Exosome composition for alleviating skin burn and preparation method thereof

Abstract

The application discloses an exosome component for relieving skin burns and a preparation method thereof, and belongs to the field of biomedical materials, and comprises the following components: sodium alginate, a multi-mechanism fluoride ion trapping agent, superoxide dismutase, trehalose, gelatin, an acid-triggered intracellular calcium delivery body, an engineered exosome and calcium gluconate; the application realizes the capture of fluoride ions through arginine-modified zirconium metal organic framework materials; the acid-triggered intracellular calcium delivery body is used to restore the intracellular calcium homeostasis damaged by fluoride ions; the engineered exosome is used to realize the accurate identification and efficient delivery of target cells on a wound surface, and significantly accelerate the wound healing; the application integrates multiple functions such as fluoride ion capture, calcium homeostasis restoration, antioxidant protection and tissue repair, and forms an intelligent biomaterial system for systematically solving the complex pathological problems of hydrogen fluoride acid burns.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and relates to an exosome component for relieving skin burns and its preparation method. Background Technology

[0002] Skin burns are a common type of tissue injury, with hydrofluoric acid burns being particularly severe due to their unique pathological mechanisms. The hydrogen ions in hydrofluoric acid can cause tissue protein coagulation and acid burns, while the fluoride ions have extremely strong tissue penetrating power, reaching deep into the subcutaneous tissue and even bone. They combine with calcium and magnesium ions to form insoluble salts, leading to progressive tissue necrosis and severe pain, and may cause fatal hypocalcemia. Traditional treatments, such as local injections or topical application of calcium gluconate, can partially neutralize fluoride ions, but they are difficult to effectively track and neutralize the continuously penetrating fluoride ions, and cannot reverse the deep cellular damage already caused.

[0003] In recent years, molecular biology research has further revealed the underlying mechanisms of hydrofluoric acid toxicity. Studies have shown that fluoride ion exposure can significantly downregulate the expression of fibroblast growth factor 21 (FGF21) and its receptor complex in myoblast models (such as C2C12 cells). FGF21 is a key metabolic regulator and cell-protective factor that promotes cell survival, proliferation, and differentiation by binding to its receptor and activating the downstream MAPK / ERK signaling pathway. The inhibition of the FGF21-receptor-MAPK / ERK signaling axis by fluoride ions directly leads to the obstruction of myoblastic differentiation in C2C12 cells, which explains at the molecular level an important reason for delayed tissue repair and poor functional recovery after hydrofluoric acid burns.

[0004] Exosomes, as nanoscale vesicles secreted by cells, have shown great potential in tissue engineering and regenerative medicine due to their natural biocompatibility, low immunogenicity, and excellent ability to penetrate biological barriers. They can serve as ideal carriers for bioactive molecules (such as proteins and nucleic acids), delivering them to lesion sites, regulating the local immune microenvironment, and promoting tissue repair. However, directly applying exosomes to the unique and harsh wound environment of hydrofluoric acid burns faces significant challenges: exosomes themselves are easily inactivated by residual fluoride ions and severe oxidative stress in the wound, and single exosome therapy is insufficient to address multiple pathological factors such as continuous fluoride ion penetration and intracellular calcium imbalance. Summary of the Invention

[0005] The purpose of this invention is to provide an exosome component for relieving skin burns and its preparation method, specifically for hydrofluoric acid burn wounds.

[0006] The technical solution adopted in this invention is as follows: An exosome component for relieving skin burns includes phase A and phase B, wherein phase A and phase B are mixed in a volume ratio of 1:1 to form a hydrogel before use; Phase A comprises the following components by weight: 1.5-2.5 parts sodium alginate, 2.0-4.0 parts multi-mechanism fluoride ion trapping agent, 0.010-0.025 parts superoxide dismutase, 0.05-0.15 parts trehalose, and deionized water to a total of 100 parts. Phase B comprises the following components by weight: 3.0-5.0 parts gelatin, 1.5-3.0 parts acid-triggered intracellular calcium delivery body, 0.00005-0.00015 parts engineered exosomes based on the amount of exosome protein they contain, 0.1-0.3 parts calcium gluconate, and phosphate buffer to a total of 100 parts. The multi-mechanism fluoride ion trapping agent is an arginine-modified zirconium-based metal-organic framework material; The acid-triggered intracellular calcium delivery system is a core-shell structured nanoparticle. Its outer shell is made of aldehyde polyethylene glycol grafted with polyacrylhydrazine via hydrazone bonds. Its core contains a thiolized casein phosphopeptide-calcium complex linked by disulfide bonds and thiol-modified cell-penetrating peptides. The engineered exosomes are genetically engineered human mesenchymal stem cell-derived exosomes with LAMP2-RGD fusion protein on their membrane surface and rich in FGF21 in their lumen.

[0007] Furthermore, the FGF21 content in the engineered exosome protein is 50-200 ng / μg exosome protein.

[0008] Furthermore, the phosphate buffer solution has a concentration of 0.01 mol / L and a pH value of 7.4.

