Plant-bacterium hybridized outer vesicle composite hydrogel microneedle as well as preparation and application thereof

Abstract

The invention relates to the technical field of biological materials, and particularly provides a plant-bacterium hybrid outer vesicle composite hydrogel microneedle as well as a preparation method and application thereof. The plant-bacterium hybrid outer vesicle composite hydrogel microneedle provided by the invention comprises a needle tip loaded with plant-bacterium hybrid outer vesicles (GBEVs-pVEGF) and a substrate containing an antibacterial material nano silver (AgNPs). After the microneedle penetrates through necrotic tissue on the surface of a diabetic wound surface, the needle point of the microneedle can quickly release the GBEVs-pVEGF, after the GBEVs-pVEGF is taken by cells, the anti-oxidation effect is achieved, meanwhile, shuttle plasmids pVEGF in the GBEVs-pVEGF are expressed by the cells to be VEGF protein, and therefore angiogenesis is promoted. In addition, a physical protection barrier formed by the composite microneedle substrate and AgNPs have a synergistic effect, and the problem of bacterial infection in the wound healing process is solved. The composite microneedle system integrates the characteristics of oxidation resistance, angiogenesis and antibiosis, effectively recovers the damaged wound surface microenvironment, and remarkably accelerates the healing of the refractory wound surface of diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and particularly relates to a plant-bacteria hybrid extracellular vesicle composite hydrogel microneedle and a preparation method and application thereof. Background Art

[0002] Diabetic chronic non-healing wounds are common chronic complications of diabetes. It is estimated that 19% - 34% of diabetic patients globally are accompanied by the problem of difficult wound healing. These wounds not only heal slowly but also are often accompanied by long-term infections, and in severe cases, the mortality rate can reach as high as 30.5%. Currently, conservative drug treatments are mainly adopted clinically, including offloading treatment, surgical debridement, and antibiotic treatment, etc. However, the treatment effects of these methods are limited and may be accompanied by adverse reactions. The healing process of diabetic chronic non-healing wounds is hindered by the complex interaction of elevated reactive oxygen species (ROS), impaired angiogenesis, and persistent bacterial infections. The elevated ROS level can directly cause tissue damage, inhibit angiogenesis, and exacerbate the risk of infection. Persistent bacterial infections further activate the production of ROS, deteriorate tissue damage, and inhibit blood vessel regeneration. In-depth understanding of these interrelated factors is crucial for developing effective treatment strategies to accelerate the healing of diabetic chronic non-healing wounds.

[0003] Extracellular vesicles (EVs) are nanoscale vesicles secreted by living cells, with good biocompatibility, low toxicity, and excellent drug-loading capacity. In the treatment of diabetic wounds, EVs have attracted attention due to their unique biological properties and treatment potential. The discovery of plant extracellular vesicles (PEVs) and bacterial extracellular vesicles (BEVs) has overcome the challenges of traditional mammalian extracellular vesicles (MEVs), such as complex purification processes and low yields, providing new possibilities for accelerating wound healing. Due to their high extraction efficiency, high yield, and significant anti-inflammatory and antioxidant effects, PEVs can effectively regulate the inflammatory response and oxidative stress. However, in the diabetic wound microenvironment, the angiogenesis ability of PEVs is limited, thus affecting their application in wound healing. Gut microbiota plays a key role in the development of diabetes by affecting the host's glucose metabolism and insulin sensitivity. Among them, the probiotic Escherichia coli Nissle 1917 (ECN), as a kind of gut microbiota, has the advantages of easy gene editing and large-scale fermentation. At the same time, ECN bacteria produce BEVs through an explosive cell lysis mechanism, and these BEVs carry the same components as inside the bacteria, making it an ideal carrier for engineering and customizing the content of BEVs. Therefore, designing shuttle plasmids and customizing the content of probiotics, and using BEVs to deliver shuttle plasmids to express target proteins may become an effective strategy to solve the problem of insufficient vascularization in diabetic chronic non-healing wounds.

