MXene modified and engineered exosome loaded GelMA hydrogel, preparation method thereof and application thereof in preparation of burn wound repair products

Abstract

The invention provides MXene modified and engineered exosome loaded GelMA hydrogel, a preparation method thereof and application of the MXene modified and engineered exosome loaded GelMA hydrogel in preparation of burn wound repair products, and belongs to the technical field of medical biology. The preparation method comprises the following steps: introducing the anti-tagomiR-192-5p into mesenchymal stem cell MSCs exosome through an electroporation technology to obtain an exosome preparation for promoting wound healing based on regulation and control of the miR-192-5p, and recording the exosome preparation as Exo-anti-tagomiR-192-5p; the preparation method comprises the following steps: mixing Exo-antitago miR-192-5p, MXene and a methacrylated gelatin solution, so as to obtain the Exo-antitago miR-192-5p (at) M-GelMA hydrogel. The preparation method comprises the following steps: mixing the Exo-antitago miR-192-5p (at) with the methacrylated gelatin solution; in a mouse burn model, after the hydrogel is used, the wound closing speed is obviously increased, the inflammatory reaction is obviously relieved, and the tissue regeneration quality is higher.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biotechnology, and specifically relates to a MXene-modified GelMA hydrogel loaded with engineered exosomes, a preparation method thereof, and an application thereof in the preparation of burn wound repair products. Background Art

[0002] The burn wound microenvironment is extremely complex, involving multiple pathological mechanisms such as oxidative stress, excessive inflammatory response, and impaired epithelial regeneration. After a burn, local tissues produce a large amount of reactive oxygen species, leading to a sharp increase in oxidative stress levels. This oxidative damage further disrupts mitochondrial function, inhibits keratinocyte migration, and induces a persistent inflammatory response, ultimately significantly delaying the process of wound re-epithelialization. Although conventional treatment strategies such as growth factor therapy and biological dressings have promoted wound healing to a certain extent, the inhibitory effect of the oxidative stress microenvironment on epidermal cell function remains a key factor limiting the efficacy of these treatments.

[0003] In recent years, microRNA (miRNA), due to its ability to precisely regulate gene expression networks, has played a crucial role in regulating epidermal cell function under conditions of oxidative stress. Studies have shown that miRNAs regulate epidermal cell proliferation, migration, apoptosis, and inflammatory responses under conditions of oxidative stress. However, current research focuses on the role of miRNAs in epidermal cell oxidative stress damage caused by conditions such as skin aging, inflammatory skin diseases, skin cancer, and diabetic wounds. However, there are no reports on how miRNAs regulate epidermal cell function within the oxidative stress microenvironment of burn wounds.

[0004] How to apply miRNA to the treatment of burn wounds is a major challenge. Free miRNAs are easily degraded, and their ability to penetrate the cell membrane is quite limited. An efficient carrier is needed to deliver miRNAs into cells. In recent years, mesenchymal stem cells (MSCs) and their exosomes have become disruptive tools in the fields of tissue engineering and regenerative medicine. As natural nano-scale vesicles, MSC-derived exosomes (MSC-Exo) are considered to be ideal drug delivery carriers due to their low immunogenicity and high biocompatibility. Mesenchymal stem cell-derived exosomes can not only protect the encapsulated miRNA from degradation, but can also be rapidly absorbed by target cells, thereby achieving efficient gene delivery. The pro-repair proteins they carry (such as VEGF, TGF-β, etc.) can also synergistically regulate the wound microenvironment to promote wound repair. However, traditional local administration of exosomes has problems such as short retention time and easy dilution by body fluids.

[0005] Gelatin methacrylate (GelMA) is a photopolymerizable hydrogel composed of modified natural extracellular matrix (ECM) components. It exhibits morphologically adjustable properties, high transparency, excellent moisturizing properties, good biocompatibility, and superior barrier properties. GelMA hydrogels have been developed for encapsulating exosomes, prolonging their retention on wound surfaces. However, GelMA hydrogels have weak mechanical properties and lack multiple functionalities, such as antioxidant and anti-inflammatory properties. MXene (Ti3C2Tx) is a novel two-dimensional nanomaterial with excellent biocompatibility, electrical conductivity, and mechanical properties. It can also generate photothermal effects under near-infrared conditions, leading to its widespread application in biological fields such as biosensing, tumor therapy, and tracer imaging. Furthermore, studies have shown that MXene exhibits excellent antibacterial and anti-inflammatory activities, as well as some reactive oxygen species (ROS) scavenging capabilities. Furthermore, MXene can enhance the crosslinking density of the GelMA network and improve its mechanical strength. These properties are highly consistent with those required for wound dressings, making MXene a popular material for skin wound dressing modification in recent years. Therefore, MXene-modified GelMA hydrogel is expected to become an ideal delivery vehicle for exosomes. Summary of the Invention

[0006] To address the current lack of miRNAs that regulate epidermal cell function in the oxidative stress microenvironment of burn wounds, the problems of direct miRNA degradation and local administration of miRNA encapsulated in exosomes, which suffer from short retention times and susceptibility to dilution by body fluids, the present invention provides a MXene-modified GelMA hydrogel loaded with engineered exosomes, a preparation method, and its application in burn wound repair products. The multifunctional hydrogel dressing described in this invention features ease of manufacture, high biosafety, excellent mechanical properties, sustained drug release, and ideal anti-inflammatory properties, making it suitable for burn wound repair and the treatment of other oxidative stress-related diseases.

