Method for preparing crgd-targeted peptide-modified endothelial progenitor cell-derived biomimetic nanovesicles

Abstract

This invention relates to the field of biomaterials technology, and more particularly to a method for preparing cRGD-targeted peptide-modified biomimetic nanovesicles derived from endothelial progenitor cells. The preparation method includes the following steps: S1, preparing EPC-NVs; S11, isolating, culturing, and identifying EPCs; S12, preparing EPCs-NVs; S13, modifying and identifying EPC-NVs with cRGD; S2, preparing ADM-Fe 3+ @PA / Ge lma hydrogel: S21, Fe synthesized in an environment with a pH of 10 at a molar ratio of 1:3. 3+ @Original catechin aldehyde solution; S22, Fe from the combined vehicle 3+ @The original catechuic aldehyde solution was mixed with ADM, and after multiple centrifugal washings, the prepared photocurable gelatin (Ge lmA) precursor solution was added; S23, Fe was carried out by ultrasonic cavitation. 3+ @PA's ADM is uniformly dispersed; S24, a double cross-linked hydrogel is formed by irradiation with 416nm blue light and self-assembly of ADM. This method provides a novel biodelivery system with pro-angiogenic, antioxidant, and antibacterial properties, improving the wound treatment effect for diabetes.

Description

Preparation method of cRGD-targeted peptide-modified endothelial progenitor cell-derived biomimetic nanovesicles Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a method for preparing biomimetic nanovesicles derived from endothelial progenitor cells modified with cRGD-targeting peptides. Background Technology

[0002] Angiogenesis is an essential step in wound healing. However, endothelial cell dysfunction in diabetic wounds, leading to reduced angiogenesis, is a key factor in their non-healing. Endothelial progenitor cells (EPCs), also known as endothelial precursor cells, possess strong paracrine capabilities. Previously, it was thought that EPCs primarily regulated endothelial cell function by secreting chemokines and cytokines. Recent studies have found that EPCs can also improve endothelial cell function and accelerate angiogenesis in diabetic wounds by secreting nanovesicles, potentially serving as a means of repairing diabetic wounds.

[0003] However, these naturally occurring exosomes secreted by cells have significant drawbacks in their translational applications. First, the yield of most natural exosomes is extremely low and unstable; it has been reported that ultracentrifugation strategies can only yield 1-10 μg of nanovesicles per milliliter of cell supernatant. Second, the isolation and purification of natural exosomes are cumbersome, costly, and time-consuming. Third, the performance of natural exosomes is unstable; the phenotype and function of nanovesicles secreted by parental cells may change significantly after multiple passages, reducing their therapeutic potential. Therefore, solutions are urgently needed.

[0004] The above content is only used to help understand the technical solution of the present invention and does not represent an admission that the above content is the closest prior art. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing cRGD-targeted peptide-modified endothelial progenitor cell-derived biomimetic nanovesicles. This method provides a novel biodelivery system with pro-angiogenic, antioxidant and antibacterial properties, which improves the wound treatment effect of diabetes.

[0006] To achieve the aforementioned objective, the technical solution of the present invention is implemented as follows: a method for preparing cRGD-targeted peptide-modified endothelial progenitor cell-derived biomimetic nanovesicles, the preparation method comprising the following steps:

[0007] S1. Preparation of EPC-NV;

[0008] S11. Perform EPCs cell isolation, culture, and identification.

[0009] S12, Prepare EPCs-NVs.

[0010] S13. Perform cRGD modification and identification of EPC-NVs;

[0011] S2, Preparation of ADM-Fe 3+ @PA / Gelma hydrogel:

[0012] S21. Synthesize Fe in a 1:3 molar ratio in an environment with a pH of 10. 3+ @Protocatechualdehyde (PA) solution;

[0013] S22, the synthesized Fe 3+ @The catechin solution was mixed with ADM and centrifuged and washed multiple times before adding the prepared photocurable gelatin (GelmA) precursor solution;

[0014] S23, Using ultrasonic cavitation to propel Fe 3+ @PA's ADM is uniformly dispersed;

[0015] S24, a double crosslinked hydrogel is formed by irradiation with 416nm blue light and self-assembly of ADM.

