Joel M. Friedman

Nitrate and nitrite in biology, nutrition and therapeutics

Inorganic nitrate and nitrite from endogenous or dietary sources are metabolized in vivo to nitric oxide (NO) and other bioactive nitrogen oxides. The nitrate-nitrite-NO pathway is emerging as an important mediator of blood flow regulation, cell signaling, energetics and tissue responses to hypoxia. The latest advances in our understanding of the biochemistry, physiology and therapeutics of nitrate, nitrite and NO were discussed during a recent 2-day meeting at the Nobel Forum, Karolinska Institutet in Stockholm.

Antimicrobial and Healing Efficacy of Sustained Release Nitric Oxide Nanoparticles Against Staphylococcus Aureus Skin Infection

Staphylococcus aureus (SA) is a leading cause of both superficial and invasive infections in community and hospital settings, frequently resulting in chronic refractory disease. It is imperative that innovative therapeutics to which the bacteria are unlikely to evolve resistance be developed to curtail associated morbidity and mortality and ultimately improve our capacity to treat these infections. In this study, a previously unreported nitric oxide (NO)-releasing nanoparticle technology is applied to the treatment of methicillin-resistant SA (MRSA) wound infections. The results show that the nanoparticles exert antimicrobial activity against MRSA in a murine wound model. Acceleration of infected wound closure in NO-treated groups was clinically shown compared with controls. The histology of wounds revealed that NO nanoparticle treatment decreased suppurative inflammation, minimal bacterial burden, and less collagen degradation, providing potential mechanisms for biological activity. Together, these data suggest that these NO-releasing nanoparticles have the potential to serve as a novel class of topically applied antimicrobials for the treatment of cutaneous infections and wounds.

Sustained release nitric oxide releasing nanoparticles: Characterization of a novel delivery platform based on nitrite containing hydrogel/glass composites ☆

A new platform using biocompatible materials is presented for generating powders comprised of nanoparticles that release therapeutic levels of nitric oxide (NO) in a controlled and sustained manner. The capacity of these particles to retain and gradually release NO arises from their having combined features of both glassy matrices and hydrogels. This feature allows both for the generation of NO through the thermal reduction of added nitrite by glucose and for the retention of the generated NO within the dry particles. Exposure of these robust biocompatible nanoparticles to moisture initiates the sustained release of the trapped NO over extended time periods as determined both fluorimetrically and amperometrically. The slow sustained release is in contrast to the much faster release pattern associated with the hydration-initialed NO release in powders derived from glassy matrices. These glasses are prepared using trehalose and sucrose doped with either glucose or tagatose as the source of thermal electrons needed to convert nitrite to gNO. Significantly, the release profiles for the NO in the hydrogel/glass composite materials are found to be an easily tuned parameter that is modulated through the specific additives used in preparing the hydrogel/glass composites. The presented data raise the prospect that these new NO releasing nanoparticles can be easily formulated for use under a wide range of therapeutic circumstances.

Demonstration of Antibiofilm and Antifungal Efficacy of Chitosan against Candidal Biofilms, Using an In Vivo Central Venous Catheter Model

Candida species are a major cause of catheter infections. Using a central venous catheter Candida albicans biofilm model, we demonstrated that chitosan, a polymer isolated from crustacean exoskeletons, inhibits candidal biofilm formation in vivo. Furthermore, chitosan statistically significantly decreased both the metabolic activity of the biofilms and the cell viability of C. albicans and Candida parapsilosis biofilms in vitro. In addition, confocal and scanning electron microscopic examination demonstrated that chitosan penetrates candidal biofilms and damages fungal cells. Importantly, the concentrations of chitosan that were used to evaluate fungal biofilm susceptibility were not toxic to human endothelial cells. Chitosan should be considered for the prevention or treatment of fungal biofilms on central venous catheters and perhaps other medical devices.

Nitric oxide releasing nanoparticles are therapeutic for Staphylococcus aureus abscesses in a murine model of infection.

Staphylococcus aureus (SA) is a leading cause of a diverse spectrum of bacterial diseases, including abscesses. Nitric oxide (NO) is a critical component of the natural host defense against pathogens such as SA, but its therapeutic applications have been limited by a lack of effective delivery options. We tested the efficacy of a NO-releasing nanoparticle system (NO-np) in methicillin-resistant SA (MRSA) abscesses in mice. The results show that the NO-np exert antimicrobial activity against MRSA in vitro and in abscesses. Topical or intradermal NO-np treatment of abscesses reduces the involved area and bacterial load while improving skin architecture. Notably, we evaluated pro- and anti-inflammatory cytokines that are involved in immunomodulation and wound healing, revealing that NO-np lead to a reduction in angiogenesis preventing bacterial dissemination from abscesses. These data suggest that NO-np may be useful therapeutics for microbial abscesses.

Nitric Oxide–Releasing Nanoparticles Accelerate Wound Healing by Promoting Fibroblast Migration and Collagen Deposition

Wound healing is a complex process that involves coordinated interactions between diverse immunological and biological systems. Long-term wounds remain a challenging clinical problem, affecting approximately 6 million patients per year, with a high economic impact. To exacerbate the problem, these wounds render the individual susceptible to life-threatening microbial infections. Because current therapeutic strategies have proved suboptimal, it is imperative to focus on new therapeutic approaches and the development of technologies for both short- and long-term wound management. In recent years, nitric oxide (NO) has emerged as a critical molecule in wound healing, with NO levels increasing rapidly after skin damage and gradually decreasing as the healing process progresses. In this study, we examined the effects of a novel NO-releasing nanoparticle technology on wound healing in mice. The results show that the NO nanoparticles (NO-np) significantly accelerated wound healing. NO-np modified leukocyte migration and increased tumor growth factor-β production in the wound area, which subsequently promoted angiogenesis to enhance the healing process. By using human dermal fibroblasts, we demonstrate that NO-np increased fibroblast migration and collagen deposition in wounded tissue. Together, these data show that NO-releasing nanoparticles have the ability to modulate and accelerate wound healing in a pleiotropic manner.

