Dermal Nanoemulsion Treatment Reduces Burn Wound Conversion and Improves Skin Healing in a Porcine Model of Thermal Burn Injury

Abstract

Burn wound progression is an inflammation-driven process where an initial partial-thickness thermal burn wound can evolve over time to a full-thickness injury. We have developed an oil-in-water nanoemulsion formulation (NB-201) containing benzalkonium chloride for use in burn wounds that is antimicrobial and potentially inhibits burn wound progression. We used a porcine burn injury model to evaluate the effect of topical nanoemulsion treatment on burn wound conversion and healing. Anesthetized swine received thermal burn wounds using a 25-cm2 surface area copper bar heated to 80°C. Three different concentrations of NB-201 (10, 20, or 40% nanoemulsion), silver sulfadiazine cream, or saline were applied to burned skin immediately after injury and on days 1, 2, 4, 7, 10, 14, and 18 postinjury. Digital images and skin biopsies were taken at each dressing change. Skin biopsy samples were stained for histological evaluation and graded. Skin tissue samples were also assayed for mediators of inflammation. Dermal treatment with NB-201 diminished thermal burn wound conversion to a full-thickness injury as determined by both histological and visual evaluation. Comparison of epithelial restoration on day 21 showed that 77.8% of the nanoemulsion-treated wounds had an epidermal injury score of 0 compared to 16.7% of the silver sulfadiazine-treated burns (P = .01). Silver sulfadiazine cream- and saline-treated wounds (controls) converted to full-thickness burns by day 4. Histological evaluation revealed reduced inflammation and evidence of skin injury in NB-201-treated sites compared to control wounds. The nanoemulsion-treated wounds often healed with complete regrowth of epithelium and no loss of hair follicles (NB-201: 4.8 ± 2.1, saline: 0 ± 0, silver sulfadiazine: 0 ± 0 hair follicles per 4-mm biopsy section, P < .05). Production of inflammatory mediators and sequestration of neutrophils were also inhibited by NB-201. Topically applied NB-201 prevented the progression of a partial-thickness burn wound to full-thickness injury and was associated with a concurrent decrease in dermal inflammation.

Figure 1.

Schematic of NB-201 nanoemulsion droplet.

Figure 2.

Visual burn wound healing. (A) Burn wound sites were created by application of a copper bar heated to 80°C in a water bath for 20 seconds. Photographs of burn wounds by topical treatment (saline, silver sulfadiazine, 10% NB-201, 20% NB-201, 40% NB-201 taken on day 1, 4, 10, and 21 postburn injury). (B) Representative hematoxylin and eosin-stained cross-sectional histology samples for normal unburned skin, 40% NB-201-treated skin, and silver sulfadiazine-treated skin sampled on day 21 (20× magnification). Burn wound sites were created by application of a copper bar heated to 80°C in a water bath for 20 seconds.

Figure 3.

Dermal expression of burn-induced inflammatory mediators at day 4 and day 21. Four-millimeter full-thickness punch biopsies of skin were homogenized and assayed using ELISA for level of IL-1β, IL-6, IL-8, and TNF-α. N = 8 separate burn wounds per treatment group. Statistics: two-way ANOVA with Sidak’s multiple comparisons test. Displayed as mean ± SD. (A) Burn wound created by application of a copper bar heated to 80°C in a water bath for 20 seconds. (B) Burn wound created by application of a copper bar heated to 80°C in a water bath for 30 seconds.

Figure 4.

Quantitative skin biopsy bacterial growth in burn wounds on day 21 postinjury. N = 8 separate burn wounds per treatment group. Results expressed as median number of colony forming units/g of tissue (bar). Statistics: ordinary one-way ANOVA with Tukey’s multiple comparisons test. *P < .05 saline vs NB-201, 10 or 20 or 40%. ^#P < .05 silver sulfadiazine vs NB-201, 10 or 20 or 40%.

Figure 5.

Pathologic scoring of skin samples stained with hematoxylin and eosin and analyzed by two independent/masked pathologists. Scores determined by pathologists were averaged and plotted. Statistics: three pigs, six burn wounds per treatment group, two-way ANOVA with Sidak’s multiple comparisons test. Displayed as mean ± SD, *P < .05. Burn wound created by application of a copper bar heated to 80°C in a water bath for 20 seconds.

Figure 6.

Evidence of neutrophil sequestration into the burn wound after thermal injury and topical treatment at day 4 and day 21 postinjury. (A) Myeloperoxidase assay. (B) Histopathologic neutrophil count. (C) Representative photomicrographs of slides from histopathologic examination (10× magnification, inset 60× magnification). N = 8 wounds per treatment group. Statistics: two-way ANOVA with Sidak’s multiple comparisons test. Displayed as mean ± SD.

Figure 7.

Hair follicle preservation in burned skin. NB-201 treatment resulted in hair follicle preservation as compared to hair follicle loss in controls on day 21 postburn. Cross-sectional skin histological samples were stained with H&E and viable hair follicles were counted. N = 4 separate burn wounds per treatment group. Statistics: ordinary one-way ANOVA with Tukey’s multiple comparisons test. Displayed as mean ± SD.