Design and Synthesis of Nitric Oxide Releasing Polymers for Biomedical Applications

Poor biocompatibility is an ongoing problem for almost all types of implanted medical devices. The adhesion of cells and proteins at implant surfaces often results in serious complications such as infection, scar tissue formation, and thrombosis. The mediation of these effects by the endogenously produced free radical nitric oxide (NO) support its use a biocompatibility agent for many types of implantable materials. My dissertation research has focused on the development of materials capable of controllably releasing NO to facilitate implant compatibility. To examine the influence of N-diazeniumdiolate structure on NO release characteristics and their physical retention within polymeric matrices, various dialkyl diamines were synthesized and covalently modified to store NO. Diazeniumdiolation reactions of the synthesized diamines however resulted in the competitive formation of both N-diazeniumdiolates and potentially carcinogenic N-nitrosamines. Amine spacing and total alkyl content of these polymer additives were investigated as a means to control the efficiency of the diazeniumdiolation reaction. The ability of these amine compounds to form stabilizing hydrogen bonds to a nitrosamine intermediate species controlled the overall efficiency of diazeniumdiolation, with compounds more capable of forming intermolecular hydrogen bonds resulting in greater nitrosamine content. Monoamine compounds were shown to predominantly form diazeniumdiolated products due to their inability to form stabilizing intermolecular hydrogen bonds. As the characteristics of biomedical implants are closely selected based on their intended application, supplementing material properties is not a universal task. To extend the therapeutic benefits of NO release to degradable materials, a group of absorbable NO-releasing polyesters was synthesized. Highly crosslinked polyesters were formed by the thermal polycondensation and curing of polyols with diacid compounds, followed by a thiol functionalization step. Thiol-modified polymers could then be modified to store and controllably release NO via S-nitrosothiol functionalities. The ability of these materials to store and release NO was dependent on the selection of starting materials, curing temperatures, and the material's glass transition temperature. Reduced bacterial adhesion was observed for all NO-releasing polyesters over controls, with materials capable of releasing higher amounts of NO providing greater antibacterial character. Synthesizing these polymers from metabolic intermediates and non-toxic compounds resulted in products with minimal toxicity to healthy mammalian cells. Further diversification of NO-releasing materials was provided by the synthesis and characterization of S-nitrosothiol-modified polyurethanes. The modification of both hard and soft segment domains of polyurethanes was shown to result in the formation of NO-releasing polyurethane species. The extent and characteristics of NO release were shown to be highly dependent on NO donor position along the polymer backbone, with more substantial NO release resulting from soft segment modified materials. Increased phase miscibilities were shown to occur as a result of specific hard segment modifications, which in turn influenced the extent of NO release. Finally, the fabrication of electrospun polymer microfibers with NO release capabilities is reported. The physical dispersion of various NO donating materials such as nanoparticles and low molecular weight compounds was investigated as a means to controllably deliver NO to physiological environments. The release of NO from these scaffolds was shown to be diffusion mediated both in terms of solution uptake into the fibers and diffusion of NO out of the fiber. Hydrophobic microfibers exhibited prolonged NO release durations compared to hydrophilic materials as they inhibited solution uptake into fibers regulating the rate of diazeniumdiolate decomposition. As a result, microfiber diameter also influenced the rate of NO release from fibers due to greater diffusion pathways required to trigger NO release via diazeniumdiolate protonation

Nitric oxide release: Part III. Measurement and reporting

Nitric oxide’s 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 NO’s 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

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

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

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 crosslinked 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 °C 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

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 using 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

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- and 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 0.87–1.78 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 in physiological buffer at 37 °C was ~23 pmol cm−2 s−1 (20% NAPTMS balance TEOS xerogel film cast using 30 µL). The biomedical utility of the photo-initiated 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

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. 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 chronic and burn wounds

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

Zwitterionic Polyurethane Hydrogels Derived from Carboxybetaine-Functionalized Diols

The synthesis of novel zwitterionic polyurethane hydrogels with tunable water uptake via the polymerization of protected carboxybetaine-functionalized diols with polyisocyanate oligomers is presented. Post-polymerization hydrolysis of a diol-segment side chain establishes zwitterionic carboxybetaine functionalities that facilitate water uptake via the enhanced hydration capacities surrounding the opposing charges of the diol component. Tunable hydration of these materials, ranging from 24 to 250% solution uptake (based on the dry polymer weight), is achieved by controlling the structural characteristics of the diol precursor, such as ammonium/carboxylate spacing and ethyl ester hydrolysis conditions (i.e., exposure time to an aqueous base)