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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.

The gold standard of care for coronary artery disease, a leading cause of death for in the world, is balloon angioplasty in conjunction with stent deployment. However, implantation injuries and long-term presence of foreign material often promotes significant luminal tissue growth, leading to a narrowing of the artery and severely restricted blood flow. A promising method to mitigate this process is the use of biodegradable metallic stents, but thus far they have either degraded too slowly (iron) or disappeared prematurely (magnesium). The present work investigates the use of a unique type of magnetic material, galfenol (iron-gallium), for postoperative wireless control of stent degradation rates. Due to its magnetoelastic property, galfenol experiences longitudinal micron-level elongations when exposed to applied magnetic fields, allowing generation of a microstirring effect that affect its degradation behavior. In vitro indirect cytotoxicity tests on primary rat aortic smooth muscle cells indicated that galfenol byproducts must be concentrated approximately seven times from collected 60 day degradation medium to cause ∼15% of death from all cells. Surface and cross-sectional characterization of the material indicate that galfenol (Fe80 Ga20 ) degradation rates (∼0.55% per month) are insufficient for stenting applications. While this material may not be ideal for comprising the entire stent, there is potential for use in combination with other materials. Furthermore, the ability to control degradation rates postimplantation opens new possibilities for biodegradable stents; additional magnetoelastic materials should be investigated for use in stenting applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 232-241, 2019.

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.

Although significant advances have been made in the development of artificial vascular grafts, small-diameter grafts still suffer from excessive platelet activation, thrombus formation, smooth muscle cell intimal hyperplasia, and high occurrences of restenosis. Recent discoveries demonstrating the excellent blood-contacting properties of the natural elastic lamina have raised the possibility that an acellular elastic lamina could effectively serve as a patent blood-contacting surface in engineered vascular grafts. However, the elastic lamina alone lacks the requisite mechanical properties to function as a viable vascular graft. Here, we have screened a wide range of biodegradable and biostable medical-grade polymers for their ability to adhere to the outer surface of the elastic lamina and allow cellular repopulation following engraftment in the rat abdominal aorta. We demonstrate a novel method for the fabrication of elastic lamina-polymeric hybrid small-diameter vascular grafts and identify poly(ether urethane) (PEU 1074A) as ideal for this purpose. In vivo results demonstrate graft patency over 21 days, with low thrombus formation, mild inflammation, and the general absence of smooth muscle cell hyperplasia on the graft's luminal surface. The results provide a new direction for developing small-diameter vascular grafts that are mass-producible, shelf-stable, and universally compatible due to a lack of immune response and inhibit the in-graft restenosis response that is common to nonautologous materials.

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.

Nitric oxide plays important roles in cardiovascular homeostasis, immune responses and wound repair. Therefore, polymers that release nitric oxide locally at the surface exhibit improved biocompatibility for biomedical implants through reducing neointimal hyperplasia and thrombosis caused by blood vessel wall damage. The objective of this article was to fabricate a nitric oxide–releasing gelatin hydrogel that can continuously generate nitric oxide at a physiologically relevant level and inhibit cell attachment and proliferation. The nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP), was successfully conjugated to the gelatin hydrogel, which showed a rapid nitric oxide release in the first 2 h and then a slower but sustained release in the next 70-h period. Human mesenchymal stem cells (hMSCs), as a model cell line with wide biomedical applications, were used to examine the cell attachment and proliferation of the nitric oxide–releasing gelatin hydrogel. Compared with the control gelatin, the nitric oxide–releasing gelatin hydrogel demonstrated a 0·35 times lower hMSCs attachment at 6 h and a 3·15 times lower hMSCs proliferation after 72-h incubation. Moreover, hMSCs on nitric oxide–releasing gelatin exhibited a rounder cell shape and covered less cellular area than their counterparts on the control gelatin hydrogel. This gelatin hydrogel with local nitric oxide release at physiological level provides a promising therapeutic approach in enhancing the performance of biomedical implants.

An S-nitroso-N-acetylpenicillamine (SNAP) derivatization approach was used to modify existing free primary amines found in fibrin (a natural protein-based biomaterial) to generate a controlled nitric oxide (NO) releasing scaffold material. The duration of the derivatization reaction affects the NO release kinetics, the induction of controlled NO-release, hydrophobicity, swelling behavior, elastic moduli, rheometric character, and degradation behavior. These properties were quantified to determine changes in fibrin hydrogels following covalent attachment of SNAP. NO-releasing materials exhibited minimal cytotoxicity when cultured with fibroblasts or osteoblasts. Cells maintained viability and proliferative character on derivatized materials as demonstrated by Live/Dead cell staining and counting. In addition, SNAP-derivatized hydrogels exhibited an antimicrobial character indicative of NO-releasing materials. SNAP derivatization of natural polymeric biomaterials containing free primary amines offers a means to generate inducible NO-releasing biomaterials for use as an antimicrobial and regenerative support for tissue engineering.

Nitric oxide (NO) plays an important role in cardiovascular homeostasis, immune responses, and wound repair. The pro-angiogenic and antimicrobial properties of NO has stimulated the development of NO-releasing materials for wound dressings. Gelatin, an abundant natural biodegradable polymer derived from collagen, is able to promote wound repair. S-Nitroso-N-acetylpenicillamine (SNAP) can release NO under physiological conditions and when exposed to light. The objective of this project was to fabricate a NO-releasing gelatin-based nanofibrous matrix with precise light-controllable ability. Results showed that under controlled phase separation fabrication conditions, the gelatin formed a highly porous matrix with the nanofiber diameter ranging from 50 to 500 nm. Importantly, the removal of the trace amount of divalent metal ions within gelatin generated a more stable nanofibrous structure. N-acetyl-D-penicillamine (NAP) was functionalized onto the matrix and nitrosated with t-butyl nitrite, yielding a SNAP-gelatin matrix. Analysis of the photoinitiated NO-release showed that the SNAP-gelatin matrices released NO in a highly controllable manner. Application of increasing light intensities yielded increased NO flux from the matrices. In addition, the dried matrices stored in dark at 4 °C maintained stable NO storage capacity, and the purified (ion-removed) gelatin preserved higher NO-releasing capacity than nonpurified gelatin. The antibacterial effect from the SNAP-gelatin matrices was demonstrated by exposing Staphylococcus aureus ( S. aureus ) to a light-triggered NO flux. This controllable NO-releasing scaffold provides a potential antibacterial therapeutic approach to combat drug resistant bacteria.

Directly monitoring physiological analytes on a continuous, real-time basis may lead to improved patient outcomes. Although the technology to fabricate robust miniature sensors has existed for decades, these sensors fail to accurately track the true physiological status of patients after implantation in the human body. Nitric oxide (NO) may be employed to mediate the biological response toward the implanted sensors allowing the sensors to accurately and reliably monitor analytes of interest such that therapeutic decisions could be made based on sensor readings. This chapter discusses details of the biological response that results in senor failure and why NO may help control this response. Three approaches for NO production (N-diazeniumdiolates, S-nitrosothiols, and catalytic NO generation from endogenous S-nitrosothiols) and NO releasing materials that utilize these approaches which have been applied in sensor fabrication are presented. NO releasing sensors have shown greatly enhanced performance in blood contacting and tissue contacting applications in vivo.

Nitric oxide (NO) release can promote healthy tissue regeneration. A PEG-fibrinogen adhesive hydrogel that would allow for inducible NO release was created with mechanical properties that could be tailored to specific applications and tissue types. PEG (4-arm)-fibrinogen hydrogels of varying ratios were derivatized with S-nitroso-N-acetyl-D, L-penicillamine (SNAP)-thiolactone to create an active NO donor material. Controlled release from gels was established using light as the activating source, although temperature, pH, and external mechanical loading are also means to induce active NO release. Gels with varying ratios of fibrinogen to PEG were made, derivatized, and tested. Gels below a ratio of 1.5:1 (fibrinogen:PEG) did not gel, while at ratio of 1.5:1 gelation occurs and NO release can be induced. Interestingly, the release from 1.5:1 gels was significantly lower compared to 2:1 and 3:1 gel formulations. Rheometric data show that lower ratio gels are more elastic than viscous. Derivatized gels exhibited linear elastic moduli, behaving more like other more synthetic hydrogels. Swelling data indicates that as the ratio of fibrinogen to PEG increases the swelling ratio decreases, likely due to the hydrophobic nature of the NO donor. Cells remain viable on both derivatized and non-derivatized gels.

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.

Nitric oxide (NO) has been heavily studied over the past two decades due to its multitude of physiological functions and its potential therapeutic promise. Of major interest is the desire to fabricate or coat implanted devices with an NO releasing material that will impart the appropriate dose and duration of NO release to positively mediate the biological response to the medical device, thereby improving its safety and efficacy. To date, this goal has not yet been achieved, despite very promising early research. Herein, we describe the synthesis and NO release properties of a novel NO donor which covalently links the S-nitrosothiol, S-nitroso-N-acetyl-D-penicillamine (SNAP), to the macrocycle, cyclam (SNAP-cyclam). This compound can then be blended into a wide variety of polymer matrices, imparting NO release to the polymer system. This release can be initiated and controlled by transition metal catalysis, thermal degradation or photolytic release of NO from the composite NO-releasing material. SNAP-cyclam is capable of releasing physiologically relevant levels of NO for up to 3 months in vitro when blended into poly(l-lactic acid) thin films.