[0009] Furthermore, the multi-mechanism fluoride ion trapping agent is prepared by the following method: A1. Dissolve zirconium chloride hexahydrate in anhydrous N,N-dimethylformamide to obtain a zirconium chloride solution; add terephthalic acid and glacial acetic acid to anhydrous N,N-dimethylformamide and dissolve completely to obtain a terephthalic acid solution; add the zirconium chloride solution dropwise to the terephthalic acid solution. After the addition is complete, transfer the solution to a high-pressure reactor, seal it, and allow it to react. After the reaction is complete, allow it to cool naturally to room temperature. Pour the reaction solution into a centrifuge tube, centrifuge, collect the precipitate, resuspend the precipitate in fresh N,N-dimethylformamide, then resuspend it in anhydrous ethanol, and dry it under vacuum to obtain solid A. A2. Disperse solid A in anhydrous ethanol, sonicate to form a uniform suspension, add L-arginine, stir magnetically until dissolved, reflux in an oil bath at 70°C under nitrogen protection, after the reaction is complete, cool to room temperature, centrifuge, wash with anhydrous ethanol, and vacuum dry to obtain a multi-mechanism fluoride ion trapping agent.

[0010] Furthermore, the acid-triggered intracellular calcium delivery body is prepared by the following method: B1. Aldehyde polyethylene glycol is dissolved in anhydrous dimethyl sulfoxide to obtain an aldehyde polyethylene glycol solution; polyacrylhydrazine is dissolved in anhydrous dimethyl sulfoxide and slowly added dropwise to the above aldehyde polyethylene glycol solution; glacial acetic acid is added to adjust the pH to 5.0~6.0; the reaction is carried out under nitrogen protection; after the reaction is completed, the reaction solution is added dropwise to pre-cooled anhydrous diethyl ether; the precipitate is collected by centrifugation, washed with anhydrous diethyl ether, and dried under vacuum to obtain an acid-sensitive polymer; B2. Casein phosphopeptide and calcium gluconate were dissolved in phosphate buffer at pH 7.4 and stirred to obtain a casein phosphopeptide-calcium complex solution. 2-Iminothiacyclopentane hydrochloride was then added, and the reaction was carried out at 25°C under nitrogen protection and in the dark. After the reaction was completed, the solution was dialyzed with deionized water. The dialyzed solution was diluted with pH 7.0 phosphate buffer to obtain a thiolized calcium complex solution. Thiol-modified cell-penetrating peptides were dissolved in pH 7.0 phosphate buffer and added dropwise to the above thiolized calcium complex solution. The reaction was carried out under light protection and stirred. After the reaction, air was slowly introduced into the solution and gently stirred overnight. The solution was dialyzed with deionized water, and the solution was filtered and sterilized to obtain the core calcium complex. B3. The acid-sensitive polymer and the core calcium complex were dissolved together in a pH 8.0 Tris-HCl buffer to obtain an aqueous phase. In an ice-water bath, the aqueous phase was added to dichloromethane containing 1 wt% Span 80 and emulsified to form a primary emulsion. The primary emulsion was immediately poured into a 1 wt% polyvinyl alcohol aqueous solution and homogenized to form a secondary emulsion. The resulting secondary emulsion was magnetically stirred at room temperature, then centrifuged, the precipitate was resuspended in deionized water and centrifuged again, and finally resuspended in a pH 7.4 phosphate buffer and filtered to obtain the acid-triggered intracellular calcium delivery body.

[0011] Furthermore, the engineered exosomes are prepared by the following method: C1. Human umbilical cord mesenchymal stem cells were infected with a lentiviral vector carrying the FGF21 gene and the LAMP2-RGD fusion gene. After 48 hours of infection, the culture medium was replaced with a selective medium containing 1 μg / mL puromycin. The culture medium was replaced every 2-3 days and the selection was continued for 2 weeks to obtain a stable transfected cell line. C2. The stably transfected cell lines were cultured in T175 culture flasks. The cells were washed twice with PBS and replaced with α-MEM medium containing 10% exosome-free fetal bovine serum. The cells were cultured for 48-72 hours. The cell culture supernatant was collected and transferred to centrifuge tubes. The following centrifugation was performed sequentially: 300g at 4°C for 10 minutes; 2,000g at 4°C for 20 minutes; and 10,000g at 4°C for 45 minutes. The supernatant was filtered, polyethylene glycol 6000 was added, and the mixture was thoroughly mixed and incubated overnight at 4°C. The supernatant was then carefully discarded and the precipitate was gently resuspended. The precipitate was washed and purified by ultracentrifugation, and the supernatant was discarded. The precipitate at the bottom of the tube was gently resuspended to obtain engineered exosomes.

[0012] The method for preparing an exosome component for relieving skin burns includes the following steps: S1. Preparation of phase A: Sodium alginate is added to deionized water and stirred at room temperature until completely dissolved. Multi-mechanism fluoride ion trapping agent, superoxide dismutase and trehalose are added in sequence. The mixture is placed in an ice-water bath and ultrasonically dispersed using an ultrasonic cell disruptor. After ultrasonication, deionized water is added, and the mixture is gently stirred and mixed. The solution is transferred to a vacuum desiccator and allowed to stand at room temperature to remove air bubbles. S2. Preparation of phase B: Gelatin is added to phosphate buffer and stirred in a 40°C water bath until completely dissolved. Acid-triggered intracellular calcium delivery body and calcium gluconate are added in sequence and stirred until uniformly dispersed. After natural cooling, engineered exosomes are added in an ice bath and mixed by gently and repeatedly inverting the container. S3. Preparation of hydrogel: Before use, put phase A and phase B into the two independent chambers of the double-barrel syringe respectively. After cleaning the burn wound, rinse it with physiological saline and dry it. Align the outlet of the static mixing head of the double-barrel syringe with the center of the wound and push the plungers on both sides evenly. At a body temperature of 37°C, it will gradually form a gel within 2-4 minutes.