[0004] However, single ginger extracellular vesicles or engineered probiotic extracellular vesicles are difficult to effectively address the complex challenges in the diabetic wound microenvironment. To solve this problem, inspired by the fusion of liposomes and exosomes, a plant-bacteria hybrid extracellular vesicle (GBEVs-pVEGF) was custom-built using a continuous extrusion membrane fusion method, which combines the antioxidant properties of plant extracellular vesicles and the angiogenesis-promoting ability of engineered probiotic extracellular vesicles. However, there has been no research on constructing plant-bacteria hybrid extracellular vesicles and using them as a treatment plan to accelerate the healing of diabetic non-healing wounds. Summary of the Invention

[0005] Aiming at the problems existing in the prior art, the present invention aims to provide a plant-bacteria hybrid extracellular vesicle composite hydrogel microneedle capable of accelerating the healing of diabetic non-healing wounds and a preparation method thereof, so as to provide a new way for the treatment of diabetic non-healing wounds. Specifically, after the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedle penetrates the necrotic tissue of the diabetic wound, the tip of the microneedle can rapidly release GBEVs-pVEGF. After being taken up by the cells at the wound site, the active substances of the retained ginger extracellular vesicles released by GBEVs-pVEGF play an antioxidant role, while the shuttle plasmid pVEGF in GBEVs-pVEGF is expressed as VEGF protein by the cells, thereby promoting angiogenesis. In addition, after the microneedle penetrates the skin, the base part of the microneedle can form a physical protection barrier for the wound tissue to prevent external bacterial infection, and at the same time, the base can continuously release the antibacterial material AgNPs, which effectively responds to the persistent bacterial infection during the wound healing process in cooperation with the physical barrier of the base.

[0006] To achieve the invention purpose, the technical solution of the present invention is as follows: In the first aspect of the present invention, a plant-bacteria hybrid extracellular vesicle is provided, which is formed by the membrane continuous extrusion fusion of plant extracellular vesicles and engineered probiotic extracellular vesicles.

[0007] Further, in the plant-bacteria hybrid extracellular vesicle, the concentrations of both the plant extracellular vesicles and the engineered probiotic extracellular vesicles are 10 10 ~10 11 particles / mL.

[0008] Further, the engineered probiotic extracellular vesicle is an extracellular vesicle of E. coli Nissle1917 (ECN) bacteria loaded with the shuttle plasmid pVEGF.

[0009] Further, the plant extracellular vesicle is a ginger extracellular vesicle.

[0010] Further, the particle size of the plant-bacteria hybrid extracellular vesicle is 50 - 200 nm, and it has a complete phospholipid bilayer structure.

[0011] In the second aspect of the present invention, a plant-bacteria hybrid extracellular vesicle composite hydrogel microneedle is provided. The plant-bacteria hybrid extracellular vesicle composite hydrogel microneedle is a two-component microneedle with a base and a tip: the base part is composed of AlgMA hydrogel loaded with antibacterial material silver nanoparticles (AgNPs), and the tip part is composed of AlgMA hydrogel loaded with plant-bacteria hybrid extracellular vesicles (GBEVs-pVEGF).

[0012] Furthermore, in the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedle, the concentration of AgNPs in the base part is 200 μg / mL, and the loading amount of GBEVs-pVEGF in the tip part is 10 9 ~10 11 particles / mL.

