A hydrogel composite material, a preparation method and application thereof

Abstract

The present application relates to the technical field of biomedical materials, and specifically discloses a hydrogel composite material, a preparation method and application thereof. The composite material is formed by organically combining the three-dimensional network support of methacrylated gelatin, the efficient drug delivery capacity and biological activity regulation function of plant-derived exosomes, and the efficient contact type antibacterial mechanism of N-chlorinated MOF, forming a multifunctional composite system with antibacterial, anti-inflammatory and healing functions. The application of the composite hydrogel in the preparation of medical dressings can solve the clinical problems of infection control difficulty, low wound repair efficiency and poor dressing adhesion in the treatment of chronic refractory wounds, significantly improve the wound care effect, and provide a new and efficient medical material for the clinical treatment of chronic refractory wounds.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a hydrogel composite material, its preparation method, and its application. Background Technology

[0002] As the largest barrier organ in the human body, the skin plays a vital role in protecting the body, resisting the invasion of external microorganisms, regulating body temperature, and maintaining fluid balance. Once injured by trauma, burns, ulcers, or other injuries, if not treated promptly and effectively, it is highly susceptible to infection, exacerbated inflammation, delayed wound healing, and scar hyperplasia. In severe cases, it can even lead to systemic complications, threatening human health. Chronic, non-healing wounds (such as diabetic foot ulcers and pressure sores) present significant challenges in treatment and care due to high oxidative stress and persistent inflammation, necessitating the development of high-performance wound dressings to meet clinical needs.

[0003] Hydrogel dressings, due to their excellent hydrophilicity, biocompatibility, and structural flexibility, can mimic the structure and function of the human extracellular matrix, providing a moist healing microenvironment for wounds. They can also absorb wound exudate and reduce wound adhesion during dressing changes, making them a research hotspot and mainstream direction in the field of wound dressings. However, most existing hydrogel dressings cannot simultaneously achieve multiple functions such as antibacterial, anti-inflammatory, scavenging of reactive oxygen species (ROS), and promotion of angiogenesis, failing to meet the diverse nursing needs of complex wounds, especially infected and chronically difficult-to-heal wounds.

[0004] Therefore, developing a hydrogel composite material with excellent mechanical properties and multiple functions such as antibacterial, anti-inflammatory, and healing-promoting properties, and applying it to the preparation of wound dressings, is of great clinical significance and application value for improving wound care, promoting rapid wound healing, and especially solving the treatment problem of chronic and refractory wounds. Summary of the Invention

[0005] To address the problem that most existing hydrogel dressings cannot simultaneously achieve multiple functions such as antibacterial, anti-inflammatory, scavenging of reactive oxygen species (ROS), and promotion of angiogenesis, and thus cannot meet the diverse nursing needs of complex wounds, especially infected and chronic non-healing wounds, this invention provides a hydrogel composite material, its preparation method, and its application.

[0006] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows: In a first aspect, the present invention provides a hydrogel composite material, comprising: The three-dimensional network matrix formed by methacrylamide gelatin; and Plant-derived exosomes carrying luteolin and N-chloroamino-functionalized metal-organic framework materials are uniformly dispersed in the methacrylamide gelatin matrix.

[0007] Compared to existing technologies, the hydrogel composite material provided by this invention, using methacrylamide gelatin as a matrix, endows the composite material with excellent biocompatibility, biodegradability, and structural flexibility. It can simulate the extracellular matrix environment of human cells, adhere to the wound surface, and absorb wound exudate, effectively avoiding wound adhesion during dressing changes. Furthermore, the three-dimensional porous network structure of methacrylamide gelatin provides suitable mechanical support for the composite system and can effectively regulate the release kinetics of active ingredients. Plant-derived exosomes carrying luteolin, uniformly dispersed in the matrix, can protect luteolin from degradation, achieving its long-term stable delivery. They can also synergistically exert anti-inflammatory, collagen-promoting, angiogenesis-promoting, and cell migration-promoting effects with luteolin by utilizing the endogenous microRNA abundant in the exosomes themselves, accelerating the wound healing process. The N-chloroamino-functionalized metal-organic framework material achieves highly efficient and low-toxicity contact sterilization through surface covalently bonded -N-Cl functional groups, effectively controlling wound infection and avoiding the potential toxicity of traditional MOF materials that rely on the release of metal ions.

