The skin serves as a crucial barrier between the internal body systems and the external environment, playing a vital role in maintaining homeostasis (1). Skin damage compromises this protective barrier, potentially exposing the body to pathogen invasion risks and substantial fluid loss. Therefore, prompt restoration of skin integrity following injury represents the most effective approach to reestablish internal equilibrium, combat infections, and prevent fluid loss and electrolyte imbalance (2). Skin wound healing constitutes a complex and precisely regulated biological process that occurs through four overlapping phases: hemostasis/inflammation, proliferation, and remodeling (3). Disruption in any of these processes may result in impaired wound healing and the development of chronic wounds, including diabetic foot ulcers and pressure injuries, which present significant clinical challenges and affect approximately 1–2% of the global population (4).

Following skin injury, necrotic cells and disrupted tissues release damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), which trigger the activation of innate immune responses. These molecular patterns stimulate resident immune cells, including mast cells, T cells, and macrophages. Specifically, activated macrophages engage in downstream inflammatory signaling pathways by binding to their cognate receptors (5), thereby initiating the release of pro-inflammatory mediators. These pro-inflammatory factors induce vasodilation and facilitate the adhesion and transmigration of neutrophils and monocytes (6). Neutrophil recruitment to the wound site is mediated through chemoattractants such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and bacterial endotoxins like lipopolysaccharide (LPS). Upon activation, neutrophils respond to inflammatory signals and pathways, including NF-κB, by secreting pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-8 (7). Additionally, neutrophils clear necrotic tissue and pathogens via phagocytosis and the release of reactive oxygen species (ROS), antimicrobial peptides, and proteolytic enzymes (8). However, excessive neutrophil infiltration may have detrimental effects on tissue repair and delay wound healing (9). During the intermediate healing phase, neutrophils undergo reverse transmigration back into the bloodstream or undergo programmed cell death and subsequent clearance by macrophages (10).

Monocytes migrate to the inflammatory site during the later stages of the inflammatory response, where they either directly participate in phagocytic immune responses or differentiate into inflammatory macrophages (11). It is important to distinguish monocyte-derived macrophages from the conventional dendritic cells residing in the skin, including Langerhans cells (LCs) in the epidermis and dermal macrophages in the dermis (12). Emerging evidence indicates that the population of LCs in the epidermal layer significantly increases during the initial phases of wound healing and in healing diabetic foot ulcers (DFUs) compared to non-healing DFUs, suggesting a potential therapeutic role of LCs in diabetic wound management (13). Due to their remarkable plasticity, macrophages exhibit more diverse functions than monocytes and assume the phagocytic roles previously performed by polymorphonuclear neutrophils (PMNs) and monocytes. This plasticity enables their functional transition from pro-inflammatory phenotypes in the early healing stages to anti-inflammatory/reparative phenotypes during the proliferative phase (14). Wound macrophages originate from two primary sources: tissue-resident macrophages, which constitute a minor proportion, and monocyte-derived macrophages, which predominate and can rapidly expand in response to inflammatory stimuli (15).

Conventional wound management approaches encompass a range of surgical interventions, non-surgical therapies, and pharmacological treatment regimens for skin wound repair (16). While surgical debridement remains the gold standard in wound management due to its clinical efficacy (17), it can induce significant patient discomfort. Pharmacological interventions have emerged as promising alternatives to promote wound healing while minimizing patient distress. Among these, growth factor-based therapies (e.g., FGF21, EGF, and PDGF) represent a promising therapeutic approach. However, several limitations, including their short biological half-life, receptor desensitization issues, and reduced efficacy in metabolic-impaired microenvironments, necessitate the development of novel therapeutic strategies (18, 19).

Fibroblast growth factor 21 (FGF21), fibroblast growth factor 19 (FGF19), and fibroblast growth factor 23 (FGF23) constitute the FGF19 subfamily, exhibiting significant structural homology (20). The human FGF21 gene, located on chromosome 19, encodes a 209-amino acid precursor protein. Following synthesis, the precursor undergoes proteolytic cleavage of its signal peptide, yielding a mature protein comprising 181 amino acids. Notably, the human FGF21 mature protein shares 75% sequence homology with its murine counterpart (21). FGF21, a recently identified metabolic regulator, demonstrates predominant expression in adipocytes, hepatocytes, and pancreatic β-cells, where it plays a pivotal role in modulating lipid and carbohydrate metabolism (22).

