Nitric Oxide-Releasing Hyaluronic Acid as an Antibacterial Agent for Wound Therapy.

Taking advantage of their respective wound-healing roles in physiology, the dual activity of hyaluronic acid (HA) and nitric oxide (NO) was combined to create a single-agent wound therapeutic. Carboxylic acid groups of HA (6 and 90 kDa) were chemically modified with a series of alkylamines via carbodiimide chemistry to provide secondary amines for subsequent N-diazeniumdiolate NO donor formation. The resulting NO-releasing HA derivatives stored 0.3-0.6 µmol NO mg-1 and displayed diverse release kinetics (5-75 min NO-release half-lives) under physiological conditions. The 6 kDa HA with terminal primary amines and intermediate release kinetics exhibited broad-spectrum bactericidal activity against common wound pathogens, including planktonic methicillin-resistant Staphylococcus aureus as well as planktonic and biofilm-based multidrug-resistant Pseudomonas aeruginosa. The treatment of infected murine wounds with NO-releasing HA facilitated more rapid wound closure and decreased the quantity of the P. aeruginosa genetic material in the remaining wound tissue. Hyaluronidase readily degraded the HA derivatives, indicating that NO donor modification did not prohibit endogenous biodegradation pathways.

Nitric Oxide-Releasing Cyclodextrins.

A series of secondary amine-modified cyclodextrin (CD) derivatives was synthesized with diverse exterior terminal groups (i.e., hydroxyl, methyl, methoxyl, and primary amine). Subsequent reaction with nitric oxide (NO) gas under alkaline conditions yielded N-diazeniumdiolate-modified CD derivatives. Adjustable NO payloads (0.6-2.4 µmol/mg) and release half-lives (0.7-4.2 h) were achieved by regulating both the amount of secondary amine precursors and the functional groups around the NO donors. The bactericidal action of these NO-releasing cyclodextrin derivatives was evaluated against Pseudomonas aeruginosa, a Gram-negative pathogen, with antibacterial activity proving dependent on both the NO payload and exterior modification. Materials containing a high density of NO donors or primary amines exhibited the greatest ability to eradicate P. aeruginosa. Of the materials prepared, only the primary amine-terminated heptasubstituted CD derivatives exhibited toxicity against mammalian L929 mouse fibroblast cells. The NO donor-modified CD was also capable of delivering promethazine, a hydrophobic drug, thus demonstrating potential as a dual-drug-releasing therapeutic.

Nitric Oxide-Releasing Alginates.

Low and high molecular weight alginate biopolymers were chemically modified to store and release potentially therapeutic levels of nitric oxide (NO). Carbodiimide chemistry was first used to modify carboxylic acid functional groups with a series of small molecule alkyl amines. The resulting secondary amines were subsequently converted to N-diazeniumdiolate NO donors via reaction with NO gas under basic conditions. NO donor-modified alginates stored between 0.4-0.6 µmol NO·mg-1. In aqueous solution, the NO-release kinetics were diverse (0.3-13 h half-lives), dependent on the precursor amine structure. The liberated NO showed bactericidal activity against Pseudomonas aeruginosa and Staphylococcus aureus with pathogen eradication efficiency dependent on both molecular weight and NO-release kinetics. The combination of lower molecular weight (∼5 kDa) alginates with moderate NO-release durations (half-life of ∼4 h) resulted in enhanced killing of both planktonic and biofilm-based bacteria. Toxicity against human respiratory epithelial (A549) cells proved negligible at NO-releasing alginate concentrations required to achieve a 5-log reduction in viability in the biofilm eradication assay.

Mode of nitric oxide delivery affects antibacterial action.

Nitric oxide (NO) is a broad-spectrum antibacterial agent, making it an attractive alternative to traditional antibiotics for treating infections. To date, a direct comparison of the antibacterial activity of gaseous NO (gNO) versus water-soluble NO-releasing biopolymers has not been reported. In this study, the bactericidal action of NO-releasing chitosan oligosaccharides was compared to gNO treatment against cystic fibrosis-relevant Gram-positive and Gram-negative bacteria. A NO exposure chamber was constructed to enable the dosing of bacteria with gNO at concentrations up to 800 ppm under both aerobic and anaerobic conditions. Bacteria viability, solution properties (i.e., pH, NO concentration), and toxicity to mammalian cells were monitored to ensure a thorough understanding of bactericidal action and reproducibility for each delivery method. The NO-releasing chitosan oligosaccharides required significantly lower NO doses relative to gNO therapy to elicit antibacterial action against Pseudomonas aeruginosa and Staphylococcus aureus under both aerobic and anaerobic conditions. Reduced NO doses required for bacteria eradication using water-soluble NO-releasing chitosan were attributed to the release of NO in solution, removing the need to transfer from gas to liquid phase and the associated long diffusion distances of gNO treatment.

