(T1130-02-12) Nitric Oxide Releasing Albumin Nanoclusters for Treating Methicillin-Resistant staphylococcus aureus Infected Cutaneous Wound

Purpose: Methicillin resistant staphylococcus aureus (MRSA) infected cutaneous wound poses a great challenge worldwide. Since MRSA infection causes delayed wound healing, the importance of eradicating MRSA in infected wound is getting highlighted. However, due to growing antibiotic resistance, treatment of the MRSA infected wound is getting difficult. Therefore, novel agents for the eradication of MRSA in infected wound healing are urgently needed. In recent decades, nitric oxide (NO) has been recognized for having good antibacterial and wound healing effects with low or no resistance because of its various working mechanisms. However, since gaseous NO has extremely low stability in physiological condition, proper NO formulations are required to use it properly. Nanoparticle based formulations have been reported to having a better potency than molecular counterparts in numerous research. Nevertheless, it has been a great challenge to encapsulate NO in nanoparticle formulation because of several reasons including high hydrophilicity and low stability of NO donors. To handle this problem, we chose to use albumin as a nano formulation material due to its hydrophilic nature which could easily encapsulate hydrophilic NO donors and its general usages in numerous research due to its good biocompatibility and biodegradability. In this study, diethylenetriamine diazeniumdiolate (NO/DETA) was synthesized and adsorbed onto albumin nanoclusters (NO/Alb NCs) during desolvation process followed by glutaraldehyde crosslinking to endow mechanical stability. After analyzing characteristics of NO/Alb NCs, in vivo MRSA infected full-thickness cutaneous wound healing study was conducted to investigate its enhanced healing effect of the infected wound.

Methods: NO/DETA was synthesized in a Parr high-pressure reactor and confirmed using Sievers Nitric Oxide Analyzer (NOA). After NO/DETA synthesis, NO/Alb NCs was fabricated via ethanol desolvation process and during which NO/DETA was adsorbed onto albumin nanoclusters followed by glutaraldehyde crosslinking to increase its stability. The presence of NO in NO/Alb NCs was confirmed using NOA and Griess reagent discoloration reaction which turns to red color in reaction with NO. In case of Alb NCs, DETA was adsorbed onto albumin nanoclusters using the same formulation method of NO/Alb NCs. The hydrodynamic size and zeta potential of NO/Alb NCs and Alb NCs were analyzed using a Zetasizer Nano ZS90. Surface morphology of NO/Alb NCs was analyzed using scanning electron microscopy (SEM). In in vivo wound size reduction study, MRSA was challenged at Day -1 and the next day, each formulation was treated every 2 days ending at Day 10. In in vivo antibacterial study, after euthanizing all mice, each wound tissue was excised, chopped and sonicated for the measurement of the amount of MRSA in the wound tissues using CFU method. For further investigation, histological assessments were also conducted: H&E for general tissue morphologies, Trichrome for collagen regeneration, Twort’s gram staining for MRSA detection.

Results: Since albumin has negative charge and DETA and NO/DETA have positive charge, DETA and NO/DETA could be adsorbed onto albumin nanoclusters as expected. 24.32 ± 1.55 nmol/mg of NO was successfully adsorbed onto NO/Alb NCs and released more than 17 hours. It showed hydrodynamic size of 364.3 ± 2.0 with narrow polydispersity index of 0.123 ± 0.02 and zeta potential of -5.46 ± 0.21 at pH 7.4. In case of Alb/NCs, it showed hydrodynamic size of 215.9 ± 5.3 with polydispersity index of 0.23 ± 0.01 and zeta potential of -5.22 ± 0.20 at pH 7.4. NO/Alb NCs treated group showed enhanced antibacterial effect of about 90% reduced CFU counts compared to those of control groups including NO/DETA, Alb NCs treated group and untreated group which showed similar 10⁶ CFU counts per wound. Wound size reduction effect showed about 50% reduction in NO/Alb NCs treated group at day 10 while nearly the same size of initial in cases of Alb NCs, NO/DETA treated group and untreated group. Histological assessments also supported the results. In case of H&E staining, NO/Alb NCs treated group showed integral epidermis and fully regenerated wound morphologies similar with healthy group while Alb NCs and NO/DETA treated group and untreated group showed destructed and immaturely regenerated morphologies. In case of Trichrome staining, NO/Alb NCs treated group showed distinct blue colored collagen region similar with healthy group while Alb NCs and NO/DETA treated group and untreated group showed small amount of collagen region. In case of Twort’s gram staining, dark blue colored MRSA colony was not detected in NO/Alb NCs treated and healthy group while detected in Alb NCs and NO/DETA treated group and untreated group.

Conclusion: In this study, NO/Alb NCs was successfully developed using desolvation method and crosslinked to endow mechanical stability. In vivo antibacterial study showed significantly enhanced antibacterial effect and in vivo wound size reduction study showed enhanced wound size reduction effect. Moreover, histological study showed fully regenerated morphologies without MRSA detection. Therefore, NO/Alb NCs could be a promising NO releasing formulation for the treatment of MRSA infected cutaneous wound.
Fig 1. Characterization of NO/Alb NCs. (a) Illustration of NO/Alb NCs fabrication process (b) Scanning electron microscope images (SEM) and particle distribution of NO/Alb NCs. Scale bar represents 500 nm. (c) Hydrodynamic size and zeta potential distribution of NO/Alb NCs at pH 7.4. (d) Griess reagent reaction of NO/Alb NCs and Alb NCs.

Fig 2. In vivo wound healing effect comparison. (a) Treatment schedule of MRSA infected full thickness cutaneous wound model. (b) Wound site morphologies by treatment schedule (c) Wound size reduction profiles after 10 days treatment schedule (n=4). (d) Antibacterial effect of NO/Alb NCs treated group, NO/DETA treated group, Alb NCs treated group and Untreated group. Bacterial counts of each group are demonstrated in dot & bar graph (n=3). # NO/Alb NCs versus NO/DETA, * NO/Alb NCs versus Untreated

Fig 3. Histology study. (a) H&E staining. (b) Masson’s trichrome staining. (c) Twort’s gram staining. Blue colored spots represent Gram-positive bacteria (MRSA). Black and red bars represent 200 µm and 20 µm, respectively.