Peter N. Coneski

Nitric oxide release: Part III. Measurement and reporting

Nitric oxides expansive physiological and regulatory roles have driven the development of therapies for human disease that would benefit from exogenous NO administration. Already a number of therapies utilizing gaseous NO or NO donors capable of storing and delivering NO have been proposed and designed to exploit NOs influence on the cardiovascular system, cancer biology, the immune response, and wound healing. As described in Nitric oxide release: Part I. Macromolecular scaffolds and Part II. Therapeutic applications, the preparation of new NO-release strategies/formulations and the study of their therapeutic utility are increasing rapidly. However, comparison of such studies remains difficult due to the diversity of scaffolds, NO measurement strategies, and reporting methods employed across disciplines. This tutorial review highlights useful analytical techniques for the detection and measurement of NO. We also stress the importance of reporting NO delivery characteristics to allow appropriate comparison of NO between studies as a function of material and intended application.

Inaccuracies of nitric oxide measurement methods in biological media.

Despite growing reports on the biological action of nitric oxide (NO) as a function of NO payload, the validity of such work is often questionable due to the manner in which NO is measured and/or the solution composition in which NO is quantified. To highlight the importance of measurement technique for a given sample type, NO produced from a small-molecule NO donor (N-diazeniumdiolated l-proline, PROLI/NO) and a NO-releasing xerogel film were quantified in a number of physiological buffers and fluids, cell culture media, and bacterial broth by the Griess assay, a chemiluminescence analyzer, and an amperometric NO sensor. Despite widespread use, the Griess assay proved to be inaccurate for measuring NO in many of the media tested. In contrast, the chemiluminescence analyzer provided superb kinetic information in most buffers but was impractical for NO analysis in proteinaceous media. The electrochemical NO sensor enabled greater flexibility across the various media with potential for spatial resolution, albeit at lower than expected NO totals versus either the Griess assay or chemiluminescence. The results of this study highlight the importance of measurement strategy for accurate NO analysis and reporting NO-based biological activity.

Degradable Nitric Oxide-Releasing Biomaterials via Post-Polymerization Functionalization of Cross-Linked Polyesters

The synthesis of diverse nitric oxide (NO)-releasing network polyesters is described. The melt phase condensation of polyols with a calculated excess of diacid followed by thermal curing generates cross-linked polyesters containing acid end groups. Varying the composition and curing temperatures of the polyesters resulted in materials with tunable thermal and degradation properties. Glass transition temperatures for the synthesized materials range from -25.5 to 3.2 °C, while complete degradation of these polyesters occurs within a minimum of nine weeks under physiological conditions (pH 7.4, 37 °C). Post-polymerization coupling of aminothiols to terminal carboxylic acids generate thiol-containing polyesters, with thermal and degradation characteristics similar to those of the parent polyesters. After nitrosation, these materials are capable of releasing up to 0.81 μmol NO cm(-2) for up to 6 d. The utility of the polyesters as antibacterial biomaterials was indicated by an 80% reduction of Pseudomonas aeruginosa adhesion compared to unmodified controls.

Nitric Oxide-Releasing Electrospun Polymer Microfibers

The preparation of electrospun polymer microfibers with nitric oxide (NO)-release capabilities is described. Polymer solutions containing disodium 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO), a low-molecular-weight NO donor, were electrospun to generate fibers ranging from 100-3000 nm in diameter capable of releasing NO upon immersion in aqueous solutions under physiological conditions (pH 7.4, 37 °C), with kinetics depending on polymer composition and fiber diameter. The NO release half-life for PROLI/NO-doped electrospun fibers was 2-200 times longer than that of PROLI/NO alone. The influence of polymer concentration, applied voltage, capillary diameter, solution conductivity, flow rate, and additives on fiber properties are reported and discussed with respect to potential applications.

Photoinitiated Nitric Oxide-Releasing Tertiary S-Nitrosothiol-Modified Xerogels

The synthesis of a tertiary thiol-bearing silane precursor (i.e., N-acetyl penicillamine propyltrimethoxysilane or NAPTMS) to enable enhanced NO storage stability at physiological temperature is described. The novel silane was co-condensed with alkoxy- or alkylalkoxysilanes under varied synthetic parameters (e.g., water to silane ratio, catalyst and solvent concentrations, and reaction time) to evaluate systematically the formation of stable xerogel films. The resulting xerogels were subsequently nitrosated to yield tertiary RSNO-modified coatings. Total NO storage ranged from 0.87 to 1.78 μmol cm(-2) depending on the NAPTMS concentration and xerogel coating thickness. Steric hindrance near the nitroso functionality necessitated the use of photolysis to liberate NO. The average NO flux for irradiated xerogels (20% NAPTMS balance TEOS xerogel film cast using 30 μL) in physiological buffer at 37 °C was ∼23 pmol cm(-2) s(-1). The biomedical utility of the photoinitiated NO-releasing films was illustrated by their ability to both reduce Pseudomonas aeruginosa adhesion by ∼90% relative to control interfaces and eradicate the adhered bacteria.

Synthesis of nitric oxide-releasing polyurethanes with S-nitrosothiol-containing hard and soft segments

Nitric oxide (NO)-releasing polyurethanes capable of releasing up to 0.20 μmol NO cm(-2) were synthesized by incorporating active S-nitrosothiol functionalities into hard and soft segment domains using thiol group protection and post-polymerization modifications, respectively. The nitrosothiol position within the hard and soft segment domains of the polyurethanes impacted both the total NO release and NO release kinetics. The NO storage and release properties were correlated to both chain extender modification and ensuing phase miscibility of the polyurethanes. Thorough material characterization is provided to examine the effects of hard and soft segment modifications on the resultant polyurethane properties.

Synergy of nitric oxide and silver sulfadiazine against gram-negative, gram-positive, and antibiotic-resistant pathogens.

The synergistic activity between nitric oxide (NO) released from diazeniumdiolate-modified proline (PROLI/NO) and silver(I) sulfadiazine (AgSD) was evaluated against Escherichia coli, Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis using a modified broth microdilution technique and a checkerboard-type assay. The combination of NO and AgSD was defined as synergistic when the fractional bactericidal concentration (FBC) was calculated to be <0.5. Gram-negative species were generally more susceptible to the individual antimicrobial agents than the Gram-positive bacteria, while Gram-positive bacteria were more susceptible to combination therapy. The in vitro synergistic activity of AgSD and NO observed against a range of pathogens strongly supports future investigation of this therapeutic combination, particularly for its potential use in the treatment of burns and chronic wounds.

Enhancing the Fouling Resistance of Biocidal Urethane Coatings via Surface Chemistry Modulation

A group of novel cross-linked polyurethane materials with varying ratios of hydroxyl-terminated macrodiols and tethered quaternary ammonium biocides have been prepared. The resulting materials had a wide range of thermal, mechanical, and surface properties, dictated by the macrodiol composition and biocide concentration. The complex interplay between surface chemistry and biocide concentration was shown to have a profound effect on the fouling resistance of these materials. While the combination of quaternary ammonium salt (QAS) diols with poly(tetramethylene oxide) macrodiols did not result in any enhancement of fouling resistance, addition of biocides to poly(ethylene glycol)-containing urethanes resulted in up to a 90% increase in biocidal activity compared to control materials while reducing the ability for microbes to adhere to the surface by an additional 60%. Materials prepared with polybutadiene macrodiols underwent a thermally induced oxidation, resulting in partial decomposition of the quaternary ammonium salt biocide and joint antimicrobial activity arising from remaining QAS and peroxide compounds.

Competitive Formation of N-Diazeniumdiolates and N-Nitrosamines via Anaerobic Reactions of Polyamines with Nitric Oxide

Reactions of amines with nitric oxide (NO) at high pressures form diverse NO donor species, highly dependent on the precursor structure. While monoamine precursors favor the formation of N-diazeniumdiolates in high yield, polyamines exhibit competitive formation of N-nitrosamines and diazeniumdiolates, resulting in mixed products containing significant percentages of undesired N-nitroso compounds.

Decontamination of chemical-warfare agent simulants by polymer surfaces doped with the singlet oxygen generator zinc octaphenoxyphthalocyanine.

Using reactive singlet oxygen (1O2), the oxidation of chemical-warfare agent (CWA) simulants has been demonstrated. The zinc octaphenoxyphthalocyanine (ZnOPPc) complex was demonstrated to be an efficient photosensitizer for converting molecular oxygen (O2) to 1O2 using broad-spectrum light (450-800 nm) from a 250 W halogen lamp. This photosensitization produces 1O2 in solution as well as within polymer matrices. The oxidation of 1-naphthol to naphthoquinone was used to monitor the rate of 1O2 generation in the commercially available polymer film Hydrothane that incorporates ZnOPPc. Using electrospinning, nanofibers of ZnOPPc in Hydrothane and polycarbonate were formed and analyzed for their ability to oxidize demeton-S, a CWA simulant, on the surface of the polymers and were found to have similar reactivity as their corresponding films. The Hydrothane films were then used to oxidize CWA simulants malathion, 2-chloroethyl phenyl sulfide (CEPS), and 2-chloroethyl ethyl sulfide (CEES). Through this oxidation process, the CWA simulants are converted into less toxic compounds, thus decontaminating the surface using only O2 from the air and light.