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 synthetic methods used recently in this laboratory to prepare a variety of novel nitric oxide (NO)-releasing hydrophobic polymers are reviewed. Nitric oxide is a well known inhibitor of platelet adhesion and activation. Thus, such NO release polymers have potential applications as thromboresistant coatings for a large number of blood-contacting biomedical devices (e.g., in vivo sensors, arteriovenous grafts, stents, catheters, extracorporeal circuits). The approaches taken to prepare NO releasing poly(vinyl chloride) (PVC), silicone rubber (SR), polymethacrylate (PM), and polyurethane (PU) materials are grouped into three categories: (1) dispersion/doping of discrete diazeniumdiolated molecules within the polymeric films; (2) chemical derivatization of polymeric filler microparticles (e.g., silicon dioxide, titanium dioxide) to possess NO release chemistry and then their dispersion within the hydrophobic polymers; and (3) covalent attachment of NO release moieties to polymer backbones. Specific chemical examples of each of these approaches are summarized and the advantages and disadvantages of each are discussed. Other related work in the field of NO release polymers is also cited. It is further shown that several of the NO-releasing polymeric materials already prepared exhibit the expected improved thromboresistivity when tested in vivo using appropriate animal models.

The widespread use of miniaturized chemical sensors to monitor clinically important analytes such as PO2, PCO2, pH, electrolytes, glucose and lactate in a continuous, real-time manner has been seriously hindered by the erratic analytical results often obtained when such devices are implanted in vivo. One major factor that has influenced the analytical performance of indwelling sensors is the biological response they elicit when in contact with blood or tissue (e.g. thrombus formation on the device surface, inflammatory response, encapsulation, etc.). Nitric oxide (NO) has been shown to be a potent inhibitor of platelet adhesion and activation as well as a promoter of wound healing in tissue. Herein, we review recent work aimed at the development of hydrophobic NO-releasing polymers that can be employed to coat catheter-type amperometric oxygen sensors without interfering with the analytical performance of these devices. Such modified sensors are shown to exhibit greatly enhanced hemocompatibility and improved analytical performance when implanted within porcine carotid and femoral arteries for up to 16 h. Further, results from preliminary studies also demonstrate that prototype fluorescent oxygen sensors, catheter-style potentiometric carbon dioxide sensors and subcutaneous needle-type enzyme-based amperometric glucose sensors can also be fabricated with new NO-release outer coatings without compromising the analytical response characteristics of these devices. The NO-release strategy may provide a solution to the lingering biocompatibility problems encountered when miniature chemical sensors are implanted in vivo.

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.

Recently, progress has been made in the development of implantable chemical sensors capable of real-time monitoring of clinically important species such as PO(2), PCO(2), pH, glucose and lactate. The need for developing truly biocompatible materials for sensor fabrication remains the most significant challenge for achieving robust and reliable sensors capable of monitoring the real-time physiological status of patients.

We have used multiple linear regression to predict either initial rate, log initial rate or specificity for enzyme-catalyzed reactions performed in non-aqueous solvents. The Subtilisin Carlsberg catalyzed transesterification of N -acetyl- l -phenylalanine ethyl ester by methanol, 1-propanol, and 1-butanol was assayed in 30 non-aqueous solvents, and the lipase catalyzed transesterification of methyl methacrylate in 23 non-aqueous solvents. Both sets of reactions were performed at fixed thermodynamic water activity. The lipase catalyzed reactions were also performed in water saturated solvents and in dry solvents. The report illustrates that regression analysis may provide insight into how solvents can alter the activity and specificity of enzymes suspended therein. A regression model for the subtilisin catalyzed reaction suggests that solvents which have a flat hydrophobic region inhibit by competing with the substrate for an enzyme cleft. In the lipase catalyzed reaction, tetrachloroethylene is an outlier (i.e., behaves differently to other solvents) for all the regression models. This deviation, together with an element of structural similarity to the substrate, suggests that tetrachloroethylene acts as a competitive inhibitor. Log P is an important descriptor and it, or an expression containing log P , appears in all the regression equations. Log initial rate is predicted by a two-descriptor model for either enzyme system in solvents of high log P at fixed thermodynamic water activity. Regression models with the same two descriptors predict initial rate for the lipase system over the entire log P range for solvents maintained at fixed thermodynamic water activity and for dry solvents, but not for water saturated solvents.

