A novel preparation method of hybrid extracellular vesicles and application thereof

Abstract

The application discloses a novel preparation method and application of hybrid extracellular vesicles. The application combines endothelial cell-derived and neutrophil-derived vesicles with deferoxamine to develop a biological mixed nano-vesicle platform (DFO@HEVs) to solve the problem of diabetic wound healing. The dual-targeting system utilizes CXCR4-mediated endothelial cell homing and beta2 integrin-dependent inflammatory tropism to accurately deliver DFO to the wound site, DFO@HEVs activate HIF-1alpha / VEGF to restore vascular regeneration, inhibit ferroptosis through Nrf2 / GPX4 signaling, and reprogram macrophages into a pro-repair M2 phenotype, effectively breaking the oxidative stress-inflammation-ferroptosis cycle.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomaterials, and particularly relates to a preparation method of a novel hybrid extracellular vesicle and application thereof. BACKGROUND

[0002] Diabetes is a chronic metabolic disease that is prevalent worldwide, and has a high incidence and mortality rate. Delayed wound healing is a common complication of diabetes, which seriously affects people's normal life.

[0003] Diabetic wounds have the characteristics of chronic inflammation, oxidative stress and epithelial cell dysfunction, and these problems delay wound healing and increase the risk of amputation. Existing targeted delivery systems for diabetic wounds focus on single endothelial cell vesicles or hybrid vesicles of multiple endothelial cell vesicles. There is a lack of neutrophil inflammation chemotaxis to target inflammatory sites. In addition, existing hybrid vesicle delivery drugs simply address oxidative stress without considering iron death and its mechanisms. SUMMARY

[0004] The purpose of the present application is to solve the above-mentioned deficiencies, and to provide a preparation method of a novel hybrid extracellular vesicle capable of solving the problem of diabetic wound healing and application thereof.

[0005] In order to solve the above technical problems, the present application adopts the following technical solution: a preparation method of a novel hybrid extracellular vesicle, comprising the following steps:

[0006] S1, preparing endothelial cell nanovesicles EVs:

[0007] culturing HUVEC, and then extruding an EV suspension by an extrusion method;

[0008] S2, preparing neutrophil nanovesicles NVs:

[0009] culturing neutrophils, and then extruding an NV suspension by an extrusion method;

[0010] S3, mixing the EV suspension obtained in S1 and the NV suspension obtained in S2, and hybridizing by ultrasonication to obtain a hybrid extracellular vesicle HEVs suspension;

[0011] S4, loading DFO into HEVs by ultrasonic perforation to obtain DFO@HEVs.

[0012] Further, before the extrusion method is used in S1, TrypLE™ Express enzyme is used to dissociate the cultured HUVEC, and is diluted to 5x10 6 cells / mL in PBS.

[0013] Further, before the extrusion method in S2, the cultured neutrophils are dissociated using TrypLE™ Express enzyme, and diluted to 5 x 10 6 cells / mL in PBS.

[0014] Further, in S1, the specific steps of culturing HUVECs include:

[0015] 1) Culturing HUVECs cells with high-glucose DMEM complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin;

[0016] 2) Using ultraviolet lamp to disinfect microorganisms in the clean bench;

[0017] 3) Taking sterile PBS solution, 0.25% trypsin solution and complete medium, and fully warming them in a 37°C water bath;

[0018] 4) Using sterile PBS solution to wash the cell culture bottle 3 times;

[0019] 5) Adding trypsin solution in the culture bottle, placing it on the table after covering the bottle cap, shaking it to spread evenly, and placing it in a 37°C cell incubator for digestion;

[0020] 6) Observing the cells in the culture bottle under a microscope, when the cells shrink into a spherical shape and float, gently pat the side wall of the culture bottle, and when more than 90% of the cells are floated, stop the digestion;

[0021] 7) Adding complete medium to the culture bottle at a ratio of 1:1, and transferring it to a centrifuge tube;

[0022] 8) Centrifuging the solution in the centrifuge tube, discarding the supernatant, mixing the complete medium by blowing, transferring it to the culture bottle, adding complete medium, and placing it in a 37°C cell incubator for culture.

