Analysis of the broad-spectrum potential of nitric oxide for antibacterial activity against clinically isolated drug-resistant bacteria.

The development of drug-resistant microorganisms is taking a heavy toll on the biomedical world. Clinical infections are costly and becoming increasingly dangerous as bacteria that once responded to standard antibiotic treatment are developing resistance mechanisms that require innovative treatment strategies. Nitric oxide (NO) is a gaseous molecule produced endogenously that has shown potent antibacterial capabilities in numerous research studies. Its multimechanistic antibacterial methods prevent the development of resistance and have shown potential as an alternative to antibiotics. However, there has yet to be a direct comparison study evaluating the antibacterial properties of NO against antibiotic susceptible and antibiotic-resistant clinically isolated bacterial strains. Herein, standardized lab and clinically isolated drug-resistant bacterial strains are compared side-by-side for growth and viability following treatment with NO released from S-nitrosoglutathione (GSNO), an NO donor molecule. Evaluation of growth kinetics revealed complete killing of E. coli lab and clinical strains at 17.5 mM GSNO, though 15 mM displayed >50% killing and significantly reduced metabolic activity, with greater dose dependence for membrane permeability. Clinical P. aeruginosa showed greater susceptibility to GSNO during growth curve studies, but metabolic activity and membrane permeability demonstrated similar effects for 12.5 mM GSNO treatment of lab and clinical strains. MRSA lab and clinical strains exhibited total killing at 17.5 mM treatment, though metabolic activity was decreased, and membrane permeation began at 12.5 mM for both strains. Lastly, both S. epidermidis strains were killed by 15 mM GSNO, with sensitivities in metabolic activity and membrane permeability at 12.5 mM GSNO. The mirrored antibacterial effects seen by the lab and clinical strains of two Gram-negative and two Gram-positive bacteria reveal the translational success of NO as an antibacterial therapy and potential alternative to standard antibiotic treatment.

Bioinspired superhydrophobic surfaces with silver and nitric oxide-releasing capabilities to prevent device-associated infections and thrombosis.

Bacteria-associated infections and thrombus formation are the two major complications plaguing the application of blood-contacting medical devices. Therefore, functionalized surfaces and drug delivery for passive and active antifouling strategies have been employed. Herein, we report the novel integration of bio-inspired superhydrophobicity with nitric oxide release to obtain a functional polymeric material with anti-thrombogenic and antimicrobial characteristics. The nitric oxide release acts as an antimicrobial agent and platelet inhibitor, while the superhydrophobic components prevent non-specific biofouling. Widely used medical-grade silicone rubber (SR) substrates that are known to be susceptible to biofilm and thrombus formation were dip-coated with fluorinated silicon dioxide (SiO2) and silver (Ag) nanoparticles (NPs) using an adhesive polymer as a binder. Thereafter, the resulting superhydrophobic (SH) SR substrates were impregnated with S-nitroso-N-acetylpenicillamine (SNAP, an NO donor) to obtain a superhydrophobic, Ag-bound, NO-releasing (SH-SiAgNO) surface. The SH-SiAgNO surfaces had the lowest amount of viable adhered E. coli (> 99.9 % reduction), S. aureus (> 99.8 % reduction), and platelets (> 96.1 % reduction) as compared to controls while demonstrating no cytotoxic effects on fibroblast cells. Thus, this innovative approach is the first to combine SNAP with an antifouling SH polymer surface that possesses the immense potential to minimize medical device-associated complications without using conventional systemic anticoagulation and antibiotic treatments.

Chemical Modification of Tiopronin for Dual Management of Cystinuria and Associated Bacterial Infections.

Cystinuria is an inherited autosomal recessive disease of the kidneys of recurring nature that contributes to frequent urinary tract infections due to bacterial growth and biofilm formation surrounding the stone microenvironment. In the past, commonly used strategies for managing cystinuria involved the use of (a) cystine crystal growth inhibitors such as l-cystine dimethyl ester and lipoic acid, and (b) thiol-based small molecules such as N-(2-mercaptopropionyl) glycine, commonly known as tiopronin, that reduce the formation of cystine crystals by reacting with excess cystine and generating more soluble disulfide compounds. However, there is a dearth of simplistic chemical approaches that have focused on the dual treatment of cystinuria and the associated microbial infections. This work strategically exploited a single chemical approach to develop a nitric oxide (NO)-releasing therapeutic compound, S-nitroso-2-mercaptopropionyl glycine (tiopronin-NO), for the dual management of cystine stone formation and the related bacterial infections. The results successfully demonstrated that (a) the antibacterial activity of NO rendered tiopronin-NO effective against the stone microenvironment inhabitants, Escherichia coli and Pseudomonas aeruginosa, and (b) tiopronin-NO retained the ability to undergo disulfide exchange with cystine while being reported to be safe against canine kidney and mouse fibroblast cells. Thus, the synthesis of such a facile molecule aimed at the dual management of cystinuria and related infections is unprecedented in the literature.

Synthesis and Characterization of Nitric Oxide-Releasing Ampicillin as a Potential Strategy for Combatting Bacterial Biofilm Formation.

