Therapeutic potential of iron scavenging protein cocktail on burn injuries
Creators: name: Reddy, Swathi
Publisher: name: No Publisher Supplied
Year: 2023

description: Approximately 500,000 burn injuries are medically treated in the USA each year, with the highest incidences of fire burns and scalds in children and young adults. Burn injuries result in hemolysis, releasing toxic extracellular hemoglobin (Hb), heme, and free iron. This may result in additional tissue damage around the initial injury site, thus causing an expansion of the burn in both area and depth. While the toxicity is naturally mitigated by the upregulation of scavenger proteins haptoglobin (Hp), hemopexin (Hpx), and transferrin (Tf), this response takes hours to peak, and in cases of more extensive burns, the proteins produced are insufficient. Moreover, free iron is a nutrient for pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus causing subsequent infection, which may further inhibit healing. Therefore, we hypothesize that supplementing exogenously these scavenger proteins in a cocktail (Hp, Hpx, and Tf) would help to reduce the burn wound expansion and thereby accelerate wound healing. To test the efficacy of the scavenger protein cocktail, small and large burn injury mouse models were created by positioning their backs under a mold to expose ~1 cm2 (for small burn wounds) and ~1x3 cm2 (for large burn wounds) of the total body surface area to 92 ± 2°C water for 10s. The wounds were then treated with the scavenger protein cocktail. Varying frequencies (single or multiple doses), dosages, and routes (topical; transdermal intraperitoneal, IP; Intravenous, IV) were tested. Images of the wounds at different time points were taken to quantify the percent wound closure. Cocktail treatment provided mixed results. In some cases, cocktail prevented the wound expansion that was observed in control wounds on postburn day 3; however, this was not always observed. Furthermore, observation of the wound closure dynamics showed that in some treated groups, wound closure took place a few days earlier than the control groups. Iron staining of wound tissue sections showed iron deposition in the burn wound, and cocktail treatment showed clearance of the iron at the margin of the burnt and unburnt tissue, in some of the treatment groups. Overall, iron scavenging cocktail treatment showed some potential ability to mitigate wound expansion and free iron deposition in mouse burn wounds; however, more studies will be needed to determine whether these effects are significant and reproducible. Next, an in vitro zone inhibition assay was performed to investigate the protein cocktail's antibacterial potential against Pseudomonas aeruginosa and Staphylococcus aureus. The bacterial strains were cultured in tryptic soy broth and were spread on tryptic soy agar plates. Filters papers loaded with gentamicin (a common antibiotic used in burn patients that is effective but has undesirable side effects), the cocktail, or a combination of cocktail and gentamicin were placed on the agar plates and were left to incubate overnight at 37ºC. The area of inhibition was assessed at the end of incubation (~24 hours). The data showed that cocktail used alone had little to no effect on bacterial growth, as evidenced by the lack of a zone of inhibition. However, adding cocktail to low dose gentamicin significantly increased the zone of inhibition, as compared to that observed with low dose gentamicin alone. Thus, the iron scavenging cocktail, while unable to inhibit bacterial growth alone, significantly potentiated the effect of low dose gentamicin on bacterial growth inhibition. Therefore, it is possible that this cocktail could be used to decrease the dosage of gentamicin used in burns. Future studies in animal models of infected burns are warranted to explore this possibility.