Diabetes wounds occur in approximately 20% of diabetic patients and have a poor prognosis, high recurrence rate, and often lead to amputation. For this reason, it is one of the most severe and expensive diabetes complications [1]. However, diabetic wound healing has complex pathogenic abnormalities caused by diabetes-induced hyperglycemia, including increased levels of oxidative stress, increased inflammatory responses, blocked angiogenesis, and other complicating systemic effects due to diabetes [2]. Among these definite mechanisms, oxidative stress and impaired angiogenesis are the two main reasons for the lasting and nonfibrotic healing of chronic diabetic wounds [3]. The high level of reactive oxygen species (ROS) can prevent the inflammatory phase from entering the proliferative phase and thus hinder the construction of new healthy tissue in diabetic wound healing [4]. In addition, impaired angiogenesis causes long-term ischemia and hypoxia in diabetic wounds, impeding neutrophil-mediated bactericidal mechanisms and the proliferative phase of wound healing. However, few therapeutic strategies affect both mechanisms of oxidative stress and impaired angiogenesis at the same time. Therefore, it is urgent to construct a strategy with two healing mechanisms for diabetic wound healing.

Acid fibroblast growth factor (aFGF) has attracted considerable attention due to its various pharmacological properties that could significantly promote angiogenesis to accelerate wounds in recent years [5]. However, aFGF is highly vulnerable to hostile circumstances that are mainly caused by hyperglycemia-induced oxidative stress in diabetic wounds. Various nanoparticle delivery systems have been widely developed to improve the stability of the protein to maximize its bioactivity in wound healing. The nanoparticle could encapsulate aFGF, prolonging the half-life of aFGF. However, the complicated preparation procedure usually involves multiple emulsification steps or the use of an organic solvent, which compromises the stability of aFGF for applications in vivo. Meanwhile, nanoparticles usually improve the drug load by changing the drug-to-carrier ratio, while the use of high quantities of carriers can result in toxicity due to poor metabolism and elimination of the carriers. Therefore, despite the emergence of various nanoparticle-based protein delivery approaches, it remains challenging to engineer a versatile delivery system capable of enhancing protein stability in diabetic wounds without the need for complex preparation and excess toxic carriers of the delivery system.

(−)-Epigallocatechin gallate (EGCG), one of the polyphenol components extracted from green tea leaves, has a series of biological functions, including anti-lipid peroxidation, antioxidant, anti-inflammatory, antibacterial, and anticancer activities [6] EGCG was reported to have a strong ROS scavenging ability to protect oxidatively injured cells and accelerate wound healing [7]. More importantly, EGCG exhibited strong interactions with biomacromolecules, including proteins, polypeptides, and Deoxyribonucleic Acid (DNA), via hydrogen bonding and hydrophobic interactions, resulting in the formation of polyphenol‐containing nanoparticles [8]. Meanwhile, owing to polyphenols having bioadhesive properties at cell interfaces, polyphenol‐based materials exhibit good cell affinity and promote cell adhesion via the phenolic hydroxyl groups of polyphenols [9, 10]. In addition, polyphenol‐containing nanoparticles could enhance intracellular protein delivery by competing for supramolecular interactions [11]. It was reported that the self-assembled nanocomplexes of EGCG derivatives and anticancer proteins could significantly enhance the anticancer effects in vitro and in vivo compared with the free protein. Therefore, the strategy of polyphenol-driven assembly of nanoparticles has great potential for improving the bioactivity of aFGF and accelerating diabetic wound healing. However, T specific formation mechanism of aFGF and EGCG has not yet been reported.

Herein, a polyphenol-driven facile assembly of nanosized coacervates (AE-NPs) composed of aFGF and EGCG was constructed and applied in the healing of diabetic wounds for the first time. (Fig. 1). First, the binding patterns of EGCG and aFGF were predicted by molecular docking analysis, suggesting that the coacervate was formed by the action of hydrogen and hydrophobic bonds. Meanwhile, the affinity constant (KD=5.3×10−7) of EGCG against native aFGF was measured by surface plasmon resonance (SPR) for the first time, strongly proving the interaction between the two substances. In addition, in vitro characterization demonstrates that the drug loading of aFGF and the particle size of the coacervate could be controlled by adjusting the EGCG/aFGF feed ratio. Therefore, we prepared a series of nanosized coacervates, and the optimal coacervate AE-NPs-3 was obtained with suitable characteristics for diabetic wound healing. Meanwhile, AE-NPs showed high aFGF activity in the harsh microenvironment of the diabetic wound. The mechanism was most likely that EGCG coupled to the active binding sites of the aFGF molecule, preventing secondary structural damage and destruction by proteases and other harmful chemicals. More importantly, the AE-NPs could enhance the binding activity of aFGF to cell surface receptors via bioadhesion between the phenolic hydroxyl group and protein on the cell surface, which significantly improved the biological effect of aFGF. In addition, the AE-NPs displayed a good ability to scavenge ROS and promote angiogenesis in vitro. Given the various characteristics of AE-NPs, we assessed the therapeutic efficacy of the nanosized coacervate system in full-thickness excisional skin wounds of diabetic mice, indicating that AE-NPs could significantly accelerate wound healing by scavenging ROS and promoting angiogenesis. Besides, the AE-NPs suppressed the early scar formation by improving collagen remodeling and the mechanism was associated with the TGF-β/Smad signaling pathway. Conclusively, the higher therapeutic quality of AE-NPs on the diabetic wound was demonstrated compared with the single aFGF solution, suggesting that AE-NPs might be a potential strategy for stabilizing protein drugs and achieving the nonfibrotic healing of diabetic wounds.