[0023] Further, in S2, the specific steps of culturing neutrophils include:

[0024] 1) Differentiating human HL-60 cells and human Jurkat clone E6 cells into neutrophils by 1 μm all-trans retinoic acid ATRA and 1.25% dimethyl sulfoxide DMSO for 5 days;

[0025] 2) Culturing neutrophils with RIPM-1600 complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin;

[0026] 3) Using ultraviolet lamp to disinfect microorganisms in the clean bench;

[0027] 4) Take sterile PBS solution, 0.25% trypsin solution and complete medium, and fully reheat in a 37℃ water bath;

[0028] 5) Wash the cell culture bottle 3 times with sterile PBS solution;

[0029] 6) Add the trypsin solution in the culture bottle, place it on the table after covering the bottle cap, shake to spread evenly, and place it in a 37℃ cell culture box for digestion;

[0030] 7) Observe the cells in the culture bottle under a microscope, when the cells shrink into a spherical shape and float, gently tap the side wall of the culture bottle, when more than 90% of the cells are floated by tapping, stop the digestion;

[0031] 8) Add complete medium to the culture bottle at a ratio of 1:1, and transfer it to a centrifuge tube;

[0032] 9) Centrifuge the solution in the centrifuge tube, discard the supernatant, mix the complete medium by blowing, transfer it to the culture bottle, add complete medium, and place it in a 37℃ cell culture box for culture.

[0033] Further, the extrusion method in S1 and S2 is: using a micro-extruder to extrude the cell suspension through a series of Nuclepore™ polycarbonate membranes, the pore size of the series of Nuclepore™ polycarbonate membranes is 10 μm, 5 μm, 1 μm, 0.4 μm and 0.2 μm in turn.

[0034] Further, in S3, the EVs suspension and the NVs suspension are in a weight ratio of 1:1.

[0035] Further, the method for obtaining hybrid extracellular vesicles in S3 is: using an ultrasonic instrument to perform ultrasonic treatment on an ice water bath at 30% intensity, turning on for 30s and turning off for 2min, and repeating 4-6 times, then extruding through a 0.2 μm polycarbonate membrane to produce a hybrid extracellular vesicle suspension.

[0036] Further, before S4, the hybrid extracellular vesicle suspension obtained in S3 is subjected to ultracentrifugation, the hybrid extracellular vesicle suspension is centrifuged at 3000xg and 10000xg at 4℃ for 30min in turn, the debris in the hybrid extracellular vesicle suspension is removed, then the supernatant is centrifuged at 140000xg at 4℃ for 70min, the nanoscale vesicles are collected, washed twice and resuspended in sterile PBS, and stored at -80℃.

[0037] The application of a new type of hybrid extracellular vesicle in the preparation of a drug for treating diabetic wounds.

[0038] Compared with the prior art, the application has the following beneficial effects:

[0039] The present application develops a biological hybrid nanovesicle platform (DFO@HEVs) by combining endothelial cell-derived and neutrophil-derived vesicles with deferoxamine to solve the problem of diabetic wound healing. The dual-targeting system uses CXCR4-mediated endothelial cell homing and β2 integrin-dependent inflammatory tropism to precisely deliver DFO to the wound site, DFO@HEVs restore vascular regeneration through HIF-1α / VEGF activation, inhibit ferroptosis through Nrf2 / GPX4 signaling, and reprogram macrophages to a pro-repair M2 phenotype, effectively breaking the oxidative stress-inflammation-ferroptosis cycle. In vivo, DFO@HEVs accelerate wound closure, reduce neutrophil infiltration, and promote collagen remodeling, demonstrating their therapeutic application prospects. This drug delivery platform integrates endothelial repair, antioxidant defense, and immune modulation, providing a new strategy for diabetic wound repair and paving the way for the next generation of nanotherapies for diabetic chronic wounds. BRIEF DESCRIPTION OF DRAWINGS

[0040] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The illustrations are shown schematically in the drawings wherein:

[0041] Figure 1 Schematic diagram for engineering and identification of DFO@HEVs of the present application.

[0042] Figure 2 Schematic diagram for uptake and targeting of DFO@HEVs of the present application.

[0043] Figure 3 Schematic diagram for cell proliferation, migration, and activity experiments of HNV of the present application.

[0044] Figure 4 Schematic diagram for DFO@HEVs of the present application reversing oxidative stress damage to cells.

[0045] Figure 5 Schematic diagram for DFO@HEVs of the present application alleviating ferroptosis through lipid peroxidation and Nrf2 pathway.

[0046] Figure 6 Schematic diagram for DFO@HEVs of the present application reducing neutrophil attachment to endothelial cells and DFO@HEVs absorbing inflammatory factors.

[0047] Figure 7 Schematic diagram for DFO@HEVs of the present application exerting anti-inflammatory effect by inhibiting ROS / NF-kB signaling pathway.