[0016] Preferably, the EPCs cell isolation, culture and identification in step S11 includes isolating EPCs from rat bone marrow cell suspension, performing primary cell culture and passage.

[0017] Preferably, the process of preparing EPCs-NVs in step S12 includes:

[0018] S12a. Primary EPCs were cultured in a medium containing exosome-free serum, and the cell supernatant was collected. Natural exosomes of the EPCs were extracted by ultracentrifugation.

[0019] S12b. Pass the EPCs suspension of 5-10 million / mL sequentially through the 10μm, 5μm, and 1μm pore membranes of the squeezer to remove cell debris and obtain cell vesicle suspension. Then, centrifuge the suspension at high speed to obtain EPC-NVs.

[0020] Preferably, the process of cRGD modification of EPC-NVs in S13 includes:

[0021] S13a. The DeEPC-NVs suspension was co-incubated with cRGD micelle solution, and the cRGD-modified EPC-NVs were purified by size exclusion chromatography to obtain cRGD@DeEPC-NVs;

[0022] Identification of S13b.cRGD@DeEPC-NVs: Identification and comparison of EPCs natural exosomes and EPC-NVs: Transmission electron microscopy (TEM) was used to observe the morphology and size of the two types of vesicles; nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) were used to detect the particle size and distribution of the two types of vesicles; Western blotting was used to detect the specific membrane proteins of the two types of vesicles, including CD9, CD63 and CD81; and zeta potential was used to detect the membrane potential.

[0023] The beneficial effects of this invention are reflected in:

[0024] (1) The biomimetic nanovesicles derived from endothelial progenitor cells prepared by the present invention through extrusion can be used as EV mimics to deliver EPC contents to the wound. Furthermore, the present invention couples cRGD targeting peptides to the surface of EPC-NVs, enabling active targeting of endothelial cells (ECs). In addition, a dual hydrogel network is provided, incorporating Fe... 3+ @PA complex-modified ADM was combined with photocurable gelatin (GelMA) to enrich and sustain the release of mEPC-NV. This hydrogel network possesses antioxidant and antibacterial properties, reducing reactive oxygen species levels and inhibiting bacterial infection in diabetic wounds, thus compensating for the functional deficiencies of EPC-NV.

[0025] (2) Based on the characteristics of low cell uptake capacity and complex types in diabetic wounds, this invention constructs a customized mEPC-NV with enhanced uptake and enhanced targeting, and for the first time applies cRGD targeting peptides to diabetic wounds, thereby improving the treatment effect.

[0026] (3) This invention constructs ADM-Fe 3+- The mNV@PA / Gelma targeted delivery system, used in conjunction with other treatments, enhances the therapeutic effect on diabetic wounds.

[0027] (4) The present invention uses an extrusion method to obtain vesicle particles or protein yields that are 5-100 times higher than those of natural exosomes under the same cell quantity conditions, and avoids cumbersome steps such as cell expansion and supernatant concentration. Secondly, due to the significant increase in yield, obtaining the same amount of nanovesicles does not require multiple passages of parental cells, thus ensuring the performance stability of biomimetic nanovesicles. Attached Figure Description

[0028] Figure 1 shows the synthesis and characterization of EPC-NV and mEPC-NV;

[0029] Figure 2 shows the EC function promoted by EPC-NV under oxidative stress.

[0030] Figure 3 shows the functional diagram of cRGD-modified EPC-NV achieving targeted EC;

[0031] Figure 4 shows the function diagram of mEPC-NV in restoring EC;

[0032] Figure 5 shows the fabrication and characterization of AFG-mNV hydrogel;

[0033] Figure 6 shows the in vitro verification of the antibacterial and antioxidant capabilities of AFG hydrogel;

[0034] Figure 7 shows how AFG-mNV hydrogel accelerates the healing of diabetic wounds.

