Preparation and properties of HPDA nanoparticles and HPDAlR
Nanoparticles are widely used in modern biomedicine due to their surface functionalization, targeting ability, degradability, and biocompatibility [31,32,33,34]. Recently, nanoparticles have attracted attention in the field of wound healing, such as the application of biomimetic elastomeric peptide-based nanofibrous matrices, engineered nanomaterial for infection control and treatment, and nanotechnology for skin wound repair [35,36,37]. Hollow colloidal particles in nanomaterial are of particular interest due to their low density, excellent surface permeability, remarkable loading capacity, and good morphology [17]. These unique properties make hollow colloidal particles widely applicable in chemical catalysis, biomedicine, optics, electronics, energy storage and conversion, environmental protection, anti-tumor treatment, anti-oxidation, drug transport, and tissue regeneration [16, 38, 39]. HPDA has all the advantages of hollow colloids but is smaller than most nanoparticles. To date, however, research on the application of such important nanoparticles in promoting skin wound regeneration remains limited.
In this study, HPDA nanoparticles were synthesized as per earlier research (Fig. 1A) [40]. TEM images (Fig. 1B) confirmed that the resultant HPDA nanoparticles showed well-defined spherical morphologies and hollow structures. Their average diameter was 52 nm (Fig. 1B). The elemental mapping patterns revealed a uniform distribution of C, N, and O elements (Fig. 1C), further confirming the formation of HPDA nanoparticles. Brunauer–Emmett–Teller (BET) was used to analyze surface physical characteristics of HPDA nanoparticles, as shown in Fig. 1D and E, the nitrogen adsorption–desorption isotherms of HPDA demonstrated hysteresis loops, which is characteristic of mesoporous materials. Besides, as listed in Additional file 1: Table S1, the prepared HPDA had a surface area of 39.1667 m2/g, pore volume of 0.252455 cm3/g, and pore diameter of 27.2621 nm. Due to their hollow structure and nanoscale spherical morphology, HPDA nanoparticles has been considered as drug carriers to ensure that drug molecules exert satisfactory therapeutic effect [41].
Wound healing involves a series of highly complicated physiological processes, including changes in capillary permeability, cell migration, fibroblast proliferation, endothelial and epithelial cell damage, and dynamic balance between cells, collagen, and capillaries [42]. Peptides that have positive effects on inflammation, proliferation, and remodeling are theoretically healing promoters, e.g., Bv8, Bombesin, and endothelial growth factor (EGF) and vascular EGF-releasing peptides [42, 43]. With the development of biotechnology and peptide synthesis, an increasing number of peptide drugs have been developed and applied clinically [44]. Amphibian skin secretes a variety of bioactive peptides with therapeutic effects, which have been extensively studied. As one of the shortest peptides, RL-QN15 has relatively low synthesis costs but significant ability to promote the healing of skin wounds [27].
Considering the advantages of HPDA nanoparticles, it might be reasonable to speculate that the prohealing potency of RL-QN15 might be enhanced by the loading and slow-releasing of HPDA nanoparticles, hence, as schemed in Fig. 1A, we successfully prepared HPDAlR. Because the FTIR spectra of bioactive substances differ from each other [45], we carried out FTIR qualitative analysis of HPDA and HPDAlR, to verify whether RL-QN15 was successfully loaded onto the HPDA shell. The two curves in Fig. 1F showed the characteristic peaks of HPDA and HPDAlR, respectively. In the HPDA spectrum, the peak at 3 417.34 cm−1 was identified as the characteristic absorption peak of N–H and O–H group stretching vibration, while the peak at 1 637.69 cm−1 was the characteristic absorption peak of C-O stretching vibration [46]. Compared with HPDA, the peak values of HPDAlR at these two sites were slightly lower, which was due to the existence of an intermolecular hydrogen bond between HPDA and RL-QN15. In addition, HPDA and HPDAlR exhibited different spectral fingerprint regions (1 800–500 cm−1). The HPDAlR showed characteristic absorption peaks at 1 629.42 cm−1, 1 359.70 cm−1, 1 072.40 cm−1, 861.11 cm−1, 545.10 cm−1, and 520.74 cm−1. At 1 637.69 cm−1, 1 071.70 cm−1, and 534.72 cm−1, HPDA had stronger absorption peaks than HPDAlR. These results indicated that RL-QN15 was successfully loaded onto the HPDA shell to form HPDAlR. X-ray photoelectron spectroscopy (XPS) was also used to analyze the surface chemical composition of the samples. As shown in Fig. 1G, strong O1s and C1s signal peaks accompanied by weak N1s signal peaks were observed in the HPDA sphere spectrum. After RL-QN15 was loaded onto HPDA, a weak S2p signal peak appeared. These results provide solid evidence that RL-QN15 containing intramolecular disulfide bonds was loaded into the HPDA nanoparticles. In summary, the HPDAlR was successfully prepared for the first time, which was verified by both FTIR and XPS.