[0013] In the third aspect of the present invention, a preparation method of plant-bacteria hybrid extracellular vesicles is provided. The specific preparation process includes: Step 1, shuttle plasmid synthesis: Using a one-step cloning kit, homologous sequences of 15 to 25 bases are added to both ends of the plasmid vector, and these sequences are completely matched with the sequences of the target cloning site. Under the action of ligase, the recombinant plasmid is integrated into the plasmid vector, thus successfully constructing the shuttle plasmid pVEGF; Step 2, preparation of engineered probiotics: The probiotics are cultured to calcium transformation competent state, and the shuttle plasmid pVEGF is transferred into the probiotics in the calcium transformation competent state, successfully constructing engineered probiotics; Step 3, extraction of engineered probiotic extracellular vesicles: The engineered probiotics are added to LB medium for primary and secondary culture, and the fermentation broth of the secondary culture is collected. Subsequently, the fermentation broth is treated by low-speed centrifugation and ultra-high-speed centrifugation to obtain engineered probiotic extracellular vesicles BEVs-pVEGF; Step 4, extraction of ginger extracellular vesicles: After ginger is juiced, it is preliminarily filtered through a gauze, and then purified by differential centrifugation and sucrose density gradient centrifugation to obtain ginger extracellular vesicles GEVs; Step 5, preparation of plant-bacteria hybrid extracellular vesicles: The mixture of the engineered probiotic extracellular vesicles BEVs-pVEGF and ginger extracellular vesicles GEVs is sequentially subjected to low-frequency ultrasound and membrane continuous extrusion to obtain the plant-bacteria hybrid extracellular vesicles GBEVs-pVEGF.

[0014] Furthermore, the plasmid vector in Step 1 can be selected as pEGFP-N1.

[0015] Further, the one-step method described in step 1 is specifically a one-step ligation reaction system for shuttle plasmid synthesis, with a total volume of 20 μL. The specific ratio is as follows: 0.5 - 1 μL of plasmid vector, 1 - 2 μL of target gene, 10 μL of one-step ligase, and the remaining part is added with ddH 2 O; the temperature is 50 °C and the time is 20 min.

[0016] Further, the step of culturing probiotics to calcium transformation competent state described in step 2 includes: inoculating probiotics into 3 - 5 mL of LB medium and culturing for 8 - 10 h. Then, transfer the culture solution to 50 - 100 mL of LB medium and continue to culture for 1.5 - 3 h until the OD600 value reaches 0.4 - 0.6. Stop culturing and perform an ice bath treatment for 20 - 30 min. Next, centrifuge at 5500 - 7500 rpm for 5 - 10 min under the condition of 0 - 4 °C to remove the supernatant. Subsequently, add 5 - 10 mL of 0.1 M CaCl 2 solution to resuspend, and repeat the centrifugation step. Finally, resuspend the bacterial solution in 0.1 M CaCl 2 solution containing 10 - 20% glycerol to obtain probiotics in calcium transformation competent state.

[0017] Further, the step of transferring the shuttle plasmid into probiotics in calcium transformation competent state described in step 2 includes: adding the shuttle plasmid pVEGF to the probiotic solution in calcium transformation competent state, mixing well and then performing an ice bath treatment for 20 - 30 min. Then, perform a heat shock at 37 - 42 °C for 90 - 120 s, and then perform an ice bath treatment for 2 - 10 min again. Next, add 900 - 1800 μL of LB medium and culture at 37 °C for 1 - 2 h. After the culture is completed, centrifuge at 5500 - 7500 rpm for 5 - 10 min under the condition of 0 - 4 °C, take 900 - 1800 μL of the supernatant and resuspend. Finally, take 100 - 200 μL of the resuspended solution for coating culture, and culture it upside down at 37 °C. Wait for single colonies to form to obtain engineered probiotics.

[0018] Further, the step of culturing the engineered probiotics described in step 2 includes: adding 50 - 100 μL of the engineered probiotics to 1.5 - 3 mL of LB medium and culturing at 180 - 280 rpm at 37 °C for 10 - 12 h to obtain a primary culture. Subsequently, transfer it to 75 - 150 mL of LB medium and continue to culture under the same conditions for 8 - 10 h to obtain a secondary culture, thus completing the culture of the engineered probiotics.

[0019] Further, the extraction step of the engineered probiotic exosomes in step 3 includes: subjecting the secondary culture fermentation broth to low-speed centrifugation at 10,000 - 15,000 g for 15 - 30 min under the condition of 0 - 4°C. Subsequently, filter the supernatant after low-speed centrifugation using a 0.22 μm sterile filter head, and subject the filtrate to ultra-high-speed centrifugation at 120,000 - 150,000 g for 120 min under the condition of 0 - 4°C, collect the precipitate to obtain the engineered probiotic exosomes BEVs-pVEGF.