[0013] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. This invention uses arginine-modified zirconium-based metal-organic framework materials to capture fluoride ions, thereby blocking the secondary toxicity of fluoride ions at the source and creating conditions for subsequent repair; and uses acid-triggered intracellular calcium delivery systems to achieve targeted delivery of calcium ions from in vitro to intracellular, restoring intracellular calcium homeostasis disrupted by fluoride ions.

[0014] 2. This invention achieves precise identification and efficient delivery of target cells to wounds through engineered exosomes (RGD targeting + FGF21 therapy). The multiple repair mechanisms of FGF21 significantly accelerate wound healing and reduce scar formation.

[0015] 3. This invention integrates multiple functions such as fluoride ion capture, calcium homeostasis restoration, antioxidant protection, and tissue repair, forming an intelligent biomaterial system that systematically solves the complex pathological problems of hydrofluoric acid burns. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort, wherein: Figure 1 This is a flowchart of the preparation of an exosome component for relieving skin burns according to Embodiment 1 of the present invention. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments. The components of the embodiments of the invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0018] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0019] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0020] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0021] Example 1: This example provides an exosome component for relieving skin burns, comprising phase A and phase B, wherein phase A and phase B are mixed in a volume ratio of 1:1 to form a hydrogel before use; Phase A comprises the following components by weight: 2.0 parts sodium alginate, 3.0 parts multi-mechanism fluoride ion trapping agent, 0.018 parts superoxide dismutase, 0.10 parts trehalose, and deionized water to a total of 100 parts. Phase B comprises the following components by weight: 4.0 parts gelatin, 2.2 parts acid-triggered intracellular calcium delivery body, 0.0001 parts engineered exosomes based on the amount of exosome protein they contain, 0.2 parts calcium gluconate, and phosphate buffer to a total of 100 parts. The multi-mechanism fluoride ion trapping agent is an arginine-modified zirconium-based metal-organic framework material; The acid-triggered intracellular calcium delivery body is a core-shell structured nanoparticle. Its outer shell is made of polyethylene glycol grafted with polyacrylamide via hydrazone bonds; its core contains a casein phosphopeptide-calcium complex linked to a cell-penetrating peptide via disulfide bonds. The engineered exosomes are genetically engineered human mesenchymal stem cell-derived exosomes with LAMP2-RGD fusion protein on their membrane surface and rich in FGF21 in their lumen.

[0022] The engineered exosomes contained 125 ng / μg of FGF21 in exosomal protein.

[0023] The phosphate buffer solution has a concentration of 0.01 mol / L and a pH of 7.4.

[0024] The multi-mechanism fluoride ion trapping agent was prepared by the following method: A1. Weigh 4.5g of zirconium chloride hexahydrate and dissolve it in 150mL of anhydrous N,N-dimethylformamide. Stir magnetically until completely dissolved to obtain a zirconium chloride solution. Separately, weigh 3.3g of terephthalic acid and 15mL of glacial acetic acid into a beaker and add them to 150mL of anhydrous N,N-dimethylformamide. Stir magnetically until completely dissolved to obtain a terephthalic acid solution. At room temperature and with continuous magnetic stirring, slowly add the zirconium chloride solution dropwise to the terephthalic acid solution through a constant pressure dropping funnel (1 drop / 2 seconds). After the addition is complete, transfer the mixture to a 500mL polytetrafluoroethylene-lined high-pressure reactor, seal it, and incubate it in a 120℃ oven. After the reaction lasted 24 hours, the oven power was turned off and the reactor was allowed to cool naturally to room temperature in the oven. The reaction solution was poured into a 50 mL centrifuge tube and centrifuged at 8000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in 50 mL of fresh N,N-dimethylformamide, vortexed, and centrifuged at 8000 rpm for 10 minutes. This step was repeated 3 times. The precipitate was then resuspended in 50 mL of anhydrous ethanol, vortexed, and centrifuged at 8000 rpm for 10 minutes. This step was repeated 3 times to completely remove residual DMF and unreacted substances. The precipitate was transferred to a vacuum drying oven and dried under vacuum at 60 °C for 12 hours to obtain solid A. A2. Weigh 2.0g of solid A and disperse it in 100mL of anhydrous ethanol. Sonicate for 30 minutes to form a uniform suspension. Add 1.5g of L-arginine and stir magnetically until dissolved. Transfer the reaction system to a 250mL three-necked flask equipped with a condenser and reflux in an oil bath at 70℃ for 6 hours. The entire process is carried out under nitrogen protection. After the reaction is completed, allow it to cool naturally to room temperature. Centrifuge at 8000rpm for 10 minutes to collect the product. Wash the product three times with anhydrous ethanol (50mL each time) and centrifuge for 10 minutes each time. Transfer the product to a vacuum drying oven and dry it under vacuum at 60℃ for 12 hours to obtain a multi-mechanism fluoride ion trapping agent.