[0048] Figure 8Schematic diagram of DFO@HEVs inhibiting neutrophil adhesion and promoting cell necrosis.

[0049] Figure 9 Schematic diagram of DFO@HEVs accelerating in vivo healing of diabetic wound. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The embodiments in the present application and the features in the embodiments can be combined with each other without conflict. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0051] Please refer to Figures 1-9 The preparation method of the novel hybrid extracellular vesicle of the present application comprises the following steps:

[0052] S1, preparing endothelial cell nanovesicle EVs:

[0053] Culturing HUVEC, and then extruding EV suspension by extrusion method;

[0054] S2, preparing neutrophil nanovesicle NVs:

[0055] Culturing neutrophil, and then extruding NV suspension by extrusion method;

[0056] S3, mixing the EV suspension obtained in S1 and the NV suspension obtained in S2, and hybridizing by ultrasonic to obtain hybrid extracellular vesicle HEVs suspension;

[0057] S4, loading DFO into HEVs by ultrasonic perforation to obtain DFO@HEVs.

[0058] In an embodiment, before the extrusion method is used in S1, TrypLE™ Express enzyme is used to dissociate the cultured HUVEC, and diluted to 5×10 6 cells / mL in PBS.

[0059] In an embodiment, before the extrusion method is used in S2, TrypLE™ Express enzyme is used to dissociate the cultured neutrophil, and diluted to 5×10 6 cells / mL in PBS.

[0060] In an embodiment, in S1, the specific steps of culturing HUVEC include:

[0061] 1) HUVECs cells were cultured in high glucose DMEM complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37℃, 5% CO2;

[0062] 2) Microorganisms in the clean bench were sterilized using an ultraviolet lamp for 30 min;

[0063] 3) Sterile PBS solution, 0.25% trypsin solution and complete medium were taken from a 4℃ refrigerator and fully warmed in a 37℃ water bath;

[0064] 4) The cell culture flask was washed three times with sterile PBS solution;

[0065] 5) The trypsin solution was added to the culture flask, which was then placed on the table and shaken to evenly spread the cells, and then placed in a 37℃ cell incubator for digestion;

[0066] 6) The cells in the culture flask were observed under a microscope. When the cells were spherical and floating, the side wall of the culture flask was gently tapped. When more than 90% of the cells were floated, the digestion was stopped;

[0067] 7) Complete medium was added to the culture flask at a ratio of 1:1, and then transferred to a 15 mL centrifuge tube;

[0068] 8) The solution in the centrifuge tube was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the complete medium was mixed by blowing and transferred to the culture flask. After adding complete medium, it was placed in a 37℃ cell incubator for culture.

[0069] In an embodiment, in the S2, the specific steps of culturing neutrophils include:

[0070] 1) Human HL-60 cells and human Jurkat clone E6 cells were differentiated into neutrophils by 1 μm all-trans retinoic acid ATRA and 1.25% dimethyl sulfoxide DMSO for 5 days;

[0071] 2) The neutrophils were cultured in RIPM-1600 complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin;

[0072] 3) Microorganisms in the clean bench were sterilized using an ultraviolet lamp;

[0073] 4) Sterile PBS solution, 0.25% trypsin solution and complete medium were taken from a 4℃ refrigerator and fully warmed in a 37℃ water bath;

[0074] 5) The cell culture flask was washed three times with sterile PBS solution;

[0075] 6) Add trypsin solution into the culture flask, place the flask on the table and shake it gently to spread the cells evenly, and then put it into the 37℃ cell culture box for digestion;

[0076] 7) Observe the cells in the culture flask under a microscope. When the cells shrink into a spherical shape and float, gently tap the side wall of the culture flask. When more than 90% of the cells are floated, stop the digestion;

[0077] 8) Add complete culture medium into the culture flask at a ratio of 1:1, and then transfer it to a 15mL centrifuge tube;

[0078] 9) Centrifuge the solution in the centrifuge tube at 1000rpm for 5min, discard the supernatant, mix the complete culture medium by blowing, transfer it to the culture flask, add complete culture medium, and then put it into the 37℃ cell culture box for culture.

[0079] In an embodiment, the extrusion method in S1 and S2 is: using a micro-extruder to extrude the cell suspension through a series of Nuclepore™ polycarbonate membranes with pore sizes of 10μm, 5μm, 1μm, 0.4μm and 0.2μm.

[0080] In an embodiment, in S3, the EVs suspension and the NVs suspension are in a weight ratio of 1:1.

