein. H 2 O 2 is a widely used disinfectant but the oxidant is hazardous to store and transport. Catechol, an adhesive moiety found in mussel adhesive proteins, was incorporated into microgels, which generated 1–5 mM of H 2 O 2 for up to four days as catechol autoxidized. The sustained release of low concentrations of H 2 O 2 was antimicrobial against both gram-positive ( Staphylococcus epidermidis ) and gram-negative ( Escherichia coli ) bacteria and antiviral against both non-enveloped porcine parvovirus (PPV) and enveloped bovine viral diarrhea virus (BVDV). The amount of released H 2 O 2 is several orders of magnitude lower than H 2 O 2 concentration previously reported for antipathogenic activity. Most notably, these microgels reduced the infectivity of the more biocide resistant non-envelope virus by 3 log reduction value (99.9% reduction in infectivity). By controlling the oxidation state of catechol, microgels can be repeatedly activated and deactivated for H 2 O 2 generation. These microgels do not contain a reservoir for storing the reactive H 2 O 2 and can potentially function as a lightweight and portable dried powder source for the disinfectant for a wide range of applications. Statement of Significance Researchers have designed bioadhesives and coatings using the adhesive moiety catechol to mimic the strong adhesion capability of mussel adhesive proteins. During catechol autoxidation, hydrogen peroxide (H 2 O 2 ) is generated as a byproduct. Here, catechol was incorporated into microgels, which can generate millimolar levels of H 2 O 2 by simply hydrating the microgels in a solution with physiological pH. The sustained release of H 2 O 2 was both antimicrobial and antiviral, inactivating even the more biocide resistant non-enveloped virus. These microgels can be repeatedly activated and deactivated for H 2 O 2 generation by incubating them in solutions with different pH. This simplicity and recyclability will enable this biomaterial to function as a lightweight and portable source for the disinfectant for a wide range of applications.

International audience; The antibacterial effect of zinc oxide (ZnO#1) as prepared and annealed (ZnO#2) at 400 o C, Cu doped ZnO (CuZnO), and Ag doped ZnO (AgZnO) nanoplates on Staphylococcus epidermidis was investigated for the inhibition and inactivation of cell growth. The results shows that pure ZnO and doped ZnO samples exhibited antibacterial activity against S. epidermidis as compared to tryptic soy broth (TSB). Also it is observed that S. epidermidis was extremely sensitive to treatment with ZnO nanoplates and it is clear that the effect is not purely depend on Cu/Ag. Phase identification of a crys­ talline material and unit cell dimensions were studied by X­ray powder diffraction (XRD). The scan­ ning electron microscopy (SEM) provides information on sample's surface topography and the EDX confirms the presence of Zn, O, Cu and Ag. X­ray photo­electron spectroscopy (XPS) was used to analyze the elemental composition and electronic state of the elements that exist within the samples. These studies confirms the formation of nanoplates and the presence of Zn, O, Ag, Cu with the oxida­ tion states +2, ­2, 0 and +2 respectively. These results indicates promising antibacterial applications of these ZnO­based nanoparticles synthesized with low­cost hydrothermal methods.

Light is a risk factor for various eye diseases, including age-related macular degeneration (AMD) and retinitis pigmentosa (RP). We aim to understand how cytoskeletal proteins in the retinal pigment epithetlium (RPE) respond to oxidative stress, including light and how these responses affect apoptotic signaling. Previously, proteomic analysis revealed that the expression levels of vimentin and serine/threonine protein phosphatase 2A (PP2A) are significantly increased when mice are exposed under continuous light for 7 days compared to a condition of 12 hrs light/dark cycling exposure using retina degeneration 1 (rd1) model. When melatonin is administered to animals while they are exposed to continuous light, the levels of vimentin and PP2A return to a normal level. Vimentin is a substrate of PP2A that directly binds to vimentin and dephosphorylates it. The current study shows that upregulation of PP2Ac (catalytic subunit) phosphorylation negatively correlates with vimentin phosphorylation under stress condition. Stabilization of vimentin appears to be achieved by decreased PP2Ac phosphorylation by nitric oxide induction. We tested our hypothesis that site-specific modifications of PP2Ac may drive cytoskeletal reorganization by vimentin dephosphorylation through nitric oxide signaling. We speculate that nitric oxide determines protein nitration under stress conditions. Our results demonstrate that PP2A and vimentin are modulated by nitric oxide as a key element involved in cytoskeletal signaling. The current study suggests that external stress enhances nitric oxide to regulate PP2Ac and vimentin phosphorylation, thereby stabilizing or destabilizing vimentin. Phosphorylation may result in depolymerization of vimentin, leading to nonfilamentous particle formation. We propose that a stabilized vimentin might act as an anti-apoptotic molecule when cells are under oxidative stress.

