Coumarin/β-Cyclodextrin Inclusion Complexes Promote Acceleration and Improvement of Wound Healing

Abstract

Coumarins have great pharmacotherapeutic potential, presenting several biological and pharmaceutical applications, like antibiotic, fungicidal, anti-inflammatory, anticancer, anti-HIV, and healing activities, among others. These molecules are practically insoluble in water, and for biological applications, it became necessary to complex them with cyclodextrins (CDs), which influence their bioavailability in the target organism. In this work, we studied two coumarins, and it was possible to conclude that there were structural differences between 4,7-dimethyl-2H-chromen-2-one (DMC) and 7-methoxy-4-methyl-2H-chromen-2-one (MMC)/β-CD that were solubilized in ethanol, frozen, and lyophilized (FL) and the mechanical mixtures (MM). In addition, the inclusion complex formation improved the solubility of DMC and MMC in an aqueous medium. According to the data, the inclusion complexes were formed and are more stable at a molar ratio of 2:1 coumarin/β-CD, and hydrogen bonds along with π-π stacking interactions are responsible for the better stability, especially for (MMC)₂@β-CD. In vivo wound healing studies in mice showed faster re-epithelialization and the best deposition of collagen with the (DMC)₂@β-CD (FL) and (MMC)₂@β-CD (FL) inclusion complexes, demonstrating clearly that they have potential in wound repair. Therefore, (DMC)₂@β-CD (FL) deserves great attention because it presented excellent results, reducing the granulation tissue and mast cell density and improving collagen remodeling. Finally, the protein binding studies suggested that the anti-inflammatory activities might exert their biological function through the inhibition of MEK, providing the possibility of development of new MEK inhibitors.

Keywords: coumarin; cyclodextrin; healing wounds; inclusion complex; skin lesions.

Figure 1

ITC for (A) DMC and (B) MMC titration with β-CD.

Figure 2

Inclusion complex geometries optimized at the B97D/6-31G(d,p) level of theory: (A) (DMC)₂@β-CD and (B) (MMC)₂@β-CD.

Figure 3

Infrared spectra at 4000–600 cm^–1 of (A) DMC, β-CD, MM (1:1), MM (2:1), FL (1:1), and FL (2:1); and (B) MMC, β-CD, MM (1:1), MM (2:1), FL (1:1), and FL (2:1).

Figure 4

Curves of (A and D) TG, (B and E) DTG, and (C and F) DTA of DMC or MMC and their inclusion complexes by MMs and FL in guest/host ratios of 1:1 and 2:1.

Figure 5

Topical application of (DMC)₂@β-CD (FL) for 5 days after excisional injury reduces granulation tissue in the wound bed. Male mice between 8 and 10 weeks of age of the Swiss lineage had an excisional lesion of 7.0 mm diameter on the median sides of the back and received (A) 20 μL of saline solution, (B) 20 μL of (DMC)₂@β-CD (FL) at 1 mg mL^–1, and (C) (MMC)₂@β-CD (FL) solution at 1 mg mL^–1 for five consecutive days in an interval of 24 h. (A–C) Panoramic view of injured skin and the granulation tissue area stained with H&E 7 days after skin injury in mice. On day 7, postwounding re-epithelialization occurred in all groups (A–C) and a small number of fibroblasts (elongated cells) and inflammatory cells (rounded cells) was present in wounds from (MMC)₂@β-CD (FL)-treated mice, as can be seen in the entire magnified image (E). The black arrows indicate the granulation tissue area in the wound bed, and small letters (indicated by white arrows) represent the following: e, epidermis; gt, granulation tissue; at, adipose tissue. (A–C) (40× magnification); (D–F) (400× magnification).

Figure 6

Topical application of coumarin for 5 days after excisional injury reduces the mast cell density in the wound bed. Morphometric analysis of mast cells after alcian blue safranine-stained sections. Mast cells were counted in 10 fields of 10,000 μm² within the wound healing area of one section per mouse, and the results from six sections per group were expressed as the mean ± SEM saline group (black bars), (DMC)₂@β-CD (FL) (open bars), and (MMC)₂@β-CD (FL) (hatched bars). Data represent mean ± SEM of the mast cell density (in μm² × 10^–3). *p ≤ 0.05 compared with the saline group, n = 6.

Figure 7

Topical application of (DMC)₂@β-CD (FL) for 5 days after excisional injury improves collagen remodeling. Male mice between 8 and 10 weeks of age of the Swiss lineage had an excisional lesion 7.0 mm in diameter on the median sides of the back and received (A) 20 μL of saline solution; (B) 20 μL of (DMC)₂@β-CD (FL) at 1 mg mL^–1; and (C) (MMC)₂@β-CD (FL) solution at 1 mg mL^–1 for five consecutive days at an interval of 24 h. Representative photomicrographs of skin from control mice with scar tissue (A, E, I); skin of mice treated with (DMC)₂@β-CD (FL) with scarless tissue (B, F, J); skin of mice treated with (MMC)₂@β-CD (FL) scar tissue (C, G, K) on day 60 after wounding and normal intact skin mice (D, H, L). On day 60 after lesion, the neodermis of mice treated with (DMC)₂@β-CD (FL) (J) closely resembles that of the normal dermis, with the collagen fibers arranged in a basket-weave pattern (L). The reorganization of the papillary dermis appears in the (DMC)₂@β-CD (FL) group (F) at 60 days compared to intact skin (H). Arrows indicate the location of the injury. Gomori’s trichrome-stained sections. (A–D), scar area (40× magnification); (E–H), epithelium and papillary dermis (400× magnification); (I–L), reticular dermis (400× magnification).

Figure 8

(A) DMC-MAPK1 and (B) MMC-MAPK1 complexes and (C) DMC-MAPK3 and (D) MMC-MAPK3 complexes. Dashed lines represent the hydrogen-bond interactions.