The loading and slow-releasing efficacy of HPDA against RL-QN15 was further determined. As shown in Fig. 1H, the loading efficacy of the HPDA against RL-QN15 showed a sharp increase at 2 h after the incubation, then a slight ascending trend along with the elapse of time and achieved a maximum value of 76.97% at 24 h. As illustrated in Fig. 1I, when dispersed in phosphate buffered saline (PBS), HPDAlR itself started to release RL-QN15 into PBS in a slow-releasing manner. The HPDAlR released almost half of the RL-QN15 peptide in ~ 4 h and reached a release peak of 75.95% at 16 h. Peptides are fragile and easily degraded by various endogenous and exogenous enzymes [44]. Therefore, coating peptides with nanoparticles could not only help to reduce the degradation induced by various enzymes on the skin surface but also sustain an effective concentration, and hence the increase in the prohealing activities of RL-QN15 might be anticipated.
The toxicity of HPDA and HPDAlR against human keratinocyte, mouse macrophage and mice
The toxic evaluation should be initially performed before the determination of biological activities of HPDA and HPDAlR. As shown in Fig. 2A, HPDA (0.01 mg/mL) had no obvious effect on the viability of keratinocyte HaCaT, and RL-QN15 (1 nM) could significantly increase the viability of HaCaT, which was consistent with the results from our previous research [27]. Interestingly, by the loading of RL-QN15, HPDA (0.01 mg/mL) lR (1 nM) obvious enhanced the viability-promoting ability of RL-QN15 (1 nM) by 37.14 ± 17.41% (n = 4). As displayed in Fig. 2B, HPDA, RL-QN15 and HPDAlR exerted no influence on the viability of mouse macrophage RAW264.7. We further confirmed the positive effects of RL-QN15 and HPDAlR on HaCaT by Live/Dead Cell Viability assay, as displayed in Fig. 2C, almost all of the keratinocyte (HaCaT) cells were stained with Calcein acetoxymethyl ester (Calcein-AM, green fluorescence) and dead cells stained with propidium iodide (PI, red fluorescence) were rarely observed. These results suggested that HPDA and HPDAlR were not cytotoxic against HaCaT and Raw 264.7. More importantly, the topical application of HPDA and HPDAlR to the dorsal skin wounds caused no any death of mice (date not shown), compared with un-treated mice, main organs, including heart, liver, spleen, lung and kidney, showed no obvious histopathological abnormalities (Fig. 2D). Previous studies had certified that the developed HPDA nanoparticles have negligible cytotoxicity [47]. These results are well consistent with the results in this research, the negligible cytotoxicity laid solid foundation for us to investigate the pro-regenerative potency of HPDAlR in vitro and in vivo.
HPDA enhanced the pro-healing activity of RL-QN15 against cell scratch of keratinocyte
Keratinocytes plays vital roles in the healing process of skin wounds for their migration fleetly to wound areas and proliferation to promote re-epithelialization of the wound [26]. RL-QN15 (1 nM and 10 nM) has been proved to significantly promote the healing of HaCaT scratch with a pro-healing rate of ≈80% and 95%, respectively [27]. The results that HPDAlR exerted better ability to promote the proliferation than RL-QN15 (Fig. 2A) indicated that HPDA might enhance the promoting effect of RL-QN15 against cell scratch of keratinocyte. As shown in Fig. 3A, when at 24 h, compared with vehicle (PBS), HPDA (0.01 mg/mL) itself didn’t, but RL-QN15 (1 nM) and HPDA (0.01 mg/mL)lR(1 nM) did promote the healing of HaCaT scratch. As quantified in Fig. 3B, the prohealing activities of RL-QN15 and HPDAlR were both time-dependent. The healing rate of RL-QN15 group was 58.89 ± 4.30% (n = 3) at 12 h and 83.07 ± 7.53% (n = 3) at 24 h, whereas the HPDAlR healing rate was 83.07 ± 6.51% (n = 3) at 12 h and 97.95 ± 1.84% (n = 3) at 24 h. Compared with our previous research, we found that the cellular pro-healing effect of HPDA (0.01 mg/mL)lR(1 nM) was equivalent to that of RL-QN15 at 10 nM. All these results indicated that even HPDA nanoparticle itself showed no prohealing activity, by the loading and slow-releasing of HPDA, the cellular prohealing potency of RL-QN15 was significantly enhanced.
