Drug-loaded vesicles for promoting healing of diabetic wounds

Abstract

This invention belongs to the field of biomedical technology and relates to a drug-loaded vesicle for promoting the healing of diabetic wounds. The drug-loaded vesicle has a core-shell structure. The core contains siRNA targeting ALKBH5 mRNA; the shell is a hybrid membrane of human umbilical vein endothelial cell membrane and milk-derived extracellular vesicle membrane, with its surface embedded with arginine-rich lipopeptides, and at least some arginine exposed outside the hybrid membrane. The core-shell structure effectively protects the siRNA from degradation and reduces off-target interference. The hybrid membrane shell has good biocompatibility, immunogenicity, and low cytotoxicity. The exposed arginine enhances tissue penetration and cellular uptake. In vitro and in vivo experiments have confirmed that this drug-loaded vesicle has good cellular uptake efficiency, effectively downregulates ALKBH5 expression, and restores mRNA levels. 6 A-modification increases the stability of C2orf69 mRNA, improves endothelial cell function, significantly promotes the healing of diabetic wounds, and has good biocompatibility and safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a drug-loaded vesicle for promoting the healing of diabetic wounds. Background Technology

[0002] Diabetic wounds are characterized by high incidence, difficulty in repair, and prolonged non-healing, severely impacting patients' quality of life and threatening human health. Despite advancements in clinical wound management and nursing strategies, the prognosis for diabetic patients remains poor. One of the core pathophysiological features contributing to this predicament is vascular dysfunction caused by endothelial cell dysfunction. Vascular dysfunction directly leads to insufficient tissue perfusion, impaired oxygen and nutrient delivery, and inefficient removal of metabolic waste. Therefore, developing novel therapeutic strategies that can precisely regulate endothelial cell function at the molecular level and promote angiogenesis has significant clinical implications.

[0003] Small interfering RNA (siRNA)-mediated RNA interference technology has become a research hotspot in the field of gene therapy due to its ability to specifically silence the expression of pathogenic genes. Therefore, siRNA therapy can provide a new direction for the precision treatment of diabetic wounds. However, its clinical translation faces many difficulties. First, its poor tissue permeability makes it difficult to effectively penetrate deep into the wound and surrounding tissues. Second, negatively charged siRNAs are difficult to spontaneously penetrate negatively charged cell membranes, resulting in low delivery efficiency and a lack of targeting. Furthermore, most siRNAs accumulate in lysosomes and degrade after cellular uptake. Although chemical modification and novel targeted delivery systems have alleviated these problems to some extent, their efficacy in delivering siRNAs intracellularly remains unsatisfactory. In summary, how to construct an siRNA delivery system with high stability, strong tissue penetration, efficient cellular uptake and lysosomal escape properties, while also possessing good biocompatibility and endothelial targeting, to overcome the multiple delivery barriers in the microenvironment of diabetic wounds, achieve precise regulation of key endothelial cell targets, and accelerate wound healing has become a crucial problem urgently needing to be solved in the field of precision treatment of diabetic wounds. Summary of the Invention

[0004] To construct an siRNA delivery system that combines high stability, strong tissue penetration, efficient cellular uptake and lysosomal escape capabilities, and good biocompatibility and endothelial targeting, thereby overcoming multiple delivery barriers in the microenvironment of diabetic wounds and achieving precise regulation of key endothelial cell targets to promote wound healing, this invention provides a drug-loaded vesicle for promoting diabetic wound healing. The structure of this drug-loaded vesicle includes an arginine-rich lipopeptide, a cell membrane derived from human umbilical vein endothelial cells, and an extracellular vesicle derived from milk. The ALKBH5 siRNA loaded within it can target the ALKBH5 / C2orf69 axis, thereby restoring mitochondrial function and promoting angiogenesis, accelerating diabetic wound healing. Its core-shell structure encapsulates the siRNA in the nuclear layer, protecting it from degradation, enhancing stability under physiological conditions, prolonging the circulating half-life, and reducing non-specific distribution of siRNA, thus minimizing off-target interference with normal tissues. The hybrid membrane used in the shell layer has good biocompatibility and can reduce immunogenicity and cytotoxicity. Furthermore, the arginine residues exposed outside the hybridization membrane can, on the one hand, enhance the interaction between the drug-loaded vesicles and the wound tissue and cell membranes due to their cationic properties, thereby improving tissue penetration and cellular uptake efficiency; on the other hand, they can avoid non-specific protein adsorption and off-target binding caused by excessive surface positive charge, achieving a balance between efficient penetration and biosafety. In vitro experiments showed that the fluorescence intensity of these drug-loaded vesicles was higher than that of ordinary liposomes and free siRNA in a high-glucose-treated cell model, and they could significantly reduce ALKBH5 expression and restore m 6 The drug-loaded vesicles improved mitochondrial function and restored endothelial cell angiogenesis by modifying the A level and C2orf69 mRNA stability. In vivo experiments showed that the drug-loaded vesicles could significantly accelerate the wound healing rate in diabetic wound model mice and had good biocompatibility and safety.

[0005] The technical solution provided by this invention is as follows: In a first aspect, the present invention provides a drug-loaded vesicle for promoting the healing of diabetic wounds, the drug-loaded vesicle having a core-shell structure, wherein: the core contains siRNA that targets and binds to ALKBH5 mRNA, the sense strand of which is shown in SEQ ID NO.1 and the antisense strand is shown in SEQ ID NO.2; the shell is a hybrid membrane of human umbilical vein endothelial cell membrane and milk-derived extracellular vesicle membrane, and the surface of the hybrid membrane is embedded with lipopeptides containing arginine, and at least a portion of the arginine is exposed outside the hybrid membrane.

[0006] In conjunction with the first aspect of the present invention, in some embodiments, the dispersion medium for the siRNA in the nucleus is PBS buffer.

[0007] In conjunction with the first aspect of the present invention, in some embodiments, the particle size of the drug-loaded vesicles is 100 nm to 200 nm.

[0008] In conjunction with the first aspect of the present invention, in some embodiments, the core-shell mass ratio of the drug-loaded vesicle is 1:(0.8~1.2).

[0009] In conjunction with the first aspect of the present invention, in some embodiments, the arginine content in the lipopeptide is 7% to 11%.

[0010] In a second aspect, the present invention provides a method for preparing drug-loaded vesicles for promoting the healing of diabetic wounds, characterized in that it comprises: Extracellular vesicles, human umbilical vein endothelial cell membrane, lipopeptides and siRNA derived from milk were initially mixed and then subjected to sonication in an ice-water bath to obtain a mixed solution. The mixed solution was extruded from a porous membrane using a micro extruder to obtain the drug-loaded vesicles for promoting the healing of diabetic wounds.

[0011] In conjunction with the second aspect of the present invention, in some embodiments, the mass ratio of milk-derived extracellular vesicles, human umbilical vein endothelial cell membrane, lipopeptides and siRNA in the mixed solution is 1:(0.9~1.1):(0.9~1.1):(2~4).

[0012] Thirdly, the present invention provides a diabetic wound healing drug comprising the above-mentioned drug-loaded vesicles for promoting diabetic wound healing.

[0013] Compared with the prior art, the present invention has at least the following beneficial effects: (1) To address the key bottlenecks in siRNA delivery, such as poor tissue permeability, difficulty in cell membrane penetration, and susceptibility to lysosomal degradation, this invention provides a drug-loaded vesicle for promoting the healing of diabetic wounds. The structure of this drug-loaded vesicle comprises an arginine-rich lipopeptide, a cell membrane derived from human umbilical vein endothelial cells, and an extracellular vesicle derived from milk. The ALKBH5 siRNA loaded within it can target the ALKBH5 / C2orf69 axis, thereby restoring mitochondrial function and promoting angiogenesis, thus accelerating the healing of diabetic wounds. In vitro experiments show that the fluorescence intensity of this drug-loaded vesicle in a high-glucose-treated cell model is higher than that of ordinary liposomes and free siRNA, and it can significantly reduce ALKBH5 expression and restore mitochondrial function. 6 The drug-loaded vesicles improved mitochondrial function and restored endothelial cell angiogenesis by modifying the A level and C2orf69 mRNA stability. In vivo experiments showed that the drug-loaded vesicles could significantly accelerate the wound healing rate in diabetic wound model mice and had good biocompatibility and safety.

[0014] (2) The hybrid membrane structure used in the shell of the drug-loaded vesicle has good biocompatibility and can reduce immunogenicity and cytotoxicity. In addition, the arginine exposed outside the hybrid membrane can enhance the interaction between the drug-loaded vesicle and the wound tissue and cell membrane by means of its cationic properties, thereby improving tissue penetration and cell uptake efficiency; on the other hand, it can avoid non-specific protein adsorption and off-target binding caused by excessive positive charge on the surface, thereby achieving a balance between efficient penetration and biosafety.

[0015] (3) The nuclear-shell structure of the drug-loaded vesicle can encapsulate siRNA in the nuclear layer, protect it from degradation, improve its stability under physiological conditions and prolong its circulating half-life, while reducing the non-specific distribution of siRNA and reducing off-target interference to normal tissues. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Schematic diagram of siALK@L-Hyb preparation and treatment. Wherein: (A) The manufacturing process of siALK@L-Hyb; (B~C) siALK@L-Hyb achieves efficient siRNA delivery through hierarchical intervention at the levels of tissue (I), cell (II), and organelles (III, IV), thereby reshaping m 6 A modifier restores endothelial cell function, ultimately accelerating the healing of diabetic wounds.