[0025] The acid-triggered intracellular calcium delivery body was prepared by the following method: B1. Weigh 2.0 g of aldehyde-based polyethylene glycol (Mn=2000), dissolve it in 50 mL of anhydrous dimethyl sulfoxide, and stir at room temperature until completely dissolved to obtain an aldehyde-based polyethylene glycol solution; weigh 1.5 g of polyacrylhydrazine, dissolve it in 20 mL of anhydrous dimethyl sulfoxide, and slowly add it dropwise to the above aldehyde-based polyethylene glycol solution. Add glacial acetic acid to adjust the pH of the reaction system to about 5.5. React at 25 °C under nitrogen protection for 24 hours. After the reaction is completed, slowly add the reaction solution dropwise to 500 mL of pre-cooled anhydrous diethyl ether, and a white precipitate is produced. Centrifuge at 4 °C and 8000 rpm for 10 minutes to collect the precipitate, wash it 3 times with anhydrous diethyl ether, and vacuum dry the precipitate for 12 hours to obtain an acid-sensitive polymer; B2. Weigh 1.0 g of casein phosphopeptide (purity ≥95%) and 0.5 g of calcium gluconate, dissolve them in 50 mL of pH 7.4 phosphate buffer, and stir magnetically until completely dissolved to obtain a casein phosphopeptide-calcium complex solution. Add 0.15 g of Traut reagent (2-iminothiacyclopentane hydrochloride) to this solution, transfer to a brown bottle, and stir for 2 hours at 25°C under nitrogen protection and in the dark to induce thiolization modification of the amino groups on the surface of the casein phosphopeptide. After the reaction is complete, transfer the solution to a dialysis bag (MWCO 3500). The solution was dialyzed with 1L of deionized water at 4℃ for 24 hours, with the dialysate changed every 6 hours (a small amount of EDTA was added to the dialysate to chelate trace metal ions and prevent thiol oxidation). The dialyzed solution was diluted with PBS at pH 7.4, and 20mL of phosphate buffer at pH 7.0 (0.1M) was added. The solution was magnetically stirred to obtain a thiolized calcium complex mixture. 0.3g of thiol-modified cell-penetrating peptide (such as thiolized TAT peptide, purity ≥95%) was weighed, dissolved in 5mL of PBS at pH 7.0, and slowly added dropwise to the above mixture. The reaction was carried out at 25℃ in the dark with gentle stirring for 6 hours. After the reaction, to form disulfide crosslinks, air was slowly introduced into the solution and gently stirred overnight. The reaction solution was then transferred to a dialysis bag (MWCO 3500). The solution was dialyzed with deionized water at 4°C for 48 hours, with the dialysate changed every 8 hours. The solution was then removed, filtered through a 0.22 μm filter membrane for sterilization, aliquoted, and stored at -20°C to obtain the core calcium complex. B3. Weigh 200 mg of acid-sensitive polymer and 100 mg of core calcium complex, dissolve them together in 10 mL of pH 8.0 Tris-HCl buffer (0.05 M), and sonicate for 10 minutes until completely dissolved to obtain an aqueous phase. In an ice-water bath, add the above aqueous phase dropwise to 50 mL of dichloromethane containing 1 wt% Span 80 through a syringe needle (22 G), while simultaneously emulsifying with a high-speed homogenizer (10,000 rpm) for 2 minutes to form a primary emulsion. Immediately pour the primary emulsion into 200 mL of 1 wt% polyvinyl alcohol aqueous solution and homogenize with a high-speed homogenizer (15,000 rpm) for 5 minutes to form a secondary emulsion. Magnetically stir the resulting secondary emulsion at room temperature for 4 hours to allow the dichloromethane to evaporate and the nanoparticles to solidify. Then, at 8000... Centrifuge at rpm for 15 minutes, discard the supernatant, resuspend the precipitate in deionized water and centrifuge again. Repeat the washing process 3 times. Finally, resuspend the nanoparticles in 10 mL of pH 7.4 PBS, filter through a 0.45 μm filter membrane, aliquot and store at 4°C in the dark to obtain the acid-triggered intracellular calcium delivery system.

[0026] The engineered exosomes were prepared by the following method: C1. Purchase lentiviral vectors carrying the FGF21 gene (driven by the CMV promoter) and the LAMP2-RGD fusion gene (LAMP2 full-length cDNA fused with the RGD tripeptide coding sequence, driven by the EF-1α promoter). Human umbilical cord mesenchymal stem cells (hUC-MSCs, passage numbers P3-P5) are cultured in α-MEM medium containing 10% FBS. When the cell density reaches 50-60% confluence, infection is performed. Lentiviral virus is added at a ratio of MOI=10, and 8 μg / mL polybrene is added to improve infection efficiency. After 24 hours of culture, the medium is replaced with fresh complete medium. After 48 hours of infection, the medium is replaced with selective medium containing 1 μg / mL puromycin. The medium is changed every 2-3 days, and the selection is continued for 2 weeks to obtain a stable transfected cell line. Select single or amplified hybrid clones and detect FGF21 expression (cell lysate and supernatant) and LAMP2-RGD expression (cell membrane protein) by Western blot. Detect the proportion of RGD-positive cells by flow cytometry (using anti-RGD antibody or integrin ligand binding assays). Select high-expression clones as production cell lines.