Antibiofilm Efficacy of Nitric Oxide-Releasing Alginates against Cystic Fibrosis Bacterial Pathogens.

Colonization of the lungs by biofilm-forming pathogens is a major cause of mortality in cystic fibrosis (CF). In CF patients, these pathogens are difficult to treat due to the additional protection provided by both the biofilm exopolysaccharide matrix and thick, viscous mucus. The antibiofilm efficacy of nitric oxide (NO)-releasing alginates was evaluated against Pseudomonas aeruginosa, Burkholderia cepacia, Staphylococcus aureus, and methicillin-resistant S. aureus biofilms in both aerobic and anaerobic environments. Varying the amine precursor grafted onto alginate oligosaccharides imparted tunable NO storage (∼0.1-0.3 µmol/mg) and release kinetics (∼4-40 min half-lives) in the artificial sputum media used for biofilm testing. The NO-releasing alginates were highly antibacterial against the four CF-relevant pathogens, achieving a 5-log reduction in biofilm viability after 24 h of treatment, with biocidal efficacy dependent on NO-release kinetics. Aerobic biofilms required greater starting NO doses to achieve killing relative to the anaerobic biofilms. Relative to tobramycin (the minimum concentration of antibacterial agent required to achieve a 5-log reduction in viability after 24 h, MBEC24h, of ≥2000 µg/mL) and vancomycin (MBEC24h ≥ 1000 µg/mL), the NO-releasing alginates proved to be more effective (NO dose ≤ 520 µg/mL) regardless of growth conditions.

Nitric oxide-releasing alginates as mucolytic agents.

The excessive production of thick, viscous mucus in severe respiratory diseases leads to obstruction of the airways and provides a suitable environment for the colonization of pathogenic bacteria. The effect of nitric oxide (NO)-releasing alginates with varying NO release kinetics on the viscoelastic properties of human bronchial epithelial (HBE) mucus was evaluated as a function of the NO-release kinetics using parallel plate rheology. Low molecular weight (~5 kDa) alginates with high NO flux (~4000 ppb/mg) and sustained release (half-life ~0.3 h) proved to be most effective in reducing both mucus elasticity and viscosity (≥60% reduction for both). The efficacy of the NO-releasing alginates was shown to be dose-dependent, with high concentrations of NO-releasing alginates (~80 mg•mL-1) resulting in greater reduction of the viscosity and elasticity of the mucus samples. Greater reduction in mucus rheology was also achieved with NO-releasing alginates at lower concentrations when compared to both NO-releasing chitosan, a similarly biocompatible cationic polymer, and N-acetyl cysteine (NAC), a conventional mucolytic agent.

Nitric Oxide-Releasing Macromolecular Scaffolds for Antibacterial Applications.

Exogenous nitric oxide (NO) represents an attractive antibacterial agent because of its ability to both disperse and directly kill bacterial biofilms while avoiding resistance. Due to the challenges associated with administering gaseous NO, NO-releasing macromolecular scaffolds are developed to facilitate NO delivery. This progress report describes the rational design and application of NO-releasing macromolecular scaffolds as antibacterial therapeutics. Special consideration is given to the role of the physicochemical properties of the NO storage vehicles on antibacterial or anti-biofilm activity.

Role of Nitric Oxide-Releasing Chitosan Oligosaccharides on Mucus Viscoelasticity.

Nitric oxide (NO)-releasing chitosan oligosaccharides were modified with ester functional groups to examine how the mucoadhesive nature of the scaffold impacts the ability of NO to degrade mucins from human bronchial epithelial cell cultures and clinical sputum samples collected from patients with cystic fibrosis (CF). Agarose gel electrophoresis experiments indicated that the mucoadhesive NO-releasing chitosan oligosaccharides degraded both the purified mucins and sputum, while control scaffolds (without NO release or mucoadhesive ligands) had no effect on mucin structure. Microscopic observations of sputum treated with the mucoadhesive NO-releasing chitosan oligosaccharide confirmed degradation of the mucin and DNA networks. Similarly, the viscosity and elasticity of sputum were reduced upon treatment with the mucoadhesive NO-releasing chitosan, demonstrating the potential utility of these NO-releasing scaffolds as mucolytic agents.

S-nitrosothiol-modified hyperbranched polyesters.

Hyperbranched polyesters with a range of exterior thiol modifications were synthesized through a Michael addition thiol-ene reaction. S-Nitrosothiol nitric oxide (NO) donors were subsequently introduced onto the scaffolds to yield NO-releasing polyesters with total NO storage of ~2.0 µmol mg-1. Multiple decomposition pathways (i.e., use of light, copper ions, and heat) triggered S-nitrosothiol NO donor breakdown and NO release under physiological conditions (37 °C, pH 7.4). The NO-releasing polyesters were characterized as a function of chemical modification and scaffold size or generation. The approaches described herein expand the scope of biodegradable NO-releasing materials with large NO payloads.