HPDAlR raised the selective modulatory ability of RL-QN15 to induce the release of cytokines from macrophage
Tissue damage induces a complex series of reactions in which macrophages clear cell fragments, activates and eliminate inflammation and promotes tissue fibrosis by releasing cytokines, such as TNF-α, TGF-β1, IL-1β, IL-6 and vascular endothelial growth factor (VEGF) [48]. TNF-α activates neutrophils and lymphocytes to increase the permeability of blood vessel endothelial cells and promote the synthesis and release of other cytokines. TGF-β1 induces macrophages to migrate to the wound sites to promote the proliferation of fibroblasts and the synthesis of cell matrix, and promote the proliferation of epidermal cells. The IL-1β and IL-6 recruit inflammatory cells to secrete pro-healing growth factors to the wound sites [27]. VEGF increase the vascular permeability, proliferation and migration of vascular endothelial cell, and angiogenesis [49]. Our previous study has shown that RL-QN15 significantly blocks the release of lipopolysaccharide (LPS)-induced pro-inflammatory factor TNF-α, induces the release of pro-healing factor TGF-β1 and pro-inflammatory factor IL-1β, but does not induce the release of IL-6 and VEGF [27]. In this research, we found that HPDAlR also played the same role by enhancing the ability of RL-QN15 to selectively regulate the release of cytokines. However, HPDA itself has no effect on the release of these factors from macrophages, as shown in Fig. 4A, compared with LPS-stimulated group, HPDA (0.01 mg/mL) had no effect on the release of LPS-induced TNF-α (793.31 ± 137.65 pg/mL vs 780.70 ± 62.80 pg/mL, n = 3). RL-QN15 (1 nM) inhibited the release of TNF-α (495.03 ± 39.25 pg/mL, n = 3), and HPDA (0.01 mg/mL)lR(1 nM) had more significantly inhibiting effects on the release of TNF-α (372.80 ± 73.73 pg/mL, n = 3). In Fig. 4B and C, compared with vehicle and HPDA (0.01 mg/mL), RL-QN15 (1 nM) promoted the release of TGF-β1 and IL-1β, and HPDA (0.01 mg/mL)lR(1 nM) had more significant effects on the release of TGF-β1 and IL-1β. On the RL-QN15 group, the release of TGF-β1 and factor IL-1β were 463.14 ± 58.86 pg/mL and 66.44 ± 13.28 pg/mL (n = 3). However, On the HPDAlR group, the release of TGF-β1 and IL-1β were 586.03 ± 56.48 pg/mL and 117.62 ± 27.17 pg/mL (n = 3).
HPDA enhanced the pro-regenerative activity of RL-QN15 on acute full-thickness injured skin wounds in mice
On the basis that HPDAlR significantly enhanced the abilities of RL-QN15 to promote the healing of HaCaT scratch and selectively regulate the release of healing-involved cytokines from macrophage, it might be reasonable to raise the speculation that the in vivo pro-regenerative of RL-QN15 could be up-graded by HPDA. Hence, we topically applied vehicle (PBS), HPDA (0.1 mg/mL), RL-QN15 (1 nM), HPDA (0.2 mg/mL)lR(1 nM), HPDA (0.1 mg/mL)lR(1 nM) to skin wounds in mice twice a day. As shown in Fig. 5A and B, compared with vehicle, HPDA (0.1 mg/mL) itself had no effect on skin wound healing, as contrary, both RL-QN15 (1 nM), HPDA (0.2 mg/mL)lR(1 nM) and HPDA (0.1 mg/mL)lR(1 nM) had significant effects on skin wound healing. On postoperative day 8, the wound healing rates in the vehicle and HPDA groups were 64.60 ± 2.67% and 59.82 ± 2.23% (n = 9), respectively. The healing rate in the RL-QN15 group was 81.05 ± 1.68% (n = 9), which was 1.25 and 1.35 times higher than that of the vehicle and HPDA groups, respectively. More importantly, by loading of HPDA, RL-QN15 achieved a much greater repair effect. On postoperative days 2, 4, 6, and 8, the wound healing rates of RL-QN15 were 56.57 ± 1.65%, 62.99 ± 1.33%, 69.19 ± 1.62%, and 81.05 ± 1.68% (n = 