Nitric Oxide-Induced Morphological Changes to Bacteria.

Antimicrobial resistance poses a serious threat to global health, necessitating research for alternative approaches to treating infections. Nitric oxide (NO) is an endogenously produced molecule involved in multiple physiological processes, including the response to pathogens. Herein, we employed microscopy- and fluorescence-based techniques to investigate the effects of NO delivered from exogenous NO donors on the bacterial cell envelopes of pathogens, including resistant strains. Our goal was to assess the role of NO donor architecture (small molecules, oligosaccharides, dendrimers) on bacterial wall degradation to representative Gram-negative bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecium) upon treatment. Depending on the NO donor, bactericidal NO doses spanned 1.5-5.5 mM (total NO released). Transmission electron microscopy of bacteria following NO exposure indicated extensive membrane damage to Gram-negative bacteria with warping of the cellular shape and disruption of the cell wall. Among the small-molecule NO donors, those providing a more extended release (t1/2 = 120 min) resulted in greater damage to Gram-negative bacteria. In contrast, rapid NO release (t1/2 = 24 min) altered neither the morphology nor the roughness of these bacteria. For Gram-positive bacteria, NO treatments did not result in any drastic change to cellular shape or membrane integrity, despite permeation of the cell wall as measured by depolarization assays. The use of positively charged quaternary ammonium (QA)-modified NO-releasing dendrimer proved to be the only NO donor system capable of penetrating the thick peptidoglycan layer of Gram-positive bacteria.

Role of Nitric Oxide-Releasing Glycosaminoglycans in Wound Healing.

Two glycosaminoglycan (GAG) biopolymers, hyaluronic acid (HA) and chondroitin sulfate (CS), were chemically modified via carbodiimide chemistry to facilitate the loading and release of nitric oxide (NO) to develop a multi-action wound healing agent. The resulting NO-releasing GAGs released 0.2-0.9 µmol NO mg-1 GAG into simulated wound fluid with NO-release half-lives ranging from 20 to 110 min. GAGs containing alkylamines with terminal primary amines and displaying intermediate NO-release kinetics exhibited potent, broad spectrum bactericidal action against three strains each of Pseudomonas aeruginosa and Staphylococcus aureus ranging in antibiotic resistance profile. NO loading of the GAGs was also found to decrease murine TLR4 activation, suggesting that the therapeutic exhibits anti-inflammatory mechanisms. In vitro adhesion and proliferation assays utilizing human dermal fibroblasts and human epidermal keratinocytes displayed differences as a function of the GAG backbone, alkylamine identity, and NO-release properties. In combination with antibacterial properties, the adhesion and proliferation profiles of the GAG derivatives enabled the selection of the most promising wound healing candidates for subsequent in vivo studies. A P. aeruginosa-infected murine wound model revealed the benefits of CS over HA as a pro-wound healing NO donor scaffold, with benefits of accelerated wound closure and decreased bacterial burden attributable to both active NO release and the biopolymer backbone.