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Topical nitric oxide releasing nanoparticles are effective in a murine model of dermal Trichophyton rubrum dermatophytosis

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Abstract

Systemic therapies are preferred for treating dermal dermatophytosis due to inadequate penetration of topical agents. However, systemic antifungals are associated with off-target effects and limited tissue penetration, and antimicrobial resistance is a growing concern. To address this, we investigated topical nitric oxide-releasing nanoparticles (NO-np), which have been used against superficial fungal infections and bacterial abscesses. In addition to enhanced penetration and permeation conferred by nanoparticles, nitric oxide, a broad-spectrum multi-mechanistic antimicrobial agent, offers decreased likelihood of resistance development. In the current study, NO-np inhibited Trichophyton rubrum in vitro, as well as in a murine model of dermal dermatophytosis. In mice, NO-np reduced fungal burden after three days, with complete clearance after seven. Furthermore, NO-np decreased tissue IL-2, 6, 10 and TNFα, indicating earlier attenuation of the host inflammatory response and decreased tissue morbidity. Thus, topical NO-np represent an attractive alternative to systemic therapy against dermal T. rubrum infection.

Graphical Abstract

Systemic therapies are preferred for treating dermal dermatophytosis due to inadequate penetration of topical agents. However, systemic antifungals are associated with off-target effects, lengthy treatment schedules and limited tissue penetration. This study investigated topical nitric oxide releasing nanoparticles (NO-np) in a murine model of dermal dermatophyosis. Nitric oxide provides broad-spectrum multi-mechanistic antimicrobial activity while the small size of nanoparticles offers enhanced penetration and permeation of tissues. Data demonstrated NO-np inhibition of Trichophyton rubrum in vitro, as well as in mice. NO-np also decreased tissue IL-2, 6, 10 and TNFα, indicating earlier attenuation of the host inflammatory response.

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Section snippets

Methods

NO-np were synthesized as previously described.6 Minimum inhibitory concentrations (MICs) were determined for two T. rubrum reference strains (ATCC MYA-4438 and ATCC 28189) and a clinical isolate using methods proposed by the Clinical and Laboratory Standards Institute (CLSI), modified by the use of a resazurin colorimetric assay.14 Minimum fungicidal concentrations (MFCs) were determined as previously described.15 Additionally, antimicrobial effects of the MIC of NO-np were visualized directly

Results

The MICs of NO-np for the T. rubrum reference strains and clinical isolate were 10 and 5 mg/mL, respectively. MFC results demonstrated fungistatic behavior of NO-np, and empty nanoparticles did not demonstrate antifungal activity at concentrations up to 20 mg/mL (Figure 1, A). On TEM, accumulation of material suggestive of intracellular vesicles and black granules within vacuoles was observed in the fungal cells following incubation with the MIC of NO-np (Figure 1, B).

CFU quantification

Discussion

Our results present topical NO-np as a potential treatment for dermal T. rubrum infection. NO exerts direct fungistatic activity via DNA damage, lipid peroxidation, and enzyme inactivation,3 as well as indirect activity via upregulation of macrophage phagocytic activity.17 Of note, NO-np exhibit trans-nitrosylation activity in addition to NO release, which likely explains the significant effects visualized on TEM.18

In addition to fungal death, NO-np were associated with decreased IL-2, 6, 10

Acknowledgments

We thank Dr. Mahmoud A. Ghannoum of Case Western Reserve University for providing the T. rubrum ATCC MYA-4438 strain.

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    Funding: CBCO received fellowship funding from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (process number: 99999.007910/2014-02) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (process number: 150261/2016-0).

    Disclosures: AJF and JMF are co-inventors of NO-np, a technology licensed to Nano Biomed Inc. for commercialization. JDN serves as an advisor to Nano Biomed Inc. Portions of this publication were presented in abstract form at the 74th Annual Meeting of the American Academy of Dermatology.

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    These authors contributed equally to the production of this work.

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