[0081] In an embodiment, the method for obtaining hybrid extracellular vesicles in S3 is: using an ultrasonic instrument to perform ultrasonic treatment on an ice water bath at 30% intensity, turning on for 30s and turning off for 2min, repeating 4-6 times, and then extruding through a 0.2μm polycarbonate membrane to produce a hybrid extracellular vesicle suspension.

[0082] In an embodiment, before S4, the hybrid extracellular vesicle suspension obtained in S3 is subjected to ultracentrifugation. The hybrid extracellular vesicle suspension is centrifuged at 3000xg and 10000xg at 4℃ for 30min, respectively, to remove debris in the hybrid extracellular vesicle suspension, and then the supernatant is centrifuged at 140000xg at 4℃ for 70min, nanoscale vesicles are collected, washed twice, resuspended in sterile PBS, and stored at -80℃.

[0083] The application of a new type of hybrid extracellular vesicle in the preparation of a drug for treating diabetic wounds.

[0084] Figure 1(A) is a schematic diagram of DFO@HEVs preparation; (B) and (C) are particle size analysis and zeta potential of vesicles in PBS; (D) is an HNV electron microscope image, scale bar 100 nm; (E) is a DFO release curve of DFO@HEVs; (F) is a laser confocal microscope image (Dil-labeled vesicles red, DiO-labeled vesicles green), scale bar 10 μm; (G) is a co-localization analysis experiment of vesicle fusion group and physical mixing group; (H) is a Fourier infrared effect confirming successful fusion of hybrid vesicles; (I) is a red fluorescent labeled HEV loading green fluorescent labeled DFO, scale bar 100 μm; (J) is a Western blotting result of EVs, NVs and HEVs specific proteins; (K) is a SDS-PAGE electrophoresis result of HUVEC, neutrophil, EVs, NVs and HEVs.

[0085] Figure 2 (A) is a fluorescence image of HUVEC uptake of 293T cell-HEVs, HEVs, anti-CXCR4 pretreated HEVs and IgG pretreated HEVs. Scale bar 10 μm; (B) is a quantitative determination result of HUVEC uptake of the above vesicles; (C) is a corresponding flow cytometry result. Three groups of results constitute a histogram. n=3; (D) is a fluorescence image of HUVEC uptake of EVs, NVs, HEVs and DFO@HEVs with or without anti-ICAM-1 antibody treatment. Scale bar 10 μm; (E) is a quantitative determination result of HUVEC uptake of vesicles; (F) is a corresponding flow cytometry result; (G) is a fluorescence image of DFO@HEVs targeting endothelial cell model; (H) is a corresponding flow cytometry result; (I) (J) is a corresponding statistical analysis of EC target efficiency under different co-culture conditions.

[0086] Figure 3Figure 6. Representative images of HUVECs treated with different vesicles. (A) Representative images of HUVECs treated with different vesicles and (B) corresponding statistical analysis (n = 5). Scale bar: 100 pm. (C) DCFH-DA staining analysis of HUVECs and (D) corresponding statistical analysis and (E) flow cytometry to measure DCF relative fluorescence intensity (n = 5). Scale bar: 100 pm. (F) DHE staining analysis of HUVECs and (G) corresponding statistical analysis of DHE relative fluorescence intensity (n = 5). Scale bar: 100 pm. (H) JC-1 staining analysis of HUVECs and (I) corresponding statistical analysis (n = 5). Scale bar: 50 pm. (J) Flow cytometry to measure TMRE staining of HUVECs. Data are shown as mean ± SD. Data were evaluated using one-way ANOVA and Tukey post-hoc, *P < 0.05, **P < 0.01, ***P < 0.001.

[0087] Figure 4 Figure 6. Representative images of HUVECs treated with different vesicles. (A) Representative images of HUVECs treated with different vesicles and (B) corresponding statistical analysis (n = 5). Scale bar: 100 pm. (C) DCFH-DA staining analysis of HUVECs and (D) corresponding statistical analysis and (E) flow cytometry to measure DCF relative fluorescence intensity (n = 5). Scale bar: 100 pm. (F) DHE staining analysis of HUVECs and (G) corresponding statistical analysis of DHE relative fluorescence intensity (n = 5). Scale bar: 100 pm. (H) JC-1 staining analysis of HUVECs and (I) corresponding statistical analysis (n = 5). Scale bar: 50 pm. (J) Flow cytometry to measure TMRE staining of HUVECs. Data are shown as mean ± SD. Data were evaluated using one-way ANOVA and Tukey post-hoc, *P < 0.05, **P < 0.01, ***P < 0.001.