[0035] Figure 8 is a statistical analysis chart of healed diabetic wounds;

[0036] Figure 9 shows the in vivo validation of the ROS scavenging and EC targeting capabilities of AFG-mNV hydrogel;

[0037] Figure 10 is a schematic diagram of the process of this invention. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1

[0040] As shown in Figure 10:

[0041] A method for preparing cRGD-targeted peptide-modified endothelial progenitor cell-derived biomimetic nanovesicles includes the following steps:

[0042] S1. Preparation of EPC-NV:

[0043] S11. Perform EPCs cell isolation, culture, and identification;

[0044] The EPCs isolation, culture, and identification in step S11 includes isolating EPCs from rat bone marrow cell suspension, performing primary cell culture, and passage.

[0045] S12, Prepare EPCs-NVs.

[0046] The preparation process of EPCs-NVs in step S12 includes:

[0047] S12a. Primary EPCs were cultured in a medium containing exosome-free serum, and the cell supernatant was collected. Natural exosomes of the EPCs were extracted by ultracentrifugation.

[0048] S12b. Pass the EPCs suspension of 5-10 million / mL sequentially through the 10μm, 5μm, and 1μm pore membranes of the squeezer to remove cell debris and obtain cell vesicle suspension. Then, centrifuge the suspension at high speed to obtain EPC-NVs.

[0049] S13. Perform cRGD modification and identification of EPC-NVs;

[0050] The process of cRGD modification of EPC-NVs in S13 includes:

[0051] S13a. The DeEPC-NVs suspension was co-incubated with cRGD micelle solution, and the cRGD-modified EPC-NVs were purified by size exclusion chromatography to obtain cRGD@DeEPC-NVs;

[0052] Identification of S13b.cRGD@DeEPC-NVs: Identification and comparison of natural exosomes of EPCs and EPC-NVs: Transmission electron microscopy (TEM) was used to observe the morphology and size of the two types of vesicles; nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) were used to detect the particle size and distribution of the two types of vesicles; Western blotting was used to detect the specific membrane proteins of the two types of vesicles, including CD9, CD63 and CD81; and zeta potential was used to detect the membrane potential.

[0053] S2, Preparation of ADM-Fe 3+ @PA / Gelma hydrogel:

[0054] S21. Synthesize Fe in a 1:3 molar ratio in an environment with a pH of 10. 3+ @Protocatechualdehyde (PA) solution,

[0055] S22, the synthesized Fe 3+ @The protocatechuic aldehyde solution was mixed with ADM, and after multiple centrifugation and washing, the prepared photocurable gelatin (GelmA) precursor solution was added.

[0056] S23, Using ultrasonic cavitation to propel Fe 3+ @PA's ADM is uniformly dispersed.

[0057] S24, a double crosslinked hydrogel is formed by irradiation with 416nm blue light and self-assembly of ADM.

[0058] Example 2

[0059] Preparation of ADM-Fe 3+ The detailed process for @PA / GelMA hydrogel includes:

[0060] 1) Irradiate the clean bench with ultraviolet light for 30 minutes;

[0061] 2) Weigh 3g of PA and 2g of ferric chloride at a molar ratio of 3:1, pour them into a 50mL centrifuge tube, add 40mL of double-distilled water, and mix well; adjust the pH value to 10 with NaOH solution;

[0062] 3) Pipette 1 ml of the above solution into a 2 ml centrifuge tube, weigh 0.1 g of ADM powder using an analytical balance and add it to the solution. Use an ultrasonic shaker to sonicate for 2 min (power: 30%, working for 2 s, stopping for 2 s).

[0063] 4) Centrifuge the above dispersion solution in a centrifuge (5000 rpm, 5 min), discard the supernatant, add 1 mL of double-distilled water, and sonicate for 2 min using an ultrasonic oscillator (power: 30%, working for 2 s, stopping for 2 s).

[0064] 5) Repeat step (4) 3 times, discard the supernatant, and remove residual Fe. 3+ @PA;

[0065] 6) Prepare the photoinitiator according to the GelMA kit. Weigh 1g of GelMA solid and put it into a 50 mL centrifuge tube. Add 10mL of the photoinitiator prepared according to the kit instructions and heat in a 70 ℃ water bath. Shake continuously to mix. After the solution is clear, filter the solution through a 0.22μm pore size filter.

[0066] 7) Add 1 ml of GelMA solution to the ADM-Fe obtained in step (5). 3+ In PA solid, ultrasonic vibration for 2 minutes (power: 30%, 2 seconds on, 2 seconds off) is used, followed by irradiation with 416nm wavelength blue light for about 10 seconds to form a gel.