[0007] The present invention provides a method for preparing a MXene-modified GelMA hydrogel loaded with engineered exosomes, which has the following characteristics: step 1, introducing antagomiR-192-5p into the exosomes of mesenchymal stem cells MSCs by electroporation technology to obtain an exosome preparation that promotes wound healing based on miR-192-5p regulation, recorded as Exo-antagomiR-192-5p; step 2, mixing Exo-antagomiR-192-5p, MXene and methacrylated gelatin solution to obtain Exo-antagomiR-192-5p@M-GelMA hydrogel.

[0008] In the preparation method of the MXene-modified and engineered exosome-loaded GelMA hydrogel provided by the present invention, it can also have the following characteristics: wherein, in step 1, the specific preparation process of Exo-antagomiR-192-5p includes: step 1, diluting MSCs exosomes in electroporation buffer at a volume ratio of 1:9, then adding antagomiR-192-5p, transferring the mixed solution to an electroporation cuvette, standing in an ice bath for 10 minutes, and then placing the cuvette into the electroporation instrument to complete the electroporation operation.

[0009] In the preparation method of the MXene-modified and engineered exosome-loaded GelMA hydrogel provided by the present invention, the method may also have the following characteristics: wherein, in step 1, the ratio of antagomiR-192-5p to MSCs exosomes is 20 nmol:1 mg.

[0010] The preparation method of the MXene-modified GelMA hydrogel loaded with engineered exosomes provided by the present invention may also have the following characteristics: during electroporation, a 200-volt square wave voltage with a pulse length of 10 milliseconds and 5 pulses with an interval of 1 second are used.

[0011] The preparation method of the MXene-modified GelMA hydrogel loaded with engineered exosomes provided by the present invention may also have the following characteristics: wherein step 2 specifically comprises: dissolving methacrylated gelatin GelMA dry powder in a photoinitiator to prepare a 10% (w / v) GelMA solution, adding 10% of the GelMA solution to each milliliter of the GelMA solution. 11 The Exo-antagomiR-192-5p and 100 μg of MXene were added, and the obtained mixed solution was then mixed by ultrasound, and finally irradiated with light of wavelength 405 nm to obtain Exo-antagomiR-192-5@M-GelMA hydrogel.

[0012] The present invention also provides a MXene-modified and engineered exosome-loaded GelMA hydrogel having the following characteristics: the hydrogel is prepared using the above-mentioned method for preparing the MXene-modified and engineered exosome-loaded GelMA hydrogel.

[0013] The present invention also provides the use of MXene-modified GelMA hydrogel loaded with engineered exosomes in the preparation of burn wound repair products.

[0014] In the application of the MXene-modified and engineered exosome-loaded GelMA hydrogel provided by the present invention in the preparation of burn wound repair products, it can also have the following characteristics: wherein, the Exo-antagomiR-192-5p in the MXene-modified and engineered exosome-loaded GelMA hydrogel significantly reduces oxidative stress and cell apoptosis by inhibiting the function of miR-192-5p.

[0015] In the application of the MXene-modified and engineered exosome-loaded GelMA hydrogel provided by the present invention in the preparation of burn wound repair products, it can also have the following characteristics: wherein, the Exo-antagomiR-192-5p in the MXene-modified and engineered exosome-loaded GelMA hydrogel enhances the proliferation and migration ability of keratinocytes, thereby promoting wound repair.

[0016] In the application of the MXene-modified and engineered exosome-loaded GelMA hydrogel provided by the present invention in the preparation of burn wound repair products, the MXene in the MXene-modified and engineered exosome-loaded GelMA hydrogel has a certain anti-inflammatory function and can induce the phenotypic transformation of macrophages.

[0017] In the application of the MXene-modified and engineered exosome-loaded GelMA hydrogel provided by the present invention in the preparation of burn wound repair products, the MXene-modified and engineered exosome-loaded GelMA hydrogel can also have the following characteristics: wherein, the MXene-modified and engineered exosome-loaded GelMA hydrogel can increase the closure speed of burn wounds, reduce inflammatory responses and improve the quality of tissue regeneration.

[0018] Functions and effects of the invention

[0019] According to the present invention, a method for preparing a MXene-modified GelMA hydrogel loaded with engineered exosomes and its application in preparing burn wound repair products includes: step 1, introducing antagomiR-192-5p into mesenchymal stem cell (MSC) exosomes via electroporation to obtain Exo-antagomiR-192-5p; step 2, mixing Exo-antagomiR-192-5p, MXene, and a methacrylated gelatin solution to obtain an Exo-antagomiR-192-5p@M-GelMA hydrogel. The hydrogel, loaded with antagomiR-192-5p, can effectively inhibit the function of miR-192-5p, significantly reduce oxidative stress and cell apoptosis, and enhance the proliferation and migration ability of keratinocytes. The MXene component in the Exo-antagomiR-192-5p@M-GelMA hydrogel has a certain anti-inflammatory effect, can induce the transformation of macrophages from M1 to M2, and promote wound repair.