Either a second order or a third polynomial equation accurately predicts R F in a mobile phase which is a binary mixture of a strong and weak solvent. The mole fraction of the strong solvent is used as the independent variable. The p -nitrobenzyl esters of fifteen dansyl amino acids were used as model solutes for planar chromatography, using each of five different mobile phases on silica gel layers. For either of the polynomial equations, a minimum of 98% of the predicted R F values, at mole fractions not used for establishing the equations, are within 0.05 or less of an R F unit (when compared to the experimental R F ), and 90% of the predictions are within 0.03 or less of an R F unit.

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.

A highly water-soluble BODIPY dye bearing electron-rich o-diaminophenyl groups at 2,6-positions was prepared as a highly sensitive and selective fluorescent probe for detection of nitric oxide (NO) in living cells. The fluorescent probe displays an extremely weak fluorescence with fluorescence quantum yield of 0.001 in 10 mM phosphate buffer (pH 7.0) in the absence of NO as two electron-rich o-diaminophenyl groups at 2,6-positions significantly quench the fluorescence of the BODIPY dye via photoinduced electron transfer mechanism. The presence of NO in cells enhances the dye fluorescence dramatically. The fluorescent probe demonstrates excellent water solubility, membrane permeability, and compatibility with living cells for sensitive detection of NO.

Despite the documented potential to leverage nitric oxide generation to improve in vivo performance of implanted devices, a key limitation to current NO releasing materials tested thus far is that there has not been a means to modulate the level of NO release after it has been initiated. We report the fabrication of a wireless platform that uses light to release NO from a polymethylmethacrylate (PMMA) optical fiber coated with an S -nitroso- N -acetylpenicillamine derivatized polydimethylsiloxane (SNAP-PDMS). We demonstrate that a VAOL-5GSBY4 LED ( λ dominant = 460 nm) can be used as a dynamic trigger to vary the level of NO released from 500 μm diameter coated PMMA. The ability to generate programmable sequences of NO flux from the surface of these coated fibers offers precise spatial and temporal control over NO release and provides a platform to begin the systematic study of in vivo physiological response to implanted devices. NO surface fluxes up to 3.88 ± 0.57 × 10 −10 mol cm −2 min −1 were achieved with ∼100 μm thick coatings on the fibers and NO flux was pulsed, ramped and held steady using the wireless platform developed. We demonstrate the NO release is linearly proportional to the drive current applied to the LED (and therefore level of light produced from the LED). This system allow the surface flux of NO from the fibers to be continuously changed, providing a means to determine the level and duration of NO needed to mediate physiological response to blood contacting and subcutaneous implants and will ultimately lead to the intelligent design of NO releasing materials tailored to specific patterns of NO release needed to achieve reliable in vivo performance for intravascular and subcutaneous sensors and potentially for a wide variety of other implanted biomedical devices.

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.

The gold standard for anticoagulation during extracorporeal circulation (ECC) remains systemic heparinization and the concomitant risk of bleeding in an already critically ill patient could lead to death. Normal endothelium is a unique surface that prevents thrombosis by the release of antiplatelet and antithrombin agents. Nitric oxide (NO) is one of the most potent, reversible antiplatelet agents released from the endothelium. Nitric oxide released from within a polymer matrix has been proven effective for preventing platelet activation and adhesion onto extracorporeal circuits. However, the critical NO release (NO flux) threshold for thrombus prevention during ECC has not yet been determined.1 Using a 4-hour arteriovenous (AV) rabbit model of ECC,2 we sought to find this threshold value for ECC circuits, using an improved NO-releasing coating ( Norel-b ). Four groups of animals were tested at variable NO flux levels. Hourly blood samples were obtained for measurement of arterial blood gases, platelet counts, fibrinogen levels and platelet function (via aggregometry). A custom-built AV circuit was constructed with 36 cm of poly(vinyl)chloride (PVC) tubing, a 14 gauge (GA) angiocatheter for arterial access and a modified 10 French (Fr) thoracic catheter for venous access. The Norel-b coating reduced platelet activation and thrombus formation, and preserved platelet function — in all circuits that exhibited an NO flux of 13.65 × 10— 10 mol·cm—2·min—1. These results were significant when compared with the controls. With the Norel-b coating, the NO flux from the extracorporeal circuit surface can be precisely controlled by the composition of the polymer coating used, and such coatings are shown to prevent platelet consumption and thrombus formation while preserving platelet function in the animal. Perfusion (2007) 22, 193—200.