[0067] Example 3

[0068] The principle behind cRGD modification of biomimetic nanovesicles to enhance endothelial cell targeting:

[0069] Biomimetic nanovesicles effectively address the shortcomings of pre-native exosomes, namely low yield, cumbersome isolation and purification procedures, and unstable functional phenotypic performance. Biomimetic nanovesicles are simple to synthesize; after rupturing parental cells using various strategies such as freeze-thaw cycles, extrusion, ultrasound, and microfluidics, cell membrane fragments self-assemble in solution to form nanovesicles. Inheriting the cell membrane structure and endogenous therapeutic molecules of parental cells, biomimetic nanovesicles are considered to possess low immunogenicity and biological activity similar to natural exosomes. Furthermore, compared to natural exosomes, biomimetic nanovesicles offer several advantages in translational applications. First, biomimetic nanovesicles exhibit significantly higher yields and simpler, faster collection and purification procedures than natural nanovesicles. Extrusion methods can achieve 5-100 times the yield of vesicle particles or proteins compared to natural exosomes under the same cell volume conditions, avoiding cumbersome cell expansion and supernatant concentration steps. Second, due to the significantly increased yield, obtaining the same amount of nanovesicles does not require multiple passages of parental cells, ensuring the performance stability of biomimetic nanovesicles.

[0070] ① Hypoxia at the wound site stimulates fibroblasts, macrophages, and endothelial cells to release VEGF and SDF-1α; ② VEGF induces the activation and phosphorylation of eNOS in the bone marrow, and SDF-1α and H2S can bind to CXCR4 to activate the PI3K / Akt / eNOS pathway. Both of these mechanisms can lead to increased NO levels in the bone marrow cavity, thereby increasing sKitL levels and causing c-kit+ EPCs to migrate from the bone marrow cavity to the peripheral blood; ③ EPCs migrating to the peripheral blood can relocate to the injury site under the action of the chemokine SDF-1α; ④ EPCs adhere to activated endothelial cells and their extracellular matrix; ⑤ EPCs proliferate and differentiate into mature endothelial cells at the ischemic site.

[0071] This invention reveals that EPC-NVs can be taken up not only by wound endothelial cells but also by fibroblasts and keratinocytes, but they do not promote the function of fibroblasts and keratinocytes. This will significantly reduce the effectiveness of EPC-NVs when applied to wounds.

[0072] Therefore, this invention considers modifying the extruded EPC-NVs to enhance their targeting and promote greater uptake of EPC-NVs by endothelial cells.

[0073] This invention has discovered that the small molecule polypeptide with the arginine-glycine-aspartic acid sequence (RGD polypeptide) can specifically bind to the avB3 receptor on the endothelial cell membrane. Furthermore, compared to linear RGD polypeptides, cyclic RGD polypeptides (cRGD) exhibit stronger specificity and affinity for avB3 and are more stable in vivo. Therefore, coupling cRGD to the surface of nanovesicles can achieve active targeting of endothelial cells by the nanovesicles.

[0074] This invention utilizes click chemistry to successfully couple cRGD to the surface of EPC-NVs membranes, and demonstrates through uptake experiments and flow cytometry that cRGD-modified EPC-NVs can be taken up by endothelial cells more efficiently and better promote endothelial cell function.

[0075] Example 4

[0076] Results Analysis

[0077] 1.1 Manufacturing and Characterization of EPC-NV and mEPC-NV

[0078] As described above, NVs from EPC were separated by extrusion and ultracentrifugation. Electron microscopy images showed that the NVs were spherical and double-membrane shaped, with a diameter of approximately 100 nm (Figure 1B). Microflow cytometry results further confirmed the size of the NVs. The diameter of EPC-NVs was approximately 54.8 ± 7.9 nm, while the diameter of mEPC-NVs was slightly larger, approximately 67.8 ± 23.8 nm (Figures 1D, 1F). The zeta potentials of EPC-NVs and mEPC-NVs were -15 ± 0.9 mV and -20 ± 0.6 mV, respectively (Figure 1E). Western blotting analysis showed that EV-specific markers were highly expressed in both types of NVs, including CD9, CD81, and TSG101 (Figure 1C). Overall, the results of this invention indicate that mEPC-NVs have similar characteristics to EPC-NVs and meet the criteria for extracellular vesicles.