[0088] Figure 5 Figure 6. Representative images of HUVECs treated with different vesicles. (A) Representative images of HUVECs treated with different vesicles and (B) corresponding statistical analysis (n = 5). Scale bar: 100 pm. (C) DCFH-DA staining analysis of HUVECs and (D) corresponding statistical analysis and (E) flow cytometry to measure DCF relative fluorescence intensity (n = 5). Scale bar: 100 pm. (F) DHE staining analysis of HUVECs and (G) corresponding statistical analysis of DHE relative fluorescence intensity (n = 5). Scale bar: 100 pm. (H) JC-1 staining analysis of HUVECs and (I) corresponding statistical analysis (n = 5). Scale bar: 50 pm. (J) Flow cytometry to measure TMRE staining of HUVECs. Data are shown as mean ± SD. Data were evaluated using one-way ANOVA and Tukey post-hoc, *P < 0.05, **P < 0.01, ***P < 0.001. 2+Quantitative analysis of (F) and corresponding flow cytometry results of (G). (H) MDA levels in endothelial cells after different treatments (n = 3). (I) GPx activity in endothelial cells after different treatments (n = 3). (J) SOD activity in endothelial cells after different treatments (n = 3). (K) Western blotting of Nrf2 total, nuclear Nrf2, HO-1, NQO-1, GPX4, ACSL4, β-actin, Lamine B in endothelial cells of each group after different treatments. Data are shown as mean ± standard deviation. Data were evaluated using one-way ANOVA and Tukey post-hoc, *P < 0.05, **P < 0.01, ***P < 0.001.

[0089] Figure 6 (B, C) Experimental schematic. (B) Fluorescence images and corresponding static analysis of HUVEC uptake of hybrid vesicles and neutrophils after pre-treatment of vesicles with anti-ITGB2 or IgG antibody, scale bar: 50 pm. (C) Adhesion ratio of neutrophils in different groups. (D) Absorbance of IL-6 and (E) TNF-a after 24 h incubation with vesicles. Data are shown as mean ± standard deviation. Data were evaluated using one-way ANOVA and Tukey post-hoc, *P < 0.05, **P < 0.01, ***P < 0.001.

[0090] Figure 7(A) is macrophage phenotype polarization study. Flow cytometry was used to detect the polarization of inflammatory iBMDMs after different vesicle treatment, iBMDMs without LPS stimulation were used as normal control group (n=3). (B) is CD80 and (C) is CD206 positive cells representing M1 and M2 subtypes after treatment, respectively. (D) is the quantitative characterization of M2 / M1 ratio after treatment. (E) is the amount of NO produced after treatment of inflammatory macrophages. (F) is fluorescence images and (G) (H) is the quantitative expression of iNOS and CD206 after treatment of inflammatory iBMDMs (n=3), scale bar: 50 μm. (I) is Western blot analysis of Arg-1 and iNOS in iBMDMs after different treatments and (J) (K) is the corresponding statistical analysis. (L) is fluorescence images and (M) is the quantitative expression of ROS after treatment of inflammatory iBMDMs (n=3), scale bar: 50 μm. (N) is the content of TNF-α, IL-10 and IL-6 in the supernatant of inflammatory macrophages after treatment (n=3). (O) is Western blot analysis of Ikkα / β, P-Ikkα / β, IκBα, P-IκBα, NF-κB, P-NF-κB and NLRP3 in iBMDMs after different treatments. Data are expressed as mean ± standard deviation, one-way ANOVA and Tukey post-hoc test, *P<0.05, **P<0.01, ***P<0.001.

[0091] Figure 8 (A) is a schematic diagram of (B), (C) and (D) experiments; (B) is the evaluation of the phagocytosis of the ex vivo experiment by laser confocal microscopy. iBMDM is labeled green with CFSE, its nucleus is labeled blue, and Jurkat suspension cells are labeled red. Scale bar: 50 um; (C) is the corresponding statistical analysis; (D) is the corresponding flow cytometry analysis (n=3); (E, F) is the detection of the total number of cells in each group by ANnexin-V apoptosis cell detection reagent in the control group, neutrophil vesicle group, HEVs, DFO-HEVs and HUVEC group of apoptotic NVs uptake.