[0020] The exosome preparation (Exo-antagomiR-192-5p) was verified by TEM, NTA and immunofluorescence, showing excellent physical and chemical properties, with the characteristics of small particle size, high encapsulation efficiency, good biocompatibility and high biological uptake rate.

[0021] Exo-antagomiR-192-5p@M-GelMA hydrogel is prepared by photocuring, showing ideal mechanical strength and porous structure. It has mechanical support, anti-inflammatory and ROS scavenging capabilities, and can continuously release drugs to play a wound protection function.

[0022] In vitro experiments showed that the exosome preparation (Exo-antagomiR-192-5p) significantly improved the function of HaCaTs cells under oxidative stress and reduced the rate of cell apoptosis. In a mouse burn model, the use of Exo-antagomiR-192-5p@M-GelMA hydrogel significantly accelerated wound closure, significantly reduced inflammatory response, and improved tissue regeneration quality.

[0023] Therefore, this invention combines miRNA regulation, exosome delivery technology, and advanced materials science to provide a novel solution for burn wound treatment. Beyond burn wounds, this technology also has broad potential for the treatment of other oxidative stress-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1Figure 1 shows the construction and sequencing analysis results of the HaCaTs oxidative stress model in Example 1 of the present invention. A. Effects of different H2O2 concentrations on HaCaTs cell viability; B and C. Effects of different H2O2 concentrations on HaCaTs cell apoptosis; D. Effects of different H2O2 concentrations on HaCaTs cell cycle; E. Fluorescence image of ROS staining after H2O2 treatment of HaCaTs; F and G. Flow cytometry detection of ROS levels in HaCaTs after H2O2 stimulation; H. Volcano plot of differential miRNAs; I. Heat map of differential miRNAs; J and K: log2FC values and BaseMean values of differential miRNAs with p < 0.05, |log2FC| > 1, and expression levels (TPM) > 1; L. RT-qPCR verification of miR-192-5p expression changes in the oxidative stress model; M. RT-qPCR detection of miR-192-5p expression levels in full-thickness skin defects and burn wound margins on day 6 after injury.

[0025] Figure 2 Figure 2 shows the preparation and characterization results of Exo-antagomiR-192-5p in Example 2 of the present invention. A. TEM images of Exo-antagomiR-192-5p and MSCs exosomes; B. Particle size distribution of Exo-antagomiR-192-5p and MSCs exosomes; C. Expression of exosomal protein markers CD81, HSP70, and TSG101; D. Loading efficiency of exosomal antagomiR-192-5p; E. Internalization of Exo-antagomiR-192-5p and MSCs exosomes by HaCaTs.

[0026] Figure 3 These are the results of in vitro functional experiments of Exo-antagomiR-192-5p in Example 3 of the present invention. A. CCK8 assay detects the effect of Exo-antagomiR-192-5p on HaCaTs cell proliferation under oxidative stress; B and C. Wound-wound assay detects the effect of Exo-antagomiR-192-5p on HaCaTs cell migration under oxidative stress; D and E. Flow cytometry assays detect the effect of ExoantagomiR-192-5p on HaCaTs cell apoptosis under oxidative stress; F. Fluorescent staining image showing the effect of Exo-antagomiR-192-5p on ROS expression under oxidative stress.

[0027] Figure 4This is a diagram showing the preparation and characterization results of Exo@GelMA hydrogel and Exo@M-GelMA hydrogel in Example 4 of the present invention. Among them, A. Preparation of photocross-linked Exo@M-GelMA hydrogel; B. Injectability of Exo@M-GelMA hydrogel; C. SEM images of Exo@GelMA and Exo@M-GelMA; D. Swelling curves of Exo@GelMA and Exo@M-GelMA; E. Degradation curves of Exo@GelMA and Exo@M-GelMA; F. Exosome release curves of Exo@GelMA and Exo@M-GelMA; G. Stress-strain curves of Exo@GelMA and Exo@M-GelMA; H. CCK8 detection of the effect of co-culture with Exo@GelMA and Exo@M-GelMA on the viability of HaCaTs cells; I. Live-dead staining detection of the effect of co-culture with Exo@GelMA and Exo@M-GelMA on the viability of HaCaTs cells; J. Phalloidin staining of the cytoskeleton of HaCaTs co-cultured with Exo@GelMA and Exo@M-GelMA.

[0028] Figure 5 Figures 1 and 2 show the results of the animal experiment evaluating the Exo-antagomiR-192-5p@M-GelMA hydrogel for promoting burn wound healing in Example 5 of the present invention. A and B show representative images and healing rate statistics of burn wounds covered by the blank group, M-GelMA group, Exo@M-GelMA group, antagomiR-192-5p@M-GelMA group, and Exo-antagomiR-192-5p@M-GelMA hydrogel; C and D show HE staining of the wound surface and statistical analysis of epidermal crawling rate on day 12 after injury. (Note: In A and C, M-GelMA is abbreviated as M-Gel, Exo@M-GelMA is abbreviated as Exo@M-Gel, antagomiR-192-5p@M-GelMA is abbreviated as ant-192@M-Gel, and Exo-antagomiR-192-5p@M-GelMA is abbreviated as Exo-ant-192@M-Gel) DETAILED DESCRIPTION

[0029] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the following examples, combined with the accompanying drawings, specifically illustrate the exosome preparation for promoting wound healing based on miR-192-5p regulation, its preparation method and application.