[0079] Related results

[0080] 3.1 Synthesis and Characterization of EPC-NV and mEPC-NV

[0081] As described above, NVs from EPC were separated by extrusion and ultracentrifugation. Electron microscopy images showed that the NVs were spherical and double-membrane shaped, with a diameter of approximately 100 nm (Fig. 1B). Microflow cytometry results further confirmed the size of the NVs. The diameter of EPC-NVs was approximately 54.8 ± 7.9 nm, while the diameter of mEPC-NVs was slightly larger, approximately 67.8 ± 23.8 nm (Fig. 1D, 1F). The zeta potentials of EPC-NVs and mEPC-NVs were -15 ± 0.9 mV and -20 ± 0.6 mV, respectively (Fig. 1E). Western blotting analysis showed that EV-specific markers were highly expressed in both types of NVs, including CD9, CD81, and TSG101 (Fig. 1C). Overall, the results of this invention indicate that the characteristics of mEPC-NVs are similar to those of EPC-NVs, meeting the criteria for extracellular vesicles.

[0082] 3.2 EPC-NV can promote EC function under oxidative stress

[0083] The internalization of EPC-NVs was assessed by incubating 20 μg of DiI-labeled EPC-NVs with human venous endothelial cells (HUVECs), human dermal fibroblast cells (HDFs), and human immortalized epidermal cells (HaCaTs), respectively.

[0084] Red fluorescent staining was observed in the cytoplasm of all cell types using laser scanning confocal microscopy, indicating that EPC-NVs were stably phagocytosed by these cells (Fig. 2A). Previous studies have shown that EPC-NVs promote proliferation and migration, enhancing the angiogenesis capacity of HUVECs under oxidative stress (47-49). In this study, HUVEC, HDF, and HaCaT cells were treated with EPC-NVs under MGO and H2O2-induced oxidative stress conditions, respectively. Control groups were treated with PBS. Cell behavior was then assessed using EdU assays, transwell assays, and in vitro tube formation assays. The results showed that, compared to the control group, EPC-NVs in HUVEC cells partially restored their proliferation, migration, and angiogenesis capacity under oxidative stress (Figs. 2B-D, 2I-K, Fig. S1). The present invention also observed that EPC-NVs failed to restore the proliferation and migration functions of HDF (Fig. 2E, F and 2L, M, Fig. S2) and HaCaT cells (Fig. 2G, H and 2N, O, Fig. S3) under oxidative stress.

[0085] These results indicate that EPC-NVs can be internalized by HUVECs, HDF, and HaCaT, but only promote HUVEC function under oxidative stress.

[0086] 3.3 cRGD-modified EPC-NV achieves EC targeting in vitro

[0087] Next, this invention considered whether modifying the surface of EPC-NVs with cRGD-targeting peptides could help avoid loss due to uptake by other cells, thereby maximizing the therapeutic function of EPC-NVs. Miniature flow cytometry analysis showed that 76.1% of the cRGD peptides bound to NVs (Figure 3G), indicating that most of the cRGD peptides successfully attached to EPC-NVs. To assess the intracellular uptake of mEPC-NVs, DiI-labeled NVs and DiI-labeled mNVs (red) were co-incubated with HUVECs for 12 hours and examined under a laser confocal microscope. Some red fluorescence was observed in the HUVECs of the EPC-NV group, while in the mEPC-NV group, this red fluorescence was clearly distributed next to the cell nucleus (blue, DAPI staining) (Figure 3A). The cellular uptake rate in the mEPC-NV group was 82.03%, while it was only 26.03% in the EPC-NV group (Figure 3B). This result indicates that the cRGD peptide helps enhance cellular uptake of mEPC-NV in vitro, a finding confirmed by flow cytometry (Figure 3E, F). The mean red fluorescence intensity (MFI) of the mEPC-NV group was significantly higher than that of the EPC-NV group.