This report describes the first hydrophobic nitric oxide (NO)-releasing material that utilizes light as an external on/off trigger to control the flux of NO generated from cured polymer films. Fumed silica polymer filler particles were derivatized with S-nitroso-N-acetyl-dl-penicillamine and blended into the center layer of trilayer silicone rubber films. Nitric oxide is generated upon irradiation with light, and fluxes increase with increasing power of incident light. The ability to precisely control NO generation from this material has the potential to answer fundamental questions about the levels of NO needed to achieve desired therapeutic affects in different biomedical applications.

Publisher Summary This chapter describes the procedure employed to construct functional NO-releasing catheter-type amperometric oxygen sensors, including coating the sensor with NO-release materials, assembly of the sensor itself, and in vivo evaluation of the analytical performance and hemocompatibility of the device. The procedure describing the specific fabrication of an intravascular catheter-type sensor that is introduced into an artery via a 14-gauge, 1.16-in. angiocath isdiscussed. In this procedure, the sensor is fixed into a four-way stopcock to allow a means to attach the sensor to the catheter securely and to introduce a saline drip to prevent blood from pooling in the catheter. The length and diameter of the sensor can be adjusted by selecting tubing of the appropriate diameters and adjusting lengths of wires and the sensor body to accommodate a wide variety of sizes and specific methods of introducing the sensor into the blood vessel or tubing to be monitored. Material and procedure for coating sensor sleeves, sensor fabrication, and concepts related to sensor calibration and use are also described.

A synthetic path for the preparation of methacrylic homo- and copolymers containing secondary amine groups that can be converted into nitric oxide (NO) releasing N-diazeniumdiolates is described. The polymers are obtained by a multistep procedure involving synthesis of methacrylate monomers containing boc-protected secondary amine sites, free radical benzoyl peroxide initiated polymerization, deprotection of the amine sites, and subsequent reaction of the polymers with NO in the presence of sodium methoxide. Monomers with both linear and cyclic pendant secondary amines are examined as polymer building blocks. In most cases, polymers are obtained for both types with compositions that agree well with initial monomer ratios and with number average molecular weights (M(n)) ranging from 1.69 to 2.58 x 10(6) Da. The final N-diazeniumdiolated methacrylic amine polymers are shown to release NO for extended periods of time with "apparent" t(1/2) values ranging from 30 to 60 min when suspended in phosphate buffer, pH 7.4. Total NO loading and release for these materials can reach 1.99 micromol per mg of polymer and is proportional to the amine content of the polymer. It is further shown that by using a dimethacrylate cross-linking agent in conjunction with the various methacrylate amines, suspension polymerization methods can be employed to create small (100-200 microm) polymeric methacrylate microbeads. Such microbeads that can be sequentially deprotected and converted to NO release particles via in-situ diazeniumdiolate formation as carried out for the non-crosslinked polymers.

Planar chromatography with electroosmotic flow is used to separate either a mixture of dyes using 80% aqueous ethanol as the mobile phase or a mixture of miscellaneous compounds using 45% aqueous acetonitrile as the mobile phase. Both mobile phases are 1.0 mM in N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS) buffer. Separations using this technique are faster and more efficient than the same separations by conventional TLC. The respective relationships between migration velocity and applied potential, and between analysis time and distance migrated, are presented.

The purpose of this work was to develop a simple and robustmethod that would allow a stable gelatin layer to form on PDMS that is suitable for cell culturing. Treating the cured PDMS layer with (3-(2-aminoethylamino) propyl) trimethoxysilane and then casting 2 layers of 2% (w/v) gelatin on the surface created a stable layer. The water contact angle of the formed layer system is in close agreement with reported water contact angle values for standard gelatin layers. The pH of water on the gelatincoated PDMS was within physiological pH range but slightly basic. A relative hardness test showed that the hardness of the film was unchanged after silanization, allowing the PDMS to retain its mechanical properties. A fluorescence test for soluble gelatin indicated that less than 8% of gelatin was released from the surface after a 24 hour period of PBS soaking at 37° C, which is nearly identical to the amount of gelatin that came off a standard gelatin coated coverslip. MOVAS-1 cells were grown for 96 hours on the modified surfaces in a manner identical to control coverslips coated with gelatin. These results validate the use of a silanizing agent, such as (3-(2-aminoethylamino) propyl) trimethoxysilane, as an interfacing method between PDMS and gelatin, and allows appropriate films that are robust to be formed,thereby allow for cell growth and proliferation on a PDMS base material.

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.