[0027] C2. Culture the stably transfected cell lines in T175 culture flasks. When the cell density reaches 80-90% confluence, wash the cells twice with PBS, then replace with α-MEM medium containing 10% exosome-depleted FBS (exosomes removed from FBS by ultracentrifugation or ultrafiltration). Continue culturing for 48-72 hours, then collect 500 mL of cell culture supernatant (from 10 T175 flasks) and transfer it to centrifuge tubes. Repeat the following steps: Centrifuge at 4℃, 300g for 10 minutes to remove suspended cells; Centrifuge at 2,000g for 20 minutes at 4℃ to remove cell debris and large vesicles; Centrifuge at 4℃, 10,000g for 45 minutes to remove microvesicles and large particulate impurities; The supernatant after centrifugation was filtered through a 0.22 μm polyethersulfone (PES) filter to remove residual large particles. Polyethylene glycol 6000 was added to the filtered supernatant to a final concentration of 10% (w / v), and the mixture was thoroughly mixed and incubated overnight (12-16 hours) at 4°C. After centrifugation at 1,500g for 30 minutes at 4°C, the supernatant was carefully discarded. A pale yellow gelatinous precipitate (exosome enrichment) was visible at the bottom of the tube. The precipitate was gently resuspended with 5 mL of pH 7.4 PBS (avoiding vigorous pipetting), transferred to an ultracentrifuge tube, and ultracentrifuged at 100,000g for 70 minutes at 4°C for washing and purification. The supernatant was discarded, and the exosome precipitate at the bottom of the tube was gently resuspended with 100 μL of pH 7.4 PBS to obtain engineered exosomes.

[0028] Exosomal protein concentration was determined using the BCA method, typically 2-5 mg / mL. Exosomal concentration and particle size distribution (peak 80-120 nm) were determined using nanoparticle tracking analysis (NTA). Exosomal marker proteins CD63, CD81, TSG101, and the LAMP2-RGD fusion protein were detected by Western blot. FGF21 content was detected by ELISA, confirming a concentration of 125±25 ng FGF21 / μg exosomal protein. After aliquoting, the exosomal protein was stored at -80°C for later use.

[0029] The method for preparing an exosome component for relieving skin burns includes the following steps: S1. Preparation of Phase A: Weigh sodium alginate powder and slowly add it to 80% deionized water (pre-cooled to 4°C). Stir continuously with a magnetic stirrer at room temperature for 4-6 hours until completely dissolved. The solution should be clear and transparent with a viscous consistency. Add multi-mechanism fluoride ion trapping agent, superoxide dismutase, and trehalose in sequence. Place the beaker in an ice-water bath and use an ultrasonic cell disruptor (equipped with a micro-tip probe) to perform ultrasonic dispersion at 300W power. The working mode is pulsed ultrasonic (3 seconds on / 2 seconds off). The total ultrasonic time is 10 minutes, during which the process is stopped and manually stirred once every 2 minutes to ensure uniform dispersion. During the ultrasonic process, the ice-water bath temperature is kept below 10°C. After ultrasonication, add the remaining deionized water and gently stir to mix. Transfer the solution to a vacuum desiccator and let it stand at room temperature for 30-60 minutes to remove bubbles. Dispense the degassed Phase A solution into brown bottles and store them in a sealed container at 4°C for later use. The storage period should not exceed 1 week. Before use, visually inspect to confirm that there is no precipitation or phase separation. S2. Preparation of Phase B: Weigh gelatin powder and add it to phosphate buffer (0.01 mol / L, pH 7.4, preheated to 40°C). Stir magnetically in a 40°C water bath for 1-2 hours until completely dissolved and the solution is clear and pale yellow. Add acid-triggered intracellular calcium delivery body and calcium gluconate in sequence, and continue stirring in a 40°C water bath for 30 minutes until uniformly dispersed. Turn off the water bath heating and allow the solution to cool naturally to about 25°C at room temperature (30-45 minutes). In an ice bath, add engineered exosomes and mix by gently and repeatedly inverting the container (20 times, holding for 2 seconds each time). Avoid vigorous shaking, vortexing, or blowing to protect the integrity of the exosome membrane structure. After mixing, dispense the Phase B solution into brown bottles and store in a sealed container in a 4°C refrigerator away from light for up to 48 hours. Before use, gently invert and mix to confirm that there is no obvious precipitation. S3. Preparation of Hydrogel: Before use, load phase A and phase B into two separate chambers of a double-barrel syringe (5-10 mL per chamber), ensuring equal volume for a 1:1 volume ratio. Install a static mixing head (spiral mixing structure, ≥12 mixing units) at the tip of the double-barrel syringe, ensuring a tight connection. After debridement, rinse the burn wound with saline and dry any surface moisture. Align the outlet of the static mixing head of the double-barrel syringe with the center of the wound and push the plungers evenly on both sides to allow phases A and B to enter the mixing head simultaneously at the same rate. Thoroughly mix and discharge the mixture in the spiral channel. Apply the mixture directly to the wound surface in a spiral or "Z" shaped pattern, 2-3 mm thick, ensuring complete coverage and extending slightly beyond the edges. The application process should be continuous and rapid to avoid interruptions that could clog the mixing head. After application, allow the hydrogel to adhere naturally to the wound surface. At a body temperature of 37°C, the mixture will gradually gel within 2-4 minutes. Once gelled, the hydrogel is translucent pale yellow, soft but not flowing, and adheres tightly to the wound tissue without gaps. The preparation process is as follows: Figure 1 As shown.

