Abstract
In acute skin injury, healing is impaired by the excessive release of reactive oxygen species (ROS). Melanin, an efficient scavenger of radical species in the skin, performs a key role in ROS scavenging in response to UV radiation and is upregulated in response to toxic insult. In a chemical injury model in mice, we demonstrate that the topical application of synthetic melanin particles (SMPs) significantly decreases edema, reduces eschar detachment time, and increases the rate of wound area reduction compared to vehicle controls. Furthermore, these results were replicated in a UV-injury model. Immune array analysis shows downregulated gene expression in apoptotic and inflammatory signaling pathways consistent with histological reduction in apoptosis. Mechanistically, synthetic melanin intervention increases superoxide dismutase (SOD) activity, decreases Mmp9 expression, and suppresses ERK1/2 phosphorylation. Furthermore, we observed that the application of SMPs caused increased populations of anti-inflammatory immune cells to accumulate in the skin, mirroring their decrease from splenic populations. To enhance antioxidant capacity, an engineered biomimetic High Surface Area SMP was deployed, exhibiting increased wound healing efficiency. Finally, in human skin explants, SMP intervention significantly decreased the damage caused by chemical injury. Therefore, SMPs are promising and effective candidates as topical therapies for accelerated wound healing, including via pathways validated in human skin.
Introduction
Skin is the organ most exposed to external environmental damaging factors. These factors include damage from UV-light exposure, traumas and chronic wounds combined with perturbance during medical procedures. With these insults in mind, it becomes imperative to develop new therapies and to keep improving approaches that are currently available1,2. Skin wound healing is a complex process requiring a coordinated interplay of multiple cell types, growth factors and signaling molecules and our understanding of these processes will lead to advancements in how we accelerate and influence healing. Indeed, recently, significant advancements have been made in our understanding of wound repair mechanisms including the role of monocytes and macrophages3,4, the differentiation of fibroblasts and the functions of myofibroblasts5, as well as the coordination between the immune system and tissue stem cells6,7.
Oxidative stress is one of the main mechanisms underlying impaired healing in skin injury and in chronic wounds8,9. While low levels of reactive oxygen species (ROS) serve essential antimicrobial and signaling roles in wound healing10,11,12,13, excessive production of free radical species overwhelms the delicate oxidant-antioxidant homeostatic balance and leads to molecular dysfunction, cellular damage, and pathologic inflammation. This makes the redox system a natural target for wound-healing therapy14,15. Indeed, topical application of compounds with antioxidant properties such as curcumin-loaded poly (lactic acid) nanofibers16, chitosan-loaded eugenol17, and citrate-based hydrogels18 have demonstrated benefits in wound healing. However, compound biocompatibility and efficient delivery continue to pose significant challenges to their therapeutic use.
This clear clinical need led to our hypothesis that nature’s own potent antioxidant and proficient radical scavenger, melanin, could be mimicked synthetically and applied topically following skin injury, where it would be effective in healing when applied after chemical or UV-light exposure at the surface of the skin. This is based on the fact that melanin’s role as a pigment in the hair and skin19,20, is intrinsically linked to its photoprotective materials properties20,21,22. In brief, upon UV exposure, melanin expression in the skin is upregulated due to its ability to scavenge the free radicals generated by the impinging radiolytic light. Similarly, recent studies have reported increases in melanin-based skin pigmentation in response to air pollutants23.