9), respectively. Interestingly, the healing rates of HPDA (0.2 mg/mL)lR(1 nM) increased to 72.29 ± 7.87%, 83.09 ± 9.04%, 87.30 ± 3.72%, and 98.23 ± 1.54% (n = 9), respectively, and the healing rates of HPDA (0.1 mg/mL)lR(1 nM) increased to 74.46 ± 2.19%, 83.58 ± 3.78%, 88.23 ± 4.41%, and 99.52 ± 0.44% (n = 9), respectively. In addition, there was no obvious difference in the prohealing potency pf HPDA (0.2 mg/mL)lR(1 nM) and HPDA (0.1 mg/mL)lR(1 nM), therefore, the concentration of HPDA used in the following experiments was 0.1 mg/mL and HPDAlR represented HPDA (0.1 mg/mL)lR (1 nM). One point should be observed was that, HPDAlR showed an equivalent prohealing activities with RL-QN15 (50 nM) that reported in our previous research [27], hence, it might be reasonable to speculate that by the loading of HPDA nanoparticles and slow-releasing efficiency of HPDAlR, the pro-regenerative potency of RL-QN15 against acute full-thickness injured skin wounds had an markedly increase of ≈ 50 times.
Hematoxylin–eosin (H&E) staining was performed after sampling on postoperative days 4 and 8 (Fig. 5C). And the results were further quantified (Fig. 5D–G). With the passage of time, new epidermis and granulation tissues were gradually formed in each group, but after the application of RL-QN15 and HPDAlR, the regeneration and reconstruction of epidermis and granulation tissues were significantly enhanced. In particular, the HPDAlR group had recovered to normal skin level on the 8th day. On postoperative days 4 and 8, the epidermal thickness of the vehicle and HPDA groups was ~ 130 μm and ~ 80 μm, and the dermal thickness was ~ 450 μm and ~ 650 μm (n = 9), respectively. In the RL-QN15 group, the epidermal thickness was ~ 90 μm and ~ 50 μm, and the dermal thickness was ~ 400 μm and ~ 500 μm (n = 9), respectively. However, in the HPDAlR group, epidermal thickness was 53.10 ± 10.50 μm and 30.87 ± 4.63 μm, and dermal thickness of was ~ 280 μm and ~ 350 μm (n = 9), which were closest to the thickness of normal skin tissue among the four groups.
In addition, previous studies have confirmed the TGF-β1 is the key factor in the healing process of skin wounds and our research has indicated that RL-QN15 induces the release of TGF-β1 from macrophage [27], which was also verified to be enhanced by HPDAlR (Fig. 4B). As shown in Fig. 5H and I, after local application of RL-QN15 (1 nM) and HPDA (0.1 mg/mL)lR(1 nM), the expression of TGF-β1 in wound tissue samples increased significantly at the early stage of wound healing (4th day after operation). Briefly, the TGF-β1 content was 184.37 ± 14.28 (n = 3) pg/mL in the vehicle group, 192.27 ± 13.02 pg/mL (n = 3) in HPDA group, 211.57 ± 7.76 pg/mL (n = 3) in RL-QN15 group, and 221.88 ± 16.19 pg/mL (n = 3) in HPDAlR group, respectively. However, in the late stage of wound repair (postoperative day 8th), compared with the vehicle group, HPDAlR colud obviously inhibited the release of TGF-β1 (167.08 ± 11.16 pg/mL vs 112.22 ± 24.21 pg/mL, n = 3) in wound tissue. TGF-β1 is an important symbol of regenerating epithelium and an important and indispensable factor of skin matrix and granulation tissue formation. In the early stage of wound healing, the rich level of TGF-β1 ensures the acceleration of the healing of skin wounds. In addition, the TGF-β1 also promotes fibroblasts chemotaxis and the fibrosis, inhibits collagen degradation [50]. Therefore, reducing TGF-β1 in the later stage of wound healing can alleviate the occurrence of fibrosis, thus the formation of the skin scar. So, HPDAlR might accelerate the healing of skin wounds on mice without or with less the formation of scar by dynamically regulating the contents of TGF-β1.