[0030] Example 2: This example is based on Example 1, but differs from Example 1 in that phase A includes the following components by weight: 1.5 parts sodium alginate, 2.0 parts multi-mechanism fluoride ion trapping agent, 0.010 parts superoxide dismutase, 0.05 parts trehalose, and deionized water to make up to 100 parts. Phase B comprises the following components by weight: 3.0 parts gelatin, 1.5 parts acid-triggered intracellular calcium delivery body, 0.00005 parts engineered exosomes based on the amount of exosome protein they contain, 0.1 parts calcium gluconate, and phosphate buffer to a total of 100 parts. The engineered exosomes contained 50 ng / μg of FGF21 in exosomal protein.

[0031] The rest are the same.

[0032] Example 3: This example is based on Example 1, but differs from Example 1 in that phase A includes the following components by weight: 2.5 parts sodium alginate, 4.0 parts multi-mechanism fluoride ion trapping agent, 0.025 parts superoxide dismutase, 0.15 parts trehalose, and deionized water to make up to 100 parts. Phase B comprises the following components by weight: 5.0 parts gelatin, 3.0 parts acid-triggered intracellular calcium delivery body, 0.00015 parts engineered exosomes based on the amount of exosome protein they contain, 0.3 parts calcium gluconate, and phosphate buffer to a total of 100 parts. The engineered exosomes contained 200 ng / μg of FGF21 in exosomal protein.

[0033] The rest are the same.

[0034] Example 4: This embodiment differs from Example 1 in that the FGF21 content in the engineered exosomes is 75 ng / μg exosomal protein. The rest are the same.

[0035] Example 5: This embodiment differs from Example 1 in that the FGF21 content in the engineered exosomes is 150 ng / μg exosomal protein. The rest are the same.

[0036] Comparative Example 1: This comparative example differs from Example 1 in that the multi-mechanism fluoride ion trapping agent is a zirconium-based metal-organic framework material without arginine modification (only step A1 is performed, step A2 is not performed). The rest are the same.

[0037] Comparative Example 2: This comparative example differs from Example 1 in that phase A does not contain a multi-mechanism fluoride ion trapping agent. The rest are identical.

[0038] Comparative Example 3: This comparative example differs from Example 1 in that phase B does not contain an acid-triggered intracellular calcium delivery system, but only an equal amount of calcium gluconate. The rest are identical.

[0039] Comparative Example 4: This comparative example differs from Example 1 in that the shell of the acid-triggered intracellular calcium delivery body does not contain pH-sensitive hydrazone bonds, but instead uses stable amide bonds to link polyethylene glycol and polyacrylhydrazide. The rest is identical.

[0040] Comparative Example 5: This comparative example differs from Example 1 in that the engineered exosomes in phase B are ordinary, unmodified human umbilical cord mesenchymal stem cell exosomes (not expressing LAMP2-RGD fusion protein and not rich in FGF21). The rest are the same.

[0041] Comparative Example 6: This comparative example differs from Example 1 in that the engineered exosomes express only the LAMP2-RGD fusion protein but are not rich in FGF21 (i.e., only the LAMP2-RGD gene is transfected, not the FGF21 gene). The rest are the same.

[0042] Comparative Example 7: This comparative example differs from Example 1 in that phase B does not contain engineered exosomes. The rest are identical.

[0043] Comparative Example 8: This comparative example is based on Example 1, except that phase A does not contain superoxide dismutase. The rest are the same.

[0044] Comparative Example 9: This comparative example is commercially available calcium gluconate gel (standard control).

[0045] Comparative Example 10: This comparative example serves as a blank control group, where the wound was covered only with sterile gauze.

[0046] Experimental Example 1: In vitro fluoride ion capture capacity test.

[0047] Detection methods and procedures: Preparation of simulated fluoride-containing wound exudate: NaF was added to PBS at pH 7.4 to prepare a solution with a fluoride ion concentration of 10 mM (simulating the fluoride ion concentration of a severe fluoride burn wound).

[0048] Equal masses (100 mg) of the lyophilized powder of phase A from each example and comparative example were weighed and added to 10 mL of the above-mentioned fluoride-containing solution. The solutions were incubated in a shaking incubator at 37°C and 150 rpm. At time points of 0.5, 1, 2, 4, 8, and 24 hours, 100 μL of the supernatant was collected, and the concentration of residual free fluoride ions was determined using a fluoride ion selective electrode method. The fluoride ion removal rate was calculated as: [(C0-C...] t [) / C0]×100%, where C0 is the initial concentration, C t Let t be the concentration at time t.

[0049] Table 1 Results of in vitro fluoride ion capture capacity test Experimental Example 2: Intracellular calcium ion recovery capacity test.

[0050] Detection methods and procedures: Cell culture and establishment of a fluoride ion damage model: Human skin fibroblasts (HDF) were cultured in DMEM medium (containing 10% FBS) and seeded in 96-well plates (1×10⁻⁶ cells / wells). 4 (Cells / well). After culturing for 24 hours to allow cell adhesion, cells were treated with 5 mM NaF for 2 hours to establish a fluoride-induced low-calcium model. NaF was removed by washing with PBS.

[0051] Treatment intervention: Each experimental group was given the phase B dilution solution (containing acid-triggered intracellular calcium delivery body or control) from the examples and comparative examples, and cultured for another 12 hours. The blank control group received no treatment.