[0030] Example 1

[0031] This example is the construction and sequencing analysis of the HaCaTs oxidative stress model.

[0032] 1.1 Experimental methods

[0033] (1) CCK-8 kit to detect cell viability

[0034] HaCaTs cells were plated in a 96-well plate at a density of approximately 4000 cells per well and cultured for 12 hours. After the cells were completely attached, complete culture medium containing 0, 0.2, 0.4, 0.6, 0.8, and 1 mmol / L H2O2 was added and cultured for 12 hours. The culture medium was then replaced with 100 μl of new complete culture medium containing 10 μl CCK-8. After incubation in the dark for 2 hours, the OD value of each well at an absorbance of 450 nm was detected using a microplate reader.

[0035] (2) Annexin V / PI flow cytometry detection of cell apoptosis

[0036] The cells were plated in 6-well plates, with approximately 5 × 10 5 Cells were cultured in complete medium to a density of approximately 70% and then various concentrations of H₂O₂ were added. After 12 hours of culture, the cells were digested to a single-cell suspension, resuspended in 300 μL of buffer, and incubated at room temperature for 5 minutes with 3 μL of FITC-Annexin V and 6 μL of propidium iodide (PI). The cells were then loaded onto a flow cytometer and the proportion of apoptotic cells was determined.

[0037] (3) Flow cytometry detection of cell cycle

[0038] The cells were plated in 6-well plates, with approximately 5 × 10 5 Cells were cultured in complete medium to a density of approximately 70% and then various concentrations of H₂O₂ were added. Culture was continued for 12 hours, then digested to a single-cell suspension and centrifuged to discard the supernatant. Washed once with PBS and centrifuged to discard the supernatant. Added 1 ml of DNA staining solution and 10 μL of permeabilization solution and vortexed for 5-10 seconds to mix. Incubated at room temperature in the dark for 30 minutes. Samples were loaded at the lowest speed and analyzed on a flow cytometer. Data were analyzed using ModFit LT to generate cell cycle distribution maps.

[0039] (4) Quantitative detection of intracellular ROS levels

[0040] Intracellular reactive oxygen species (ROS) levels in HaCaTs cells were measured using the oxidation-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). HaCaTs cells were seeded in 24-well plates and treated with 0.6 mmol / L H2O2 for 12 hours the following day. After treatment, the cells were washed with DMEM to remove residual residues. The cells were then incubated with 10 μM DCFH-DA dye for 10 minutes. After staining, the cells were washed again with PBS to remove unbound dye. Intracellular fluorescence signals were detected using flow cytometry to quantify ROS levels. Fluorescence signals were observed and captured using a fluorescence microscope to visualize ROS expression.

[0041] (5) Small RNA sequencing

[0042] HaCaT cells were stimulated with 0.6 mmol / L H₂O₂ for 12 hours to establish an oxidative stress model. Three replicates were set up for the oxidative stress model and the control group. Total RNA was extracted using Trizol reagent according to the manufacturer's instructions. The total amount and integrity of RNA were assessed using a NanoDrop One and an Agilent 2100 Bioanalyzer, respectively. One microgram of total RNA from each sample was used to construct a small RNA library using the NEBNext Small RNA Library Prep Set for Illumina. Briefly, sequencing adapters were ligated to both ends of the RNA, and the adapter-ligated RNA was reverse transcribed into cDNA, followed by PCR amplification. Subsequently, 140-160 bp PCR products were isolated and purified by agarose gel electrophoresis to form the small RNA library. After library quality assurance using an Agilent 2100 Bioanalyzer, the library was sequenced using the Illumina Novaseq 6000 platform to generate 150 bp paired-end reads.

[0043] (6) Bioinformatics analysis

[0044] The raw image data files obtained by high-throughput sequencing are converted into raw sequencing sequences through base calling analysis, which are called RawReads. Low-quality reads are further filtered to remove reads containing 5' primers and poly(A) tails. Reads without 3' adapters and tag sequences, and reads with lengths less than 15nt or greater than 41nt are filtered to obtain clean reads.

[0045] First, the length distribution of clean reads in the reference genome was analyzed. Then, the sequences were aligned with the Rfam v10.1 database using Bowtie software. rRNA, scRNA, Cis-reg, snRNA, and tRNA sequences were annotated and filtered. Subsequently, Bowtie software was used to sequentially align and annotate with cDNA sequences, the Repbase database, and the miRBase database to remove degraded transcript sequences and repetitive sequences. This allowed the identification and annotation of known miRNAs, and also analyzed the expression patterns of known miRNAs in different samples. Unannotated reads were then analyzed using miRDeep2 to predict novel miRNAs. Based on the hairpin structure of pre-miRNAs and the miRBase database, miRDeep2 can identify mature and star sequences of corresponding miRNAs.