[0008] In summary, the hydrogel composite material of this invention organically integrates the supporting and conforming properties of the matrix, the synergistic healing properties of exosomes and luteolin, and the antibacterial properties of functionalized MOFs. It solves the technical pain points of existing wound dressings, such as difficulty in achieving both antibacterial and biocompatibility, limited healing effects, and unstable drug delivery. It is especially suitable for the repair of chronic and difficult-to-heal wounds and has good clinical application value and promotion prospects.

[0009] Furthermore, the swelling rate of the hydrogel composite material in PBS buffer is 55%~60%, and the fracture strain is 230%~280%.

[0010] Furthermore, the mass ratio of the methacrylamide gelatin matrix, the plant-derived exosomes loaded with luteolin, and the N-chloroaminofunctionalized metal-organic framework material is 50:(1~5):(1~10).

[0011] Furthermore, the plant-derived exosomes are exosomes derived from cucumber fruits.

[0012] Furthermore, the N-chloroamino-functionalized metal-organic framework material is UIO-66-NHCl.

[0013] It should be noted that the methacrylamide gelatin described in this invention can be obtained directly from commercially available materials or prepared according to conventional methods in the art, such as reacting gelatin with methacrylic anhydride to prepare methacrylamide gelatin.

[0014] As a specific embodiment of the present invention, the preparation method of the methacrylamide gelatin includes the following steps: Gelatin is added to water and heated until completely dissolved to obtain a gelatin solution. Methacrylic anhydride is added to the gelatin solution and stirred at 50℃~60℃ for 2h~3h. After the reaction is completed, the reaction solution is transferred to a dialysis bag for dialysis, centrifuged, and the supernatant is collected to obtain methacrylamide gelatin.

[0015] Methacrylamide gelatin (GelMA) contains photocrosslinkable methacryloyl groups on its molecular chain, which can form a hydrogel with a three-dimensional porous network structure through crosslinking reaction under ultraviolet light irradiation. This hydrogel matrix provides the necessary mechanical support for the composite system described in this invention, maintains a moist wound healing environment, and effectively regulates the release kinetics of the active ingredients.

[0016] Specifically, the gelatin is gelatin derived from pigskin.

[0017] Specifically, the molar ratio of gelatin to methacrylic anhydride is 1:(0.5~1.0).

[0018] As a specific embodiment of the present invention, the method for preparing the plant-derived exosomes loaded with luteolin includes the following steps: Cucumber fruit juice was extracted, and after differential centrifugation to remove cell debris and large particulate impurities, the juice was filtered through a microporous membrane. The filtrate was then subjected to ultracentrifugation, and the precipitate was collected to obtain cucumber-derived exosomes. The cucumber-derived exosomes were dispersed in a buffer solution to obtain a cucumber-derived exosome suspension. Luteolin was dissolved in an alcohol solvent and added to the cucumber-derived exosome suspension. The mixture was incubated to allow luteolin to enter the exosomes. Then, the unloaded luteolin was removed to obtain the plant-derived exosomes loaded with luteolin.

[0019] Cucumber-derived exosomes can serve as carriers for the hydrophobic drug luteolin, enhancing its stability. Furthermore, their membrane fusion capabilities allow them to penetrate wound barriers and precisely deliver luteolin to deep wound tissues. Additionally, cucumber-derived exosomes carry multiple endogenous microRNAs (miRNAs), and high-throughput sequencing has confirmed that the expression levels of miR-159, miR-156, and miR-166 are all above 10-1. 5 Copy / μg. Further analysis showed that the above miRNAs can participate in the wound repair process through specific molecular pathways: miR-159 inhibits excessive cell proliferation by downregulating cyclin expression through MYC, while promoting collagen synthesis; miR-166 reduces the inflammatory response by targeting and inhibiting the expression of pro-inflammatory factors such as TNF-α and IL-6, inhibiting the activation of the NF-κB signaling pathway; miR-156 can promote the migration of L929 fibroblasts and HaCaT keratinocytes, upregulate the expression of VEGF and FGF2, and enhance angiogenesis.