[0020] Further, the extraction step of the ginger exosomes in step 4 includes: after juicing the ginger, first perform preliminary filtration through a gauze; then, subject the ginger filtrate to differential centrifugation, successively: centrifuge at 3000 g for 30 min, 8000 g for 60 min, 10000 g for 60 min, 12000 g for 60 min, and finally centrifuge at 150,000 g for 120 min; finally, purify by sucrose density gradient centrifugation to obtain the ginger exosomes.

[0021] Even further, the specific steps of the sucrose density gradient centrifugation in the extraction process of the ginger exosomes in step 4 are: transfer the bottom precipitate solution obtained after differential centrifugation to the sucrose density gradient solution, and the sucrose concentration gradients are successively 8%, 15%, 30%, 45% and 60%; at 4°C, centrifuge at 150,000 g for 120 min, collect the band between the 30% and 45% concentration gradients to obtain the ginger exosomes.

[0022] Further, the preparation process of the plant-bacteria hybrid exosomes in step 5 includes: subjecting the mixed solution of the ginger exosomes and the engineered probiotic exosomes to low-frequency ultrasound and membrane continuous extrusion to obtain the plant-bacteria hybrid exosomes.

[0023] Further, the preparation process of the plant-bacteria hybrid exosomes in step 5 includes: after mixing the ginger exosomes and the engineered probiotic exosomes with equal concentrations, incubate at 4°C for 30 min; then subject the vesicle mixture to low-frequency ultrasound for 10 min; finally, repeatedly extrude through a porous polycarbonate membrane, starting from 400 nm, proceeding to 200 nm, and finally to 100 nm, and repeat each size setting 10 - 20 times to obtain the plant-bacteria hybrid exosomes.

[0024] Even closer, the concentrations of the ginger exosomes and the engineered probiotic exosomes in step 5 are both 10 10 ~10 11 particles / mL.

[0025] In the fourth aspect of the present invention, a method for preparing a plant-bacteria hybrid extracellular vesicle composite hydrogel microneedle is provided. The specific preparation process includes: Mix the plant-bacteria hybrid extracellular vesicles (GBEVs-pVEGF) and silver nanoparticles (AgNPs) with AlgMA hydrogel respectively and conduct co-incubation; then inject the AlgMA hydrogel loaded with GBEVs-pVEGF into a microneedle mold, and perform vacuum degassing and thermal concentration; then fill the AlgMA hydrogel loaded with AgNPs into the upper layer (base) part of the microneedle mold, and after thermal concentration and photocuring treatment, dry and demold at room temperature to obtain the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedle.

[0026] Further, the preparation process of the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedle includes: adding 0.08 - 0.4 g of AlgMA powder into 1 - 5 mL of sterile ultrapure water, incubating at room temperature for 30 min; then adding GBEVs-pVEGF and AgNPs respectively, incubating at room temperature for 30 min, and then adding 2.5 - 12.5 mg of photoinitiator LAP respectively, incubating at 37°C at 300 - 500 rpm for 30 min; then, inject 100 - 150 μL of the AlgMA hydrogel containing GBEVs-pVEGF into the microneedle mold, perform vacuum degassing multiple times and thermally concentrate at 37°C for 4 - 6 h; then, inject 50 - 100 μL of the AlgMA hydrogel loaded with AgNPs into the upper layer part (base layer part) of the microneedle mold, thermally concentrate at 37°C for 4 - 6 h; finally, irradiate with a UV lamp with a wavelength of 450 nm for 30 s for photocuring, and dry at room temperature for 2 days and then demold to obtain the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedle.

[0027] In the fifth aspect of the present invention, an application of the plant-bacteria hybrid extracellular vesicle or the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedle of the present invention in accelerating the healing of diabetic refractory wounds is provided.

[0028] Further, the plant-bacteria hybrid extracellular vesicle or the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedle of the present invention can promote collagen deposition and fibrosis at the wound surface, and effectively accelerate wound healing.

[0029] Further, the plant-bacteria hybrid extracellular vesicle or the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedle of the present invention can also be used to prepare a drug for accelerating the healing of diabetic refractory wounds.