[0018] Figure 2 m in vascular endothelial cells of diabetic wounds and high-glucose treatment 6 A levels were decreased and ALKBH5 expression was increased. Specifically: (A) dot blot staining detected m in the wound tissues of control mice and diabetic model mice. 6 A) Modification level, with methylene blue staining as the RNA loading control, n = 6; (B) EpiQuik m 6 A kit was used to quantitatively detect mRNA methylation in wound tissues of control mice and diabetic model mice. 6 A. Modification level, n = 6; (C) Dot blot analysis of m in wound tissues of non-diabetic and diabetic patients 6 A modifies the level, n = 6; (D) EpiQuik m 6 A kit for the quantitative detection of RNA methylation in wound tissues from both non-diabetic and diabetic patients. 6A modification level, n = 6; (E) m in HUVEC cells of the control group, mannitol group or high glucose group as detected by dot blot assay 6 A Modification Level; (F) EpiQuikm 6 A quantitative detection kit was used to detect m in HUVEC cells from the control group, mannitol group, or high glucose group. 6 A modification level, n = 3; (G) 30 m in the wound tissue of control mice and diabetic model mice 6 A. Related mRNA gene heatmap; (H) qRT-PCR detection of mRNA expression levels of 5 dysregulated genes in wound tissues of non-diabetic and diabetic patients; (I) Quantitative results of ALKBH5 protein expression level in wound tissues of control mice and diabetic model mice using Western blot; (J) qRT-PCR detection of mRNA expression levels of 5 dysregulated genes in HUVEC cells of control, mannitol, or high glucose groups; (K) Quantitative results of ALKBH5 protein expression level in wound tissues of non-diabetic controls and diabetic patients using Western blot; (L) ALKBH5 protein expression level in wound tissues of control mice and diabetic model mice using Western blot; (M) Quantitative results of ALKBH5 protein expression level in HUVEC cells of control, mannitol, or high glucose groups using Western blot; (N) ALKBH5 protein expression level in wound tissues of non-diabetic controls and diabetic patients using Western blot; (O) Western blot... The expression level of ALKBH5 protein in HUVEC cells of the control group, mannitol group, or high glucose group was detected by blot. (P) Representative immunofluorescence of ALKBH5 and CD31 in wound tissues of non-diabetic and diabetic patients, scale bar: 50 μm. ns indicates no significance, *p indicates <0.05, **p indicates <0.01, ***p indicates <0.001, **** indicates p<0.0001.

[0019] Figure 3 Knocking down ALKBH5 attenuates mitochondrial dysfunction and restores angiogenesis in endothelial cells treated with high glucose. Specifically: (A) Western blot analysis to detect the knockdown efficiency of shALKBH5; (B) Western blot analysis to quantify the knockdown efficiency of shALKBH5; (C) Dot blot analysis to detect the m6A modification level in HUVEC cells under different treatment conditions; (D) EpiQuik m 6 A kit for RNA methylation assay was used to quantitatively detect m in HUVEC cells under different treatment conditions. 6(A) Modification level; (E) Representative confocal images of reactive oxygen species (ROS) in mitochondria of HUVEC cells under different treatment conditions detected by MitoSOX staining, scale bar: 25 μm; (F) Quantitative results of ROS in mitochondria of HUVEC cells under different treatment conditions detected by MitoSOX staining; (G) Flow cytometry measurement of cytoplasmic ROS levels in HUVEC cells under different treatment conditions; (H) Representative confocal images of JC-1 staining for mitochondrial membrane potential difference ΔΨm in HUVEC cells under different treatment conditions, with green and red fluorescence representing JC-1 monomers and JC-1 dimers, respectively, scale bar: 25 μm; (I) Quantitative results of representative confocal images of JC-1 fluorescence staining for mitochondrial membrane potential difference ΔΨm in HUVEC cells under different treatment conditions; (J) Quantitative results of cytoplasmic ROS levels in HUVEC cells under different treatment conditions measured by flow cytometry; (K) Microscopic images of HUVEC cells under different treatment conditions for angiogenesis experiments processed by ImageJ, scale bar: 200 μm; (L) Quantitative analysis results of HUVEC angiogenesis experiments under different treatment conditions, using total tube length as an indicator; (M) Enzyme activity of mitochondrial respiratory chain complex I in HUVEC cells under different treatment conditions; (N) Enzyme activity of mitochondrial respiratory chain complex IV in HUVEC cells under different treatment conditions; (O) Enzyme activity of mitochondrial respiratory chain complex V in HUVEC cells under different treatment conditions; (P) Oxygen consumption rate of HUVECs under different treatment conditions measured by extracellular flux analyzer. ns indicates no significance, *p indicates <0.05, **p indicates <0.01, ***p indicates <0.001, **** indicates p<0.0001.

[0020] Figure 4 ALKBH5 depends on m 6 A-IGF2BP2 regulates the stability of C2orf69 mRNA. Among them: (A) Volcano plot of differentially expressed genes in the transcriptome of HUVEC cells between the ALKBH5 knockdown group and the control group (fold change ≥ 1.2, p ≤ 0.05, red dots represent upregulated mRNA, blue dots represent downregulated mRNA, and gray dots represent mRNA with no significant difference in expression; (B) m 6 (A) Distribution density of peak A in different functional regions of mRNA (5'UTR, CDS, 3'UTR); (C) mRNA identified using HOMER 6 A) Analysis of conserved motifs modified by A; (D) RNA-seq differentially expressed genes, m 6A. Venn diagram of the intersection of upregulated modification genes and MitoCarta 3.0 mitochondrial genes; (E) qRT-PCR detection of C2orf69 mRNA expression levels in HUVEC cells under different treatment conditions; (F) MeRIP-qPCR detection of mRNA expression levels in C2orf69. 6 Enrichment level of A modification; (G)C2orf69 gene structure and its m 6 A. Schematic diagram of the site; (H) Effect of ALKBH5 knockdown on the activity of wild-type or mutant C2orf69 reporter gene in HUVEC cells under high glucose treatment, detected by dual-luciferase reporter assay; (I) Effect of mannitol or high glucose treatment on the activity of wild-type or mutant C2orf69 reporter gene in HUVEC cells, detected by dual-luciferase reporter assay; (J) C2orf69 mRNA expression level in HUVEC cells with knockdown of IGF2BP1, IGF2BP2, and IGF2BP3; (K) Half-life (t) of C2orf69 mRNA in HUVEC cells under different treatment conditions. 1 / 2 (L) RIP-qPCR was used to detect the enrichment level of IGF2BP2 in HUVEC cells of the control group and the ALKBH5 knockdown group under high glucose treatment. ns indicates no significance, *p indicates <0.05, **p indicates <0.01, ***p indicates <0.001, **** indicates p<0.0001.

[0021] Figure 5The ALKBH5 / C2orf69 axis regulates mitochondrial function in HUVEC cells under high glucose treatment. The study included: (A) Western blot analysis of ALKBH5 and C2orf69 expression in HUVEC cells under different treatments; (B) flow cytometry measurement of reactive oxygen species (ROS) levels in the cytoplasm of HUVEC cells under different treatments; (C) extracellular flux analysis of oxygen consumption in HUVEC cells under different treatments; (D) representative confocal microscopy images of mitochondrial ROS stained with MitoSOX in HUVEC cells under different treatments (scale bar: 25 μm); (E) enzyme activity of mitochondrial respiratory chain complex I in HUVEC cells under different treatments; and (F) representative confocal images of mitochondrial membrane potential (ΔΨm) detected by JC-1 fluorescent probe staining in HUVEC cells under different treatments (green and red fluorescence represent JC-1 monomers and J-dimers, respectively, scale bar: 25 μm). (G) Enzyme activity of mitochondrial respiratory chain complex IV in HUVEC cells under different treatments; (H) Microscopic images of angiogenesis experiments in HUVEC cells under different treatments and their ImageJ processed images, scale bar: 200 μm; (I) Enzyme activity of mitochondrial respiratory chain complex V in HUVEC cells under different treatments. ns indicates no significance, * p indicates <0.05, ** p indicates <0.01, *** p indicates <0.001, **** indicates p<0.0001.