The biological activities of FGFs are primarily mediated through fibroblast growth factor receptors (FGFRs). However, FGFs cannot directly interact with FGFRs, requiring co-activator binding for effective activation of FGFR signaling pathways (23). Specifically, FGF21 must first interact with β-klotho, a transmembrane protein, before forming a ternary complex with FGFRs to initiate downstream signaling cascades (24). This molecular interaction is particularly significant in adipose tissue, liver, and pancreatic cells, where both FGF21 and β-klotho are predominantly co-expressed (25). Although FGF21 is principally synthesized and secreted by hepatic and adipose tissues (26), however, emerging experimental evidence has revealed that FGF21 exhibits significant upregulation in injured skin tissues (27). Following skin injury, the expression levels of both FGF21 and its co-receptor β-klotho demonstrate continuous elevation, reaching up to 2.2-fold and 4-fold increases, respectively, compared to baseline levels (28). Despite the observed high expression of FGF21 during cutaneous wound healing, its precise mechanistic role in promoting tissue repair remains incompletely understood. In wound healing assays, Western blot analysis has demonstrated that FGF21 expression is modulated by Wnt pathway activators. These investigations have revealed that FGF21 facilitates fibroblast migration and enhances skin wound repair processes. Notably, these studies have identified a reciprocal regulatory loop between FGF21 and Wnt signaling pathways, mediated through β-catenin in dermal fibroblasts (29). The interplay between FGF21 and Wnt signaling during cutaneous wound healing has been further corroborated by subsequent research (30). Persistent inflammation is a critical impediment to cutaneous wound repair, significantly hindering the healing process. FGF21 plays a pivotal role in modulating inflammatory responses, as evidenced by the development of various inflammatory disorders in FGF21-deficient conditions, including non-alcoholic fatty liver disease (NAFLD) and pancreatitis (31, 32). FGF21 has demonstrated potent anti-inflammatory properties across multiple pathological models. Experimental studies have shown that FGF21 can mitigate free fatty acid-induced nephritis in mice, reduce monosodium glutamate (MSG)-induced hepatic steatosis in obese mice, alleviate high-fat diet (HFD)-induced NAFLD, and attenuate lipopolysaccharide (LPS)-induced sepsis-associated inflammation (33–36). In the context of corneal epithelial wound healing, FGF21 has been shown to enhance epithelial repair while significantly reducing the levels of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, in diabetic mouse epithelium (37). Beyond its cellular functions, FGF21 interacts with multiple signaling pathways, such as Nrf2 and SIRT1, which regulate metabolism and fibrosis. Investigating these pathways could provide deeper insights into FGF21’s tissue-specific effects and its potential role in skin wound healing, an area that remains poorly understood.

The skin, constituting approximately one-third of human tissue, encompasses a surface area of about two square meters (72). This complex organ is composed of three distinct layers: the epidermis, dermis, and subcutaneous tissue. As a primary delivery organ for dermatological therapeutics, the skin allows drug penetration primarily through passive diffusion across the stratum corneum into the inter-follicular region (73). Following penetration, drugs subsequently diffuse to target tissues to exert their pharmacological effects. However, this process is restricted by the skin’s epidermal barrier, which limits the passage of molecules exceeding 500 Da, thereby imposing significant constraints on the therapeutic application of high molecular weight compounds.

Since the landmark discovery of insulin in 1921, remarkable progress has been achieved in the development of therapeutic proteins and peptides, encompassing antibodies, hormones, cytokines, and vaccines (74). To date, over 240 protein and peptide-based therapeutics have received FDA approval for disease treatment (75). Unlike small-molecule drugs, protein therapeutics exhibit high specificity and complex biological functions due to their intricate structural configurations.

Despite the development of various non-injectable drug delivery strategies, including oral, transdermal, nasal, mucosal, pulmonary, and rectal routes, significant challenges persist. Oral delivery, the most commonly employed strategy, is particularly hindered by gastrointestinal barriers characterized by gastric pH variations, proteolytic enzyme activity, and inefficient intestinal absorption mechanisms (76). Consequently, intravenous administration remains the predominant clinical route for protein and peptide delivery (77), despite its associated limitations of patient discomfort, high costs, and logistical challenges.

The Transdermal Drug Delivery System (TDDS) has emerged as a prominent non-invasive method for the systemic administration of therapeutic agents through the skin. This innovative system has demonstrated significant clinical utility across diverse therapeutic areas, including pain management, hormone replacement therapy, and the treatment of cardiovascular and central nervous system disorders (78–80). One of the key advantages of TDDS lies in its ability to bypass the gastrointestinal tract, thereby eliminating first-pass metabolism and its associated limitations on drug bioavailability. This unique delivery mechanism ensures that therapeutic agents are absorbed systemically without interference from gastric pH variations, enzymatic degradation, or intestinal microbiota. Specifically, TDDS facilitates drug absorption through the skin’s epidermal and dermal layers, where agents subsequently enter the microcirculation and systemic circulation at controlled, sustained rates (81). Furthermore, TDDS has exhibited remarkable versatility in enabling the permeation of diverse therapeutic agents across the skin barrier, including lipid-based compounds, proteins, nucleic acids, and collagen-based formulations. This systemic delivery approach offers distinct advantages over traditional administration routes, particularly for compounds that are susceptible to gastrointestinal degradation or rapid hepatic metabolism.