The current state-of-the-art with respect to the preparation, characterization and biomedical applications of novel nitric oxide (NO) releasing or generating polymeric materials is reviewed. Such materials show exceptional promise as coatings to prepare a new generation of medical devices with superior biocompatiblity. Nitric oxide is a well-known inhibitor of platelet adhesion and activation, as well as a potent inhibitor of smooth muscle cell proliferation. Hence, polymers that release or generate NO locally at their surface exhibit greatly enhanced thromboresistivity and have the potential to reduce neointimal hyperplasia caused by device damage to blood vessel walls. In this review, the use of diazeniumdiolates and nitrosothiols as NO donors within a variety polymeric matrixes are summarized. Such species can either be doped as discrete NO donors within polymeric films, or covalently linked to polymer backbones and/or inorganic polymeric filler particles that are often employed to enhance the strength of biomedical polymers (e.g., fumed silica or titanium dioxide). In addition, very recent efforts to create catalytic polymers possessing immobilized Cu(II) sites capable of generating NO from endogenous oxidized forms of NO already present in blood and other physiological fluids (nitrite and nitrosothiols) are discussed. Preliminary literature data illustrating the efficacy of the various NO release/generating polymers as coatings for intravascular sensors, extracorporeal blood loop circuits, and arteriovenous grafts/shunts are reviewed.

A new type of nitric oxide (NO)-releasing material is described that utilizes S-nitrosothiols anchored to tiny fumed silica (FS) particles as the NO donor system. The synthetic procedures suitable for tethering three different thiol species (cysteine, N-acetylcysteine, and N-acetylpenicillamine) to the surface of FS polymer filler particles are detailed. The thiol-derivatized particles are converted to their corresponding S-nitrosothiols by reaction with t-butylnitrite. The total NO loading on the resulting particles range from 21-138 nmol/mg for the three different thiol-derivatized materials [S-nitrosocysteine-(NO-Cys)-FS, S-nitroso-N-acetylcysteine (SNAC)-FS, and S-nitroso-N-acetylpenicillamine (SNAP)-FS], with SNAP-FS yielding the highest NO loading. NO can be generated from these particles when suspended in solution via the addition of copper(II) ions, ascorbate, or irradiation with visible light. The SNAC-FS and SNAP-FS particles can be blended in polyurethane and silicone rubber matrixes to create films that release NO at controlled rates. Polyurethane films containing SNAC-FS submerged in phosphate-buffered saline (pH 7.4) generate NO surface fluxes approximately 0.1-0.7x10(-10) mol cm-2 min-1 and SNAP-FS films generate NO fluxes of approximately 0-7.5x10(-10) mol cm-2 min-1 upon addition of increasing amounts of copper ions. Silicone rubber films containing SNAC-FS or SNAP-FS do not liberate NO upon exposure to copper ions or ascorbate in phosphate-buffered saline solution. However, such films are shown to release NO at rates proportional to increasing intensities of visible light impinging on the films. Such photoinitiated NO release from these composite materials offers the first NO-releasing hydrophobic polymers with an external on/off trigger to control NO generation.

The in vivo biocompatibility and analytical performance of amperometric oxygen-sensing catheters prepared with a new type of nitric oxide (NO)-releasing silicone rubber polymer (DACA/N2O2 SR) is reported. The NO-release silicone rubber coating contains diazeniumdiolated secondary amine sites covalently anchored to a dimethylsiloxane matrix. Narrow diameter (0.9 mm, o.d.) silicone rubber tubing coated with this polymer can be employed to construct functional oxygen-sensing catheters that release NO continuously at levels1 x 10(-10) mol/cm2-min for more than 20 h. In vivo evaluation of such sensors within the carotid and femoral arteries of swine over a 16-h time period demonstrates that sensors prepared with the new NO-release coating exhibit no significant platelet adhesion or thrombus formation, but control sensors (non-NO release) implanted within the same animals do show a high propensity for cell adhesion and bulk clot formation. Furthermore, the in vivo analytical data provided by sensors fabricated with NO-release coatings (N = 9) are shown to be statistically equivalent to PO2 levels measured in vitro on discrete samples of blood. Control sensors (N = 9) placed within the same animals yield average PO2 values that are statistically different (por = 0.05) (lower) from both the levels measured on discrete samples and those provided by the NO-release sensors over a 16-h in vivo monitoring period.