[0046] When calculating differentially expressed miRNAs, the default thresholds for filtering were q-value < 0.05 and FC > 2 or FC < 0.5. For samples with biological replicates, q-values were calculated using the DEG algorithm in the R package, while for samples without biological replicates, q-value statistics were calculated using the Audic Claverie algorithm.

[0047] 1.2 Experimental Results

[0048] The results showed that H2O2 could significantly inhibit the cell viability of HaCaTs ( Figure 1 A), promote cell apoptosis ( Figure 1 B and Figure 1 C) and arrest the cell cycle at S phase ( Figure 1 D), and has a concentration-dependent effect; Based on the above experimental results, the modeling condition was selected as 0.6mmol / L H2O2 stimulation of HaCaTs for 12 hours. After staining the cells with a reactive oxygen species detection kit and observing them using fluorescence microscopy and flow cytometry, the results showed that the ROS content in the modeling group was significantly increased, indicating that the model was successfully established ( Figure 1 EG). Then, miR-192-5p ( Figure 1 HK), and its expression was verified by RT-qPCR in HaCaTs oxidative stress model and burn wound ( Figure 1 L and M).

[0049] Example 2

[0050] This embodiment provides an exosome preparation (Exo-antagomiR-192-5p) that promotes wound healing based on miR-192-5p regulation, and its preparation method and characterization.

[0051] 2.1 Experimental methods

[0052] (1) Preparation of Exo-antagomiR-192-5p

[0053] By introducing antagomiR-192-5p into mesenchymal stem cell (MSC) exosomes (Exo) through electroporation technology, an engineered exosome encapsulated with antagomiR-192-5p was obtained, i.e., an exosome preparation (Exo-antagomiR-192-5p) that promotes wound healing based on miR-192-5p regulation. Among them, antagomiR-192-5p is an RNA sequence that antagonizes miR-192-5p, playing a role in inhibiting miR-192-5p.

[0054] The preparation process is as follows:

[0055] MSCs exosomes were diluted in Gene Pulser electroporation buffer (Bio-Rad) at a volume ratio of 1:9, and then antagomiR-192-5p and antagomiR-NC (as a control) were added. The mixture was transferred to a 4mm GenePulser / MicroPulser electroporation cuvette, and after standing in an ice bath for 10 minutes, the cuvette was placed in the Gene PulserXcell TM Total System instrument. Electroporation was performed using a square wave voltage (200 V), a 10-millisecond pulse length, and five pulses at 1-second intervals. The buffer was removed and the exosomes purified by ultrafiltration to obtain Exo-antagomiR-192-5p and its control.

[0056] In this example, MSCs exosomes were purchased from Beijing Enze Kangtai Biotechnology Co., Ltd. and antagomiR-192-5p was purchased from Aikerui Biotechnology Co., Ltd.

[0057] (2) Characterization of Exo-antagomiR-192-5p

[0058] The size distribution and typical morphology of exosomes were characterized by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Exosome marker proteins, including tumor susceptibility gene 101 (TSG101), CD81, and heat shock protein 7 (HSP70), were detected by Western blot.

[0059] (3) Loading efficiency of Exo-antagomiR-192-5p

[0060] According to the above method, Cy3-labeled antagomiR-192-5p was encapsulated into Exo by electroporation, and the obtained liquid was diluted to 100 μL with PBS; an equal amount of Cy3-labeled antagomiR-192-5p was diluted to 100 μL with PBS; the diluted Exo-cy3-antagomiR-192-5p and cy3-antagomiR-192-5p were added to a 96-well plate, and three replicate wells were set up for each group. The SpectraMax microplate reader was used for detection with an excitation wavelength of 532 nm and an emission wavelength of 580 nm. The ratio of encapsulated antagomiR-192-5p to the initial total antagomiR-192-5p was calculated by the obtained absorbance.

[0061] (4) Uptake of Exo-antagomiR-192-5p

[0062] PKH67 was used to stain the Cy3-labeled antagomiR-192-5p engineered exosomes and natural exosomes. The two were co-incubated with HaCaTs for 4 hours and then washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 10 minutes and then washed three times with PBS. The cell nuclei were stained with DAPI for 10 minutes and washed, and then observed under a fluorescence microscope.

[0063] 2.2 Experimental Results

[0064] Electroporated engineered exosomes encapsulating antagomiR-192-5p (Exo-antagomiR-192-5p) were compared with untreated MSC exosomes. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot analysis were used to characterize the morphology, size distribution, and biomarkers of the two exosomes.

[0065] TEM showed that the exosomes used in the experiment showed the typical morphology of exosomes ( Figure 2 A). NTA results showed that the particle size distribution of the exosomes used in the experiment was consistent with the size characteristics of exosomes ( Figure 2 B). Western blot analysis characterized exosomal protein markers, showing the presence of positive markers CD81, HSP70, and TSG101, while the negative marker Calnexin was absent ( Figure 2 C). The loading efficiency of Exo-Cy3-antagomiR-192-5p was 35.22±0.34% ( Figure 2 D). The red fluorescence of antagomiR-192-5p appears around the cell nucleus, indicating that the engineered exosomes can be successfully internalized into the perinuclear region of the cell and release antagomiR-192-5p ( Figure 2 E).