[0020] In summary, plant-derived exosomes carrying luteolin can promote the healing of chronic wounds by synergistically interacting with miRNA to achieve both anti-inflammatory and repair-promoting effects.

[0021] Furthermore, the cucumber-derived exosomes prepared above have an average particle size of 142 nm to 150 nm and a zeta potential of -25 mV to -10 mV, exhibiting a typical spherical bilayer membrane structure.

[0022] Furthermore, the mass ratio of luteolin to cucumber-derived exosomes is 1:(5~20), preferably 1:10.

[0023] This ratio ensures efficient drug loading without affecting the structural integrity of exosomes.

[0024] Furthermore, the incubation temperature is 35℃~38℃, and the incubation is carried out in the dark for 2h~4h. During the incubation process, the stirring is continuously carried out at a stirring speed of 80rpm~120rpm.

[0025] The encapsulation rate of luteolin in the plant-derived exosomes loaded with luteolin prepared above was 75%~80%.

[0026] As a specific embodiment of the present invention, the preparation method of the N-chloroamino-functionalized metal-organic framework material includes the following steps: The N-chloroation reaction of UIO-66-NH2 and sodium p-toluenesulfonyl chloride yields the N-chloroamino-functionalized metal-organic framework material (UIO-66-NHCl).

[0027] UIO-66-NHCl utilizes its surface-covalently bonded -N-Cl functional groups to undergo nucleophilic substitution reactions with amino and thiol groups on bacterial cell membrane proteins. This increases cell membrane permeability, leading to leakage of contents and achieving highly efficient contact sterilization. This avoids the traditional MOF's metal ion-dependent antibacterial mechanism and its potential toxicity. This mechanism allows UIO-66-NHCl to maintain highly efficient antibacterial activity while exhibiting greater safety for normal tissue cells, making it particularly suitable for biomedical applications such as wound dressings.

[0028] The N-chloroamino-functionalized metal-organic framework materials prepared above have a particle size of 100nm~200nm, an average particle size of 125nm, and exhibit a regular rhombic dodecahedral structure.

[0029] Furthermore, the mass ratio of UIO-66-NH2 to sodium p-toluenesulfonyl chloride is 1:(3~8).

[0030] Furthermore, the N-chloroation reaction is carried out at a temperature of 20°C to 30°C for a reaction time of 1 hour to 6 hours.

[0031] It should be noted that the UIO-66-NH2 described in this invention can be prepared directly using commercially available products or using conventional methods in the art, such as preparing UIO-66-NH2 by reacting zirconium salt and 2-aminoterephthalic acid.

[0032] For example, the preparation method of UIO-66-NH2 includes the following steps: Zirconium tetrachloride and 2-aminoterephthalic acid were dissolved in N,N-dimethylformamide, glacial acetic acid was added, and the mixture was then transferred to an autoclave and reacted at 110℃~130℃ for 20h~25h to obtain UIO-66-NH2.

[0033] Secondly, the present invention provides a method for preparing the above-mentioned hydrogel composite material, comprising the following steps: S1, dissolve methacrylamide gelatin in buffer solution, add photoinitiator to obtain prepolymer solution; S2, plant-derived exosomes loaded with luteolin and N-chloroamino-functionalized metal-organic framework materials are added to the prepolymer liquid, mixed evenly, and photocrosslinked and cured to obtain a hydrogel composite material.

[0034] The method for preparing hydrogel composite materials provided by this invention has significant advantages such as simple steps, easy operation, mild conditions, and good repeatability, providing strong support for the large-scale production and clinical application of hydrogel composite materials.

[0035] Further, in S1, the buffer solution is PBS buffer with a pH of 7.2-7.4 and a concentration of 0.01 mol / L-0.02 mol / L.

[0036] Further, in S1, the mass-to-volume ratio of the methacrylamide gelatin to the buffer solution is (5~15) g:100 mL.

[0037] Further, in S1, the mass ratio of the photoinitiator to the methacrylamide gelatin is 1:(15~30).

[0038] Specifically, the photoinitiator is a PBS dispersion system of Irgacure 2959, and the concentration of the PBS dispersion system of the photoinitiator is 0.25%~0.5% (w / v).

