L-ENK (YGGFL) was synthesized and provided by BIO-FD&C, Co., Ltd. Based on L-ENK, AGGFL (E10) was synthesized using the conventional solid-phase peptide synthesis method with 9-fluorenylmethoxycarbonyl (Fmoc) as a protector of amino acids. Fmoc-Leu-Wang resin was used (Wang resin attached to leucine, which is an amino group protected by Fmoc). Extension of the amino acid residue was achieved by using N-hydroxybenzotriazole (HOBt) and N,N´-diisopropylcarbodiimide (DIC) as activators. The amino acids, HOBt, and DIC, used in the step-by-step binding process, were combined with five equivalents of resin and reacted at room temperature for 2 hours. After the reaction was complete, it was washed six times with dimethylformamide, six times with dichloromethane, and then dried. Furthermore, AGGFL-resin, a dried pentapeptide, was reacted with a combination of trifluoroacetic acid: triisopropylsilane: H2O (90:5:5 [v/v]) for 2 hours at room temperature to separate the peptide from the resin. Further, the peptide was precipitated with cold ether to obtain the crude peptide, using a centrifuge. Finally, the obtained AGGFL (E10) derivative was purified by reverse-phase high-performance liquid chromatography (column: Gemini, C18; 5 μ; 110 Å, 250×21.2 mm) using 0.1% trifluoroacetic acid and acetonitrile as the solvent. Other enkephalin derivatives were synthesized in the same manner as described above.

Fifteen C57BL/6N mice (Orient Bio Inc.), which were 6 weeks old and weighed 18 to 24 g, were used. All animal procedures were approved by the Institutional Animal Care and Use Committee. Mice were anesthetized with an intraperitoneal injection of 0.006 mL/10 g Zoletil and 0.004 mL/10 g Rompun. After hair removal, two full-thickness skin defects with a diameter of 10 mm were made on both sides of the back of the mouse. In each mouse, the right-side defect was treated with an enkephalin derivative (enkephalin group), and the left-side defect was treated with phosphate-buffered saline (PBS; control group). All skin defects were covered with a semipermeable film (Tegaderm; 3M Health Care), and prepared enkephalin was administered between the skin defect and semipermeable film using a syringe and a 26G needle. In the enkephalin group, 100 μL of E10 (AGGFL; 200 μg/mL) was injected into the skin defect site, and 100 μL of sterile PBS was injected in the control group. To evaluate changes in wound size, digital photographs were taken on days 2, 4, 7, and 10, starting from the first experimental day. The camera lens was positioned vertically on the wounds. The camera was fixed at a certain distance using a stand to minimize errors in the position of the camera. Digital images of the wound were analyzed, and the number of pixels in each wound was determined using ImageJ software (National Institutes of Health). The proportion of wounds on days 2, 4, 7, and 10 were calculated and compared to the initial defect size (Fig. 1).

The mouse excisional model described above was used to evaluate the effect of the enkephalin derivative on cicatrization. After 21 days, full-thickness scar tissue was harvested from the surrounding normal tissue. The tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Tissue sections were observed under a light microscope and evaluated using CaseViewer (3DHISTECH Ltd.). The width and thickness of the depressed scar were measured and expressed using the scar depression index (SDI) (Fig. 2). The SDI was defined as the difference in area between the unwounded dermis and wounded dermis divided by the unwounded dermis. The SDI value is less than 1 by definition, and a value closer to 0 indicates a lower degree of dermal depression.

Wounded skin fragments of the mice were homogenized in RIPA buffer (10 mM Tris-Cl, pH 8.0; 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, and 1 mM PMSF) containing a mixture of protease inhibitors for 30 minutes on ice. Homogenates were centrifuged at 20,000×g for 15 minutes at 4 °C, and supernatants were collected and measured using a Pierce protein bicinchoninic acid assay (Thermo Fisher). EGF production was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (PeproTech). Samples and standards (100 µL in each well) were added to washed plates in duplicate. The plates were then covered and incubated for 2 hours at room temperature. The plates were washed before the addition of the antibody detection solution and incubation for 2 hours at room temperature. The washed plates were incubated with streptavidin-horseradish peroxidase conjugate solution (Amersham) for 30 minutes. The plates were washed with PBS and incubated with 100 µL of tetramethylbenzidine solution for 20 minutes at room temperature. The staining reaction was stopped using a stop-solution, and absorbance was measured at 490 nm. EGF levels were expressed as picograms per milligram of total protein.

Statistical analyses were performed using the IBM SPSS version 20. Representative data were presented as mean±standard deviation. Statistical significance was set at P<0.05. The Kolmogorov-Smirnov test was used to verify data normality. The independent two-sample t-test and Mann-Whitney U test were used for parametric and non-parametric tests, respectively.

Based on L-ENK, enkephalin derivatives (AGGFL, YAGFL, YGAFL, YGGAL, and YGGFA) were synthesized using the alanine scan method by substituting the position of each amino acid with alanine (Table 1). Among these, E10 (AGGFL) and E13 (YGGAL) were the most effective in promoting wound healing in our preliminary studies [14,15]. Furthermore, a pilot test showed that after complete wound healing, the total collagen content of the E10 group was lower than that of the other groups. Therefore, only the E10 derivative was assessed in this study.

All 15 mice survived until day 10. In the control group, the percentage areas of the skin defects were 98.4%±17.9%, 83.2%±24.0%, 39.7%±17.4%, and 16.2%±10.0% on days 2, 4, 7, and 10, respectively. In the enkephalin group (E10 derivative), the percentage areas of the skin defects were 86.1%±15.0%, 61.4%±11.6%, 26.6%± 8.8%, 16.4%±8.8% on days 2, 4, 7, and 10, respectively. The wound healing rate of the enkephalin group was much higher than that of the control group, and the enkephalin group showed a statistically significant reduction in the wound area on days 2, 4, and 7 (P=0.023, P=0.006, and P =0.040, respectively) (Fig. 3A). Changes in the wound area over time were also confirmed in the gross photographs (Fig. 3B). The residual wound areas in the control group were statistically significantly larger on days 2, 4, and 7.

One of the 15 mice was lost before day 21, and the scar tissues of 14 mice were evaluated to prove the effectiveness of the enkephalin derivative on cicatrization. After the wound was completely healed on day 21, the SDI values were 0.22±0.13 in the control group and 0.06±0.19 in the enkephalin group. The SDI values of the enkephalin group were significantly lower than those of the control group (P<0.001) (Fig. 4A). Given the difference in SDI values, it could be interpreted that the enkephalin group had flatter skin surfaces in scar tissue. In the representative H&E-stained slides, the degree of depression of the wounded dermis between the two groups was markedly different (Fig. 4B).

The study was conducted using 14 samples from each group. To compare the concentrations of EGF in the enkephalin and control groups, an ELISA kit was used with excised scar tissues on day 21. The EGF levels of the control group and the enkephalin group were 42.1±20.5 pg/mL and 102.2±22.6 pg/mL, respectively, which showed a statistically significant difference (P<0.001) (Fig. 5).