Biofilm formation on biomaterial interfaces and the development of antibiotic-resistant bacteria have decreased the effectiveness of traditional antibiotic treatment of infections. In this project, ampicillin, a commonly used antibiotic, was conjugated with S-nitroso-N-acetylpenicillamine (SNAP), an S-nitrosothiol compound (RSNO) used for controlled nitric oxide (NO) release. This novel multifunctional molecule is the first of its kind to provide combined antibiotic and NO treatment of infectious pathogens. Characterization of the molecule included NMR, FTIR, and mass spectrometry. NO release behavior was also measured and compared to pure, unmodified SNAP. When evaluating the antimicrobial efficacy, the synthesized SNAPicillin molecule showed the lowest MIC value against Gram-negative Pseudomonas aeruginosa and Gram-positive methicillin-resistant Staphylococcus aureus compared to ampicillin and SNAP alone. SNAPicillin also displayed enhanced biofilm dispersal and killing of both bacterial strains when treating a 48 h biofilm preformed on a polymer surface. The antibacterial results combined with the biocompatibility of the molecule show great promise for infection prevention and treatment of polymeric interfaces to reduce medical device-related infections.

Nanoarchitectonics of nitric oxide releasing supramolecular structures for enhanced antibacterial efficacy under visible light irradiation.

Light-controlled therapies offer a promising strategy to prevent and suppress infections caused by numerous bacterial pathogens. Excitation of exogenously supplied photosensitizers (PS) at specific wavelengths elicits levels of reactive oxygen intermediates toxic to bacteria. Porphyrin-based supramolecular nanostructure frameworks (SNF) are effective PS with unique physicochemical properties that have led to their widespread use in photomedicine. Herein, we developed a nitric oxide (NO) releasing, biocompatible, and stable porphyrin-based SNF (SNF-NO), which was achieved through a confined noncovalent self-assembly process based on π-π stacking. Characterization of the SNFs via scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis showed the formation of three-dimensional, well-defined octahedral structures. These SNF-NO were shown to exhibit a red shift due to the noncovalent self-assembly of porphyrins, which also show extended light absorption to broadly cover the entire visible light spectrum to enhance photodynamic therapy (PDT). Under visible light irradiation (46 J cm-2), the SNF generates high yields of singlet oxygen (1O2) radicals, hydroxyl radicals (HO), superoxide radicals (O2), and peroxynitrite (ONOO-) radicals that have shown potential to enhance antimicrobial photodynamic therapy (APDT) against Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative Escherichia coli (E. coli). The resulting SNFs also exhibit significant biofilm dispersion and a decrease in biomass production. The combination of robust photosensitizer SNFs with nitric oxide-releasing capabilities is dynamic in its ability to target pathogenic infections while remaining nontoxic to mammalian cells. The engineered SNFs have enormous potential for treating and managing microbial infections.

Biomimetic gasotransmitter-releasing alginate beads for biocompatible antimicrobial therapy.

HYPOTHESIS: Alginate is widely used in biomedical applications due to its high biocompatibility as well as structural and mechanical similarities to human tissue. Further, simple ionic crosslinking of alginate allows for the formation of alginate beads capable of drug delivery. S-nitrosoglutathione is a water-soluble molecule that releases nitric oxide in physiological conditions, where it acts as a potent antimicrobial gas, among other functions. As macrophages and endothelial cells endogenously produce nitric oxide, incorporating nitric oxide donors into polymers and hydrogels introduces a biomimetic approach to mitigate clinical infections, including those caused by antibiotic-resistant microorganisms. The incorporation of S-nitrosoglutathione into macro-scale spherical alginate beads is reported for the first time and shows exciting potential for biomedical applications. EXPERIMENTS: Herein, nitric oxide-releasing crosslinked alginate beads were fabricated and characterized for surface and cross-sectional morphology, water uptake, size distribution, and storage stability. In addition, the NO release was quantified by chemiluminescence and its biological effects against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus were investigated. The biocompatibility of the alginate beads was tested against 3T3 mouse fibroblast cells. FINDINGS: Overall, nitric oxide-releasing alginate beads demonstrate biologically relevant activities without eliciting a cytotoxic response, revealing their potential use as an antimicrobial material with multiple mechanisms of bacterial killing.

Dual Action Nitric Oxide and Fluoride Ion-Releasing Hydrogels for Combating Dental Caries.

Demineralization and breakdown of tooth enamel are characterized by a condition called dental caries or tooth decay, which is caused by two main factors: (1) highly acidic food intake without proper oral hygiene and (2) overactive oral bacteria generating acidic metabolic byproducts. Fluoride treatments have been shown to help rebuild the hydroxyapatite structures that make up 98% of enamel but do not tackle the bacterial overload that continues to threaten future demineralization. Herein, we have created a dual-function Pluronic F127-alginate hydrogel with nitric oxide (NO)- and fluoride-releasing capabilities for the two-pronged treatment of dental caries. Analysis of the hydrogels demonstrated porous, shear-thinning behaviors with tunable mechanical properties. Varying the weight percent of the NO donor S-nitrosoglutathione (GSNO) within the hydrogel enabled physiologically actionable NO release over 4 h, with the fabricated gels demonstrating storage stability over 21 days. This NO-releasing capability resulted in a 97.59% reduction of viable Streptococcus mutans in the planktonic state over 4 h and reduced the preformed biofilm mass by 48.8% after 24 h. Delivery of fluoride ions was confirmed by a fluoride-sensitive electrode, with release levels resulting in the significant prevention of demineralization of hydroxyapatite discs after treatment with an acidic demineralization solution. Exposure to human gingival fibroblasts and human osteoblasts showed cytocompatibility of the hydrogel, demonstrating the potential for the successful treatment of dental caries in patients.