[0039] Furthermore, in S2, the light wavelength for photocrosslinking curing is 320nm~400nm, and the light intensity is 5mW / cm². 2 ~15mW / cm 2 The illumination time is 8 to 15 minutes.

[0040] Preferably, in S2, the light wavelength for photocrosslinking curing is 365 nm, and the light intensity is 10 mW / cm². 2 The illumination time is 10 minutes.

[0041] The optimized reaction conditions can protect the membrane structure integrity of plant-derived exosomes and the bioactivity of their endogenous miRNAs, prevent the degradation of luteolin, retain the antibacterial activity of N-chloroaminofunctionalized metal-organic framework materials, and ensure that the active components are uniformly dispersed in the methacryloylated gelatin matrix, thus guaranteeing the overall performance consistency of the hydrogel composite material.

[0042] Thirdly, the present invention also provides the application of the above-mentioned hydrogel composite material in the preparation of medical dressings.

[0043] Fourthly, the present invention provides a medical dressing comprising the hydrogel composite material described in any of the preceding claims.

[0044] The medical dressing is prepared by cutting the hydrogel composite material described in any of the preceding claims into specific shapes and sizes as needed, sterilizing it, and then sealing and packaging it. Alternatively, the dressing can also be provided in the form of a prepolymer liquid, which is injected into a mold or directly applied to the wound surface during use, and then photocrosslinked in situ to form a hydrogel dressing.

[0045] The medical dressings are available in forms including, but not limited to, sheets, films, sponges, or injectable hydrogels. These dressings possess good flexibility and adhesion, allowing them to adhere closely to the wound while maintaining a suitable moist environment to promote wound healing.

[0046] Furthermore, the medical dressing is a medical dressing used to treat chronic, non-healing wounds. These chronic, non-healing wounds include, but are not limited to, diabetic foot ulcers, pressure ulcers, venous ulcers, or bacterially infected wounds. In addition, the dressing can also be used to cover and protect acute wounds (such as trauma, abrasions, and surgical incisions) to prevent infection and promote healing.

[0047] In summary, this invention provides a hydrogel composite material that organically combines the three-dimensional network support of methacrylamide gelatin, the efficient drug delivery capability and bioactivity regulation function of plant-derived exosomes, and the efficient contact antibacterial mechanism of N-chloroMOFs, forming a multifunctional composite system with antibacterial, anti-inflammatory, and healing-promoting functions. Applying this composite hydrogel to the preparation of medical dressings can specifically address clinical pain points in the treatment of chronic, refractory wounds, such as difficulty in infection control, low wound repair efficiency, and poor dressing adherence, significantly improving wound care outcomes and providing a novel and efficient medical material option for the clinical treatment of chronic, refractory wounds. Attached Figure Description

[0048] Figure 1 This is a transmission electron microscope image of cucumber-derived exosomes prepared in Example 1 of the present invention; Figure 2 This is a tracking analysis diagram of the cucumber-derived exosomes prepared in Example 1 of this invention. Figure 3 This is a bar chart showing the miRNA expression levels of cucumber-derived exosomes prepared in Example 1 of this invention. Figure 4 This is a transmission electron microscope image of UIO-66-NHCl prepared in Example 3 of the present invention; Figure 5 The XRD pattern of UIO-66-NHCl prepared in Example 3 of this invention; Figure 6 The infrared spectrum of UIO-66-NHCl prepared in Example 3 of this invention; Figure 7 Scanning electron microscope images of the hydrogel composite materials prepared in Example 4 and Comparative Examples 5-7 of the present invention; wherein, (a) Comparative Example 5, (b) Comparative Example 6, (c) Comparative Example 7, and (d) Example 4; Figure 8 The stress-strain curves of the hydrogel composite materials prepared in Example 4 and Comparative Examples 1-7 of this invention are shown. Figure 9 The bonding performance test diagram of the hydrogel composite material prepared in Example 4 is shown. Figure 10 The weight changes of the hydrogel composite material prepared in Example 4 of this invention at different swelling times; Figure 11 The degradation performance diagram of the hydrogel composite material prepared in Example 4 of this invention is shown. Figure 12 The antibacterial effects of the hydrogel composite materials prepared in Example 4 and Comparative Examples 1-7 of this invention against Escherichia coli and Staphylococcus aureus are shown in the figure. Figure 13 These are scanning electron microscope images showing the morphological changes of bacteria caused by the hydrogel composite materials prepared in Example 4 and Comparative Examples 3-4 of this invention. Figure 14 The figures show the cytotoxicity test results of the hydrogel composite materials prepared in Example 4 and Comparative Examples 3-4 of this invention. Figure 15 A photograph showing how the hydrogel composite material prepared in Example 4 of this invention promotes the healing of full-thickness skin defects in rats. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0050] To better illustrate the present invention, further examples are provided below.