[0022] Figure 6Preparation, characterization, and multi-stage drug loading evaluation of siALK@L-Hyb. The results include: (A) Electron microscopy images of mEV, CMN, and siALK@L-Hyb, scale bar: 100 nm; (B) Particle size distribution of mEV, CMN, and siALK@L-Hyb detected by particle size tracer; (C) Protein composition analysis of mEV, CMN, and siALK@L-Hyb using SDS-polyacrylamide gel electrophoresis combined with Coomassie brilliant blue staining; (D) Expression of HUVEC-specific membrane proteins (CD31 and ICAM-1) and mEV-specific proteins (TSG101 and CD81) in mEV, CMN, and siALK@L-Hyb by Western blot; (E) Loading efficiency of siALKBH5 in siALK@L-Hyb; (F) Representative confocal microscopy images of the fusion experiment of DiI-labeled mEV and DiO-labeled CMN, scale bar: 10 nm. μm; (G) Flow cytometry detection of the uptake efficiency of different types of nanovesicles and carriers by HUVEC cells under mannitol or high glucose treatment; (H) Quantitative results of the uptake efficiency of different types of nanovesicles and carriers by HUVEC cells under mannitol or high glucose treatment; (I) Flow cytometry detection of the uptake efficiency of free siALKBH5 or different types of drug-loaded vesicles by HUVEC cells under high glucose treatment; (J) Quantitative results of the uptake efficiency of free siALKBH5 or different types of drug-loaded vesicles by HUVEC cells under high glucose treatment; (K) The left figure shows the co-localization of different types of nanovesicles / carriers with lysosomes in HUVEC cells observed by confocal laser scanning microscopy. Lysosomes are marked with red fluorescent markers, siALKBH5 loaded with different types of nanovesicles or carriers is marked with green fluorescent markers, and cell nuclei are marked with blue fluorescent markers. Scale bar: 10 μm; The right figure shows the results of fluorescence intensity co-localization analysis along the selected yellow line in the figure. (L) Representative confocal images of the penetration of different types of nanovesicles or carriers into the dermis of diabetic wounds. The white and red dashed lines represent the wound edge and the furthest penetration range, respectively. Green fluorescence represents FAM-labeled siALKBH5. Scale bar: 100 μm. ns indicates no significance, * p indicates <0.05, ** p indicates <0.01, *** p indicates <0.001, **** indicates p <0.0001.

[0023] Figure 7siALK@L-Hyb alleviates mitochondrial dysfunction in HUVEC cells caused by high glucose damage and restores their function. The results included: (A) Western blot analysis of ALKBH5 and C2orf69 protein expression levels in HUVEC cells under different treatment conditions; (B) Quantitative analysis of ALKBH5 protein expression levels in HUVEC cells under different treatment conditions using Western blot analysis; (C) Quantitative analysis of C2orf69 protein expression levels in HUVEC cells under different treatment conditions using Western blot analysis; (D) Flow cytometry measurement of reactive oxygen species (ROS) levels in the cytoplasm of HUVEC cells under different treatment conditions; (E) Representative confocal images of ROS in the mitochondria of HUVEC cells under different treatment conditions using MitoSOX staining, scale bar: 25 μm; (F) Enzyme activity of mitochondrial respiratory chain complex I in HUVEC cells under different treatment conditions; (G) Representative confocal images of mitochondrial membrane potential (ΔΨm) detected by JC-1 fluorescent probe staining in HUVEC cells under different treatment conditions, with green and red fluorescence representing JC-1 monomers and JC-1 dimers, respectively, scale bar: 25 μm. (H) Enzymatic activity of mitochondrial respiratory chain complex IV in HUVEC cells under different treatments; (I) Microscopic images of angiogenesis experiments in HUVEC cells under different treatments and their ImageJ processed images, scale bar: 200 μm; (J) Enzymatic activity of mitochondrial respiratory chain complex V in HUVEC cells under the same treatment. ns indicates no significance, * p indicates <0.05, ** p indicates <0.01, *** p indicates <0.001, **** indicates p<0.0001.

[0024] Figure 8 The therapeutic effect of siALK@L-Hyb on diabetic wounds, including: (A) a schematic diagram of diabetic wound modeling and treatment procedures; (B) representative wound images of different treatment groups at specified time points; (C) a schematic diagram of the wound healing process of different treatment groups from day 1 to day 14, scale bar: 5 mm; (D) a line graph showing the trend of wound area changes in different treatment groups from day 1 to day 14; (E) histological analysis of wound tissue by H&E staining on day 14 of treatment, with black arrows indicating epithelialized edges; black dashed lines indicating dermal-epidermal junctions; and blue dashed lines indicating wound boundaries, scale bar: 500 μm; (F) quantitative analysis of the length of non-epithelialized wound gaps in Figure E; and (G) quantitative analysis of wound contraction width in Figure E. ns indicates no significance, * p indicates <0.05, ** p indicates <0.01, *** p indicates <0.001, and **** indicates p <0.0001.

[0025] Figure 9Analysis of Masson's trichrome staining and fluorescence staining in tissue sections from diabetic wounds. Specifically: (A) Masson's trichrome staining to assess collagen deposition in wound tissues under different treatment conditions 14 days after treatment (scale bar: 100 μm); (B) Ethidium dihydrochloride staining to detect reactive oxygen species (ROS) levels in wound tissue sections under different treatment conditions after 14 days of treatment (scale bar: 50 μm); (C) Immunofluorescence staining of CD31 and α-SMA in wound tissue sections under different treatment conditions (scale bar: 50 μm); (D) Quantitative results of collagen deposition in representative wound tissues under different treatment conditions; (E) Quantitative results of ethidium dihydrochloride staining for ROS fluorescence intensity in wound tissue sections from different treatment groups; (F) Quantitative results of CD31-positive areas in wound tissue sections under different treatment conditions; (G) Quantitative results of α-SMA-positive areas in wound tissue sections under different treatment conditions. ns indicates no significance, * p indicates <0.05, ** p indicates <0.01, *** p indicates <0.001, **** indicates p <0.0001. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] As an epigenetic modification, N6-methyladenosine (m 6 A) is the most common internal chemical modification in eukaryotic mRNA. Its dynamic and reversible methylation process is jointly regulated by methyltransferases, demethylases, and ribonucleic acid-binding proteins, playing a crucial role in gene expression. Among them, α-ketoglutarate-dependent dioxygenase alkB homologue 5 (ALKBH5), as a demethylase, can specifically recognize and remove methylation from mRNA. 6 Methylation caused by ALKBH5 reverses the effects mediated by methylation modification. ALKBH5 is widely involved in many biological processes, such as cell differentiation, immune responses, and tumorigenesis, by regulating the splicing, translation efficiency, and stability of target mRNAs. However, the fundamental role of ALKBH5 in vascular endothelial cell dysfunction in diabetic wounds has not been reported to date. This invention is the first to reveal the key role of ALKBH5 in the pathogenesis of diabetic wounds: elevated ALKBH5 expression leads to m... 6 Reduced A methylation leads to decreased mRNA stability encoding the mitochondrial protein C2orf69. This posttranscriptional repression results in impaired mitochondrial function in endothelial cells.

[0028] Based on this mechanism, this invention provides a drug-loaded vesicle for promoting the healing of diabetic wounds, which has a core-shell structure: the core loads siRNA targeting and binding to ALKBH5 mRNA; the shell is a hybrid membrane of human umbilical vein endothelial cell membrane and milk-derived extracellular vesicle membrane, with arginine-containing lipopeptides embedded on the membrane surface, and at least a portion of the arginine exposed outside the hybrid membrane. The core-shell structure encapsulates the siRNA in the core layer, protecting it from degradation, improving its stability under physiological conditions, and prolonging its circulating half-life, while reducing non-specific distribution of siRNA and minimizing off-target interference with normal tissues. The hybrid membrane used in the shell layer has good biocompatibility, reducing immunogenicity and cytotoxicity. Furthermore, the arginine exposed outside the hybrid membrane, on the one hand, can enhance the interaction between the drug-loaded vesicle and wound tissue and cell membranes through its cationic properties, improving tissue penetration and cellular uptake efficiency; on the other hand, it can avoid non-specific protein adsorption and off-target binding caused by excessive surface positive charge, achieving a balance between efficient penetration and biosafety. The siRNA targeting ALKBH5 mRNA in the core can inhibit m... 6 Demethylation of α enhances the stability of C2orf69 mRNA, reverses mitochondrial damage in diabetic wounds, and ultimately accelerates wound healing. Simultaneously, the outer shell components of this drug-loaded vesicle possess multiple functions: human umbilical vein endothelial cell membrane components promote specific uptake of the vesicles by endothelial cells; human umbilical vein endothelial cells, as natural vascular endothelial cells, can achieve precise targeted delivery and long-lasting sustained release of siRNA through drug-loaded vesicles encapsulated by their cell membranes, while also exhibiting excellent biocompatibility and low immunogenicity; milk-derived extracellular vesicles facilitate lysosomal escape; and arginine-rich lipopeptides significantly enhance the tissue penetration ability of the vesicles.

[0029] In some implementations, the sense strand of the siRNA targeting and binding ALKBH5 mRNA is shown in SEQ ID NO.1, and the antisense strand is shown in SEQ ID NO.2; In some embodiments, the core also contains a dispersion medium for the siRNA. Specifically, the dispersion medium for the siRNA in the core is PBS buffer. The core advantage of using PBS buffer as the dispersion medium for siRNA lies in its excellent physiological compatibility and stability. The pH and osmotic pressure of PBS buffer are highly matched with the human internal environment, which can minimize irritation or damage caused by injection. At the same time, its good buffering capacity can provide a stable environment for drug-loaded vesicles and prevent their degradation.

[0030] In some embodiments, the particle size of the drug-loaded vesicles is 100 nm to 200 nm. If the size of the drug-loaded vesicles is too small, they are easily degraded by lysosomes, while if the size is too large, they are difficult to pass through cell membrane pores.

[0031] In some embodiments, the core-shell mass ratio of the drug-loaded vesicle is 1:(0.8~1.2). Preferably, the core-shell mass ratio of the drug-loaded vesicle is 1:1.

[0032] In some embodiments, the arginine content in the lipopeptide is 7% to 11%. Preferably, the lipopeptide content loaded on the hybridization membrane is 9.6%. Due to the positively charged nature of arginine, it can be strongly adsorbed onto the negatively charged cell membrane through electrostatic interactions, thereby significantly enhancing the efficiency of drug-loaded vesicles being taken up and endocytosed by cells.