[0030] Compared with the prior art, the present invention has the following technical effects: First, the plant-bacteria hybrid extracellular vesicles loaded with antibacterial material composite hydrogel microneedles provided by the present invention offer a new safe and effective treatment strategy for accelerating the healing of diabetic refractory wounds. After the multifunctional hydrogel microneedles are implanted into diabetic refractory wounds, the tip part can rapidly release the plant-bacteria hybrid extracellular vesicles to achieve antioxidant and pro-angiogenic effects. At the same time, the base part of the microneedles closely adheres to the wound surface to form a physical barrier and synergistically slowly releases the antibacterial material silver nanoparticles (AgNPs) to effectively address the persistent bacterial infection in diabetic refractory wounds.

[0031] Secondly, the plant-bacteria hybrid extracellular vesicles loaded with antibacterial material composite hydrogel microneedles provided by the present invention exhibit excellent antioxidant, antibacterial, and pro-angiogenic properties, significantly shortening the inflammatory phase, proliferation phase, and remodeling phase during the healing process of diabetic refractory wounds, significantly promoting the rate of wound collagen deposition and angiogenesis, accelerating the healing process of diabetic refractory wounds, and having significant biocompatibility and a wound repair ability with rapid and good healing effects. Description of the Drawings

[0032] Figure 1 . Transmission electron microscopy (TEM) image of the plant-bacteria hybrid extracellular vesicles prepared in Example 1.

[0033] Figure 2 . Detection result graph of the dynamic light scattering (NTA) of the plant-bacteria hybrid extracellular vesicles prepared in Example 1.

[0034] Figure 3 . Effect graph of the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedles prepared in Example 1 acting on RAW264.7 cells to reduce the ROS level.

[0035] Figure 4 . Scratch test effect graph after the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedles prepared in Example 1 induce the proliferation of HUVECs for 24 h.

[0036] Figure 5 . Transwell migration test effect graph after the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedles prepared in Example 1 induce the migration of HUVECs for 24 h.

[0037] Figure 6 . Vascular node effect graph after the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedles prepared in Example 1 induce the tube formation of HUVECs for 6 h.

[0038] Figure 7 . Vascular length effect graph after the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedles prepared in Example 1 induce the tube formation of HUVECs for 6 h.

[0039] Figure 8. Experimental effect diagram of coating after the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedles prepared in Example 1 acted on Escherichia coli for 24 h.

[0040] Figure 9 . Experimental effect diagram of coating after the plant-bacteria hybrid extracellular vesicle composite hydrogel microneedles prepared in Example 1 acted on Staphylococcus aureus for 24 h.

[0041] Figure 10 . Effect diagram of the relative wound area on the 3rd, 7th, and 14th days after the intervention protocol treatment of type II diabetic skin defect mice in each group in Example 2. Detailed implementation manners

[0042] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those of ordinary skill in the art can make several deformations and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0043] Example 1 Preparation of plant-bacteria hybrid extracellular vesicle composite hydrogel microneedles Synthesis of shuttle plasmid: Introduce 15 - 25 bp homologous sequences identical to the cloning sites at both ends of the plasmid vector pEGFP-N1, and then configure a 20 μL reaction system, which includes 0.5 - 1 μL of linearized vector, 1 - 2 μL of target gene, 10 μL of HB-infusion one-step ligase, and finally make up to 20 μL with ddH 2 O. Under the catalytic action of the one-step ligase, transfer the VEGF recombinant plasmid into the plasmid vector to construct the shuttle plasmid pVEGF.