[0066] Example 3

[0067] This example is an experiment to improve the in vitro function of Exo-antagomiR-192-5p prepared in Example 2.

[0068] 3.1 Experimental methods

[0069] (1) CCK-8 kit to detect cell viability

[0070] HaCaTs cells were plated in a 96-well plate at a density of approximately 4,000 cells per well and cultured in complete medium for 24 hours. The cells were then stimulated with 0.6 mmol / L H₂O₂ for 12 hours. Exo-antagomiR-192-5p prepared in Example 2 and its control were then added and cultured for a further 24 hours. To assess cell viability, the medium was replaced with 100 μL of new complete medium containing 10 μL of CCK-8. After incubation in the dark for 2 hours, the OD value of each well was measured using a microplate reader at an absorbance of 450 nm.

[0071] (2) Scratch assay to detect cell migration ability

[0072] Cells were plated in 6-well plates. After reaching 100% cell density, HaCaTs were stimulated with 0.6 mmol / L H₂O₂ for 12 hours. A 200 μL pipette tip was then used to scratch the cell layer. The culture medium was then replaced with complete medium containing the Exo-antagomiR-192-5p prepared in Example 2 and a control. Images of the scratched area were captured using an inverted microscope at designated time points, and the wound healing rate was calculated using ImageJ software.

[0073] (3) Annexin V / PI flow cytometry detection of cell apoptosis

[0074] Cells were plated in 6-well plates and cultured to a density of 70% in complete culture medium. HaCaTs were stimulated with 0.6 mmol / L H₂O₂ for 12 hours. Exo-antagomiR-192-5p prepared in Example 2 and its control were then added and cultured for another 24 hours. The cells were then digested to prepare a single-cell suspension. The cells were resuspended in 300 μL of buffer, and 3 μL of FITC-Annexin V and 6 μL of propidium iodide (PI) were added, followed by incubation at room temperature for 5 minutes. The percentage of apoptotic cells was determined by flow cytometry.

[0075] (4) Reactive oxygen detection

[0076] HaCaTs cells were seeded in a 6-well plate and cultured for 12 hours in complete medium containing 0.6 mmol / L H2O2 when the cell density reached 70%. Exo-antagomiR-192-5p prepared in Example 2 and its control were added and cultured for another 24 hours. The cells were then washed with DMEM to remove residues. The cells were then incubated with 10 μM DCFH-DA stain for 10 minutes. After staining, the cells were washed again with PBS to remove unbound dye. Intracellular fluorescence signals were observed and captured using a fluorescence microscope to visually display ROS expression.

[0077] 3.2 Experimental Results

[0078] The results showed that the preparation significantly improved the viability of HaCaTs cells under oxidative stress conditions ( Figure 3 A), scratch test showed its promoting effect on cell migration ability ( Figure 3 B and Figure 3 C). In addition, Exo-antagomiR-192-5p can also reduce the apoptosis rate of epidermal cells ( Figure 3 D and Figure 3 E) and reduce the production of ROS ( Figure 3 F).

[0079] Example 4

[0080] This example is the preparation and characterization of exosome-loaded hydrogel.

[0081] 4.1 Experimental methods

[0082] (1) Preparation of exosome-loaded hydrogel

[0083] The GelMA powder was dissolved in the photoinitiator to prepare a 10% (w / v) GelMA solution. In the first group, only 10 11 In the second group, 10 MSC Exo were added per ml of GelMA solution. 11 MSC Exo and 100 μg MXene; the third group, only 10 11 The fourth group, each milliliter of GelMA solution was added with 10 11The Exo-antagomiR-192-5p prepared in Example 2 and 100 μg of MXene were prepared. The mixed solutions obtained from Groups 1 to 4 were ultrasonically mixed and finally irradiated with 405 nm wavelength light to obtain Exo@GelMA hydrogel, Exo@M-GelMA hydrogel, Exo-antagomiR-192-5p@GelMA hydrogel, and Exo-antagomiR-192-5p@M-GelMA hydrogel, respectively.

[0084] (2) Characterization of Exo@GelMA hydrogel and Exo@M-GelMA hydrogel

[0085] After the hydrogel is formed, the original weight of the hydrogel is weighed. d The hydrogel was then immersed in PBS and placed in a 37°C constant temperature shaker. After the preset time point, the hydrogel was taken out and the free liquid on the surface was absorbed with filter paper. The wet weight of the hydrogel after expansion (W t ) Calculate the swelling ratio SR of the hydrogel. SR=(W t -W d ) / W d .

[0086] The original weight of the sample was weighed and recorded as W0. The hydrogel was soaked in PBS solution for 24 consecutive days, and the PBS solution was replaced every day. After reaching the preset time point, the free liquid on the sample surface was removed and weighed and recorded as W0. t The percentage of the remaining mass is calculated as W t / W0×100%.