[0033] This invention provides a method for preparing drug-loaded vesicles for promoting the healing of diabetic wounds, characterized by comprising: Extracellular vesicles derived from milk, human umbilical vein endothelial cell membrane, lipopeptides, and siRNA are initially mixed and then subjected to sonication in an ice-water bath to obtain a mixed solution. Preferably, the extracellular vesicles used are obtained from raw milk using differential centrifugation. Milk-derived extracellular vesicles are chosen because they are abundant, inexpensive, and readily available for large-scale, stable production, facilitating clinical translation and production scale-up. Furthermore, they exhibit good biocompatibility, low immunogenicity, and high biosafety, are well-tolerated in humans, have a low risk of immune rejection, and can effectively escape lysosomes. The mixed solution is then extruded from a porous membrane using a micro-extruder to obtain the drug-loaded vesicles for promoting diabetic wound healing.

[0034] In some embodiments, the mass ratio of milk-derived extracellular vesicles, human umbilical vein endothelial cell membrane, lipopeptide, and siRNA in the mixed solution is 1:(0.9~1.1):(0.9~1.1):(2~4). Preferably, the mass ratio of milk-derived extracellular vesicles, human umbilical vein endothelial cell membrane, lipopeptide, and siRNA is 1:1:1:3. In the embodiments of the present invention, the drug-loaded vesicles prepared using this mass ratio exhibit good membrane fusion and good encapsulation efficiency.

[0035] The present invention also provides a diabetic wound healing drug comprising the above-mentioned drug-loaded vesicles for promoting diabetic wound healing.

[0036] In the following examples, free siALK represents free si-ALKBH5; siALK@mEV is a milk-derived extracellular vesicle loaded with si-ALKBH5; siALK@CMN is a purified HUVEC cell membrane component vesicle loaded with si-ALKBH5; siALK@Hyb is a drug-loaded vesicle constructed by loading si-ALKBH5 onto a hybrid membrane carrier formed by co-assembling milk-derived extracellular vesicles, HUVEC cell membrane components, and ordinary lipopeptides; siALK@L-Hyb is a drug-loaded vesicle obtained by loading si-ALKBH5 onto a hybrid membrane carrier formed by co-assembling milk-derived extracellular vesicles, HUVEC cell membrane components, and arginine-rich lipopeptides. Correspondingly, Hyb and L-Hyb are blank hybrid membrane vesicles without si-ALKBH5, and their membrane compositions correspond to siALK@Hyb and siALK@L-Hyb, respectively.

[0037] Example 1: m in HUVEC cells from diabetic wounds and high glucose treatment 6 A expression level decreased (1) Constructing a mouse model of diabetic wounds Eight-week-old male C57BL / 6 mice, weighing approximately 20g, were acclimatized for one week in an SPF environment with a standard diet. The mice were then randomly divided into two groups of 10 mice each. The specific groupings were as follows: i. Control group: 100 μL PBS was injected intraperitoneally once a day for 4 consecutive days.

[0038] ii. Diabetes model group: 100 μL of 50 mg / kg streptozotocin (purchased from Beyotime) was injected intraperitoneally once a day for 4 consecutive days.

[0039] Four days after injection, blood glucose levels were monitored by tail vein sampling in mice from both the control and diabetes model groups. A diabetes model was considered successfully established when the blood glucose level remained consistently above 16.7 mM. Prior to the wound modeling surgery, all mice were anesthetized with 1% sodium pentobarbital (50 mg / kg, purchased from Beyotime). The skin on their backs was then dehaired and disinfected, and a 10 mm diameter full-thickness skin defect was created using a sterile biopsy punch. After the wound was created, it was covered with medical gauze using a purse-string suture method to prevent contact with the bedding or moisture, keeping the wound clean until the experimental endpoint.

[0040] (2) Detection of m in diabetic wounds 6 A level of expression After establishing the diabetic wound model, mice were euthanized, and total RNA was extracted from wound tissues of both the control and diabetic model groups, followed by mRNA purification. Specifically, the total RNA was heated at 65°C for 10 minutes, and then hybridized with a biotinylated oligonucleotide probe (purchased from Millipore, USA) in 20X sodium citrate hydrochloride buffer at room temperature. The resulting hybrids were then captured using streptavidin MagneSphere® paramagnetic particles (purchased from MagneSphere®) and a magnetic scaffold. The bound mRNA was eluted in RNase-free water to produce a formulation free of other nucleic acids. The purity of the isolated mRNA was assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), with an A260 / A280 ratio ≥2.0 indicating successful purification.

[0041] The purified RNA sample was first denatured at 95°C for 3 minutes, then immediately cooled on ice to eliminate secondary structures. The denatured RNA was spotted onto a nylon membrane (purchased from Millipore, USA) and cross-linked by UV irradiation. The membrane was blocked with 5% skim milk at room temperature for 1 hour, and then coated with an anti-m... 6 The primary antibody (Proteintech, China) was incubated overnight at 4°C. After washing, the membrane was incubated with HRP-conjugated secondary antibody at room temperature for 1 hour, and the signal was detected using an ECL kit (Biosharp, China). Results showed that, compared to the control group, the m... 6 A modification level significantly decreased ( Figure 2 A). EpiQuik m 6 A quantitative RNA methylation kit (Epigentek, Cat# P-9005, USA) was used to quantitatively analyze RNA methylation in wound tissues of control and diabetic model mice. 6 The percentage of A-modified RNA in total RNA was determined, and the results showed that, compared to the control group, the m-modified RNA in the wound tissue of diabetic model mice was significantly lower. 6 A modification level significantly decreased ( Figure 2 B). With the approval of the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, discarded wound tissue from non-diabetic and diabetic patients was collected from clinical debridement surgeries (sourced from Union Hospital, Tongji Medical College, Huazhong University of Science and Technology). Similarly, m 6 A spot imprint and EpiQuik m 6 Quantitative RNA methylation results showed that, compared with the non-diabetic group, the m-methylation concentration in the wound tissue of the diabetic group was significantly higher. 6 A modification level significantly decreased ( Figure 2 C~D).

[0042] (3) Detection of m in HUVEC cells under high glucose treatment conditions 6 A level of expression Human umbilical vein endothelial cells (HUVECs) were purchased from Carlsbad Scientific Cell Research Laboratory in the United States. To ensure consistent and robust phenotypes, cells from passage 2 to passage 8 were selected for subsequent experiments. 1×102 6 HUVEC cells were seeded in T25 culture flasks and cultured for 24 hours in ECM medium (purchased from Sciencell) containing 10% fetal bovine serum, 1% endothelial cell growth supplement, and 1% penicillin / streptomycin solution until the cell density reached 2 × 10⁶ cells / year. 6 After that, they were randomly divided into three groups for subsequent processing. The specific groupings are as follows: i. Control group: HUVEC cells were treated for 72 hours by adding glucose to a final concentration of 5.5 mM in the culture flasks by changing the culture medium.

[0043] ii. Mannitol group: HUVEC cells were treated for 72 hours by adding glucose to a final concentration of 5.5 mM and mannitol to a final concentration of 24.5 mM by changing the culture medium. iii. High glucose group: HUVEC cells were treated for 72 hours by adding glucose to a final concentration of 30 mM in the culture flask by changing the culture medium.

[0044] Cells were collected after 72 hours, RNA was extracted, and mRNA was purified for subsequent mRNA processing. 6 A spot imprint and EpiQuik m 6 A quantitative detection of RNA methylation. Results showed that, compared to the low-glucose and mannose control groups, m... 6 A modification level significantly decreased ( Figure 2 E~F).

[0045] Example 2: Increased ALKBH5 expression in HUVEC cells from diabetic wounds and high glucose treatment Differentially expressed genes in wound tissues of control and diabetic model mice were detected using RNA-seq, and 30 genes related to m were screened. 6 Genes involved in A modification regulation included METTL3 and HNRNPA2B1, which were significantly downregulated in wound tissues of diabetic mice, while WTAP, ALKBH5, and YTHDF2 were significantly upregulated in wound tissues of diabetic mice. Figure 2 G). Subsequently, qRT-PCR was used to detect the above-mentioned genes in wound tissues of non-diabetic and diabetic patients. The results showed that the expression levels of ALKBH5 and YTHDF2 were significantly increased in the wound tissues of diabetic patients. Figure 2H). Similarly, qRT-PCR was used to detect the expression of METTL3, HNRNPA2B1, WTAP, ALKBH5, and YTHDF2 in HUVEC cells from the control group, mannitol group, and high glucose group. The results showed that the expression of ALKBH5 was significantly increased in HUVEC cells from the high glucose group. Figure 2 J). Furthermore, Western blot analysis validated ALKBH5 protein expression at the mouse, patient, and cellular levels. Specifically, compared to their respective control groups, ALKBH5 expression was significantly higher in the wound tissue of diabetic model mice (J). Figure 2 L and 2I), wound tissue of diabetic patients ( Figure 2 N and Figure 2 K) and high-glucose-treated HUVEC cells ( Figure 2 Both O and 2M were significantly upregulated. Immunofluorescence staining of wound tissues from diabetic and non-diabetic patients revealed that in the non-diabetic state, ALKBH5 expression was low in vascular endothelial cells and showed no significant co-localization with CD31, while in the diabetic state, ALKBH5 was specifically highly expressed in vascular endothelial cells and co-localized with CD31. Figure 2 P).