[0088] To further validate the targeted delivery of mEPC-NV in diabetic wounds, an in vitro skin cell model was established. HUVECs were distinguished from spindle-shaped or fused HDF and HaCaT cells using green fluorescent protein (GFP) fluorescence. Subsequently, 293T cell-derived NVs (293T-NV) were prepared as a negative control using a pre-defined protocol. 293T-NV, EPC-NV, and mEPC-NV were labeled with DiI dye. Laser confocal microscopy imaging was used to evaluate the internalization of these different nanovesicles into GFP-labeled HUVECs (HUVECGFP). As shown in Figures 3C and D, mEPC-NVs were more likely to be internalized by ECs compared to other typical skin cells (HDF and HaCaT), demonstrating the targeting ability of cRGD.

[0089] These data confirm that mEPC-NV exhibits excellent endothelial cell EC targeting ability, which is attributed to the presence of membrane cRGD peptide.

[0090] 3.4 mEPC-NV restores EC function

[0091] To investigate whether mEPC-NVs could restore the proliferation, migration, and tube formation function of HUVECs in diabetic wounds, HUVECs were cultured under methylglyoxal (MGO) and H2O2-induced oxidative stress. First, the proliferation of HUVECs was assessed, and it was noted that EdU-positive cell nuclei were significantly reduced after PBS treatment under oxidative stress, a trend partially offset by EPC-NVs. Notably, the number of EdU-positive cell nuclei significantly increased under oxidative stress after pretreatment with mEPC-NVs (Fig. 4A, D). Subsequently, transwell assays were performed to further confirm the effect of mEPC-NVs on EC migration. As shown in Fig. 4B, E, cell migration was significantly enhanced after 24 hours of mEPC-NV exposure. Furthermore, tube formation assays showed that under oxidative stress, the tubular structure of the mEPC-NV group was improved, with increased tube formation and junctional point numbers, exceeding those of the PBS and EPC-NV groups (Fig. 1, Fig. 4C, F). These results indicate that mEPC-NVs restored the proliferation, migration, and tube formation functions of ECs under oxidative stress, which may be due to the higher degree of aggregation of functional mEPC-NVs near ECs.

[0092] 3.5 Preparation and Characterization of AFG-mNV Hydrogel

[0093] The hydrogel design of this invention aims to preserve the natural three-dimensional extracellular matrix structure and activity of internal bioactive factors of ADM, as well as its antioxidant and antibacterial properties, and to sustainably release mNVs for diabetic wound repair. The AFG-mNV hydrogel was synthesized at room temperature. Figure 5B shows the morphology of GelMA and the AFG-mNV hydrogel. Figure 5C shows the porous structure of the AFG-mNV hydrogel visualized by scanning electron microscopy (SEM). As shown in Figure 5D, mEPC-NVs are uniformly distributed within the hydrogel. This invention also tested the degradation rate of the AFG hydrogel, which exceeded 70% within 10 days, achieving the expected target. Furthermore, hemolysis tests showed a degree of hemolysis similar to that of blood incubated with PBS, with a low erythrocyte rupture rate (<5%) (Figure 5F, G). Rheological measurements were used to evaluate the mechanical properties of the hydrogel. The relationship between the storage modulus (G′) and loss modulus (G″) of the AFG hydrogel was examined using a rheometer (Figure 5H). The results showed that the storage modulus of the AFG hydrogel exceeded its loss modulus, indicating sufficient mechanical strength. To confirm the sustainable release of mNVs from the AFG-mNV hydrogel, the daily mNV release was quantified using the microBCA microprotein assay. Approximately 50% of the mNVs in the AFG-mNV hydrogel were released within 5 days, and nearly 80% were released within 14 days, indicating rapid release of mNVs from the hydrogel (Figure 5I). These findings collectively confirm the successful synthesis of the AFG hydrogel loaded with mEPC-NVs.