[0051] Example 1 This invention provides a method for preparing cucumber-derived exosomes: Select 800g of fresh cucumbers, wash the surface with deionized water, remove the peel, and juice them using a homogenizer. Filter the juice through four layers of gauze to remove large particles and obtain cucumber juice. Centrifuge the cucumber juice sequentially at 800×g (10min), 2000×g (20min), 8000×g (30min), and 10000×g (40min) at 4℃, collect the final supernatant, filter it through 0.45μm and 0.22μm filter membranes, and then ultracentrifuge it at 150000×g for 1.5h. Discard the supernatant, resuspend the precipitate in 10mL of PBS buffer (pH 7.2~7.4) to obtain cucumber-derived exosome suspension (CELNS).

[0052] Transmission electron microscopy image of cucumber-derived exosome nanoparticles as shown below Figure 1 As shown in the figure, the cucumber-derived exosomes prepared in this embodiment have a spherical double membrane (coffee saucer-like) structure, with no obvious aggregation and uniform morphology.

[0053] The hydrated particle size of cucumber-derived exosome nanoparticles was determined using a Malvern laser particle size analyzer, such as... Figure 2 As shown in the figure, the particle size is concentrated in the range of 142-150 nm. The average particle size was measured to be 148.9 nm using a nanoparticle tracking analyzer (NTA), and the concentration was stable at 10. 6 Microparticles / mL. The Zeta potential of cucumber-derived exosomes is -22.85 mV.

[0054] The cucumber-derived exosomes prepared in this embodiment were subjected to miRNA sequencing analysis. The specific steps are as follows: (1) Total RNA extraction: Cucumber-derived exosome samples were taken and total RNA was extracted using the Trizol method. The specific operation was performed according to the kit instructions.

[0055] (2) RNA quality testing: RNA integrity was detected by agarose gel electrophoresis, and RNA purity was detected by Nanodrop 2000. 260 / A 280 The ratio), the RIN value (RNA integrity value) of RNA was detected by the Agilent 2100 Bioanalyzer.

[0056] (3) Small RNA library construction: Select qualified RNA samples and construct the library using the Illumina small RNA library construction kit. The specific steps include: RNA fragmentation, reverse transcription to synthesize cDNA, PCR amplification, and library purification.

[0057] (4) High-throughput sequencing: The constructed small RNA library was sequenced using the Illumina NovaSeq6000 sequencing platform, and the sequencing strategy was single-end sequencing (SE50).

[0058] (5) Data analysis: The sequencing data were filtered for quality control to remove low-quality reads, adapter sequences, etc.; the clean reads after quality control were compared with the miRBase database to identify known miRNAs and predict new miRNAs; the expression levels of miRNAs were statistically analyzed and the expression levels were standardized using the TPM (Transcripts Per Million) method; the target gene prediction and functional enrichment analysis (GO enrichment and KEGG pathway analysis) of highly expressed miRNAs were performed using the TargetScan database.

[0059] The results are as follows Figure 3 As shown, miRNA sequencing analysis revealed that cucumber-derived exosomes are rich in various functional miRNAs closely related to wound healing. Among them, miR-159a had the highest expression level (211,042 copies / μg), while miR-166a and miR-156a expression levels were both above 1.0 × 10⁻⁶. 5 The concentration of 1 copy / μg meets the effective concentration requirements for bioactive molecules. This result confirms that cucumber-derived exosomes are not simply drug carriers; their own high-abundance functional miRNAs can form a synergistic healing unit with luteolin, creating a "carrier-drug-active molecule" triad. This provides multiple signal regulation for chronic wound repair, further enhancing the wound repair effect.