The usage of gelatin hydrogel is limited due to its instability and poor mechanical properties, especially under physiological conditions. Divalent metal ions present in gelatin such as Ca2+ and Fe2+ play important roles in the gelatin molecule interactions. The objective of this study was to determine the impact of divalent ion removal on the stability and mechanical properties of gelatin gels with and without chemical crosslinking. The gelatin solution was purified by Chelex resin to replace divalent metal ions with sodium ions. The gel was then chemically crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Results showed that the removal of divalent metal ions significantly impacted the formation of the gelatin network. The purified gelatin hydrogels had less interactions between gelatin molecules and form larger-pore network which enabled EDC to penetrate and crosslink the gel more efficiently. The crosslinked purified gels showed small swelling ratio, higher crosslinking density and dramatically increased storage and loss moduli. The removal of divalent ions is a simple yet effective method that can significantly improve the stability and strength of gelatin hydrogels. The in vitro cell culture demonstrated that the purified gelatin maintained its ability to support cell attachment and spreading.

Nitric oxide (NO) plays a critical role in the regulation of a wide variety of physiological processes. It is a potent inhibitor of platelet adhesion and aggregation, inhibits bacterial adhesion and proliferation, is implicated in mediating the inflammatory response toward implanted devices, plays a role in tumor growth and proliferation, and is a neurotransmitter. Herein, we describe the synthesis and NO-release properties of a modified polydimethylsiloxane that contains S-nitroso-N-acetyl-D-penicillamine covalently attached to the cross-linking agent (SNAP–DMS). Light from a C503B-BAN-CY0C0461 light-emitting diode (470 nm) was used as an external trigger to allow precise control over level and duration of NO release ranging from a surface flux of zero to approximately 3.5×10−10 mol cm-2 min-1. SNAP–PDMS films stored in the dark released NO after 297 days, indicating the long-term stability of SNAP–PDMS.

Nitric oxide (NO) plays a critical role in the regulation of a wide variety of physiological processes. It is a potent inhibitor of platelet adhesion and aggregation, inhibits bacterial adhesion and proliferation, is implicated in mediating the inflammatory response toward implanted devices, plays a role in tumor growth and proliferation, and is a neurotransmitter. Herein, we describe the synthesis and NO-release properties of a modified polydimethylsiloxane that contains S-nitroso-N-acetyl-D-penicillamine covalently attached to the cross-linking agent (SNAP–DMS). Light from a C503B-BAN-CY0C0461 light-emitting diode (470 nm) was used as an external trigger to allow precise control over level and duration of NO release ranging from a surface flux of zero to approximately 3.5×10−10 mol cm-2 min-1. SNAP–PDMS films stored in the dark released NO after 297 days, indicating the long-term stability of SNAP–PDMS.

Nitric oxide (NO) plays an important role in inhibiting the development of hepatic fibrosis and its ensuing complication of portal hypertension by inhibiting human hepatic stellate cell (HSC) activation. Here we have developed a gold nanoparticle and silica nanoparticle mediated drug delivery system containing NO donors, which could be used for potential therapeutic application in chronic liver disease. The gold nanoconjugates were characterized using several physico-chemical techniques such as UV-visible spectroscopy and transmission electron microscopy. Silica nanoconjugates were synthesized and characterized as reported previously. NO released from gold and silica nanoconjugates was quantified under physiological conditions (pH = 7.4 at 37 degrees C) for a substantial period of time. HSC proliferation and the vascular tube formation ability, manifestations of their activation, were significantly attenuated by the NO released from these nanoconjugates. This study indicates that gold and silica nanoparticle mediated drug delivery systems for introducing NO could be used as a strategy for the treatment of hepatic fibrosis or chronic liver diseases, by limiting HSC activation.

Previous studies have shown that many S-nitrosothiols (RSNOs) rapidly degrade, with half-lives from minutes to seconds in aqueous solution [1]. The research presented in this paper presents data that the RSNO 1,3-benzenedinitrosothiol has been relatively stable for over 1 year. This RSNO still releases nitric oxide (NO) when subjected to ultraviolet light and has the same characteristic absorbance peak as a freshly made RSNO. Developing this stable RSNO potentially provides a venue for further investigation into using this NO donor to improve the biocompatibility of implanted optical sensors.

Measuring the blood gases of patients in the intensive care unit or undergoing major surgical procedures in real time would help healthcare providers diagnose and manage base excess and base deficient disorders. Optical fibers provide a platform upon which an intravascular blood gas sensor may be built and has been by various companies. Unfortunately, thrombosis on the sensor surface interferes with the blood gas measurements and also poses the risk of creating emboli. Nitric oxide inhibits platelet adhesion and activation, which can reduce thrombus formation on the sensor surface. An optically based pH sensor is fabricated as a first step to show that nitric oxide can be used with blood gas sensors to reduce thrombosis and not interfere with the measurements. pH sensors fabricated using glass microscope slides suffered from leaching of the dye from the sol-gel matrix. The dye readily leached out when the dye was in its appropriate protonated or deprotonated form. To reduce the leaching of the dye, methods of covalently linking the dye to fumed silica have been investigated, and one is presented here.