[0052] Intracellular calcium ion detection: Fluo-4 AM, a fluorescent probe for calcium ions, was used. The culture medium was removed, and 5 μM Fluo-4 AM working solution was added. The cells were incubated at 37°C for 30 minutes. The cells were washed three times with PBS to remove the extracellular fluorescent probe. Intracellular fluorescence intensity was detected using fluorescence microscopy (excitation wavelength 494 nm, emission wavelength 516 nm) or flow cytometry to reflect the intracellular free calcium ion concentration. The calcium ion recovery rate was calculated for each group, with the untreated normal cell group as 100%. Calcium ion recovery rate = [(Experimental group - Fluoride-damaged group) / (Normal control - Fluoride-damaged group)] × 100%.

[0053] Table 2 Results of intracellular calcium ion recovery capacity assay Experimental Example 3: Evaluation of the therapeutic effect of hydrofluoric acid burns in animal models.

[0054] Detection methods and procedures: Experimental animals and grouping: SPF-grade male SD rats, weighing 200-220g, were randomly divided into 10 groups (Examples 1-5, Comparative Examples 1-7, Comparative Example 9, Comparative Example 10), with n=8 rats in each group, and were acclimatized for 1 week.

[0055] Establishment of a hydrofluoric acid burn model: Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg), and their backs were shaved and the skin was disinfected. Filter paper (2 cm in diameter) soaked in 40% HF solution was applied to the skin of the rat's back for 30 seconds, and immediately flushed with a large amount of physiological saline for 5 minutes to form a standard deep second-degree hydrofluoric acid burn wound.

[0056] Treatment intervention: Treatment began 1 hour after modeling. Examples 1-5: Hydrogels were prepared on-site according to the above method and applied to the wound (2 mm thickness); Comparative Examples 1-8: Applied the corresponding hydrogels; Comparative Example 9: Commercially available calcium gluconate gel was applied; Comparative Example 10: Only sterile gauze was used for covering. A transparent dressing was applied for fixation, and the dressing was changed every 2 days.

[0057] Monitoring indicators: 1. Wound healing rate: The wound area was photographed and recorded on days 0, 3, 7, 10, 14, and 21. The wound area was analyzed using ImageJ software, and the healing rate was calculated as [(initial area - current area) / initial area] × 100%.

[0058] 2. Tissue fluoride ion content: On day 3, tissue samples were taken from the area surrounding the wound (a 5 mm circumferential region outside the wound edge). After digestion, the tissue fluoride content (μg F) was measured using a fluoride ion-selective electrode. - / g tissue wet weight).

[0059] 3. Histopathological scoring: On days 7 and 14, some animals were sacrificed and full-thickness skin tissue from the wound was taken. HE staining was performed to assess indicators such as inflammatory cell infiltration, tissue necrosis, re-epithelialization, angiogenesis, and collagen deposition. A 5-point scoring system was used (0=none, 1=mild, 2=mild, 3=moderate, 4=severe, 5=major).

[0060] 4. Scar hyperplasia index: Animals were sacrificed on day 21, and the thickness of the healed scar and the thickness of the surrounding normal skin were measured. The scar hyperplasia index was calculated as scar thickness / normal skin thickness.

[0061] Table 3 Results of therapeutic efficacy testing for hydrofluoric acid burns in animal models The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An exosome component for relieving skin burns, characterized in that, It includes phase A and phase B, which are mixed in a volume ratio of 1:1 to form a hydrogel before use; Phase A comprises the following components by weight: 1.5-2.5 parts sodium alginate, 2.0-4.0 parts multi-mechanism fluoride ion trapping agent, 0.010-0.025 parts superoxide dismutase, 0.05-0.15 parts trehalose, and deionized water to a total of 100 parts. Phase B comprises the following components by weight: 3.0-5.0 parts gelatin, 1.5-3.0 parts acid-triggered intracellular calcium delivery body, 0.00005-0.00015 parts engineered exosomes based on the amount of exosome protein they contain, 0.1-0.3 parts calcium gluconate, and phosphate buffer to a total of 100 parts. The multi-mechanism fluoride ion trapping agent is an arginine-modified zirconium-based metal-organic framework material; The acid-triggered intracellular calcium delivery system is a core-shell structured nanoparticle. Its outer shell is made of aldehyde polyethylene glycol grafted with polyacrylhydrazine via hydrazone bonds. Its core contains a thiolized casein phosphopeptide-calcium complex linked by disulfide bonds and thiol-modified cell-penetrating peptides. The engineered exosomes are genetically engineered human mesenchymal stem cell-derived exosomes with LAMP2-RGD fusion protein on their membrane surface and rich in FGF21 in their lumen.

2. The exosome component for relieving skin burns according to claim 1, characterized in that, The engineered exosomal protein contains 50-200 ng / μg of FGF21.

3. The exosome component for relieving skin burns according to claim 1, characterized in that, The phosphate buffer solution has a concentration of 0.01 mol / L and a pH of 7.

4.

4. The exosome component for relieving skin burns according to claim 1, characterized in that, The multi-mechanism fluoride ion trapping agent was prepared by the following method: A1. Dissolve zirconium chloride hexahydrate in anhydrous N,N-dimethylformamide to obtain a zirconium chloride solution; add terephthalic acid and glacial acetic acid to anhydrous N,N-dimethylformamide and dissolve completely to obtain a terephthalic acid solution; add the zirconium chloride solution dropwise to the terephthalic acid solution. After the addition is complete, transfer the solution to a high-pressure reactor, seal it, and allow it to react. After the reaction is complete, allow it to cool naturally to room temperature. Pour the reaction solution into a centrifuge tube, centrifuge, collect the precipitate, resuspend the precipitate in fresh N,N-dimethylformamide, then resuspend it in anhydrous ethanol, and dry it under vacuum to obtain solid A. A2. Disperse solid A in anhydrous ethanol, sonicate to form a uniform suspension, add L-arginine, stir magnetically until dissolved, reflux in an oil bath at 70°C under nitrogen protection, after the reaction is complete, cool to room temperature, centrifuge, wash with anhydrous ethanol, and vacuum dry to obtain a multi-mechanism fluoride ion trapping agent.