[0060] Example 2 This invention provides a method for preparing cucumber-derived exosomes loaded with luteolin: Weigh 5 mg of luteolin and dissolve it in 25 mL of methanol. Add 50 mg (based on the dry weight of exosomes) of cucumber-derived exosomes prepared in Example 1 above. Incubate at 37 °C, 100 rpm, and in the dark for 3 h. Then transfer to a 100 kDa ultrafiltration tube and centrifuge at 3500 × g for 10 min to remove unloaded free luteolin. Invert the inner tube and centrifuge at 1000 × g for 2 min to obtain cucumber-derived exosomes loaded with luteolin (Lut@CELNs, which can be abbreviated as Lut@C).

[0061] The encapsulation efficiency of luteolin was determined by HPLC. The chromatographic conditions were as follows: Innoval C18 column (5 μm, 4.6 × 150 mm), detection wavelength 350 nm, column temperature 30 ℃, injection volume 10 μL, flow rate 1.0 mL / min, and mobile phase methanol-0.2% phosphoric acid aqueous solution (58:42, v / v).

[0062] HPLC analysis showed that the encapsulation efficiency of luteolin in Lut@CELNs was 78.2%. Transmission electron microscopy characterization revealed that the exosomes encapsulated with luteolin had a slightly increased particle size of 148.6 nm, while their shape remained unchanged.

[0063] Experiments have verified that the mass ratio of luteolin to cucumber-derived exosomes can be flexibly adjusted within the range of 1:5 to 1:20, specifically ratios such as 1:5, 1:8, 1:10, 1:15, and 1:20, all achieving drug loading efficiency and exosome bioactivity comparable to the aforementioned examples. Within this range, changes in the mass ratio did not significantly affect the particle size distribution, stability, drug release behavior, or synergistic healing-promoting function of luteolin-loaded cucumber-derived exosomes.

[0064] Example 3 This embodiment provides a method for preparing UIO-66-NHCl: Weigh 4.5 mmol of zirconium tetrachloride and 4.5 mmol of 2-aminoterephthalic acid, dissolve them in 40 mL of N,N-dimethylformamide, sonicate for 20 min, add 0.3 mmol of glacial acetic acid, sonicate for another 20 min, transfer the mixture to a stainless steel autoclave, react at 120 °C for 24 h, cool to room temperature, centrifuge at 12000 × g for 25 min, collect the precipitate, wash the precipitate three times each with N,N-dimethylformamide and anhydrous methanol, centrifuge at 8000 × g for 10 min after each wash, and then dry in a vacuum drying oven at 100 °C for 24 h to obtain UIO-66-NH2; Take 0.5g of the prepared UIO-66-NH2, add 20mL of ultrapure water, sonicate for 30min, add 2.5g of sodium p-toluenesulfonyl chloramine, stir magnetically for 4h under light-protected conditions, after the reaction is completed, centrifuge at 8000×g for 10min to collect the precipitate, wash the obtained precipitate three times with ultrapure water, and vacuum dry at 60℃ for 12h to obtain UIO-66-NHCl.

[0065] Figure 4 The image shows a transmission electron microscope (TEM) image of the UIO-66-NHCl prepared in this embodiment. As can be seen from the image, UIO-66-NHCl has a regular rhombic dodecahedral shape, a smooth and dense surface, a uniform morphology, good dispersibility, and no obvious agglomeration.

[0066] The hydrated particle size of UIO-66-NHCl was determined by dynamic light scattering (DLS), and the results showed that its particle size ranged from 100 to 200 nm, with an average particle size of 125 nm.

[0067] X-ray diffraction detection ( Figure 5 The results showed characteristic peaks at 2θ = 7.4°, 8.5°, 12.1°, and 14.2°, with no extraneous peaks, indicating good purity. Fourier transform infrared spectroscopy detection (…) Figure 6 This confirms that it contains -NH2 and -COO. - The presence of characteristic functional groups such as C, C / C-Cl, and Zr-O indicates the successful synthesis of UIO-66-NHCl.

[0068] In this embodiment, the experimental conditions can also be other conditions defined by the present invention. As long as they are within the scope defined by the present invention, the same effect as the UIO-66-NHCl prepared above can be achieved.