Next, in order to directly observe the biodistribution and clearance of HPDA and HPDAlR, we successfully prepared ICG-labeled HPDA and HPDAlR, which was topically applied to the skin wounds on mice. By tracking the fluorescence of ICG labeled samples using the IVIS® Spectrum in vivo optical imaging system at 0.5, 2, 4, 8, 12, 24, 48 h, as shown in Additional file 1: Figure S1A, it was found that ICG-labeled HPDA and ICG-labeled HPDAlR almost distributed to the whole wounds area. Notably, the remaining ICG-labeled HPDA and ICG-labeled HPDAlR nanoparticles in the whole wounds area rapidly decayed over time, indicating that ICG-labeled HPDA and ICG-labeled HPDAlR nanoparticles might be removed from the skin metabolism. Due to their small size, HPDA still had chance to enter into the blood, so the distribution and clearance of ICG-labeled HPDA and HPDAlR administrated by intraperitoneal injection to mice were also observed. As shown in Additional file 1: Figure S1B and C, after intraperitoneal injection, the sample rapidly distributed to the entire intraperitoneal area. Abdomen images of mice were taken and results showed that 4 h after the injection, both ICG-labeled HPDA and HPDAlR were mainly distributed in the abdominal cavity of the mice, particularly the liver and kidneys. Then, the fluorescence intensity of ICG-labeled HPDA and HPDAlR nanoparticles gradually decayed with time, indicating that ICG-labeled HPDA and ICG-labeled HPDAlR nanoparticles might be removed from the liver and kidneys metabolism.
HPDA raised the pro-healing activity of RL-QN15 on scald wounds in mice
A skin scald mouse model was also employed to evaluate whether HPDA loading could improve the pro-healing activity of RL-QN15. Here, vehicle (PBS), HPDA, RL-QN15, HPDAlR were topically applied to treat scald on the dorsal skins of mice twice a day. As shown in Fig. 6A, the scalded skin injury progressed from waxy white to brown to scab peeling off. On day 12, skin scabs in the vehicle and HPDA groups were the largest, and HPDA nanoparticles showed no positive effect against the healing of scald. Followed by the RL-QN15 group, while those in the HPDAlR group had mostly fallen off. On postoperative day 12, the repair rates in the vehicle and HPDA groups were 69.35 ± 5.30% and 72.41 ± 1.89% (n = 9), respectively, while that in the RL-QN15 group was increased to 81.10 ± 3.05% (1.67 and 1.12 times higher than the vehicle and HPDA groups, respectively). Notably, HPDAlR almost completely healed the scald wound, with an average repair rate of 99.24 ± 0.92% (n = 9), almost 1.22 times that of the RL-QN15 group (Fig. 6B).
We also sampled scalded skin of mice for H&E staining on postoperative days 8 and 12 (Fig. 6C). On postoperative day 8, all experimental groups were still in the inflammatory phase, but new epithelial tissue and granulation tissue appeared in the HPDAlR group. On postoperative day 12, epidermal regeneration and reconstruction were almost finished in the HPDAlR group, and the epidermal status was consistent with that of normal mice. As shown in Fig. 6D and E, in the process of wound healing, the level of re-epithelialization in the HPDAlR group (99.21 ± 1.41%, n = 9) was higher than that in the other groups, and new epithelium completely covered the injured area. In contrast, the rates of re-epithelialization in the vehicle (53.87 ± 23.86%, n = 9) and HPDA groups (58.24 ± 36.24%, n = 9) were only half that of the HPDAlR group, although new epithelium was also found in the RL-QN15 group (83.91 ± 33.27%, n = 9).
The most common sequela of scald is skin scar, which affects the appearance and joint function. If the burn is serious, sepsis, lung infection and acute kidney failure may also occur, which may be fatal [51]. Our results indicated that RL-QN15 had the ability to promote the healing of scalded skin injury. More importantly, the loading of HPDA significantly improved the healing ability of RL-QN15.
HPDA increased the pro-healing activity of RL-QN15 against oral ulcers in rats
Oral ulcers, a common oral mucosal disease, manifest periodically and are accompanied by burning pain. Ulcer tissue is prone to infection, inflammation, and tissue necrosis. There are many causes of oral ulcers, including immune disorders, drug stimulation, and bacterial infections [52]. Our previous research has proved the pro-regenerative effects of RL-QN15 against oral ulcers in rats [27], in the current research, we further to verify whether the load of HPDA could increase the pro-regenerative effects of RL-QN15 against oral ulcers in rats. Oral ulcer recovery in normal rats takes about 12 days, showed by our previous research [27], which was also verified in the current research, here, as shown in Fig. 7A and B, compared with vehicle, HPDA nanoparticles showed no positive effect against the healing of oral ulcers, as contrary, local application of RL-QN15 and HPDAlR accelerated the healing of oral ulcers. When treated with RL-QN15 (1 nM) or HPDAlR, it took 6 and 5 days, respectively, for all oral ulcers to heal completely. In our previous research, the total recovery of oral ulcers in 7 days required the treatment of RL-QN15 with 10 nM [27]. Thus, it might be reasonable to speculate that the therapeutic effects of HPDAlR were 10 times greater than RL-QN15 alone.