[0044] Preparation of engineered probiotics: Inoculate the ECN strain into 3 mL of LB medium and culture for 8 h, then transfer it to 50 mL of LB medium and continue to culture for 1.5 h. Stop culturing when the OD600 reaches 0.4 - 0.6, and perform a 20 min ice bath treatment. Subsequently, centrifuge at 5500 rpm for 5 min at 4°C to remove the supernatant, resuspend in 5 mL of 0.1 M CaCl 2 solution, and repeat the centrifugation and resuspension steps. Finally, use 0.1 M CaCl containing 10% glycerol 2Resuspend the solution to obtain calcium transformation-competent ECN bacteria. Add the shuttle plasmid pVEGF into the calcium transformation-competent ECN bacteria. After mixing, incubate on ice for 20 min. After heat shock at 42 °C for 90 s and ice bath treatment for 5 min, add 900 μL of LB medium and culture at 37 °C for 1 h. Then, centrifuge the bacterial cells at 5500 rpm for 5 min at 4 °C and resuspend them. Take 100 μL of the bacterial suspension and spread it on an LB solid plate. Incubate the plate upside down at 37 °C until single colonies are formed to obtain engineered probiotics.

[0045] Extraction of engineered probiotic extracellular vesicles: Take 100 μL of engineered probiotics transformed with the shuttle plasmid and inoculate them into 3 mL of LB medium. Culture them under aerobic conditions at 37 °C and 280 rpm for 12 h to obtain the primary fermentation broth. Subsequently, transfer the primary fermentation broth to 150 mL of LB medium and continue to culture it under the same conditions for 10 h to obtain the secondary fermentation broth. The secondary fermentation broth is centrifuged at 10000 g for 20 min at 4 °C. Collect the supernatant and filter it through a 0.22 μm sterile filter head. The filtrate is ultracentrifuged at 150000 g for 120 min. The precipitate is resuspended with sterile PBS to obtain purified engineered probiotic extracellular vesicles BEVs-pVEGF.

[0046] Extraction of ginger extracellular vesicles: After juicing ginger, filter it with gauze to remove large particles and collect the ginger juice filtrate. The filtrate is processed by stepwise differential centrifugation at 4 °C: centrifuge at 3000 g for 30 min, 8000 g for 60 min, 10000 g for 60 min, and 12000 g for 60 min in sequence to gradually remove the precipitate and retain the supernatant. The final supernatant is ultracentrifuged at 150000 g for 120 min. Collect the bottom precipitate and resuspend it with sterile PBS buffer. Subsequently, transfer the precipitate resuspension to a sucrose density gradient solution (concentrations are 8%, 15%, 30%, 45%, and 60% in sequence), and ultracentrifuge it again at 150000 g for 120 min. Collect the band between the 30% and 45% concentration gradients to obtain purified ginger extracellular vesicles.

[0047] Preparation of plant-bacteria hybrid extracellular vesicles: Take 1 mL of GEVs and BEVs-pVEGF respectively and mix them. Incubate at 4 °C for 30 min; then, perform low-frequency ultrasound on the mixed vesicles for 10 min; subsequently, repeatedly extrude the sonicated vesicle mixture through a porous polycarbonate membrane, starting from 400 nm, proceeding to 200 nm, and finally to 100 nm, with each size setting repeated 15 times to obtain plant-bacteria hybrid extracellular vesicles (the concentrations of GEVs and BEVs-pVEGF are both 10 10 ~10 11 particles / mL).

[0048] Figure 1 and Figure 2 are the transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) detection results of the prepared plant-bacterial hybrid extracellular vesicles; it can be seen from the figure that the particle size of the plant-bacterial hybrid extracellular vesicles is between 50 and 200 nm.

[0049] Preparation of plant-bacterial hybrid extracellular vesicle composite hydrogel microneedles: Add 0.4 g of AlgMA powder to 5 mL of sterile ultrapure water and incubate at room temperature for 30 min; then, add AgNPs and GBEVs-pVEGF respectively and incubate at room temperature for 30 min. Subsequently, add 12.5 mg of photoinitiator LAP and incubate at 45 °C and 450 rpm for 30 min; then, add 100 - 150 μL of AlgMA hydrogel containing GBEVs-pVEGF to the microneedle mold, degas under vacuum multiple times, and heat-concentrate at 37 °C for 4 - 6 h; then, add 50 - 100 μL of AlgMA hydrogel loaded with AgNPs to the microneedle mold and heat-concentrate at 37 °C for 4 - 6 h; then, irradiate with a UV lamp with a wavelength of 450 nm for 30 s for photocuring; finally, dry at room temperature for 2 days and demold to obtain plant-bacterial hybrid extracellular vesicle composite hydrogel microneedles (the concentration of AgNPs is 200 μg / mL; the loading amount of GBEVs-pVEGF is 10 9 ~10 11 particles / mL).