[0046] Example 3: Knockdown of ALKBH5 alleviates mitochondrial dysfunction and restores angiogenesis in HUVEC cells treated with high glucose.

[0047] First, the knockdown efficiency of the ALKBH5-targeting shRNA (purchased from ObioTechnology Shanghai, China) was detected by qRT-PCR and Western blot. The results showed that the shRNA could effectively reduce the expression of ALKBH5 at both the protein and mRNA levels. Figure 3 (A~B). Human umbilical vein endothelial cells (HUVECs) were purchased from Scientific Cell Research Laboratory (Carlsbad, USA). To ensure consistent and robust phenotypes, cells from passage 2 to passage 8 were selected for subsequent experiments. 1×10 6 HUVEC cells were seeded in T25 culture flasks and cultured for 24 hours in ECM medium (purchased from Sciencell) containing 10% fetal bovine serum (FBS), 1% endothelial cell growth supplement (ECGS), and 1% penicillin / streptomycin solution, until the cell density reached 1×10⁶ cells / year. 6 These individuals were randomly divided into three groups for subsequent processing. The specific groupings are as follows: i. Mannose + empty vector control group: The empty vector was transfected into HUVEC cells, and after 24 hours the medium was replaced with ECM medium containing 5.5 mM glucose and 24.5 mM mannitol, and the treatment continued for 72 hours.

[0048] ii. High glucose + empty vector control group: The empty vector was transfected into HUVEC cells, and after 24 hours, the medium was replaced with ECM medium containing 30 mM glucose and the treatment continued for 72 hours.

[0049] iii. High glucose + ALKBH5 knockdown group: shRNA targeting ALKBH5 was transfected into cells, and after 24 hours the medium was replaced with ECM medium containing 30 mM glucose and the treatment continued for 72 hours.

[0050] Cells were collected after treatment and analyzed using dot blot and EpiQuik m assays. 6 A quantitative RNA methylation kit (Epigentek, Cat# P-9005, USA) was used to quantify RNA methylation in HUVEC cells. 6 Qualitative and quantitative analyses of the A modification level were performed, with the specific detection steps being the same as in Example 1. The results showed that, compared to the mannose + empty vector control group, the m... 6 The level of A modification decreased significantly, indicating that high glucose treatment inhibits the m-modification of cells. 6 A modification; compared to the high glucose + empty vector control group, the m of cells in the high glucose + shALKBH5 group was significantly higher. 6 The level of A modification was significantly increased. This result indicates that knockdown of ALKBH5 can significantly reverse the effects of high glucose treatment on m in HUVEC cells. 6 A decrease in the level of modification ( Figure 3 C~D). Next, mitochondrial ROS in HUVEC cells under different treatments was detected by MitoSOX fluorescence staining. The results showed that knockdown of ALKBH5 could significantly reverse the abnormal increase in mitochondrial ROS caused by high glucose treatment. Figure 3 E~F). Similarly, flow cytometry was used to detect the cytoplasmic ROS content of HUVEC cells under different treatments. The results showed that knocking down ALKBH5 could significantly reverse the abnormal increase in cytoplasmic ROS caused by high glucose treatment. Figure 3 G and 3J). The mitochondrial membrane potential difference ΔΨm was detected by JC-1 staining, where green and red fluorescence represented JC-1 monomers and JC-1 dimers, respectively. The results showed that knockdown of ALKBH5 significantly reversed the abnormal decrease in mitochondrial membrane potential induced by high glucose treatment, meaning that knockdown of ALKBH5 could alleviate high glucose-induced mitochondrial dysfunction. Figure 3 H and 3I). Subsequently, HUVEC angiogenesis experiments confirmed that knocking down ALKBH5 could restore the angiogenesis ability of HUVEC cells. Compared with the high glucose + empty vector control group, the total vessel length of the high glucose + ALKBH5 knockdown group was significantly increased (3K-3L). Then, the mitochondrial respiratory chain complex I (H and 3I) of HUVEC cells under different treatment conditions was analyzed. Figure 3 M), mitochondrial respiratory chain complex IV (M), Figure 3 N) and mitochondrial respiratory chain complex V ( Figure 3 The activity of ALKBH5 enzymes was detected. The results showed that knockdown of ALKBH5 restored the decrease in mitochondrial respiratory chain complex enzyme activity induced by high glucose treatment, indicating that knockdown of ALKBH5 can improve mitochondrial oxidative phosphorylation function. Finally, the oxygen consumption rate of HUVEC cells under different treatment conditions was measured using an extracellular flux analyzer. Knockdown of ALKBH5 significantly reversed the high glucose-induced decrease in HUVEC oxygen consumption rate. Figure 3 P), meaning that knocking down ALKBH5 can enhance mitochondrial respiration under high glucose conditions.

[0051] Example 4: ALKBH5 depends on m 6 A-IGF2BP2 regulates the stability of C2orf69 mRNA HUVEC cells from the high glucose + empty vector control group (HG shCtrl) and the high glucose + ALKBH5 knockdown group (HG shALK) were collected, and their differentially expressed genes in the transcriptome were analyzed. The results showed that, compared with HG shCtrl, 512 genes were downregulated and 463 genes were upregulated in HG shALK. Figure 4 A). Additionally, under high glucose treatment conditions, m in cells with knocked-down ALKBH5 6 The A modification remains predominantly enriched in the coding region, but the sequence preference of its core conserved motif (GGACU) has changed, indicating that ALKBH5 is involved in maintaining m 6 Motif specificity of A-modification sites ( Figure 4 B~C). Next, differentially expressed genes identified based on RNA sequencing and m-type genes screened by MeRIP-seq will be... 6 Genes upregulated by A modification, along with mitochondrial-related genes obtained from the MitoCarta 3.0 database, were plotted using a Venn diagram, and the intersection of the three was taken. The overlapping area showed that the common differentially expressed genes in multiple groups were ALDH1B1 and C2orf69. Figure 4 D), qRT-PCR validation revealed that C2orf69 expression was significantly increased in the high glucose + empty vector control group compared to the high glucose + ALKBH5 knockdown group. Figure 4 E). MeRIP-qPCR analysis revealed that, compared to the high glucose + empty vector control group, the m of the C2orf69 gene in the high glucose + ALKBH5 knockdown group was significantly lower. 6 A-modification significantly increased the enrichment level ( Figure 4 F), the m 6 The A site (GGACU) is located between the 5' UTR and the intron of the C2orf69 mRNA, and immediately upstream of the coding sequence. Figure 4 G). Subsequently, the m of C2orf69 mRNA was... 6 A modified sequence GGACU was mutated to GGCCU, and a C2orf69 mutant reporter plasmid was constructed (purchased from HanbioBiotechnology, China). Dual-luciferase reporter gene assays were used to detect the effect. The results showed that under high glucose treatment conditions, knockdown of ALKBH5 significantly increased the reporter gene activity of wild-type C2orf69 in HUVEC cells, while having no significant effect on the reporter gene activity of mutant C2orf69. Figure 4 H). Furthermore, dual-luciferase reporter gene assays showed that, compared to the mannose-treated group, high-glucose treatment significantly increased the reporter gene activity of wild-type C2orf69 in HUVEC cells, while having no significant effect on the reporter gene activity of mutant C2orf69. Figure 4 I). Extensive literature review revealed that the IGF2BP family may play a regulatory role in C2orf69. Therefore, HUVEC cells were transfected with siRNAs targeting IGF2BP1, IGF2BP2, and IGF2BP3 (purchased from Hanbio Biotechnology, China), and C2orf69 mRNA levels were detected by qRT-PCR. Results showed that, under both mannitol and high glucose treatment conditions, compared to the small interfering RNA knockdown control group, the IGF2BP1 group, and the IGF2BP3 group, knocking down IGF2BP2 significantly downregulated C2orf69 mRNA expression. Figure 4 J). An IGF2BP2 overexpression plasmid (purchased from Hanbio Biotechnology, China) was further constructed and transfected into HUVEC cells. The effect of IGF2BP2 overexpression on the half-life of C2orf69 mRNA in HUVEC cells under high glucose treatment was examined. The results showed that IGF2BP2 overexpression significantly enhanced the stability of C2orf69 mRNA (J). Figure 4 Finally, RIP-qPCR further confirmed that IGF2BP2 is ALKBH5-mediated C2orf69 m. 6 A demethylated target mRNA ( Figure 4 L).