[0092] Figure 9 (A) is a representative image, (B) is monitoring data, (C) (D) (E) is the statistical result of wound closure on day 0, 2, 6, 12 (n = 5). (F) is the whole HE staining result on day 12 and (H) is the Masson staining result. Scale bar: 50 μm. Corresponding statistical analysis includes (G) is the length of granulation tissue (n = 5) and (I) is the amount of collagen deposition (n = 5). *P<0.05, ****P<0.0001. Data are expressed as mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns no significant. Data are expressed as mean ± standard deviation.

[0093] Identification of DFO@HEVs

[0094] To evaluate the release rate of DFO from DFO@HEVs, the release curve was established by measuring the absorbance at 430 nm, which corresponds to the reaction between DFO and ferric chloride Figure 1 E). To measure the fusion of HNVs, Dil-labeled EVs and DiO-labeled NVs were fused on membrane and compared with simple physical mixing as control. Laser confocal microscopy (CLSM) images and co-localization analysis confirmed that HNVs had membrane fusion but physical mixing did not Figure 1 D,F). To further determine the successful fusion of EVs and NVs, fluorescence resonance energy transfer (FRET) effect assay was performed. EVs and NVs were labeled with fluorescent donor DiO and fluorescent acceptor Dil, respectively. FRET signal of fused HEVs was read by fluorescence microplate reader Figure 1 G). FAM-labeled DFO (green) was successfully loaded into Dil-labeled DFO@HEVs Figure 1 I). Western blotting confirmed that HEVs retained membrane proteins CXCR4 and ITGB2, which can target endothelial cells and neutrophils Figure 1 J). To evaluate the release of DFO from DFO@HEVs, the release curve was established by measuring the absorbance at 430 nm, which corresponds to the reaction between DFO and ferric chloride Figure 1 E). Finally, the biocompatibility of DFO@HEVs was verified by CCK-8, Calcein-AM / PI red-green staining and comprehensive ex vivo hemolysis experiment. The results confirmed that functional membrane proteins were manufactured and DFO was loaded into the hybrid vesicles.

[0095] Uptake and targeting of DFO@HEVs

[0096] To verify the uptake of vesicles, laser confocal microscopy analysis of Dil-labeled HEVs revealed that HUVEC cells could efficiently uptake EVs, NVs and HEVs Figure 2 ). In addition, drug inhibition was used to confirm the endocytosis pathway: NVs mainly used the clathrin-dependent pathway and macropinocytosis, while HEVs relied on the lipid raft-mediated pathway, clathrin-dependent pathway and caveolin-related endocytosis Figure 2 A,B).

[0097] Studies have shown that endothelial cell extracellular vesicles (EVs) inherit the homing specificity of the parent cell, thus enabling targeting of endothelial tissues. This homing ability is mediated by the CXCR4 membrane protein, which is maintained on the surface of the vesicles during extrusion. To assess the endothelial targeting of EV components in HEVs, a comparative study was conducted using two hybrid vesicles: hybrid EVs / NVs from 293T cells (293T-HEVs) and EV / NV fusion vesicles (HEVs). The CXCR4-mediated targeting mechanism was then verified by antibody neutralization experiments, in which HEVs were pre-incubated with a CXCR4-specific antibody or an isotype-matched IgG control. Quantitative assessment by flow cytometry combined with fluorescence microscopy evaluation (Figure 2A-C) showed that endothelial cell uptake of HEVs was significantly enhanced relative to 293T-HEVs, and that this uptake-enhancing effect was significantly attenuated following CXCR4 blockade, thus confirming the key role of EVs-associated CXCR4 in targeted delivery.

[0098] To further validate the endothelial targeting of EVs, confocal microscopy experiments using CFSE-labeled ECs and DAPI-stained HDFs or HaCaTs showed that, in a co-culture model, ECs preferentially took up vesicles (Fig. 2D). Confocal experiments showed that HDFs or HaCaTs took up very few EVs, confirming endothelial targeting (Fig. 2D). CXCR4 neutralizing antibody pretreatment reduced EC uptake of EVs, linking this specificity to CXCR4-mediated interactions (Fig. 2H). Flow cytometry quantitative experiments confirmed the observations: in contrast, ECs took up more EVs than HDFs or HaCaTs (Fig. 2I, J). This stark contrast emphasizes the dependence of ECs on the CXCR4 mechanism for EV uptake. The above studies establish that EC-EVs home to endothelial cells via CXCR4, laying the foundation for their therapeutic application in vascular repair. Figure 2 I,J}). This stark contrast emphasizes the dependence of ECs on the CXCR4 mechanism for EV uptake. The above studies establish that EC-EVs home to endothelial cells via CXCR4, laying the foundation for their therapeutic application in vascular repair.