[0050] Figure 3 is the effect diagram of the plant-bacterial hybrid extracellular vesicle composite hydrogel microneedles acting on RAW264.7 cells to reduce the ROS level; where the ordinate represents the intracellular ROS fluorescence intensity. It can be seen from the figure that the plant-bacterial hybrid extracellular vesicle composite hydrogel microneedles can significantly reduce the intracellular ROS level.

[0051] Figure 4 and Figure 5 are the effect diagrams of the scratch test and Transwell migration test of the plant-bacterial hybrid extracellular vesicle composite hydrogel microneedles inducing the proliferation of HUVECs for 24 h; where Figure 4 the ordinate is the scratch coverage rate, Figure 5 and the ordinate represents the number of Transwell cell migrations. It can be seen from Figure 4 and Figure 5 that the plant-bacterial hybrid extracellular vesicle composite hydrogel microneedles can effectively promote the proliferation and migration of vascular endothelial cells.

[0052] Figure 6 and Figure 7The effect diagram of the number of vascular nodes and length after 6 h of tube formation of HUVECs induced by the prepared plant-bacteria hybrid exosome composite hydrogel microneedles; among which Figure 6 The vertical coordinate is the number of vascular nodes, Figure 7 The vertical coordinate is the vascular length. It can be seen from the figure that the plant-bacteria hybrid exosome composite hydrogel microneedles can effectively promote angiogenesis of vascular endothelial cells.

[0053] Figure 8 and Figure 9 The effect diagram of the coating experiment after the prepared plant-bacteria hybrid exosome composite hydrogel microneedles act on Escherichia coli and Staphylococcus aureus for 24 h; among which Figure 8 The vertical coordinate represents the antibacterial rate of Escherichia coli, Figure 9 The vertical coordinate represents the antibacterial rate of Staphylococcus aureus. It can be seen from the figure that the plant-bacteria hybrid exosome composite hydrogel microneedles can effectively achieve antibacterial.

[0054] Example 2 Intervention experiment on mice with type II diabetic skin defects Full-thickness skin defect model of type II diabetic mice: Select male C57BL / 6 mice at 6 weeks of age to establish a full-thickness wound model of type II diabetes. The mice were fed a high-fat and high-sugar diet for 4 weeks to induce insulin resistance, and then streptozotocin (STZ) (10 mg / kg) was intraperitoneally injected twice after a 12-h fasting, with an interval of one week between injections. When the blood glucose level remained above 16.7 mmol / L for two weeks, the type II diabetic mouse model was considered to be successfully established. After the induction of diabetes in mice was successful, the mice were anesthetized, and a 10-mm full-thickness wound was created on the back surface using a skin punch to establish a full-thickness skin defect model of type II diabetic mice.

[0055] Group experiment: The wound surface was treated with AgNPs@MNs, BEVs-pVEGF / AgNPs@MNs, GEVs / AgNPs@MNs and GBEVs-pVEGF / AgNPs@MNs, and fixed with medical tape, with the untreated group as the control group (control).

[0056] After treatment according to the above intervention plan, pictures of the wound surfaces of each group were taken at 0, 3, 7, and 14 days, and the wound surface area was quantified to evaluate the effect of the material on accelerating the healing of diabetic refractory wounds. Figure 10 The effect diagram of the relative wound surface area of the skin defect mice in each group in Example 2 on the 3rd, 7th, and 14th days after the intervention plan; among which the vertical coordinate represents the unhealed wound surface area. It can be seen from the figure that the plant-bacteria hybrid exosome composite hydrogel microneedles can effectively accelerate the healing of diabetic refractory wounds.

