Investigating therapeutic potential of Zeb2 inhibition and oxidized pullulan in diabetic wound healing

Abstract

Diabetic foot ulcers are a serious and challenging consequence of diabetes, characterized by chronic sores that defy healing. These ulcers occur from diabetes-induced microvascular problems that restrict blood flow, consequently reducing the supply of oxygen and vital nutrients to the wound site. The outcome is a reduced immune response and a delayed healing process. At the root of this issue is hyperglycaemia, which exacerbates inflammation by boosting levels of pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6. The issue is further compounded by hypoxia inside the ulcer, which boosts NF-κB signalling pathways, resulting to an enhanced inflammatory response that hampers the healing process. The combination of hyperglycaemia and hypoxia not only disrupts the normal processes of wound healing but also creates conditions that are conducive to chronic ulceration. The continuous presence of pro-inflammatory cytokines keeps the wound in a prolonged state of inflammation, this makes diabetic foot ulcers not only difficult to treat but also prone to complications such as infections, which can further deteriorate the condition. Macrophages, are critical for shifting from an initial inflammatory phase to a reparative phase, facilitating tissue regeneration and wound healing. However, in the setting of diabetes, macrophages stay imprisoned in a proinflammatory state, unable to properly convert to their repairing duty. The persistent hyperglycaemic environment and the associated chronic inflammation trap macrophages in their pro-inflammatory state, rather than transitioning to their reparative role, leads to continued production of proinflammatory cytokines that adds to the chronicity of diabetic foot ulcers. Functional dysregulation at the molecular level, is the root cause of pathophysiological disturbances. This study, for the first time, uncovers transcription factor Zeb2, , as a crucial regulator of macrophage polarity in hyperglycaemic conditions. Increased glucose levels promote Zeb2 expression caused by hyperacetylation, which supports and maintains the proinflammatory phenotype of macrophages. Notably, downregulating Zeb2 expression leads to a decrease in wound inflammation, showing its critical engagement in chronic wound etiology. We developed an advanced biopolymer-based hydrogel system for effective Zeb2 ASO delivery by addressing the complexities of biomacromolecule delivery in chronic wound environments. This multifunctional hydrogel combines oxidized pullulan (OP) with quaternized chitosan (QC), leveraging their inherent antioxidant and antibacterial properties. A strategic single-step modification of the pullulan backbone demonstrated impressive antibacterial efficacy against Staphylococcus aureus, while ensuring biocompatibility and preventing infections. The OP@QC hydrogel not only supports controlled drug release but also exhibits significant antibioflim activity. Diabetic mouse wound upon treated with OP@QC hydrogel loaded with Zeb2 ASO, notably enhanced the healing rate, mitigated inflammation, and promoted tissue regeneration by shifting macrophages from a pro-inflammatory to an antiinflammatory phenotype. Overall, our study highlights the therapeutic potential of targeting Zeb2 to manage chronic inflammation in diabetic wounds. The OP@QC hydrogel, with its antibacterial properties and ability to deliver therapeutics, represents a significant advancement in wound care technology.