Biomedical devices that contact blood and tissue universally inspire a host response that often compromises the function of the device (i.e., intravascular sensors become coated with thrombi, artificial vascular grafts become coated with thrombi, artificial vascular grafts become occluded with thrombus formation and neointimal hyperplasia). Nitric oxide (NO) has been shown to be a potent inhibitor of platelet adhesion and activation and has been implicated in mediating the inflammatory response and promoting would healing. We are currently developing NO-releasing compounds based on S-nitrosothiols derived from substituted aromatic compounds that utilize light as an external on/off trigger capable of releasing precisely controlled surface fluxes of NO. The level of NO generated is dependent on the wavelength and intensity of light shown on the compounds. Data will be presented that show the synthesis and NO-release properties of three novel compounds, S-nitroso-2-methoxybenzene, S-nitroso-3-methoxybenzene and S-nitroso-2-chlorobenzene. Ultimately, these compounds will be tethered to the surface of polymer fillers that will then be blended into hydrophobic polymers and used as coatings on biomedical devices. A model system that will be used to demonstrate the utility of this approach will be a multi-element fiber optic sensors that will contain sensing elements capable of measuring blood gases and NO-releasing fibers that locally generate enough NO to inhibit clot formation on the sensor surface, thus allowing the sensor to function reliably in vivo.

Nitric Oxide (NO) is small, free radical gas that has been shown to have a wide variety of physiological functions, including the ability to hinder tumor angiogenesis at high, but non lethal, concentrations [1]. Previous work suggests that if NO could be effectively delivered in vivo to tumors of patients currently undergoing chemotherapy treatments at the appropriate levels, less damaging chemotherapy treatments could be used against cancer [2]. This could increase the overall survivability of cancer patients, especially in those prone to the harmful effects of chemotherapy: children, elderly, and those of weak immune systems. If NO is especially successful at preventing and eliminating tumor growth, angiogenesis, and carcinogenesis the need for stressful chemotherapy treatments could be eliminated altogether. This project is focused on developing novel photosensitive NO donors that can be incorporated into polymeric systems and used in a fiber optic drug delivery system. Development of these NO-releasing polymers will allow continued investigation of NO's role in tumor death by precisely controlling the surface flux of NO that cells are exposed to. Generating specific surface fluxes of NO from polymer films has been demonstrated by using polymer films that contain photoinitiated NO donors [3], prepared by synthesizing S-nitrosothiol (RSNO) derivitized polymer fillers that are blended into hydrophobic polymers and cast into a film. These films generate and sustain a surface flux of NO based on the wavelength and intensity of light used [3]. Polymers releasing NO are more promising as an NO donor than simply injecting NO into samples because they allow for spatial and temporal control of NO delivery. The specific concentration of NO needed to produce desirable effects on tumor cells (i.e., apoptosis) is not known. Data will be presented that show the synthesis and NO-release properties of novel RSNOs based on the nitrosation of benzyl mercaptan thiols. Specifically, UV-Vis spectrum of benzyl mercaptan in toluene and S-nitrosobenzyl mercaptan after the addition of t-butyl nitrite will be presented. We are currently investigating the effects of varying NO-surface fluxes generated from photolytic NO donating polymer films on aortic smooth muscle cell cultures obtained from mice. Once we have established that we can quantitatively determine the effects of different levels of NO on the proliferation of smooth muscle cell cultures, work will begin to apply this methodology and these novel NO-releasing polymeric systems to begin investigating what durations and surface fluxes of NO are necessary to have tumorcidal effects on specific cancer cells.

Nitric oxide (NO) plays a critical role in the regulation of a wide variety of physiological processes. It is a potent inhibitor of platelet adhesion and aggregation, inhibits bacterial adhesion and proliferation, is implicated in mediating the inflammatory response toward implanted devices, plays a role in tumor growth and proliferation, and is a neurotransmitter. Herein, we describe the synthesis and NO-release properties of a modified polydimethylsiloxane that contains S-nitroso-N-acetyl-D-penicillamine covalently attached to the cross-linking agent (SNAP–DMS). Light from a C503B-BAN-CY0C0461 light-emitting diode (470 nm) was used as an external trigger to allow precise control over level and duration of NO release ranging from a surface flux of zero to approximately 3.5×10−10 mol cm-2 min-1. SNAP–PDMS films stored in the dark released NO after 297 days, indicating the long-term stability of SNAP–PDMS.