Claims

1. A plant-bacteria hybrid exovesicle, characterized in that The plant-bacteria hybrid exovesicles are formed by continuous membrane extrusion fusion of plant exovesicles and engineered probiotic exovesicles.

2. The hybrid extracellular vesicle according to claim 1, characterized in that In the plant-bacteria hybrid exosomes, the concentrations of the plant exosomes and the engineered probiotic exosomes are both 10 10 ~10 11 particles / mL.

3. The hybrid extracellular vesicle according to claim 1, characterized in that The engineered probiotic extracellular vesicles are E. coli Nissle 1917 extracellular vesicles loaded with shuttle plasmid pVEGF; and the plant extracellular vesicles are ginger extracellular vesicles.

4. The hybrid extracellular vesicle according to claim 1, characterized in that The plant-bacteria hybrid exosome has a particle size of 50-200 nm and has a complete phospholipid bilayer structure.

5. A plant-bacteria hybrid exosome composite hydrogel microneedle, characterized in that: The plant-bacteria hybrid exosome composite hydrogel microneedle is a base-needle tip two-component microneedle: the base part is composed of AlgMA hydrogel loaded with antibacterial material nanosilver AgNPs, and the needle tip part is composed of AlgMA hydrogel loaded with plant-bacteria hybrid exosome GBEVs-pVEGF.

6. The composite hydrogel microneedle according to claim 5, characterized in that: In the plant-bacteria hybrid exosome composite hydrogel microneedle, the concentration of AgNPs in the base part is 200 μg / mL, and the loading amount of GBEVs-pVEGF in the needle tip part is 10 9 ~10 11 particles / mL.

7. A method for preparing plant-bacteria hybrid exosomes, characterized in that: The specific preparation process includes: Step 1, shuttle plasmid synthesis: using a one-step cloning kit, by adding 15 to 25 bases of homologous sequences at both ends of the plasmid vector, these sequences completely match the sequence of the target cloning site. Under the action of ligase, the recombinant plasmid is integrated into the plasmid vector, thus successfully constructing the shuttle plasmid pVEGF; Step 2, preparation of engineered probiotics: culturing the probiotics to calcium-competent state, and transferring the shuttle plasmid pVEGF into the probiotics in the calcium-competent state, thereby successfully constructing the engineered probiotics; Step 3, extraction of engineered probiotic extracellular vesicles: adding the engineered probiotics to LB medium, performing primary and secondary culture, and collecting the fermentation broth of the secondary culture. Subsequently, the fermentation broth is treated by low-speed centrifugation and ultracentrifugation to obtain engineered probiotic extracellular vesicles BEVs-pVEGF; Step 4, ginger extracellular vesicle extraction: squeeze ginger juice, filter it through gauze, and then purify it by differential centrifugation and sucrose density gradient centrifugation to obtain ginger extracellular vesicles (GEVs); Step 5, preparation of plant-bacteria hybrid exovesicles: subjecting the mixed solution of the engineered probiotic exovesicles BEVs-pVEGF and ginger exovesicles GEVs to low-frequency ultrasound and membrane continuous extrusion in sequence to obtain the plant-bacteria hybrid exovesicles GBEVs-pVEGF.

8. A method for preparing plant-bacteria hybrid exosome composite hydrogel microneedles, characterized in that: The specific preparation process includes: The plant-bacteria hybrid exosome GBEVs-pVEGF and nanosilver AgNPs are mixed with AlgMA hydrogel respectively and co-incubated; the AlgMA hydrogel loaded with GBEVs-pVEGF is then injected into a microneedle mold, and vacuum degassing and heat concentration are performed; the AlgMA hydrogel loaded with AgNPs is then filled into the upper layer of the microneedle mold, and after heat concentration and photocuring, it is dried and demolded at room temperature to obtain the plant-bacteria hybrid exosome composite hydrogel microneedle.

9. Use of the plant-bacteria hybrid exosomes according to claim 1 to claim 4 in accelerating the healing of refractory diabetic wounds.

10. Use of the plant-bacteria hybrid exosome composite hydrogel microneedle according to claim 5 in accelerating the healing of refractory diabetic wounds.

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