Milk-derived exosomes carrying siRNA-KEAP1 promote diabetic wound healing by improving oxidative stress

Abstract

Diabetic wounds are a serious complication of diabetes mellitus (DM) that can lead to persistent infection, amputation, and even death. Prolonged oxidative stress has been widely recognized as a major instigator in the development of diabetic wounds; therefore, oxidative stress is considered a promising therapeutic target. In the present study, Keap1/Nrf2 signaling was confirmed to be activated in streptozotocin (STZ)-induced diabetic mice and methylglyoxal (MGO)-treated human umbilical vein endothelial cells (HUVECs). Knockdown of Keap1 by siRNA reversed the increase in Keap1 levels, promoted the nuclear translocation of Nrf2, and increased the expression of HO-1, an antioxidant protein. To explore therapeutic delivery strategies, milk-derived exosomes (mEXOs) were developed as a novel, efficient, and non-toxic siRNA carrier. SiRNA-Keap1 (siKeap1) was loaded into mEXOs by sonication, and the obtained mEXOs-siKeap1 were found to promote HUVEC proliferation and migration while relieving oxidative stress in MGO-treated HUVECs. Meanwhile, in a mouse model of diabetic wounds, injection of mEXOs-siKeap1 significantly accelerated diabetic wound healing with enhanced collagen formation and neovascularization. Taken together, these data support the development of Keap1 knockdown as a potential therapeutic strategy for diabetic wounds and demonstrated the feasibility of mEXOs as a scalable, biocompatible, and cost-effective siRNA delivery system. Graphical Abstract The therapeutic effect of siKeap1-loaded mEXOs on diabetic wound healing was assessed. First, we found that the expression of Keap1 was upregulated in the wounds of diabetic mice and in human umbilical vein endothelial cells (HUVECs) pretreated with methylglyoxal (MGO). Next, we extracted exosomes from raw milk by differential centrifugation and loaded siKeap1 into milk-derived exosomes by sonication. The in vitro application of the synthetic complex (mEXOs-siKeap1) was found to increase the nuclear localization of Nrf2 and the expression of the antioxidant protein HO-1, thus reversing oxidative stress. Furthermore, in vivo mEXOs-siKeap1 administration significantly accelerated the healing rate of diabetic wounds (Scheme 1). Scheme 1 Schematic diagram. A Synthesis of mEXOs-siKeap1 complex. B Mechanism of mEXOs-siKeap1 in vitro. C The treatment effect of mEXOs-siKeap1 on an in vivo mouse model of diabetic wounds Supplementary Information The online version contains supplementary material available at 10.1007/s13346-023-01306-x.

Milk-Derived Extracellular Vesicles: Biomedical Applications, Current Challenges, and Future Perspectives

“…19 In another study, researchers investigated the potential of miEVs as siRNA carriers to target the Keap1 gene for diabetic wound healing and found that their formulation increases collagen synthesis and neovascularization, which leads to the great healing of diabetic wounds in mice models. 137 Although unmodified miEVs have shown good potential for the delivery of various therapeutics, several studies have recently focused on the surface functionalization of miEVs to direct drugs precisely to the target tissue and thereby reduce off-target effects and increase efficacy 138,139 (Table 6). Because the folate receptor is overexpressed in various cancer cells, its ligand, folic acid (FA), has been used to target drug carriers.…”

Milk Extracellular Vesicles: Natural Nanoparticles for Enhancing Oral Drug Delivery against Bacterial Infections

“…These methods typically involve ultracentrifugation or other purification techniques to isolate the EVs from other milk components . One potential application of bovine milk-derived EVs is in the development of therapies for inflammatory diseases, such as inflammatory bowel disease and arthritis. , Studies have shown that mEVs can suppress inflammation and promote tissue repair in animal models of these diseases. In addition, mEVs have been shown to enhance the immune response in vitro, suggesting their potential as immunomodulatory agents .…”

“…Extracellular vesicles have garnered significant interest as potential vehicles for drug delivery, due to their ability to transfer their contents between cells and tissues . Scientists are investigating ways to modify extracellular vesicles to target specific cells or tissues and to load them with therapeutic payloads such as drugs or genetic material. , The use of EVs as drug delivery systems has been hindered by several factors. One of the main issues is their low gastrointestinal stability, which makes it difficult for them to survive in the harsh environment of the digestive system .…”

Small extracellular vesicles: Yields, functionalization and applications in diabetic wound management

“…Besides, to relieve oxidative stress at diabetic wound sites, siRNA‐Keap1 (siKeap1) functionalized milk‐released sEVs were fabricated by ultrasonicating the mixed PBS solution of siRNA‐Keap1 and sEVs. SiKeap1‐enriched sEVs silenced Keap1 gene and increased the expression of antioxidant protein (HO‐1) in vitro and accelerated diabetic wound healing by promoting collagen formation and neovascularization 74 . Yerneni et al.…”

“…SiKeap1-enriched sEVs silenced Keap1 gene and increased the expression of antioxidant protein (HO-1) in vitro and accelerated diabetic wound healing by promoting collagen formation and neovascularization. 74 Yerneni et al loaded the antiinflammatory drug (curcumin, CA) inside sEVs with the assistance of albumin under milder sonication. The pre-introduction of albumin provided abundant bindingsites of CA inside sEVs.…”