[0052] Example 5: ALKBH5 / C2orf69 axis regulates mitochondrial function and angiogenesis in vascular endothelial cells under high glucose conditions To investigate the role of the ALKBH5 / C2orf69 axis in regulating mitochondrial function in vascular endothelial cells treated with high glucose, 1×10 6HUVEC cells were seeded in T25 culture flasks and cultured for 4 hours in ECM medium (Sciencell) containing 10% fetal bovine serum (FBS), 1% endothelial cell growth supplement (ECGS), and 1% penicillin / streptomycin solution, until the cell density reached 2 × 10⁶ cells / year. 6 These individuals are randomly divided into three groups for subsequent processing, as shown below: Ⅰ: Mannose + shCtrl1 + shCtrl2: Transfect shCtrl1 and shCtrl2 plasmids, and after 24 hours, treat HUVEC cells with ECM medium containing 24.5 mM mannose for 72 hours; II: High glucose + shCtrl1 + shCtrl2: Transfect shCtrl1 and shCtrl2 plasmids, and after 24 hours, treat HUVEC cells with ECM medium containing 30 mM glucose for 72 hours; III: High glucose + shALKBH5 + shCtrl2: After transfecting shALKBH5 and shCtrl2 plasmids for 24 hours, HUVEC cells were treated with ECM medium containing 30 mM glucose for 72 hours. IV: High glucose + shCtrl1 + shC2orf69: Transfect HUVEC cells with shCtrl1 and shC2orf69 plasmids. After 24 hours, replace the medium with ECM medium containing 30 mM glucose and treat for 72 hours. V: High glucose + shALKBH5 + shC2orf69: Transfect HUVEC cells with shALKBH5 and shC2orf69 plasmids. After 24 hours, replace the medium with ECM medium containing 30 mM glucose and treat for 72 hours. The expression levels of ALKBH5 and C2orf69 in HUVEC cells under different treatment conditions were detected by Western blot. The results showed that, compared to group I, group II had significantly increased ALKBH5 protein expression and significantly decreased C2orf69 expression; compared to group II, group III had significantly decreased ALKBH5 protein expression and significantly increased C2orf69 protein expression; compared to group III, group IV had significantly increased ALKBH5 protein expression and significantly decreased C2orf69 expression; and compared to group IV, group V had significantly decreased ALKBH5 protein expression and significantly increased C2orf69 protein expression. These results indicate that under high glucose treatment, knocking down ALKBH5 can significantly upregulate C2orf69 protein expression. Figure 5 A). The shRNA targeting and knocking down C2orf69 mRNA was purchased from ObioTechnology Shanghai, China.

[0053] Subsequently, flow cytometry was used to measure the cytoplasmic ROS levels in HUVEC cells after the above different treatment groups. The results showed that, compared with group I, the ROS content in group II was significantly increased; compared with group II, the ROS content in group III, which knocked down ALKBH5 alone, was significantly decreased; compared with group II, the ROS content in group IV, which knocked down C2orf69 alone, was significantly increased; compared with group II, the ROS content in group V did not change significantly, that is, ALKBH5 can regulate the ROS content by regulating C2orf69. Figure 5 B). Similarly, MitoSOX staining was used to detect the content of mitochondrial reactive oxygen species in HUVEC cells after different treatments. The results also demonstrated that ALKBH5 can affect the content of mitochondrial ROS and thus its function by regulating C2orf69. Figure 5 D). Next, an extracellular flux analyzer was used to measure the oxygen consumption rate of HUVEC cells after different treatments. During the basal respiration phase (0–25 minutes), the oxygen consumption rate in group II was significantly lower than that in group I, indicating that high glucose inhibited basal mitochondrial respiration. During the stress respiration phase (approximately 50–75 minutes), group III, which had ALKBH5 knocked down alone, showed the highest OCR peak, indicating that its maximum respiratory capacity was restored. However, when C2orf69 was knocked down simultaneously, the OCR peak of group V returned to a level similar to that of group II. Figure 5 (C) This result indicates that ALKBH5 protects mitochondrial respiratory function by upregulating C2orf69 expression. Furthermore, JC-1 staining was used to detect the mitochondrial membrane potential (ΔΨm) in HUVEC cells after different treatments. Compared to group I, ΔΨm was significantly decreased in group II, indicating that high glucose treatment reduces mitochondrial membrane potential in HUVEC cells, leading to impaired mitochondrial function. Compared to group II, ΔΨm was significantly increased in group III (ALKBH5 knockdown alone) and significantly decreased in group IV (C2orf69 knockdown alone). In group V (simultaneous knockdown of ALKBH5 and C2orf69), the membrane potential level was between that of groups III and IV. This suggests that knockdown of C2orf69 exacerbates mitochondrial functional impairment and can counteract the mitochondrial membrane potential recovery effect caused by ALKBH5 knockdown, indicating that C2orf69 is a key downstream molecule of ALKBH5 regulating mitochondrial membrane potential. Figure 5 F). Additionally, mitochondrial respiratory chain complex I (F) in HUVEC cells after different treatments... Figure 5 E), mitochondrial respiratory chain complex IV ( Figure 5 G) and mitochondrial respiratory chain complex V (G) Figure 5The enzyme activities of HUVEC cells were detected. Compared with group I, the enzyme activity of mitochondrial respiratory chain complex in group II was significantly reduced. Compared with group II, the enzyme activity of mitochondrial respiratory chain complex in HUVEC cells in group III, which knocked down ALKBH5 alone, was significantly increased. Compared with group II, the enzyme activity of mitochondrial respiratory chain complex in HUVEC cells in group IV, which knocked down C2orf69 alone, was significantly reduced. Compared with group III, which knocked down ALKBH5 alone, the enzyme activity of mitochondrial respiratory chain complex in HUVEC cells in group V, which knocked down both ALKBH5 and C2orf69, was significantly reduced. Compared with group IV, which knocked down C2orf69 alone, the enzyme activity of mitochondrial respiratory chain complex in HUVEC cells in group V, which knocked down both ALKBH5 and C2orf69, was significantly increased. This indicates that ALKBH5 can regulate mitochondrial oxidative phosphorylation by regulating C2orf69.

[0054] Finally, angiogenesis experiments in HUVEC cells confirmed that ALKBH5 can regulate the angiogenesis capacity of HUVEC cells by modulating C2orf69. Specifically, compared with group I, the total blood vessel length in group II was significantly reduced; compared with group II, the total blood vessel length in group III, which knocked down ALKBH5 alone, was significantly increased; compared with group II, the total blood vessel length in group IV, which knocked down C2orf69 alone, was significantly reduced; compared with group III, which knocked down ALKBH5 alone, the total blood vessel length in group V, which knocked down both ALKBH5 and C2orf69 simultaneously, was significantly reduced; compared with group IV, which knocked down C2orf69 alone, the total blood vessel length in group V, which knocked down both ALKBH5 and C2orf69 simultaneously, was significantly increased. Figure 5 This indicates that ALKBH5 can regulate angiogenesis in HUVEC cells by modulating C2orf69.

[0055] Example 6: Preparation of siALK@L-Hyb (1) Preparation of mEV Raw milk was centrifuged at 13,000 × g for 30 minutes at 4°C to remove fat globules, cells, and cell debris. After removing the precipitate and the upper fat layer, the supernatant was collected and centrifuged a second time at 100,000 × g for 60 minutes at 4°C to remove large particles and microvesicles. The resulting supernatant was then ultracentrifuged at 140,000 × g for 90 minutes at 4°C to obtain the precipitate, which was the extracellular vesicle precipitate. The precipitate was resuspended in PBS, washed three times, and then filtered through a 0.22 μm filter membrane. The finally purified milk extracellular vesicle suspension was stored at -80°C for later use and named mEV (mEV). Figure 1 A).

[0056] (2) Preparation of CMN HUVEC cells grown to approximately 80% confluence were collected, resuspended in hypotonic buffer containing a mixture of protease and phosphatase inhibitors (Sigma Aldrich), and lysed using a Dounce homogenizer. The lysed homogenate was then subjected to differential centrifugation at 4°C to separate the cell membrane fraction: first, centrifugation at 3,200 × g for 5 minutes, collecting the supernatant; then centrifugation at 20,000 × g for 30 minutes, collecting the supernatant; finally, ultracentrifugation at 150,000 × g for 1.5 hours. The supernatant was discarded, and the resulting membrane precipitate was washed twice with PBS to obtain the purified cell membrane fraction, which was named CMN (Cellular Membrane). Figure 1 A).

[0057] (3) Preparation of siALK@mEV mEV and si-ALKBH5 were added to PBS at a mass ratio of 1:3 until fully dissolved and mixed. The mixture was sonicated in an ice-water bath (30% amplitude, 30 s sonication / 60 s interval, 10 cycles), and then extruded sequentially through polycarbonate membranes with decreasing pore sizes (1000 nm, 400 nm, 200 nm) using a microextruder (Avanti Polar Lipids). Finally, the extrudate was collected by ultracentrifugation at 150,000 × g for 90 min at 4 °C. The obtained vesicles were named siALK@mEV and stored at -80 °C for later use.

[0058] (4) Preparation of siALK@CMN mEV, CMN, and si-ALKBH5 (forward primer sequence as shown in SEQ ID NO.1, reverse primer sequence as shown in SEQ ID NO.2) were added to PBS at a mass ratio of 1:1:2 until fully dissolved and mixed. The mixture was sonicated in an ice-water bath (30% amplitude, 30 s sonication / 60 s interval, 10 cycles), and then extruded sequentially through polycarbonate membranes with decreasing pore sizes (1000 nm, 400 nm, 200 nm) using a microextruder (Avanti PolarLipids). Finally, the extrudate was collected by ultracentrifugation at 150,000 × g for 90 min at 4 °C. The obtained vesicles were named siALK@CMN and stored at -80 °C for later use.

[0059] (5) Preparation of siALK@Hyb mEV, CMN, lipopeptide (purchased from Heyuan Biotechnology), and si-ALKBH5 were added to PBS at a mass ratio of 1:1:1:3 until fully dissolved and mixed. The mixture was sonicated in an ice-water bath (30% amplitude, 30 s sonication / 60 s interval, 10 cycles), and then extruded sequentially through polycarbonate membranes with decreasing pore sizes (1000 nm, 400 nm, 200 nm) using a microextruder (Avanti Polar Lipids). Finally, the extrudate was collected by ultracentrifugation at 150,000 × g for 90 min at 4 °C. The obtained vesicles were named siALK@Hyb and stored at -80 °C for later use.