The use of chitosan to damage Cryptococcus neoformans biofilms

The use of indwelling medical devices (e.g. pacemakers, prosthetic joints, catheters, etc) continues to increase, yet these devices are all too often complicated by infections with biofilm-forming microbes with increased resistance to antimicrobial agents and host defense mechanisms. We investigated the ability of chitosan, a polymer isolated from crustacean exoskeletons, to damage biofilms formed by the pathogenic fungus Cryptococcus neoformans. Using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay and CFU determinations, we showed that chitosan significantly reduced both the metabolic activity of the biofilms and cell viability, respectively. We further demonstrated that chitosan penetrated biofilms and damaged fungal cells using confocal and scanning electron microscopy. Notably, melanization, an important virulence determinant of C. neoformans, did not protect cryptococcal biofilms against chitosan. The chitosan concentrations used in this study to evaluate fungal biofilm susceptibility were not toxic to human endothelial cells. Our results indicate that cryptococcal biofilms are susceptible to treatment with chitosan, suggesting an option for the prevention or treatment of fungal biofilms on indwelling medical devices.

Nitric oxide releasing nanoparticles are therapeutic for Acinetobacter baumannii wound infections

Acinetobacter baumannii (Ab) is a frequent cause of hospital acquired pneumonia and recently has increased in incidence as the causative agent of severe disease in troops wounded in Afghanistan and Iraq. Ab clinical isolates are frequently extremely resistant to antimicrobials, significantly complicating our capacity to treat infections due to this pathogen. Hence, the development of innovative therapeutics targeting mechanisms to which the bacteria are unlikely to evolve resistance is urgently needed. We examined the capacity of a nitric oxide -releasing nanoparticle (NO-np) to treat wounds infected with Ab. We found that the NO-nps were therapeutic in an experimental Ab murine wound model. Treatment with NO-nps significantly accelerated healing of infected wounds. Histological study demonstrated that NO-np treatment reduced suppurative inflammation, decreased microbial burden, and reduced the degradation of collagen. Furthermore, NO-np treatment alters the local cytokine milieu. In sum, we demonstrated that the NO-nps are an easily administered topical antimicrobial for the treatment of Ab wound infections, and our findings suggest that NO-nps may also be ideal for use in combat or disaster situations.

Susceptibility of Gram-positive and -negative bacteria to novel nitric oxide-releasing nanoparticle technology

The rapidly evolving crisis of antibiotic resistance among microorganisms has contributed to the rise of patient morbidity and mortality from nosocomial and community-acquired infections. Therefore, innovative antimicrobial technology targeting mechanisms to which the bacteria are unlikely to evolve resistance is urgently needed. We have previously described a nitric oxide-releasing nanoparticle (NO-np) with efficacy against methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii in vitro and in murine wound and abscess models. Although the prior findings suggest that the NO-np can be a useful therapeutic for skin and soft tissue infections, the antimicrobial spectrum of NO-np has yet to be fully elucidated. In the current study, we investigated the efficacy of a NO-np against several Gram-positive and -negative clinical isolates. We found that the NO-np were uniformly active against all of the Streptococcus pyogenes, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa clinical isolates examined, including strains that were both sensitive and resistant to commonly used antibiotics. We concluded that the NO-np have the potential to serve as a novel broad spectrum antimicrobial agent.

Sustained release nitric oxide from long lived circulating nanoparticles

The current limitations of nitric oxide (NO) delivery systems have stimulated an extraordinary interest in the development of compounds that generate NO in a controlled and sustained manner with a heavy emphasis on the treatment of cardiovascular disease states. This work describes the positive physiological response to the infusion of NO-releasing nanoparticles prepared using a new platform based on hydrogel/glass hybrid nanoparticles. When exposed to moisture, these nanoparticles slowly release therapeutic levels of NO, previously generated through thermal reduction of nitrite to NO trapped within the dry particles. The controlled and sustained release of NO observed from these nanoparticles (NO-np) is regulated by its hydration over extended periods of time. In a dose-dependent manner, circulating NO-np both decreased mean arterial blood pressure and increased exhaled concentrations of NO over a period of several hours. Circulating NO-np induced vasodilatation and increased microvascular perfusion during their several hour circulation lifetime. Control nanoparticles (control-np; without nitrite) did not induce changes in arterial pressure, although a decrease in the number of capillaries perfused and an increase in leukocyte rolling and immobilization in the microcirculation were observed. The NO released by the NO-np prevents the inflammatory response observed after infusion of control-np. These data suggest that NO release from NO-np is advantageous relative to other NO-releasing compounds, because it does not depend on chemical decomposition or enzymatic catalysis; it is only determined by the rate of hydration. Based on the observed physiological properties, NO-np has clear potential as a therapeutic agent and as a research tool to increase our understanding of NO signaling mechanisms within the vasculature.