[0099] DFO@HEVs promote macrophage efferocytosis

[0100] Macrophage efferocytosis, the clearance of apoptotic cells (ACs), is a fundamental biological process that maintains tissue homeostasis and promotes wound repair by avoiding pro-inflammatory, secondary necrosis. This process is driven by the secretion of anti-inflammatory factors such as TGF-β and IL-10, which suppress inflammation and promote tissue resolution. Crucially, these factors build an exocytosis-resolution positive feedback loop: they enhance the ability of macrophages to continuously phagocytose multiple ACs, thus promoting anti-inflammatory signaling pathways and accelerating tissue repair. In chronic wounds, efferocytosis defects allow inflammation to persist and impede healing, and intervening in this cycle is a promising therapeutic strategy to restore macrophage function and address pathological microenvironments.

[0101] To assess the effect of this process in promoting therapy, a model of co-culture of CFSE-labeled apoptotic Jurkat cells with conditioned macrophages was established Figure 8 A). Confocal imaging revealed that DFO@HEV group macrophages phagocytosed more fluorescently labeled apoptotic cells than control or damage model groups, confirming their enhanced phagocytic capacity. While EVs, NVs, and HEVs also slightly promoted macrophage phagocytic capacity, flow cytometry confirmed that DFO@HEVs were more efficient in promoting macrophage clearance of apoptotic cells Figure 8 B-D).

[0102] Mechanistic studies focused on phosphatidylserine (PS) surface exposure, a key "eat-me" signal for phagocytosis. Annexin V-FITC labeling showed minimal PS expression on HUVECs not treated with vesicles, while HEVs and DFO@HEVs expressed PS levels comparable to apoptotic neutrophil vesicles (positive control) Figure 8 D, E). The observed increase in PS exposure on EVs can be due to membrane integrity disruption during the extrusion process, which induces random redistribution of inner and outer membrane components. This phenomenon is consistent with previous studies showing that extruded EV membranes have a random orientation, with inner and outer surfaces being non-specifically remodeled. This enrichment in PS is directly linked to the enhanced phagocytic capacity, suggesting that vesicle-associated PS promotes macrophage recognition and phagocytosis of apoptotic cells.

[0103] DFO@HEVs accelerate wound healing in vivo in diabetes

[0104] By integrating a comprehensive analysis of gross morphology, histopathology, and molecular assessments, we systematically validated the therapeutic advantage of DFO@HEVs in diabetic wound repair. Serial wound imaging revealed that DFO@HEVs achieved the most pronounced acceleration of wound closure in all experimental groups, with almost complete re-epithelialization observed in diabetic mice by day 12 post-injury Figure 9 A). Quantitative analysis of wound margins Figure 9 B) and statistical comparisons Figure 9 C-E) further confirmed the exceptional healing kinetics of DFO@HEVs, exhibiting a significantly faster rate of contraction than other vesicle formulations.

[0105] Histopathological assessments highlighted the superior regenerative capacity of DFO@HEVs. Hematoxylin and eosin (H&E) stained sections Figure 9 F, G) showed complete epithelial repair and minimal inflammatory infiltration unique to the DFO-HEV group, while Masson's trichrome staining Figure 9H, I) show more organized collagen deposition and mature connective tissue structure in DFO-HEV treated wounds compared to other groups, reflecting advanced extracellular matrix remodeling. To assess the pro-angiogenic role of DFO@HEVs in diabetic wounds, immunofluorescence staining for CD31 and a-SMA was performed. The results show increased neovascularization and mature vessel density in all extracellular vesicle treated groups, with the DFO@HEVs group showing the most significant vascular regenerative capacity.

[0106] On the basis of these findings, we further validated the antioxidant, ferroptosis and anti-inflammatory effects observed in vitro through in vivo experiments. Dihydroethidium (DHE) staining at day 12 showed a significant reduction in reactive oxygen species (ROS) levels in DFO@HEVs treated wounds, consistent with in vitro data. GPX4 immunofluorescence confirmed enhanced ferroptosis resistance in the DFO@HEVs group.

[0107] Flow cytometry and immunofluorescence analysis of wound tissue showed a significant reduction in neutrophil infiltration (CD11b+ / Ly6G+ cells) following treatment with NVs, HEVs and DFO@HEVs. While EVs showed a modest impact that can be attributed to local oxidative stress alleviation, similar observations were made by Ly6G immunofluorescence.