[0060] (6) Preparation of siALK@L-Hyb mEV, CMN, arginine-rich lipopeptides (purchased from Heyuan Biotechnology), and si-ALKBH5 were added to PBS at a mass ratio of 1:1:1:3 until fully dissolved and mixed. The mixture was sonicated in an ice-water bath (30% amplitude, 30 s sonication / 60 s interval, 10 cycles), and then extruded sequentially through polycarbonate membranes with decreasing pore sizes (1000 nm, 400 nm, 200 nm) using a microextruder (Avanti Polar Lipids). Finally, the extrudate was collected by ultracentrifugation at 150,000 × g for 90 min at 4 °C. Figure 1 A) Hybridized nanovesicles loaded with siALKBH5 were obtained, named siALK@L-Hyb, and stored at -80℃ for later use.

[0061] Example 7: Characterization of siALK@L-Hyb First, the morphological characterization of the prepared siALK@L-Hyb was performed using a transmission electron microscope (Hitachi, Japan) and a NanoSight LM10 system (Malvern Instruments, UK). The microscopic images showed that siALK@L-Hyb consisted of uniform vesicle structures with a particle size of 100 nm to 200 nm. Figure 6 A~B). Coomassie Brilliant Blue was used to detect the protein expression of mEV, CMN, and siALK@L-Hyb. The results showed that siALK@L-Hyb and mEV had three protein bands at the same position around 25~35kD. Figure 6C), and no other obvious endogenous protein bands were observed, which verified the successful construction and high purity of siALK@L-Hyb. Subsequently, Western blot was used to detect the expression of HUVEC-derived cell-specific membrane proteins and milk extracellular vesicle-specific proteins in mEVs, CMNs, and siALK@L-Hyb. The results showed that specific proteins derived from the HUVEC cell membrane (CD31 and ICAM-1), as well as milk extracellular vesicle-specific proteins (TSG101 and CD81), were stably expressed in siALK@L-Hyb. Figure 6 D). Next, the encapsulation efficiency of siRNA in siALK@L-Hyb was quantified using the RiboGreen RNA detection kit (purchased from Invitrogen, USA). The encapsulation efficiency of si-ALKBH5 in siALK@L-Hyb was approximately 45%. Figure 6 E). To confirm the membrane fusion effect of the hybrid vesicles prepared by ultrasonic fusion-sequential extrusion, mEV and CMN were pre-labeled with fluorescent dyes DiI (red) and DiO (green), respectively. By comparing the fluorescence signal distribution in samples prepared by physical mixing and ultrasonic fusion-sequential extrusion, it was found that the red and green fluorescence signals were clearly separated in the samples prepared by simple physical mixing; while after ultrasonic fusion-sequential extrusion, the vesicles showed a significant superposition of yellow fluorescence signals. Figure 6 F). These results indicate that the ultrasonic fusion-sequential extrusion process can effectively promote the membrane fusion of mEVs and CMNs, thereby achieving uniform mixing of their membrane components. Flow cytometry was used to detect the cellular uptake of different types of nanovesicles in HUVEC cells treated with mannitol or high glucose. The results showed that under high glucose treatment, L-Hyb had a higher uptake efficiency than mEVs, CMNs, and Hyb, indicating that L-Hyb can reverse high glucose-induced uptake inhibition, thus maintaining a high uptake level comparable to that of the mannitol group. Figure 6 G~H).

[0062] 1×10 under high glucose stimulation conditions 6 HUVEC cells were seeded in T25 culture flasks and cultured in ECM medium containing 5% FBS, 1% ECGS, and 1% penicillin / streptomycin solution for 12 hours until their growth density reached 2×10⁶ cells / year. 6 After that, they were randomly divided into six groups for processing: (1) High glucose treatment control group (HG + PBS): Add the same volume of PBS as the experimental group and treat for 24 hours.

[0063] (2) High glucose treatment + si-ALKBH5 (HG + siALK): Cells were transfected with si-ALKBH5 and incubated for 24 hours.

[0064] (3) High sugar treatment + siALK@mEV (HG + siALK@mEV): Add siALK@mEV suspension to a final concentration of 100 μg / mL and treat for 24 hours.

[0065] (4) High sugar treatment + siALK@CMN (HG + siALK@CMN): Add siALK@CMN suspension with a final concentration of 100 μg / mL and treat for 24 hours.

[0066] (5) High sugar treatment + siALK@Hyb (HG + siALK@Hyb): Add siALK@Hyb suspension to a final concentration of 100 μg / mL and treat for 24 hours.

[0067] (6) High sugar treatment + siALK@L-Hyb (HG + siALK@L-Hyb): Add siALK@L-Hyb suspension to a final concentration of 100 μg / mL and treat for 24 hours.

[0068] Flow cytometry was used to detect the uptake of si-ALKBH5 by HUVEC cells under high glucose treatment. The results showed that, compared with the HG + PBS group, HG + free siALK group, HG + siALK@mEV group, HG + siALK@CMN group, and HG + siALK@Hyb group, the HG + siALK@L-Hyb group had a higher efficiency in uptake of si-ALKBH5 by HUVEC cells. Figure 6 I~J). Lysosomes and nuclei were then labeled using LysoTracker Green (from Beyotime) and Hoechst 33342 (from Beyotime), respectively. Colocalization analysis of free siALKBH5 (green) and lysosomes (red) was performed using laser confocal microscopy. Compared to CMN and L-Hyb, the colocalization signals of the mEV and siALK@L-Hyb groups were weaker, indicating that mEV and L-Hyb possess highly efficient lysosomal escape capabilities. Figure 6 K). In the dermal layer of the wound, the penetration of different types of nanovesicles varies. The white and red dashed lines represent the wound edge and the furthest penetration range, respectively. The green fluorescence represents FAM-labeled siALKBH5. Among them, the furthest penetration range of the siALK@L-Hyb group is much greater than that of the siALK@mEV group, siALK@CMN group, and siALK@Hyb group ( Figure 6 Therefore, siALK@L-Hyb assembled using L-Hyb as a carrier exhibits good uptake efficiency, lysosomal escape ability, and a wider wound penetration range.

[0069] Example 8: siALK@L-Hyb alleviates mitochondrial dysfunction in endothelial cells caused by high glucose damage and restores cell function. To investigate the role of the siALK@L-Hyb axis in regulating mitochondrial function in vascular endothelial cells treated with high glucose, the cells were subjected to the following grouping treatments: Ⅰ: Mannitol + PBS: HUVEC cells were treated with ECM medium containing 24.5 mM mannitol for 72 hours, followed by the addition of an equal volume of PBS buffer to replace the medium and treatment for 24 hours.

[0070] II: High glucose + PBS: HUVEC cells were treated with ECM medium containing 30 mM glucose for 72 hours, followed by the addition of an equal volume of PBS buffer to replace the medium and treatment for 24 hours.

[0071] III: High glucose + siALKBH5: HUVEC cells were treated with ECM medium containing 30 mM glucose for 72 hours, and then si-ALKBH5 was introduced into the cells by transfection and treated for 24 hours.

[0072] IV: High glucose + L-Hyb: HUVEC cells were treated with ECM medium containing 30 mM glucose for 72 hours, followed by the addition of L-Hyb suspension to a final concentration of 100 μg / mL by changing the medium, and treated for 24 hours.

[0073] V: High glucose + siALK@mEV: HUVEC cells were treated with ECM medium containing 30 mM glucose for 72 hours, followed by adding siALK@mEV suspension to a final concentration of 100 μg / mL and treating for 24 hours after changing the medium.

[0074] VI: High glucose + siALK@CMN: HUVEC cells were treated with ECM medium containing 30 mM glucose for 72 hours, followed by the addition of siALK@CMN suspension to a final concentration of 100 μg / mL for 24 hours after changing the medium.

[0075] VII: High glucose + siALK@Hyb: HUVEC cells were treated with ECM medium containing 30 mM glucose for 72 hours, followed by the addition of siALK@Hyb suspension to a final concentration of 100 μg / mL for 24 hours after changing the medium.

[0076] VIII: High glucose + siALK@L-Hyb: HUVEC cells were treated with ECM medium containing 30 mM glucose for 72 hours, followed by the addition of siALK@L-Hyb suspension to a final concentration of 100 μg / mL for 24 hours after changing the medium.