To test this hypothesis, we synthesized two types of nanoscale Synthetic Melanin Particle (SMP) with Low and High Surface Areas (SMPLo and SMPHi, respectively)24. As a result of their higher surface area, SMPHi were expected to exhibit a superior radical scavenging capability that would be reflected in wound healing efficacy compared to SMPLo. The two types of SMPs were tested by topical application following chemical (nitrogen mustard, NM) and ultraviolet (UV)-induced skin wounds in vivo using mouse models25. In addition, SMPs were tested ex vivo using a human skin explant model for chemical-induced skin wounding. The data show that SMP treatment mitigates inflammation and accelerates wound healing by decreasing Mmp9 expression26, rescuing skin superoxide dismutase (SOD) activity27, and suppressing chemical injury-induced MAPK signaling by inhibiting ERK1/2 phosphorylation. In a critical finding, SMPs were found to modulate the immune response by increasing the number of reparative and anti-inflammatory cells in the wound area and regulating the systemic immune response. Furthermore, in human skin explants SMPLo were shown to efficiently counteract the harmful effects of NM insult. The high-surface-area particles allowed for more exposure of the wound site to the melanin material and conferred stronger wound-healing benefits observable visually and pathologically in mice, and at the molecular level compared to the low-surface-area particles with both exhibiting efficacy over vehicle controls in a model-dependent fashion. Overall, the results demonstrate that biomimetic SMPs are an effective topical intervention for the acceleration of acute chemical- and UV-wound healing.
Results
SMP syntheses and characterization
Melanin mimetics were synthesized through the oxidative polymerization of dopamine, based on previously reported methods (Fig. 1)24. The surface area and morphology of the particles were characterized using N2 sorption, dynamic light scattering, and ultraviolet-visible (UV-Vis) spectroscopy (Supplementary Fig. 1). The High-surface-area Synthetic Melanin Particles (SMPHi) had a BET area of 190 m2/g with approximately 14 and 30 Å pores while the Low-surface-area Synthetic Melanin Particles (SMPLo) were non-porous with a BET area of 20 m2/g (Supplementary Fig. 1a, b)24. We note that the surface area of SMPLo is similar to that of natural melanin which has been measured to have a BET area of 6 m2/g as previously reported for melanin extracts from Sepia officinalis—squid ink24. By dynamic light scattering (DLS), SMPHi had a hydrodynamic diameter of 220 ± 50 nm. SMPLo had a hydrodynamic diameter of 320 ± 10 nm (Supplementary Fig. 1c). These scattering data are consistent with that observed by bright field TEM (Fig. 1a, b) Additionally, SMPLo and SMPHi exhibited similar absorption spectra by UV–Vis spectroscopy (Supplementary Fig. 1d) with both particles exhibiting an absorption maximum of approximately 200 nm with a shoulder at 300 nm and a broad absorption tail. Other than porosity, the two particles had similar characteristics. Neither SMPHi nor SMPLo penetrated the stratum corneum when applied topically to skin (Fig. 1c, d).
The scavenging activity of SMPHi and SMPLo was assessed via the 2.2-diphenyl-1-picrylhydrazyl (DPPH) assay (Fig. 1e). SMPHi achieved higher scavenging activity at lower particle concentrations than SMPLo. The scavenging activity of both particles plateaued at approximately 100 μg, with SMPHi achieving a maximum at 90% scavenging activity and SMPLo reaching 80%. Scavenging activity was consistent across different batches of particles and was maintained but dampened over multiple use cycles (Supplementary Fig. 2). The scavenging activities of SMPs, especially SMPHi, are similar or superior to radical scavenging activities of other antioxidants as determined via the DPPH assay, such as butylated hydroxytoluene28, luteolin29, eugenol30, and ascorbic acid31.
SMP intervention improves skin healing after nitrogen mustard-induced injury
To test the hypothesis that intervention with topical SMP would improve wound healing, we utilized an NM-induced chemical injury mouse model (Fig. 2). NM is an alkylating agent that induces cell apoptosis via the formation of DNA interstrand crosslinks32. The clinical application of NM as a topical chemotherapeutic for the treatment of skin lymphoma is known to cause severe skin reactions including irritant dermatitis and blister formation33. In this experimental model, SMPs were applied topically to the NM-induced wound site 2 h after NM-induced injury to ensure that no residual NM was present on the skin surface at the time of SMP application. SMP application was then repeated at 24- and 48-h post-injury. The mice were monitored for up to 16 days.