[0087] Exo@GelMA and Exo@M-GelMA hydrogels were immersed in PBS buffer and placed on a 37°C shaker at 80 rpm. Supernatants were collected every two days for 24 days and protein levels were measured using a BCA protein quantification kit. Protein release rates were calculated by dividing the measured protein level in PBS by the initial loading amount.

[0088] To evaluate the mechanical properties of Exo@GelMA and Exo@M-GelMA hydrogel samples, stress-strain curves were obtained by compressing the samples using an electronic universal testing machine. The morphologies of the Exo@GelMA and Exo@M-GelMA hydrogel samples were photographed using a scanning electron microscope.

[0089] (3) Biocompatibility testing of Exo@GelMA hydrogel and Exo@M-GelMA hydrogel

[0090] Cell viability was detected using the CCK-8 kit: HaCaTs were seeded in a 24-well plate and co-cultured with Exo@GelMA and Exo@M-GelMA hydrogels for 24 and 48 hours, and then the CCK-8 reagent was replaced in the lower chamber for incubation for 2 hours. The liquid in the lower chamber was then transferred to a 96-well plate, and the OD value of each well at an absorbance of 450 nm was detected using a microplate reader.

[0091] Live / dead staining assay: To evaluate the biocompatibility of Exo@GelMA and Exo@M-GelMA hydrogels, a live / dead staining assay was performed using a staining kit. HaCaTs were seeded in 24-well plates and co-cultured with Exo@GelMA and Exo@M-GelMA hydrogels. After 24, 72, and 120 hours, the upper chamber was removed, and the prepared working solutions of live cell dye (calcein AM, 1:500) and dead cell dye (EthD-1, 1:2000) were added to the lower chamber. The cells were incubated in the dark for 30 minutes, washed three times with PBS, and observed and photographed using a fluorescence microscope.

[0092] Cytoskeleton staining using phalloidin: HaCaTs cells were seeded in a 24-well plate and co-cultured with Exo@GelMA and Exo@M-GelMA hydrogels for 72 hours. The cells were then washed twice with PBS to remove residual culture medium. Subsequently, the cells were fixed with 4% paraformaldehyde for 15 minutes (room temperature) and then treated with 0.1% Triton X-100 in PBS for 5 minutes to increase the permeability of the cell membrane. 1 μL of 1000× phalloidin staining solution was added to 1 mL of PBS containing 1% BSA and mixed evenly to prepare a staining working solution. 200 μL of the staining working solution was added to each well and incubated at room temperature for 90 minutes in the dark. After staining, the cells were washed again three times with PBS. Subsequently, the cell nuclei were counterstained with DAPI for 5 minutes. Finally, fluorescence microscopy was used for observation.

[0093] 4.2 Experimental Results

[0094] Figure 4 A shows that the hydrogel solidifies after being irradiated with light at a wavelength of 405 nm. Figure 4 B shows the injectability of the hydrogel. SEM results show that the prepared Exo@GelMA hydrogel and Exo@M-GelMA hydrogel have rich porous structures ( Figure 4 C). The swelling curves of Exo@GelMA and Exo@M-GelMA hydrogels are similar ( Figure 4 D). Degradation tests showed that the degradation rates of the two hydrogels were similar ( Figure 4E). In addition, BSA was used to detect and plot the exosome release curve of the hydrogel. The results showed that the exosome release rate of Exo@M-GelMA was slower than that of Exo@GelMA ( Figure 4 F). Stress-strain curves show that Exo@M-GelMA hydrogel has stronger mechanical properties ( Figure 4 G). CCK8( Figure 4 H), live-dead staining ( Figure 4 I) and phalloidin ( Figure 4 J) Staining showed that Exo@GelMA and Exo@M-GelMA were non-cytotoxic and highly biosafe.

[0095] In addition, since the introduction of antagomiR has almost no effect on the performance of MSCs Exo hydrogel, the characterization results of the above-mentioned Exo@GelMA hydrogel and Exo@M-GelMA hydrogel are similar to those of Exo-antagomiR-192-5p@GelMA hydrogel and Exo-antagomiR-192-5p@M-GelMA hydrogel.

[0096] Example 5

[0097] This example is an in vivo functional experiment of the Exo-antagomiR-192-5p@M-GelMA hydrogel prepared in Example 4.

[0098] 5.1 Experimental methods

[0099] (1) Establishment of mouse burn model

[0100] After isoflurane is used to anesthetize C57BL / 6 mice (male, 8-10 week old), the hair on the back of the mice is removed. The copper rod that is 8mm in diameter heated in boiling water is gently pressed vertically on the back of the mice for 10 seconds, causing deep burns. Immediately after the burn, the wound surface is wet-hatched with the gauze soaked with chlorhexidine. Subsequently, the wound is bandaged with gauze and covered with iodine film. After two days, escharectomy is performed.

[0101] (2) Preparation of hydrogel

[0102] M-GelMA: 100 μg of MXene was added to 1 ml of GelMA solution, the resulting mixed solution was mixed by ultrasound, and finally irradiated with light at a wavelength of 405 nm to obtain M-GelMA hydrogel.

