Antimicrobial and Healing Efficacy of Sustained Release Nitric Oxide Nanoparticles Against Staphylococcus Aureus Skin Infection  Luis R. Martinez, George.

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Antimicrobial and Healing Efficacy of Sustained Release Nitric Oxide Nanoparticles Against Staphylococcus Aureus Skin Infection  Luis R. Martinez, George Han, Manju Chacko, Mircea Radu Mihu, Marc Jacobson, Phil Gialanella, Adam J. Friedman, Joshua D. Nosanchuk, Joel M. Friedman  Journal of Investigative Dermatology  Volume 129, Issue 10, Pages 2463-2469 (October 2009) DOI: 10.1038/jid.2009.95 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Amperometric real-time detection of NO-np released. Kinetics of nitric oxide (NO) release in solution was measured using amperometric analysis. The inset TEM photograph shows uniform nitric oxide through nanoparticles (NO-np) of ∼10nm in diameter. Bar=100nm. Journal of Investigative Dermatology 2009 129, 2463-2469DOI: (10.1038/jid.2009.95) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 NO-releasing nanoparticles have antimicrobial activity against SA. TEM of SA exposed to sustained release of NO by nanoparticles revealed cell wall damage and lysis. (a) Growth of SA in the absence of NO-np showed intact cell wall architecture (control). Growth of SA in the presence NO-np after 1 (b), 4 (c), 7 (d), and 24hours (e) showed increasing destruction of cell wall architecture, edema, and cell lysis. White and black arrows denote bacterial cross-wall and cell wall damage after NO treatment, respectively. Bar=0.2μm. Journal of Investigative Dermatology 2009 129, 2463-2469DOI: (10.1038/jid.2009.95) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 NO-np increased the wound healing rate in mice. (a) Wounds of Balb/c mice uninfected and untreated, uninfected treated with nanoparticles without nitric oxide (NO) (np), uninfected treated with nitric oxide through nanoparticle (NO-np,) untreated methicillin-resistant Staphylococcus aureus (MRSA)-infected, np-treated MRSA-infected, and MRSA-infected treated with NO-np, days 3 and 7. Bar=5mm. (b) Wound size analysis of Balb/c mice skin lesions. Wounds were uninfected or infected with MRSA and untreated or treated in the absence or presence of NO. Time points are the averages of the results for ten measurements, and error bars denote SDs. *P<0.05 in comparing the NO-treated groups with uninfected or MRSA-infected and untreated groups. Journal of Investigative Dermatology 2009 129, 2463-2469DOI: (10.1038/jid.2009.95) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 NO-np killed MRSA in superficial skin lesions. (a) Wound bacterial burden (CFU; colony forming unit) in NO-treated mice infected intradermally with 107 methicillin-resistant Staphylococcus aureus is significantly lower than untreated or NP-treated mice (n=10 per group). Four days after infection, wounds were swabbed and bacteria burden examined. (b) Seven days after infection, infected skin tissue was removed from mice and bacterial burden determined. Uninfected mice untreated, NP-, and NO-treated were used as a control. Bars are the averages of the results for three measurements, and error bars denote standard daviations. Asterisks denote P-value significance (**P<0.01) calculated by analysis of variance and adjusted by use of the Bonferroni correction. (c) Histological analysis of BALB/C mice uninfected and untreated, uninfected treated with nanoparticles without NO (NP), uninfected treated with NO-NP (NO), untreated MRSA-infected, NP-treated MRSA-infected, and MRSA-infected treated with NO, day 7. Mice were infected with 107 MRSA bacterial cells. Representative H&E-stained sections of the skin lesions are shown with the insets representing Gram staining specific for MRSA cells (shown in purple). Scale bars = 25μm. Journal of Investigative Dermatology 2009 129, 2463-2469DOI: (10.1038/jid.2009.95) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 NO-nps decrease collagen degradation in skin lesions of Balb/c mice. (a) Histological analysis of Balb/c mice uninfected and untreated (Untr), uninfected treated with nanoparticles without nitric oxide (NO) (np), uninfected treated with NO-nps (NO), untreated methicillin-resistant Staphylococcus aureus (MRSA)-infected, np-treated MRSA-infected, and MRSA-infected treated with NO, day 7. Mice were infected with 107 bacterial cells. The blue stain indicates collagen. Bar = 25μm. (b) Quantitative measurement of collagen intensity in 16 representative fields of the same size for uninfected and untreated, uninfected treated with nanoparticles without NO (np), uninfected treated with NO, untreated MRSA-infected, np-treated MRSA-infected, and MRSA-infected treated with NO wounds. Bars are the averages of the results, and error bars denote SDs. *P<0.01 in comparing the untreated groups with the uninfected np- and NO-treated groups; **P<0.001 in comparing the untreated groups with the MRSA+NO group. Journal of Investigative Dermatology 2009 129, 2463-2469DOI: (10.1038/jid.2009.95) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions