Preparation method of cRGD targeting peptide modified endothelial progenitor cell-derived bionic nano-vesicles

Abstract

The invention relates to the technical field of biological materials, in particular to a preparation method of cRGD targeting peptide modified endothelial progenitor cell derived bionic nano-vesicles. The preparation method comprises the following steps: S1, preparing EPC-NV; the method comprises the following steps: S11, carrying out EPCs cell separation, culture and identification, S12, preparing EPCs-NVs, and S13, carrying out cRGD modification and identification on the EPCs-NVs; s2, preparing ADM-Fe < 3 + > PA / Ge lma hydrogel; S21, synthesizing a Fe < 3 + > protocatechualdehyde solution according to a molar ratio of 1: 3 in an environment with a pH value equal to 10; s22, mixing the synthesized Fe < 3 + > at protocatechualdehyde solution with ADM, carrying out centrifugal washing for multiple times, and then adding a prepared photocuring gelatin (Ge lmA) precursor solution; s23, uniformly dispersing ADM carrying Fe < 3 + >-coated PA through ultrasonic cavitation; and S24, carrying out 416 nm blue light illumination and ADM self-assembly so as to form the bi-crosslinked hydrogel. According to the method, a novel biological delivery system with the functions of promoting angiogenesis, resisting oxidation and resisting bacteria is provided, and the wound surface treatment effect of diabetes mellitus is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a method for preparing cRGD targeting peptide-modified endothelial progenitor cell-derived bionic nanovesicles. Background Art

[0002] Angiogenesis is an essential part of wound healing. However, endothelial cell dysfunction in diabetic wounds leads to reduced angiogenesis, which is a key factor in their non-healing. Endothelial progenitor cells (EPCs), also known as endothelial precursor cells, have strong paracrine capacity. Previously, it was believed that EPCs mainly regulate 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, which can be used as a potential means of repairing diabetic wounds.

[0003] However, this type of natural exosomes secreted by cells have obvious defects in translational applications. First, most natural exosomes have extremely low yields and are unstable. It is reported that ultra-high-speed centrifugation-based strategies can only harvest 1-10 μg of nanovesicles from each milliliter of cell supernatant. Second, the separation and purification steps 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 after multiple passages may change significantly, reducing their therapeutic potential. Therefore, it is urgent to solve this problem.

[0004] The above contents are only used to assist in understanding the technical solution of the present invention, and do not constitute an admission that the above contents are 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 bionic nanovesicles derived from endothelial progenitor cells modified with cRGD targeting peptide, which provides a novel biological delivery system with pro-angiogenesis, anti-oxidation and antibacterial properties, thereby improving the wound treatment effect of diabetes.

[0006] To achieve the above object, the technical solution of the present invention is implemented as follows: a method for preparing bionic nanovesicles derived from endothelial progenitor cells modified with cRGD targeting peptide, the preparation method comprising the following steps:

[0007] S1. Preparation of EPC-NV;

[0008] S11, Isolate, culture and identify EPCs cells.

[0009] S12, preparing EPCs-NVs,

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

[0011] S2. Preparation of ADM-Fe3+ @PA / Gelma hydrogel:

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

[0013] S22, the Fe 3+ @The protocatechuic aldehyde solution was mixed with ADM, and after multiple centrifugal washings, the prepared photocurable gelatin (GelmA) precursor solution was added;

[0014] S23, through ultrasonic cavitation to carry Fe 3+ @PA’s ADM is evenly dispersed;

[0015] S24, double cross-linked hydrogel was formed by 416nm blue light irradiation and ADM self-assembly.

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

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

[0018] S12a. Primary EPCs were cultured with a medium containing exosome-depleted serum, and the cell supernatant was collected to extract the natural exosomes of EPCs by ultracentrifugation;

[0019] S12b. Pass 5-10 million / mL of EPCs suspension through the 10μm, 5μm, and 1μm pore membranes of the squeezer in turn to remove cell debris, obtain cell vesicle suspension, and then ultracentrifuge the suspension to obtain EPC-NVs.

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

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

[0022] S13b. Identification of 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 vesicles; Nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) were used to detect the particle size and distribution of the two vesicles; Western-Blot was used to detect the specific membrane proteins of the two vesicles, such as CD9, CD63 and CD81; and zeta potential was used to detect membrane potential.

[0023] The beneficial effects of the present invention are embodied in:

[0024] (1) The bionic nanovesicles derived from endothelial progenitor cells prepared by the extrusion method of the present invention can be used as EV mimics to deliver EPC contents to wounds. In addition, the present invention couples the cRGD targeting peptide to the surface of EPC-NV to achieve active targeting of endothelial cells (ECs). In addition, a dual hydrogel network is provided to couple Fe 3+ ADM modified with @PA complex was combined with photocurable gelatin (GelMA) to enrich and release mEPC-NV. The hydrogel network has antioxidant and antibacterial properties, which can reduce the level of reactive oxygen species and inhibit bacterial infection in diabetic wounds, making up for the lack of EPC-NV function.

[0025] (2) Based on the characteristics of low cell uptake capacity and complex types of diabetic wound cells, the present invention constructed a customized mEPC-NV with enhanced uptake and targeting, and applied the cRGD targeting peptide to diabetic wounds for the first time, thus improving the therapeutic effect.

[0026] (3) The present invention constructs ADM-Fe 3+- The mNV@PA / Gelma targeted delivery system is used for synergistic treatment, enhancing the therapeutic effect of diabetic wounds.

[0027] (4) The present invention uses an extrusion method to obtain a yield of 5-100 times the amount of vesicle particles or proteins of natural exosomes under the same cell mass conditions, and avoids cumbersome steps such as cell expansion and supernatant concentration. Secondly, due to the significant increase in yield, it is not necessary to pass the parent cells multiple times to obtain the same amount of nanovesicles, which ensures the performance stability of the biomimetic nanovesicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Figure 2 shows the synthesis and characterization of EPC-NV and mEPC-NV;

[0029] Figure 2 Figure 2 shows that EPC-NV promotes EC function under oxidative stress;

[0030] Figure 3 Functional mapping of EC targeting was achieved for cRGD-modified EPC-NV;

[0031] Figure 4 The functional map of mEPC-NV can restore EC;

[0032] Figure 5 Figure 1 shows the fabrication and characterization of AFG-mNV hydrogel;

[0033] Figure 6 This is the in vitro validation diagram of the antibacterial and antioxidant capabilities of AFG hydrogel;

[0034] Figure 7 This is a picture showing that AFG-mNV hydrogel accelerates diabetic wound healing;

[0035] Figure 8 This is a statistical analysis chart of diabetic wound healing;

[0036] Fig. 9 Figure 2 shows the in vivo validation of the ability of AFG-mNV hydrogel to scavenge ROS and target ECs.

[0037] Fig.10 It is a schematic diagram of the process of the present invention. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. In the absence of conflict, the embodiments in this application and the features in the embodiments can be combined with each other. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0039] Example 1

[0040] like Fig.10 As shown:

[0041] The preparation method of cRGD targeting peptide-modified endothelial progenitor cell-derived biomimetic nanovesicles comprises the following steps:

[0042] S1. Preparation of EPC-NV:

[0043] S11, isolate, culture and identify EPCs cells;

[0044] The EPCs cell separation, culture and identification in step S11 include isolating EPCs from rat bone marrow cell suspension, performing primary cell culture and passaging.

[0045] S12, preparing EPCs-NVs,

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

[0047] S12a. Primary EPCs were cultured with a medium containing exosome-depleted serum, and the cell supernatant was collected to extract the natural exosomes of EPCs by ultracentrifugation;

[0048] S12b. Pass 5-10 million / mL of EPCs suspension through the 10μm, 5μm, and 1μm pore membranes of the squeezer in turn to remove cell debris, obtain cell vesicle suspension, and then ultracentrifuge the suspension to obtain EPC-NVs.

[0049] S13, 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 the cRGD micelle solution, and the cRGD-modified EPC-NVs were purified by size exclusion chromatography to obtain cRGD@DeEPC-NVs;

[0052] S13b. Identification of 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 vesicles; Nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) were used to detect the particle size and distribution of the two vesicles; Western-Blot was used to detect the specific membrane proteins of the two vesicles, such as CD9, CD63 and CD81; and zeta potential was used to detect membrane potential.

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

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

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

[0056] S23, through ultrasonic cavitation to carry Fe 3+ @PA's ADM is evenly dispersed,

[0057] S24, double cross-linked hydrogel was formed by 416nm blue light irradiation and ADM self-assembly.

[0058] Example 2

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

[0060] 1) Expose the clean bench to ultraviolet light for 30 minutes;

[0061] 2) Weigh 3 g PA and 2 g ferric chloride at a molar ratio of 3:1, pour them into a 50 mL centrifuge tube, add 40 mL 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, and ultrasonically oscillate for 2 min using an ultrasonic oscillator (power: 30%, work 2 s, stop 2 s);

[0063] 4) The dispersion solution was centrifuged in a centrifuge (5000 rpm, 5 min), the supernatant was discarded, 1 mL of double distilled water was added, and ultrasonic vibration was performed 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 1 g of GelMA solid and put it into a 50 mL centrifuge tube, add 10 mL of the photoinitiator prepared according to the instructions of the kit, heat in a 70°C water bath, shake continuously to mix, and filter the solution with a 0.22 μm pore size filter after the solution is clarified;

[0066] 7) Take 1 ml of GelMA solution and add it to the ADM-Fe 3+ @PA solid was ultrasonically vibrated for 2 minutes (power: 30%, working for 2 seconds and stopping for 2 seconds) by ultrasonic oscillator, and then irradiated with 416nm wavelength blue light for about 10 seconds to form gel.

[0067] Example 3

[0068] The mechanism of cRGD modification of biomimetic nanovesicles to improve endothelial cell targeting:

[0069] Bionic nanovesicles can well solve the defects of natural exosomes, namely low yield, cumbersome separation and purification steps, and unstable functional phenotype performance of exosome vesicles. Bionic nanovesicles are easy to synthesize. After the parent cells are broken by various strategies such as freeze-thaw, extrusion, ultrasound, and microfluidics, the cell membrane fragments can be formed by self-assembly in the solution. Bionic nanovesicles inherit the cell membrane structure and endogenous therapeutic molecules of the parent cells, so they are considered to have low immunogenicity and biological activity similar to natural exosomes. In addition, compared with natural exosomes, biomimetic nanovesicles have many advantages in translational applications. First, biomimetic nanovesicles have a significantly higher yield than natural nanovesicles and simpler and faster collection and purification steps. The extrusion method can obtain 5-100 times the yield of vesicle particles or proteins relative to natural exosomes under the same cell mass conditions, and avoids cumbersome steps such as cell amplification and supernatant concentration. Secondly, due to the significant increase in yield, it is not necessary to pass the parent cells multiple times to obtain the same amount of nanovesicles, which ensures the performance stability of biomimetic nanovesicles.

[0070] ① Hypoxia at the wound surface stimulates fibroblasts, macrophages and endothelial cells to release VEGF and SDF-1a; ② VEGF induces the activation and phosphorylation of eNOS in the bone marrow, and SDF-1a and H2S can bind to CXCR4 to activate the PI3K / Akt / eNOS pathway. Both of these methods can lead to an increase in NO levels in the bone marrow cavity, thereby increasing sKitL levels and causing c-kit+EPCs to transfer from the bone marrow cavity to the peripheral blood; ③ EPCs that migrate to the peripheral blood can be relocated to the site of injury under the action of the chemokine SDF-1a; 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] The present invention has found that EPC-NVs can be taken up not only by wound endothelial cells, but also by fibroblasts and keratinocytes, but have no promoting effect on the functions of fibroblasts and keratinocytes, which will greatly reduce the effect of EPC-NVs when applied to wounds.

[0072] Therefore, the present invention considers modifying and transforming the EPC-NVs obtained by extrusion to enhance their targeting and promote more uptake of EPC-NVs by endothelial cells.

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

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

[0075] Example 4

[0076] Results Analysis

[0077] 1.1 Fabrication and characterization of EPC-NV and mEPC-NV

[0078] As described above, NVs from EPCs were isolated by extrusion and ultracentrifugation. Electron microscopy images showed that NVs were spherical and bimembranous with a diameter of approximately 100 nm (Fig. Figure 1 B). Micro-flow cytometry results further verified the size of NVs. The diameter of EPC-NVs was about 54.8±7.9nm, and the diameter of mEPC-NVs was slightly larger, about 67.8±23.8nm ( Figure 1 D, 1F). The zeta potentials of EPC-NVs and mEPC-NVs were -15±0.9mV and -20±0.6mV, respectively. Figure 1 E). Western blotting analysis showed that EV-specific markers including CD9, CD81 and TSG101 were highly expressed in both NVs ( Figure 1 C). Overall, the results of the present invention indicate that the properties of mEPC-NVs are similar to those of EPC-NVs and meet the criteria of extracellular vesicles.

[0079] Related results

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

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

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

[0083] The internalization of EPC-NVs was evaluated by incubating 20 μg DiI-labeled EPC-NV with human umbilical vein endothelial cells (HUVEC), human dermal fibroblast cells (HDF) and human immortalized epidermal cells (HaCaT), respectively.

[0084] By laser scanning confocal microscopy, red fluorescent staining was observed in the cytoplasm of all cell types, indicating that EPC-NVs were stably engulfed by these cells ( Figure 2 A). Previous studies have shown that EPC-NVs promote proliferation and migration and enhance the angiogenic ability of HUVEC under oxidative stress (47-49). In this study, the present invention treated HUVEC, HDF and HaCaT cells with EPC-NVs under oxidative stress conditions induced by MGO and H2O2, respectively. The control group was treated with PBS. Next, the present invention evaluated cell behavior by EdU detection, transwell assay and in vitro tube formation assay. The results showed that EPC-NVs partially restored the proliferation, migration and angiogenic ability of HUVEC cells under oxidative stress compared with the control group ( Figure 2 BD, 2I-K). The present invention also observed that EPC-NVs failed to restore HDF ( Figure 2 E, F and 2L, M) and HaCaT cells ( Figure 2 G, H and 2N, O) proliferation and migration functions under oxidative stress.

[0085] These results suggest that EPC-NVs can be internalized by HUVECs, HDFs, and HaCaTs but promote the function of HUVECs only under oxidative stress.

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

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

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

[0089] These data confirmed that mEPC-NVs exhibited excellent EC targeting ability, attributed to the presence of membrane cRGD peptide.

[0090] 3.4mEPC-NV restores EC function

[0091] To investigate whether mEPC-NVs can restore the proliferation, migration, and tube formation functions of HUVECs in diabetic wounds, HUVECs were cultured under oxidative stress induced by methylglyoxal (MGO) and H2O2. First, the proliferation of HUVECs was evaluated and it was noted that EdU-positive nuclei were significantly reduced after PBS treatment under oxidative stress, while this trend could be partially offset by EPC-NVs. Notably, after pretreatment with mEPC-NVs, the number of EdU-positive nuclei increased significantly under oxidative stress ( Figure 4 A, D). Subsequently, transwell assays were performed to further confirm the effect of mEPC-NV on EC migration. Figure 4 As shown in B and E, cell migration was significantly enhanced after 24 hours of mEPC-NV exposure. In addition, the results of the tube formation test showed that under oxidative stress, the tubular structure of the mEPC-NV group was improved, and the number of tubes formed and the number of connection points increased, both exceeding the PBS group and the EPC-NV group. ( Figure 1 ) Figure 4 C, F). These results suggest that mEPC-NVs restored the proliferation, migration, and tube-forming functions of ECs under oxidative stress, which may be due to the higher aggregation of functional mEPC-NVs near ECs.

[0092] 3.5 Preparation and characterization of AFG-mNV hydrogel

[0093] The hydrogel of the present invention is designed to retain the natural three-dimensional extracellular matrix structure of ADM and the activity of internal bioactive factors, as well as the antioxidant and antibacterial properties, and can sustainably release mNVs for diabetic wound repair. AFG-mNV hydrogel is synthesized at room temperature. Figure 5 B shows the morphology of GelMA and AFG-mNV hydrogels. Figure 5 C shows the porous structure of AFG-mNV hydrogel visualized by scanning electron microscopy (SEM). Figure 5 As shown in D, mEPC-NV is evenly distributed in the hydrogel. The present invention also tested that the degradation rate of AFG hydrogel exceeded 70% within 10 days, achieving the expected goal. In addition, the hemolysis test showed that the degree of hemolysis was similar to that of blood incubated with PBS, and the red blood cell rupture rate was low (<5%) ( Figure 5 F). Rheological measurements were used to evaluate the mechanical properties of the hydrogels. The relationship between the storage modulus (G′) and loss modulus (G″) of the AFG hydrogels was examined using a rheometer ( Figure 5). The results showed that the energy storage modulus of the AFG hydrogel exceeded the loss modulus, indicating sufficient mechanical strength. To confirm that mNVs could be sustainably released from the AFG-mNV hydrogel, the daily mNV release was quantified using the microBCA microprotein assay. About 50% of the mNVs in the AFG-mNV hydrogel were released within 5 days, and nearly 80% of the mNVs were released within 14 days, indicating that mNVs can be rapidly released from the hydrogel ( Figure 5 ). These findings collectively confirmed the successful synthesis of mEPC-NV-loaded AFG hydrogels.

[0094] 3.6 Biocompatibility of AFG hydrogel

[0095] The application of medical biomaterials depends largely on excellent biocompatibility. Therefore, the present invention investigated whether the AFG hydrogel of the present invention has any cytotoxic effect on HUVEC, HDF and HaCaT cells by CCK-8 assay and LIVE / DEAD staining. After incubation with AFG hydrogel, the present invention found no cytotoxicity to any cell type, and cell viability remained above 90% ( Figure 5 A and C). Calcein-AM (green) and propidium iodide (red) stains were used to distinguish live from dead cells. In addition, the biocompatibility of the degradation products of AFG hydrogels was evaluated to ensure that there was no cytotoxic effect during the hydrogel degradation process. Figure 5 As shown in B and D, degradation of AFG hydrogels was non-toxic and promoted the proliferation of HUVEC, HDF, and HaCaT cells. Notably, degradation of AFG hydrogels and cells cultured with the hydrogels both increased the survival of HUVEC, HDF, and HaCaT cells, as measured by CCK-8 assay ( Figure 5 E and F). These findings together emphasize the excellent biocompatibility of the AFG hydrogels of the present invention.

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

[0097] After incubating the bacteria with AFG hydrogel for 4 hours, the colony formation assay showed that the AFG hydrogel group significantly reduced the viability of Staphylococcus aureus and Escherichia coli ( Figure 6 A). In particular, the activity inhibition rates of AFG hydrogel against Staphylococcus aureus and Escherichia coli were 70.7% and 40.5%, respectively ( Figure 6 B). The present invention further studied the bacterial growth after 24 hours. In the AFG hydrogel group, the absorbance curve of the bacterial suspension showed an obvious peak decrease and rightward shift ( Figure 6C). In addition, the present invention conducted a bacterial inhibition ring formation assay, and the results showed that there was no bacterial growth in the ring area, and the diameter of the inhibition ring was approximately 60% of the positive control of Escherichia coli and 50% of that of Staphylococcus aureus ( Figure 6 D, E). These results indicate that AFG hydrogel has sufficient antibacterial properties. Figure 6 F and G show that the abundance of ROS in HUVEC, HDF and HaCaT cells under the AFG hydrogel effect was significantly reduced compared with the MGO group (P<0.001), indicating that the anti-ROS property of AFG hydrogel may contribute to the healing ability of AFG hydrogel in diabetic wounds. These results verified in vitro that AFG hydrogel has good antibacterial and ROS-reducing abilities in diabetic wounds.

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

[0099] To further verify the clinical potential of AFG-mNV hydrogel, the present invention established a full-thickness wound model on the back of normal and diabetic mice, and divided them into 6 groups: untreated normal mice served as the control group, and the remaining 5 groups of diabetic mice were treated with PBS, AFG, NV, mNV, and AFG-mNV, respectively. Figure 7 A). Figure 7 As shown in BE, wounds of normal mice and AFG-mNV mice were completely healed at day 12 after surgery, while diabetic wounds treated with PBS showed delayed wound healing and were far from closed. Notably, at days 4, 8, and 12 after injury, mice treated with AFG-mNV showed the lowest rate of wound non-closure among various treatment groups, approaching the level observed in the normal group.

[0100] Next, wound sections from all groups were dissected on postoperative day 12 and downstream histological analysis was performed. H&E and Masson staining analysis showed that AFG, NV, mNV, and AFG-mNV reduced the length of granulation tissue and accelerated collagen deposition in diabetic wounds compared with the PBS group. The re-epithelialization rate and collagen deposition rate were significantly higher in the mNV group and the AFG-mNV group, among which the re-epithelialization rate and collagen deposition rate in the AFG-mNV group were lower than those in the mNV and AFG-mNV groups ( Figure 8 AD). In addition, wounds treated with AFG-mNV showed collagen arrangement very similar to normal wound tissue.

[0101] To detect and quantify angiogenesis in the wound beds of the 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 8 EH).

[0102] These findings together suggest that ADM-Fe 3+ PA and mEPC-NV can synergistically promote diabetic wound healing in vivo. In addition, AFG-mNV treatment achieved better wound neovascularization than mNV in diabetic wound healing.

[0103] 3.9 In vivo ROS scavenging and EC targeting ability of AFG-mNV hydrogel

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

[0105] The EC targeting ability and distribution of mNVs in diabetic wounds were further explored by in vivo immunofluorescence examination. Fig. 9 As shown in E, the DiI-labeled mNV group was more widely distributed in the diabetic wound on day 4 (red signal) compared with the DiI-labeled NV. In addition, the present invention noted that the co-localization of mNV with blood vessels was greater than that of NV ( Fig. 9 F). The results showed that mNVs could 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 replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing biomimetic nanovesicles derived from endothelial progenitor cells modified with cRGD targeting peptide, characterized in that: The preparation method comprises the following steps: S1. Preparation of EPC-NV: S11, isolate, culture and identify EPCs cells; S12, preparing EPCs-NVs; S13, cRGD modification and identification of EPC-NVs; S2. Preparation of ADM-Fe 3+ @PA / Gelma hydrogel: S21, synthesize Fe at a molar ratio of 1:3 in an environment with a pH value of 10 3+ @Protocatechualdehyde solution; S22, the Fe 3+ @The protocatechuic aldehyde solution was mixed with ADM, and after multiple centrifugation washes, the prepared GelmA precursor solution was added; S23, through ultrasonic cavitation to carry Fe 3+ @PA’s ADM is evenly dispersed; S24, double cross-linked hydrogel was formed by 416nm blue light irradiation and ADM self-assembly.

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

3. The method for preparing the 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 were cultured with a medium containing exosome-depleted serum, and the cell supernatant was collected to extract the natural exosomes of EPCs by ultracentrifugation; S12b. Pass 5-10 million / mL of EPCs suspension through the 10μm, 5μm, and 1μm pore membranes of the squeezer in turn to remove cell debris, obtain cell vesicle suspension, and then ultracentrifuge the suspension to obtain EPC-NVs.

4. The method for preparing the cRGD targeting peptide-modified endothelial progenitor cell-derived biomimetic nanovesicles according to claim 3, characterized in that: The process of cRGD modification of EPC-NVs in S13 includes: S13a. The DeEPC-NVs suspension was co-incubated with the cRGD micelle solution, and the cRGD-modified EPC-NVs were purified 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: Transmission electron microscopy was used to observe the morphology and size of the two vesicles; nanoflow cytometry and dynamic light scattering were used to detect the particle size and distribution of the two vesicles; Western-Blot was used to detect specific membrane proteins of the two vesicles, such as CD9, CD63 and CD81; and zeta potential was used to detect membrane potential.

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