As shown in Fig. 7C, on postoperative day 2, oral ulcer formation and inflammatory cell accumulation were observed in the vehicle, HPDA, RL-QN15, and HPDAlR groups. With the progress of treatment, the therapeutic effects of RL-QN15 and HPDAlR became increasingly significant, and at postoperative day 5, the surface of the oral ulcer was completely covered by new oral mucosal epithelium and was considered to have healed. In contrast, significant oral ulcers with only partial mucosal epithelium were observed in the PBS and HPDA groups.
Periodic and repeated attacks of oral ulcers can have a serious impact on a patient’s work and life. Because of the complicated etiology and unknown pathogenesis of oral ulcers, there is no specific therapeutic drug in clinical practice. Severe patients usually receive local symptomatic treatment supplemented with systemic treatment, which can produce marked side effects and unsatisfactory outcomes [53]. Compared with the use of peptide alone, the HPDAlR used in this study improved the oral ulcer repair rate by nearly 20%, indicating that HPDAlR might be novel option for the treatment of oral ulcers.
HPDA up-graded the pro-regenerative activity of RL-QN15 in acute full-thickness injured skin wounds in swine
Previous studies have shown that skin thickness in mice is less than 50 μm, while that in swine and humans is 70–140 μm and 50–120 μm, respectively [54]. In addition, given its similar structure to human skin, swine skin is often used as an ideal model for studying skin trauma, evaluating dermatology, and developing cosmetic drugs [54]. In the current research, the effects of HPDA and HPDAlR against acute full-thickness injured skin wounds on swine were also evaluated.
As shown in Fig. 8A and B, both PBS and HPDA treatment did not promote swine wound healing, whereas RL-QN15 and HPDAlR treatment significantly promoted swine wound healing. On postoperative days 7, 14, 21, and 28, the average healing rates of the vehicle group were 9.85 ± 1.09%, 25.95 ± 6.12%, 36.87 ± 6.09%, and 47.06 ± 7.36%, respectively (n = 6), and those for the HPDA group were 10.74 ± 5.24%, 23.55 ± 7.61%, 37.39 ± 10.00%, and 47.83 ± 14.56% (n = 6), respectively. In contrast, on postoperative days 7, 14, 21, and 28, the average healing rates in the RL-QN15 group were 22.61 ± 9.34%, 31.68 ± 11.66%, 43.38 ± 9.82%, and 60.05 ± 5.02% (n = 6), respectively, which were ~ 1.28 times that of the vehicle group. Thus, RL-QN15 showed pro-regenerative potential in the full-thickness injured skin model in swine. In comparison, the healing rate in the HPDAlR group was significantly higher, with an average rate of 87.52 ± 2.74% (n = 6) on postoperative day 28, which was 1.86 and 1.46 times higher than that in the vehicle and RL-QN15 groups, respectively (Fig. 8B).
On postoperative day 28, the swine skin wounds were sampled for H&E and Masson staining. Histological analysis showed more neovascularization and hair follicles in the HPDAlR group compared with the other groups (Fig. 8C). In addition, as shown in Fig. 8D and E, the thickness of the new epidermis was 195.03 ± 20.3 μm in the vehicle group and 171.37 ± 10.91 μm (n = 6) in HPDA group. However, new epidermis was significantly thinner in the RL-QN15 (72.74 ± 4.87 μm) and HPDAlR groups (40.56 ± 11.18 μm, n = 6). These results indicated that RL-QN15 not only accelerates wound healing, but also effectively alleviated the increase in skin thickness, which was beneficial for the repair of scars. According to the Masson trichrome staining results, there was no significant difference in collagen content between the vehicle (40.52 ± 5.87%, n = 6) and HPDA groups (39.64 ± 3.2%, n = 6). However, collagen content in the HPDAlR group (82.96 ± 5.13%, n = 6) was 1.3 times higher than that in the RL-QN15 group alone (64.98 ± 16.21%, n = 6).