In recent years, TDDS has emerged as a pivotal strategy in disease prevention and therapeutic intervention. Nevertheless, the intrinsic barrier properties of the skin continue to present significant challenges to TDDS development and optimization. Notably, substantial advancements have been achieved in recent decades across three principal domains of TDDS technology: physical, chemical, and biological enhancement methods.

Physical enhancement methods have demonstrated significant efficacy in improving skin permeability for drug and biomolecule delivery through the application of various external energy modalities, including electrical, mechanical, and physical stimuli (82). These techniques offer dual advantages: ensuring rapid and reliable drug penetration through the skin barrier while simultaneously enhancing the therapeutic efficacy of delivered agents (83, 84).

Chemical enhancement methods in TDDS are primarily characterized by the utilization of synthetic mixtures containing transdermal penetration enhancers combined with active pharmaceutical ingredients. These formulations are specifically designed to optimize the percutaneous absorption rate and therapeutic efficacy of administered drugs (95). The chemical enhancement approach is particularly effective for pharmaceutical compounds that exhibit specific physicochemical properties: low molecular weight (<500 Da), appropriate lipophilicity (logP value between 1 and 3), limited skin irritation potential, and relatively short biological half-life. The research focus in transdermal technology has progressively evolved, transitioning from the development of traditional chemical penetration enhancers to the integration of advanced physical enhancement strategies. Contemporary TDDS approaches aim to enhance drug permeation through innovative mechanisms, including the optimization of drug diffusion coefficients and solubility parameters in the stratum corneum. This has been achieved through the development of advanced drug delivery vehicles such as microemulsions, nanoemulsions, and novel vesicular systems, including liposomes, niosomes, and transfersomes (96). These technological advancements have significantly expanded the range of therapeutic agents that can be effectively administered through the transdermal route.

Cell-penetrating peptides (CPPs), alternatively referred to as protein transduction domains or membrane-penetrating peptides, were first implemented as agents for transdermal delivery (105). These peptides are principally composed of cationic or amphipathic membrane-interactive sequences that demonstrate the capacity to traverse cellular membranes (106). The category of CPPs has been extended to include protein transduction domains, arginine-rich peptides, bioportides, and numerous other peptide sequences as identified through experimental and computational predictions (107).

The utility of CPPs has been demonstrated in the delivery of diverse therapeutic agents, including but not limited to fluorophores, nucleic acids, peptides, and peptide nucleic acids (108–110). It has been well established that extracellular proteins and peptides exhibit inherent impermeability to cellular membranes, necessitating the implementation of delivery vectors for intracellular transport, as exemplified by the requirement for delivery systems for therapeutic proteins (111).

The association of proteins or peptides with CPPs is achieved through multiple strategies: chemical cross-linking, genetic fusion through cloning and expression of CPP-conjugated proteins, or through non-covalent interactions (112, 113). The enhancement of peptide internalization by CPPs has been substantiated in various studies, including the improved cellular uptake of glucagon-like peptide-2 following conjugation with CPP R8 (114). Similarly, the translocation efficiency of reporter proteins, such as EGFP, has been significantly enhanced using CPPs like Pas2r12 (115). This methodology has been successfully extended to other reporter and model proteins of varying molecular weights and physicochemical properties, including β-galactosidase.

TD1 (ACSSSPSKHCG), a transdermal peptide identified through phage display screening technology, has been demonstrated to facilitate the targeted delivery of therapeutic proteins, including insulin, growth hormone, and human epidermal growth factor (hEGF), to the deep dermal layers via the follicular pathway (116–118). When TD1 is genetically fused with hEGF, not only is the biological activity of hEGF preserved, but its transdermal delivery efficiency is also significantly enhanced (119). These experimental observations have confirmed the efficacy of TD1 as a potent transdermal delivery enhancer for therapeutic proteins.

A short peptide designated as SPACE has been employed in phage display screening technology to identify its transdermal delivery capabilities. Through systematic in vitro transdermal studies, SPACE has been demonstrated to form stable conjugates with both small molecules and proteins, facilitating their penetration through the dermal layers within 30 min and enabling their delivery to deep dermal capillaries within 2 h, thereby promoting systemic absorption of therapeutic agents (120).