[0103] Exo@M-GelMA: prepared according to the preparation steps of Exo@M-GelMA hydrogel described in Example 4.

[0104] antagomiR-192-5p@M-GelMA: 16.7 nmol of antagomiR-192-5p and 100 μg of MXene were added to 1 ml of GelMA solution. The resulting mixed solution was mixed using ultrasound and finally irradiated with light at a wavelength of 405 nm to obtain antagomiR-192-5p@M-GelMA hydrogel.

[0105] Exo-antagomiR-192-5p@M-GelMA: prepared according to the preparation steps of Exo-antagomiR-192-5p@M-GelMA hydrogel described in Example 4.

[0106] (2) Hydrogel covering the wound

[0107] Given that Exo@M-GelMA hydrogel exhibited superior exosome sustained-release performance and mechanical properties to Exo@GelMA, M-GelMA, Exo@M-GelMA, antagomiR-192-5p@M-GelMA, and Exo-antagomiR-192-5p@M-GelMA hydrogels were used to cover the burn wound after eschar removal. The wounds were cleaned, the dressings were changed, photographed, and samples were collected at predetermined time points until they were completely healed. The wound healing rate was calculated using ImageJ software.

[0108] 5.2 Experimental Results

[0109] The experimental results showed that the wound surface of the Exo-antagomiR-192-5p@M-GelMA group had the fastest re-epithelialization speed and was almost completely healed on the 12th day after injury, with a healing rate of 97.49±0.844% ( Figure 5 A and B). H&E staining confirmed the results. On day 12 after injury, complete re-epithelialization of the wound surface was observed in the Exo-antagomiR-192-5p@M-GelMA group, while the re-epithelialization process was delayed in the control group ( Figure 5 C and D).

[0110] Those skilled in the art will appreciate that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a MXene-modified GelMA hydrogel loaded with engineered exosomes, characterized in that: include: Step 1: antagomiR-192-5p is introduced into exosomes of mesenchymal stem cells (MSCs) by electroporation to obtain an exosome preparation that promotes wound healing based on miR-192-5p regulation, which is recorded as Exo-antagomiR-192-5p. Step 2: Mix the Exo-antagomiR-192-5p, MXene, and methacrylated gelatin solution to obtain Exo-antagomiR-192-5p@M-GelMA hydrogel.

2. The method for preparing the MXene-modified GelMA hydrogel loaded with engineered exosomes according to claim 1, characterized in that: in, In step 1, the specific preparation process of Exo-antagomiR-192-5p includes: Step 1: Dilute MSCs exosomes in electroporation buffer at a volume ratio of 1:9, then add antagomiR-192-5p, transfer the mixture to the electroporation cuvette, let it stand in an ice bath for 10 minutes, and then place the cuvette into the electroporation instrument to complete the electroporation operation.

3. The method for preparing the MXene-modified GelMA hydrogel loaded with engineered exosomes according to claim 2, characterized in that: in, In step 1, the ratio of antagomiR-192-5p to MSCs exosomes was 20 nmol:1 mg.

4. The method for preparing the MXene-modified GelMA hydrogel loaded with engineered exosomes according to claim 2, wherein: in, For electroporation, a square wave voltage of 200 V was used with a pulse length of 10 msec and 5 pulses with an interval of 1 second.

5. The method for preparing the MXene-modified GelMA hydrogel loaded with engineered exosomes according to claim 1, wherein: in, Step 2 specifically includes: Methacrylated gelatin GelMA powder was dissolved in photoinitiator to prepare 10% (w / v) GelMA solution. 10 11 The Exo-antagomiR-192-5p and 100 μg of MXene were added, and the obtained mixed solution was mixed by ultrasound, and finally irradiated with 405 nm wavelength light to obtain the Exo-antagomiR-192-5p@M-GelMA hydrogel.

6. The MXene-modified and engineered exosome-loaded GelMA hydrogel according to claim 5, characterized in that It is prepared by the preparation method of the MXene-modified and engineered exosome-loaded GelMA hydrogel according to any one of claims 1 to 5.

7. Application of MXene-modified GelMA hydrogel loaded with engineered exosomes in the preparation of burn wound repair products.

8. Use of the MXene-modified GelMA hydrogel loaded with engineered exosomes according to claim 7 in the preparation of a burn wound repair product, characterized in that: in, Exo-antagomiR-192-5p in the MXene-modified and engineered exosome-loaded GelMA hydrogel significantly reduces oxidative stress and cell apoptosis by inhibiting the function of miR-192-5p.

9. Use of the MXene-modified GelMA hydrogel loaded with engineered exosomes according to claim 7 in the preparation of a burn wound repair product, characterized in that: in, Exo-antagomiR-192-5p in the MXene-modified and engineered exosome-loaded GelMA hydrogel enhances the proliferation and migration ability of keratinocytes, thereby promoting wound repair.

10. Use of the MXene-modified GelMA hydrogel loaded with engineered exosomes according to claim 7 in the preparation of a burn wound repair product, characterized in that: in, The MXene-modified GelMA hydrogel loaded with engineered exosomes can accelerate the closure of burn wounds, reduce inflammatory responses, and improve the quality of tissue regeneration.

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