5. The exosome component for relieving skin burns according to claim 1, characterized in that, The acid-triggered intracellular calcium delivery body was prepared by the following method: B1. Aldehyde polyethylene glycol is dissolved in anhydrous dimethyl sulfoxide to obtain an aldehyde polyethylene glycol solution; polyacrylhydrazine is dissolved in anhydrous dimethyl sulfoxide and slowly added dropwise to the above aldehyde polyethylene glycol solution; glacial acetic acid is added to adjust the pH to 5.0~6.0; the reaction is carried out under nitrogen protection; after the reaction is completed, the reaction solution is added dropwise to pre-cooled anhydrous diethyl ether; the precipitate is collected by centrifugation, washed with anhydrous diethyl ether, and dried under vacuum to obtain an acid-sensitive polymer; B2. Casein phosphopeptide and calcium gluconate were dissolved in phosphate buffer at pH 7.4 and stirred to obtain a casein phosphopeptide-calcium complex solution. 2-Iminothiacyclopentane hydrochloride was then added, and the reaction was carried out at 25°C under nitrogen protection and in the dark. After the reaction was completed, the solution was dialyzed with deionized water. The dialyzed solution was diluted with pH 7.0 phosphate buffer to obtain a thiolized calcium complex solution. Thiol-modified cell-penetrating peptides were dissolved in pH 7.0 phosphate buffer and added dropwise to the above thiolized calcium complex solution. The reaction was carried out under light protection and stirred. After the reaction, air was slowly introduced into the solution and gently stirred overnight. The solution was dialyzed with deionized water, and the solution was filtered and sterilized to obtain the core calcium complex. B3. The acid-sensitive polymer and the core calcium complex were dissolved together in a pH 8.0 Tris-HCl buffer to obtain an aqueous phase. In an ice-water bath, the aqueous phase was added to dichloromethane containing 1 wt% Span 80 and emulsified to form a primary emulsion. The primary emulsion was immediately poured into a 1 wt% polyvinyl alcohol aqueous solution and homogenized to form a secondary emulsion. The resulting secondary emulsion was magnetically stirred at room temperature, then centrifuged, the precipitate was resuspended in deionized water and centrifuged again, and finally resuspended in a pH 7.4 phosphate buffer and filtered to obtain the acid-triggered intracellular calcium delivery body.

6. The exosome component for relieving skin burns according to claim 1, characterized in that, The engineered exosomes were prepared by the following method: C1. Human umbilical cord mesenchymal stem cells were infected with a lentiviral vector carrying the FGF21 gene and the LAMP2-RGD fusion gene. After 48 hours of infection, the culture medium was replaced with a selective medium containing 1 μg / mL puromycin. The culture medium was replaced every 2-3 days and the selection was continued for 2 weeks to obtain a stable transfected cell line. C2. The stably transfected cell lines were cultured in T175 culture flasks. The cells were washed twice with PBS and replaced with α-MEM medium containing 10% exosome-free fetal bovine serum. The cells were cultured for 48-72 hours. The cell culture supernatant was collected and transferred to centrifuge tubes. The following centrifugation was performed sequentially: 300g at 4°C for 10 minutes; 2,000g at 4°C for 20 minutes; and 10,000g at 4°C for 45 minutes. The supernatant was filtered, polyethylene glycol 6000 was added, and the mixture was thoroughly mixed and incubated overnight at 4°C. The supernatant was then carefully discarded and the precipitate was gently resuspended. The precipitate was washed and purified by ultracentrifugation, and the supernatant was discarded. The precipitate at the bottom of the tube was gently resuspended to obtain engineered exosomes.

7. A method for preparing an exosome component for relieving skin burns according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Preparation of phase A: Sodium alginate is added to deionized water and stirred at room temperature until completely dissolved. Multi-mechanism fluoride ion trapping agent, superoxide dismutase and trehalose are added in sequence. The mixture is placed in an ice-water bath and ultrasonically dispersed using an ultrasonic cell disruptor. After ultrasonication, deionized water is added, and the mixture is gently stirred and mixed. The solution is transferred to a vacuum desiccator and allowed to stand at room temperature to remove air bubbles. S2. Preparation of phase B: Gelatin is added to phosphate buffer and stirred in a 40°C water bath until completely dissolved. Acid-triggered intracellular calcium delivery body and calcium gluconate are added in sequence and stirred until uniformly dispersed. After natural cooling, engineered exosomes are added in an ice bath and mixed by gently and repeatedly inverting the container. S3. Preparation of hydrogel: Before use, put phase A and phase B into the two independent chambers of the double-barrel syringe respectively. After cleaning the burn wound, rinse it with physiological saline and dry it. Align the outlet of the static mixing head of the double-barrel syringe with the center of the wound and push the plungers on both sides evenly. At a body temperature of 37°C, it will gradually form a gel within 2-4 minutes.

Images