[0077] The expression of ALKBH5 and C2orf69 in HUVEC cells after different treatments was detected by Western blot. Compared with control group I, the expression of ALKBH5 protein in high glucose treatment control group II was significantly increased, while the expression of C2orf69 was significantly decreased. Compared with control group II and experimental groups III, IV, V, and VI, the expression of ALKBH5 in experimental groups VII and VIII was significantly decreased, while the expression of C2orf69 was significantly increased (7A~C). Subsequently, the cytoplasmic ROS level in HUVEC cells after different treatments was measured by flow cytometry. Compared with control group I, the ROS content in control group II was significantly increased. Compared with control group II and experimental groups III, IV, V, and VI, the cytoplasmic ROS content in experimental groups VII and VIII was significantly decreased (7A~C). Figure 7 D). Similarly, after MitoSOX staining, confocal microscopy was used to detect the content of mitochondrial ROS in HUVEC cells after different treatments. Compared with control group I, the fluorescence intensity in control group II was significantly increased; compared with control group II and experimental groups III, IV, V, and VI, the ROS fluorescence intensity in experimental groups VII and VIII was significantly decreased. Figure 7 E). All the above results indicate that siALK@Hyb and siALK@L-Hyb can effectively reduce ROS production and thus affect mitochondrial function. JC-1 staining was used to detect the mitochondrial membrane potential ΔΨm in HUVEC cells after different treatments. Compared with control group I, ΔΨm was significantly decreased in control group II; compared with control group II and experimental groups III, IV, V, and VI, ΔΨm was significantly increased in experimental groups VII and VIII. Figure 7 G). Next, the mitochondrial respiratory chain complex I (G) of HUVEC cells after different treatments was analyzed. Figure 7 F), mitochondrial respiratory chain complex IV ( Figure 7 H) and mitochondrial respiratory chain complex V ( Figure 7 The enzyme activity of siALK@Hyb was detected. Compared with control group I, the enzyme activity of mitochondrial respiratory chain complex in HUVEC cells in control group II was significantly reduced. Compared with control group II and experimental groups III, IV, V, and VI, the enzyme activity of mitochondrial respiratory chain complex in experimental groups VII and VIII was significantly increased. This indicates that siALK@Hyb and siALK@L-Hyb can regulate mitochondrial oxidative phosphorylation. Finally, the angiogenesis experiment was conducted to detect the angiogenesis capacity of HUVEC cells under the above different treatments. Compared with control group I, the total blood vessel length in control group II was significantly reduced. Compared with control group II and experimental groups III, IV, V, and VI, the total blood vessel length in experimental groups VII and VIII was significantly increased. Figure 7 I). The above results indicate that siALK@Hyb and siALK@L-Hyb can promote angiogenesis in HUVEC cells.

[0078] Example 9: The therapeutic effect of siALK@L-Hyb on diabetic wounds The wound modeling of diabetic mice was the same as in Example 1. After modeling, the diabetic mice were randomly divided into 8 groups of 10 mice each. The specific grouping information for the in vivo experiment is as follows: Ⅰ: Non-diabetic mice + PBS: 25 μL of PBS was evenly sprayed onto the wound surface of the mouse skin flap using a syringe. The dressing was changed every 3 days, and the gauze was changed each time. The treatment was continued for 14 days.

[0079] II: Diabetic mice + PBS: 25 μL of PBS was evenly sprayed onto the wound surface of the mouse skin flap using a syringe. The dressing was changed every 3 days, and the gauze was changed each time. The treatment was continued for 14 days.

[0080] III: Diabetic mice + siALK: 25 μL of si-ALKBH5 (450 μg / mL) was evenly sprayed onto the wound surface of the mouse skin flap using a syringe. The dressing was changed every 3 days, and the gauze was changed each time. The treatment was continued for 14 days.

[0081] IV: Diabetic mice + L-Hyb: 25 μL of L-Hyb (1 mg / mL) was evenly sprayed onto the wound surface of the mouse skin flap using a syringe. The dressing was changed every 3 days, and the gauze was changed each time. The treatment was continued for 14 days.

[0082] V: Diabetic mice + siALK@mEV: 25 μL of siALK@mEV (1 mg / mL) was evenly sprayed onto the wound surface using a syringe at the mouse skin flap. The dressing was changed every 3 days, and the gauze was changed each time. The treatment was continued for 14 days.

[0083] VI: Diabetic mice + siALK@CMN: Apply 25 μL of siALK@CMN (1 mg / mL) evenly to the wound surface using a syringe on the mouse skin flap. Change the dressing every 3 days, changing the gauze each time, for a total of 14 days.

[0084] VII: Diabetic mice + siALK@Hyb: Apply 25 μL of siALK@Hyb (1 mg / mL) evenly to the wound surface using a syringe at the mouse skin flap. Change the dressing every 3 days, changing the gauze each time, for a total of 14 days.

[0085] VIII: Diabetic mice + siALK@L-Hyb: 25 μL of siALK@L-Hyb (1 mg / mL) was evenly sprayed onto the wound surface of the mouse skin flap using a syringe. The dressing was changed every 3 days, and the gauze was changed each time. The treatment was continued for 14 days.

[0086] The above treatment was performed on each group of wounds at different time points. Figure 8 A), during which photos were taken ( Figure 8B), and a quantitative analysis of the wound area was performed ( Figure 8 (D) The results showed that, compared with group I, the healing speed of diabetic wounds in group II was significantly slower, and the wound area was larger after 14 days of treatment; compared with group II and groups III, IV, V, VI, and VII, the healing speed of diabetic wounds in group VIII was significantly faster, and the wound area was smaller after 14 days of treatment. These results indicate that siALK@L-Hyb can significantly accelerate the healing of diabetic wounds. Furthermore, the dynamic contour diagram of wound healing showed that, with the extension of treatment time, the wound contraction amplitude in groups I, II, III, IV, V, VI, and VII was limited, while the wound boundary in group VIII showed a significant contraction towards the center. Figure 8 C). H&E staining on day 14 post-treatment ( Figure 8 E) showed that groups I-IV had insufficient epithelial extension, wide unepithelialized gaps, and incomplete dermal-epithelial junctions; while groups V-VIII showed significant epithelialization progress, with the unepithelialized area rapidly shrinking, and group VIII achieving near-full-thickness epithelialization and highly contracted wound edges. Quantitative analysis of the length of unepithelialized wound gaps ( Figure 8 F) and wound contraction width ( Figure 8 Quantitative analysis of G) further proved that there was no statistically significant difference among groups I to IV. From group V onwards, the length of the non-epithelialized gap and the width of wound contraction both decreased. Among them, the length of the epithelialized gap and the width of wound contraction in group VIII decreased significantly. The above results indicate that siALK@L-Hyb has the best effect in promoting wound contraction and epithelialization.

[0087] Masson's trichrome staining was performed on the wound sections of the mice treated in the above groups. The results showed that, compared with control group II and experimental groups III, IV, V, VI, and VII, the amount of collagen deposited in the diabetic wounds of experimental group VIII was greater after treatment on day 14. Figure 9 (A and 9D), indicating that siALK@L-Hyb has a significant promoting effect on the healing of diabetic wounds. Subsequent ethidium dihydrogen ionization staining assays were performed to detect reactive oxygen species (ROS) levels in the tissues. The results showed that, compared to control group I, the ROS level in diabetic wounds in control group II was significantly increased; compared to control group II and experimental groups III, IV, V, VI, and VII, the ROS level in diabetic wounds in experimental group VIII was significantly decreased after treatment on day 14. Figure 9 B and 9E), indicating that siALK@L-Hyb can effectively inhibit ROS generation in diabetic wounds. Finally, immunofluorescence staining of CD31 and α-SMA was performed on each group of wound sections (B and 9E). Figure 9C), used to assess angiogenesis on day 14 post-treatment, showed that compared to control group I, the area of ​​CD31-positive and α-SMA-positive areas in diabetic wounds in control group II was significantly reduced; compared to control group II and experimental groups III, IV, V, VI, and VII, the area of ​​CD31-positive and α-SMA-positive areas in diabetic wounds in experimental group VIII was significantly increased after day 14 of treatment. Figure 9 (F~G) indicates that siALK@L-Hyb has a significant promoting effect on angiogenesis in diabetic wounds.

[0088] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0089] It should be noted that in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0090] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A drug-loaded vesicle for promoting the healing of diabetic wounds, characterized in that: The drug-loaded vesicles have a core-shell structure, wherein: The nucleus contains siRNA that targets and binds to ALKBH5 mRNA, the sense strand of which is shown in SEQ ID NO.1 and the antisense strand in SEQ ID NO.2; The shell is a hybrid membrane of human umbilical vein endothelial cell membrane and milk-derived extracellular vesicle membrane, and the surface of the hybrid membrane is embedded with lipopeptides containing arginine, and at least a portion of the arginine is exposed outside the hybrid membrane.

2. The drug-loaded vesicle for promoting diabetic wound healing according to claim 1, characterized in that: The core also contains a dispersion medium for the siRNA.

3. The drug-loaded vesicle for promoting diabetic wound healing according to claim 1, characterized in that: The particle size of the drug-loaded vesicles is 100 nm to 200 nm.

4. The drug-loaded vesicle for promoting diabetic wound healing according to claim 1, characterized in that: The core-shell mass ratio of the drug-loaded vesicle is 1:(0.8~1.2).

5. The drug-loaded vesicle for promoting diabetic wound healing according to claim 1, characterized in that: The arginine content in the lipopeptide is 7% to 11%.

6. A method for preparing drug-loaded vesicles for promoting diabetic wound healing according to claim 1, characterized in that: include: Extracellular vesicles, human umbilical vein endothelial cell membrane, lipopeptides and siRNA derived from milk were initially mixed and then subjected to sonication in an ice-water bath to obtain a mixed solution. The mixed solution was extruded from a porous membrane using a micro extruder to obtain the drug-loaded vesicles for promoting the healing of diabetic wounds.

7. The preparation method according to claim 6, characterized in that: The mass ratio of milk-derived extracellular vesicles, human umbilical vein endothelial cell membrane, lipopeptides, and siRNA in the mixed solution is 1:(0.9~1.1):(0.9~1.1):(2~4).

8. A wound healing agent for diabetes, characterized in that: It includes the drug-loaded vesicles for promoting the healing of diabetic wounds as described in any one of claims 1 to 5.

Images