[0094] 3.6 Biocompatibility of AFG hydrogel

[0095] The application of medical biomaterials largely depends on excellent biocompatibility. Therefore, this invention investigated whether the AFG hydrogel of this invention exhibited any cytotoxic effects on HUVEC, HDF, and HaCaT cells using CCK-8 assays and LIVE / DEAD staining. After incubation with the AFG hydrogel, this invention found no cytotoxicity in any cell type, with cell viability remaining above 90% (Figures 5A and 5C). Live and dead cells were distinguished using calcein-AM (green) and propidium iodide (red) staining agents. Furthermore, to ensure no cytotoxic effects during hydrogel degradation, the biocompatibility of the AFG hydrogel degradation products was evaluated. As shown in Figures 5B and 5D, AFG hydrogel degradation was non-toxic and promoted the proliferation of HUVEC, HDF, and HaCaT cells. Notably, degradation of both the AFG hydrogel and cells cultured in the hydrogel improved the survival rate of HUVEC, HDF, and HaCaT cells, as measured by the CCK-8 assay (Figures 5S, 5E, and 5F). These findings collectively highlight the excellent biocompatibility of the AFG hydrogel of this invention.

[0096] 3.7 Antibacterial and antioxidant capabilities of AFG hydrogel in vitro

[0097] After incubating bacteria with AFG hydrogel for 4 hours, colony formation assays showed that the AFG hydrogel group significantly reduced the activity of Staphylococcus aureus and Escherichia coli (Figure 6A). Specifically, the AFG hydrogel showed inhibition rates of 70.7% and 40.5% against Staphylococcus aureus and Escherichia coli, respectively (Figure 6B). The bacterial growth was further investigated after 24 hours. In the AFG hydrogel group, the absorbance curve of the bacterial suspension showed a significant decrease in peak size and a rightward shift (Figure 6C). Furthermore, bacterial inhibition ring formation assays were performed, showing no bacterial growth in the ring region, with the diameter of the inhibition ring approximately 60% of that of the Escherichia coli positive control and 50% of that of Staphylococcus aureus (Figures 6D and 6E). These results indicate that the AFG hydrogel possesses sufficient antibacterial properties. Figures 6F and G show that, compared with the MGO group, the ROS abundance in HUVEC, HDF, and HaCaT cells was significantly reduced under the AFG hydrogel effect (P<0.001), indicating that the anti-ROS properties of AFG hydrogel may contribute to its ability to promote healing in diabetic wounds. These results validate in vitro that AFG hydrogel has good antibacterial and ROS-reducing capabilities in diabetic wounds.

[0098] 3.8 AFG-mNV hydrogel accelerates diabetic wound healing in vivo.

[0099] To further validate the clinical potential of AFG-mNV hydrogel, a full-thickness wound model was established on the backs of normal and diabetic mice, and they were divided into 6 groups: untreated normal mice served as the control group, while the remaining 5 groups of diabetic mice were treated with PBS, AFG, NV, mNV, and AFG-mNV, respectively (Figure 7A). As shown in Figures 7B-E, the wounds of normal mice and AFG-mNV mice healed completely on day 12 postoperatively, while the wounds of diabetic mice treated with PBS showed delayed healing, far from closure. Notably, on days 4, 8, and 12 post-injury, mice treated with AFG-mNV showed the lowest wound non-closure rate among all treatment groups, approaching the level observed in the normal group.

[0100] Next, wound sections from all groups were dissected on postoperative day 12 for downstream histological analysis. H&E and Masson staining analysis showed that, compared with the PBS group, AFG, NV, mNV, and AFG-mNV reduced granulation tissue length and accelerated collagen deposition in diabetic wounds. The reepithelialization rate and collagen deposition rate were significantly higher in the mNV and AFG-mNV groups, with the AFG-mNV group showing lower rates than both the mNV and AFG-mNV groups (Figures 8A-D). Furthermore, wounds treated with AFG-mNV exhibited collagen arrangement very similar to normal wound tissue.

[0101] To detect and quantify angiogenesis in the wound bed of different groups, immunofluorescence staining analysis of α-SMA and immunohistochemical staining of CD31 were performed. These analyses highlighted that the AFG-mNV hydrogel group exhibited the highest α-SMA fluorescence intensity and the highest vascular density, even approaching the levels observed in the normal group (Figure 8E-H).

[0102] These findings collectively indicate that ADM-Fe 3+ @PA and mEPC-NV can synergistically promote wound healing in diabetes in vivo. Furthermore, AFG-mNV treatment achieved better wound angiogenesis than mNV in terms of diabetic wound healing.