[0108] Finally, macrophage polarization kinetics were assessed. Immunofluorescence showed reduced iNOS expression (M1 marker) and increased Arg-1 levels (M2 marker) in the vesicle treated groups, with DFO@HEVs exhibiting superior M2 polarization efficacy, consistent with in vitro results. Overall, in vivo results indicate that DFO@HEVs accelerate diabetic wound healing by reducing neutrophil infiltration (CD11b+ / Ly6G+ cells) and promoting macrophage polarization towards a pro-repair M2 phenotype (increased Arg-1, decreased iNOS), consistent with improved vascular regeneration and inflammation resolution observed in treated wounds.

[0109] CONCLUSIONS

[0110] A biohybrid nanovesicle platform (DFO@HEVs) was developed by combining endothelial- and neutrophil-derived vesicles with deferoxamine to address diabetic wound healing. This dual-targeting system utilizes CXCR4-mediated endothelial cell homing and β2 integrin-dependent inflammatory chemotaxis to precisely deliver DFO to the wound site. DFO@HEVs restore vasoregeneration through HIF-1α / VEGF activation, inhibit ferroptosis through Nrf2 / GPX4 signaling, and reprogram macrophages to a pro-repair M2 phenotype, effectively breaking the oxidative stress-inflammation-ferroptosis cycle. In vivo, DFO@HEVs accelerate wound closure, reduce neutrophil infiltration, and promote collagen remodeling, demonstrating their therapeutic application prospects. This drug delivery platform integrates endothelial repair, antioxidant defense, and immune modulation, providing a new strategy for diabetic wound repair and paving the way for the next generation of nanotherapies for diabetic chronic wounds.

[0111] It is apparent that the present application is not limited to the details of the foregoing exemplary embodiments, and thus modifications and variations can be made in light of the above teachings or can be acquired from practice of the application. Therefore, it is to be understood that all such modifications and variations that fall within the scope of the claims are to be embraced by the scope of the application. Moreover, it is to be understood that the specific order or hierarchy of steps in the processes disclosed is an illustration of exemplary processes. Based upon the disclosure and teachings of this specification, a person of ordinary skill in the art will appreciate that the specific sequence in which steps are presented is illustrative only and is not a limitation of the present application. Accordingly, the present application is not to be limited as to just the foregoing examples, but is intended to embrace any and all applications within the scope of the following claims.

Claims

1. Use of a hybrid extracellular vesicle in the preparation of a medicament for the treatment of a diabetic wound, characterized in that, The preparation method of the hybrid extracellular vesicles comprises the following steps: S1, preparing endothelial cell nanovesicles EVs: HUVECs cells were cultured with high glucose DMEM complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, TrypLE™ Express enzyme was used to dissociate the cultured HUVEC, and diluted to 5 x 10 6 cells / mL in PBS, then the EVs suspension was extruded by extrusion method; S2, preparing neutrophil nanovesicles NVs: Human HL-60 cells and human Jurkat clone E6 cells were differentiated into neutrophils by 1 μm all-trans retinoic acid ATRA and 1.25% dimethyl sulfoxide DMSO for 5 days, the cultured neutrophils were dissociated using TrypLE™ Express enzyme and diluted to 5 x 10 6 cells / mL in PBS, after which the NVs suspension was extruded by the extrusion method; S3, mixing the EV suspension obtained in S1 and the NV suspension obtained in S2 according to a weight ratio of 1:1, performing ultrasonic treatment on an ice water bath by using an ultrasonic instrument at a strength of 30%, turning on for 30 s and turning off for 2 min, and after 4-6 cycles, extruding the hybrid extracellular vesicle HEVs suspension through a 0.2 μm polycarbonate membrane; S4, loading deferoxamine DFO into the HEVs by ultrasonic perforation to obtain DFO@HEVs; The extrusion method in the S1 and S2 is: using a micro-extruder to extrude the cell suspension through a series of Nuclepore™ polycarbonate membranes, and the pore sizes of the series of Nuclepore™ polycarbonate membranes are 10 μm, 5 μm, 1 μm, 0.4 μm and 0.2 μm in turn; Before the S4, performing ultracentrifugation on the hybrid extracellular vesicle suspension obtained in the S3, centrifuging the hybrid extracellular vesicle suspension at 3000xg and 10000xg at 4°C in turn for 30 min, removing the debris in the hybrid extracellular vesicle suspension, and then centrifuging the supernatant at 140000xg at 4°C for 70 min, collecting the nanoscale vesicles, resuspending the nanoscale vesicles in sterile PBS after washing twice, and storing at -80°C.

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