A novel delivery complex, SE-siRNA, has been developed through the conjugation of SPACE with EGF, which has been established as an effective carrier for siRNA delivery across the stratum corneum into viable cells, subsequently inducing programmed cell death in malignant cells (121). The delivery efficiency of the SE-siRNA complex has been shown to significantly exceed that of control siRNA formulations, with the enhanced transdermal penetration capacity being attributed to the SPACE-mediated transport mechanism (122).

The development of transdermal peptides has been recognized as an effective solution to the challenges of poor drug absorption caused by restrictive skin barrier properties in TDDS. These peptides have been demonstrated to significantly enhance the bioavailability of macromolecular drugs. When compared to physical and chemical enhancement methods, biological transdermal strategies have been characterized by milder application conditions and superior preservation of protein bioactivity, while eliminating the potential risks of skin tissue damage associated with alternative methods. The biological transdermal delivery system has been identified as a promising research direction for advancing the therapeutic development and clinical application of FGF21.

For a comprehensive overview of the benefits and potential drawbacks of these approaches, please refer to Table 1, which summarizes the key aspects of each method.

FGF21, as a newly identified metabolic regulator, is primarily expressed in hepatic and adipose tissues where it has been shown to play a crucial role in metabolic regulation. However, its therapeutic potential for metabolic disorders has been limited due to insufficient expression levels in pancreatic β-cells. To address this limitation, a recombinant FGF21 construct containing a protein tag has been developed through protein fusion labeling technology. The incorporation of a protein tag has been demonstrated to significantly enhance both the production yield and solubility of FGF21. Furthermore, the utilization of this recombinant FGF21 protein has been shown to simplify purification processes and improve the overall protein purity, thereby facilitating its potential therapeutic applications.

Histidine tags, typically composed of six histidine residues in tandem, are extensively utilized for the purification of recombinant proteins. These tags can be incorporated at either the N-terminal or C-terminal position of target proteins without significant disruption to their structural integrity or biological function. In a recent study, a histidine-tagged smGFP-FGF21 fusion protein was successfully engineered and expressed in Nicotiana benthamiana. The recombinant protein was found to be predominantly expressed in its soluble form, facilitating efficient purification through Ni-NTA affinity chromatography. Notably, protein yields of up to 2 mg per gram of fresh leaves have been achieved using this expression system (123).

The small ubiquitin-like modifier (SUMO) tag, an approximately 11 kDa protein encoded by the yeast Smt3 gene (124), is frequently employed for monitoring the intracellular localization of recombinant proteins. When attached at the N-terminus of target proteins through genetic fusion, the SUMO tag has been demonstrated to facilitate proper protein folding. Furthermore, the hydrophobic core of the SUMO tag has been shown to confer proteolytic stability to the fusion protein. In recent studies, FGF21 has been successfully fused with the SUMO tag using overlapping PCR technology, and the resultant fusion protein has been expressed in Escherichia coli. Through IPTG induction, SUMO-FGF21 expression levels have been observed to reach approximately 30% of total cellular protein, with the soluble fraction accounting for over 95% of the expressed protein (125).

Maltose-binding protein (MBP), a macromolecular protein tag, has been characterized by its ability to adopt both open and closed conformational states, which have been demonstrated to exhibit distinct binding properties. When in the open conformation, the MBP tag has been shown to specifically interact with gel resin, while the closed conformation has been found to prevent such binding interactions (126). The solubility of FGF21 has been significantly enhanced through fusion with the MBP tag, which has been observed to reduce the accumulation of FGF21 in inclusion bodies during expression in Escherichia coli. FGF21 sequestered in inclusion bodies has generally been considered non-functional due to its lack of biological activity. Notably, the MBP tag has been demonstrated to maintain FGF21 solubility at 37°C under culture conditions, with approximately 8.1 mg of pure FGF21 having been obtained from a 500 mL culture after tag removal (127). The MBP fusion strategy has been conclusively shown to improve FGF21 solubility and substantially facilitate the purification process of recombinant FGF21, making it a valuable tool in protein production and purification workflows.

The fusion of FGF21 with protein tags has been demonstrated to significantly enhance the solubility of the expressed protein, thereby establishing a foundation for optimized purification processes, increased production yields, and reliable protein production protocols. The functional benefits of protein fusion technology have been shown to extend beyond simplifications in purification and solubility enhancement.

Through fusion strategies, target proteins have been observed to exhibit enhanced functional characteristics. Furthermore, fusion proteins have been demonstrated to acquire several advantageous properties, including controlled release through biological barriers, targeted transport mechanisms, and structural stabilization. These properties have been found to significantly improve the pharmacological and biological performance of therapeutic proteins.