[0103] 3.9AFG-mNV hydrogel's ability to scavenge ROS and target EC in vivo

[0104] To confirm the potential role of inflammation in diabetic wound healing, immunofluorescence staining of IL-1β was performed. Results showed that the wounds in the PBS and NV groups exhibited significant inflammation compared to the negative control group, while inflammation was downregulated in the AFG and AFG-mNV groups (Fig. 9A, B). ROS levels in the wound were assessed by DHE staining. The red fluorescence signal intensity was significantly reduced in the AFG-mNV group compared to other groups (Fig. 9C, D). These results indicate that AFG-mNV hydrogel can effectively reduce inflammatory responses and oxidative stress.

[0105] The EC-targeting ability and distribution of mNVs in diabetic wounds were further investigated using in vivo immunofluorescence assays. As shown in Figure 9E, compared with DiI-labeled NVs, the DiI-labeled mNV group had a wider distribution in the diabetic wound on day 4 (red signal). Furthermore, this invention noted that mNVs exhibited greater co-localization with blood vessels compared to NVs (Figure 9F). These results indicate that mNVs can effectively adhere to ECs due to the membrane-modified cRGD-targeting peptide.

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing hydrogels containing cRGD-targeting peptide-modified endothelial progenitor cell-derived biomimetic nanovesicles, characterized in that, The preparation method includes the following steps: S1, Preparation of EPC-NV: S11, Isolation, culture, and identification of EPCs cells; S12, Preparation of EPCs-NVs; S13, cRGD modification and identification of EPC-NVs; S2, Preparation of ADM-Fe 3+ @PA / GelmA hydrogel: S21, Fe synthesized in an environment with a pH of 10 at a molar ratio of 1:

3. 3+ @Original catechin solution; S22, synthesized Fe 3+ @The original catechuic aldehyde solution was mixed with ADM, and after multiple centrifugal washings, the prepared GelmA precursor solution was added; S23, Fe was carried out by ultrasonic cavitation. 3+ @PA's ADM is uniformly dispersed; S24, a double crosslinked hydrogel is formed by 416nm blue light irradiation and ADM self-assembly; S3, in ADM-Fe 3+ @PA / GelmA hydrogel was used to synthesize ADM-Fe loaded with EPC-NV. 3+ @PA / GelmA hydrogel.

2. The method for preparing hydrogels containing cRGD-targeting peptide-modified endothelial progenitor cell-derived biomimetic nanovesicles according to claim 1, characterized in that, The EPCs cell isolation, culture, and identification in step S11 includes: isolating EPCs from rat bone marrow cell suspension, performing primary cell culture, and passage.

3. The method for preparing hydrogels containing cRGD-targeting peptide-modified endothelial progenitor cell-derived biomimetic nanovesicles according to claim 2, characterized in that, The preparation process of EPCs-NVs in step S12 includes: S12a. primary EPCs are cultured in a culture medium containing exosome-free serum and the cell supernatant is collected. Natural exosomes of EPCs are extracted by ultracentrifugation; S12b. EPCs suspension of 5-10 million / mL is passed sequentially through 10μm, 5μm, and 1μm pore membranes of a squeezer to remove cell debris and obtain cell vesicle suspension. The suspension is then ultracentrifuged to obtain EPC-NVs.

4. The method for preparing hydrogels containing cRGD-targeting peptide-modified endothelial progenitor cell-derived biomimetic nanovesicles according to claim 3, characterized in that, The process of cRGD modification and identification of EPC-NVs in S13 includes: S13a. Co-incubating the DeEPC-NVs suspension with cRGD micelle solution, purifying the cRGD-modified EPC-NVs by size exclusion chromatography to obtain cRGD@DeEPC-NVs; S13b. Identification of cRGD@DeEPC-NVs: Identification and comparison of EPCs natural exosomes and EPC-NVs: morphology and size of the two vesicles observed by transmission electron microscopy; particle size and distribution of the two vesicles detected by nanoflow cytometry and dynamic light scattering; detection of specific membrane proteins of the two vesicles by Western blotting, including CD9, CD63 and CD81; and detection of membrane potential using zeta potential.

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