[0069] Example 4 This embodiment provides a method for preparing hydrogel composite materials: S1, weigh 5g of gelatin from pigskin, add it to 100mL of deionized water, stir in a constant temperature water bath at 55℃ for 30min until completely dissolved, add methacrylic anhydride with a molar ratio of 1:0.8 to gelatin, and continue stirring for 2h. Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 14kDa, dialyze in deionized water at 4℃ for 72h, changing the deionized water every 8h. After dialysis, centrifuge at 8000×g for 10min, collect the supernatant, and obtain a methacrylamide gelatin prepolymer solution with a total volume of 100mL. S2, Irgacure 2959 was added to the methacrylamide gelatin prepolymer solution and dispersed with PBS buffer. The mixture was vortexed to achieve a final concentration of 0.3% (w / v) of Irgacure 2959 in the prepolymer solution system. Subsequently, 0.3 g of Lut@CELNs prepared in Example 2 and 0.5 g of UIO-66-NHCl prepared in Example 3 were added, wherein the mass ratio of methacrylamide gelatin:Lut@CELNs:UIO-66-NHCl was 50:3:5, and the final concentration of UIO-66-NHCl in the system was 0.5% (w / v). The mixture was magnetically stirred for 30 min until uniformly dispersed. S3. Quickly inject the above mixed solution into a circular polydimethylsiloxane mold with a diameter of 10 mm and a height of 2 mm. Immediately place the mold under a UV lamp at a wavelength of 365 nm and a light intensity of 10 mW / cm². 2 Irradiate for 10 minutes under the specified conditions. Immediately after irradiation, remove the cross-linked and cured hydrogel from the mold to obtain the hydrogel composite material, denoted as Lut@C / UIO-66-NHCl@GelMA.

[0070] Comparative Example 1 The only difference between this comparative example and Example 2 is that the cucumber-derived exosomes loaded with luteolin are replaced with cucumber-derived exosomes loaded with curcumin; otherwise, they are exactly the same.

[0071] The method for preparing cucumber-derived exosomes loaded with curcumin includes the following steps: Weigh 5 mg of curcumin and dissolve it in 25 mL of methanol. Add 50 mg (based on the dry weight of exosomes) of cucumber-derived exosomes prepared in Example 1 above. Incubate at 37 °C, 100 rpm, and in the dark for 3 h. Then transfer to a 100 kDa ultrafiltration tube and centrifuge at 3500 × g for 10 min to remove unloaded curcumin. Invert the inner tube and centrifuge at 1000 × g for 2 min to obtain cucumber-derived exosomes loaded with curcumin (Cur@CELNs).

[0072] The above-mentioned Cur@CELNs were prepared using the same method as in Example 4 to obtain a hydrogel composite material, denoted as Cur@C / UIO-66-NHCl@GelMA.

[0073] Comparative Example 2 The only difference between this comparative example and Example 2 is that the cucumber-derived exosomes loaded with luteolin are replaced with cucumber-derived exosomes loaded with quercetin; otherwise, they are exactly the same.

[0074] The method for preparing cucumber-derived exosomes loaded with quercetin includes the following steps: Weigh 5 mg of quercetin and dissolve it in 25 mL of methanol. Add 50 mg (based on the dry weight of exosomes) of cucumber-derived exosomes prepared in Example 1 above. Incubate at 37 °C, 100 rpm, and in the dark for 3 h. Then transfer to a 100 kDa ultrafiltration tube and centrifuge at 3500 × g for 10 min to remove unloaded quercetin. Invert the inner tube and centrifuge at 1000 × g for 2 min to obtain cucumber-derived exosomes loaded with quercetin (Que@CELNs).

[0075] The above-prepared Que@CELNs were used to prepare a hydrogel composite material, denoted as Que@C / UIO-66-NHCl@GelMA, using the same method as in Example 4.

[0076] Comparative Example 3 The only difference between this comparative example and Example 4 is that the hydrogel composite material does not contain UIO-66-NHCl. The specific steps are as follows: S1, same as Example 4; S2, Irgacure 2959 was added to the methacrylamide gelatin prepolymer solution and dispersed with PBS buffer. The mixture was vortexed to achieve a final concentration of 0.3% (w / v) of Irgacure 2959 in the prepolymer solution system. Subsequently, 0.3 g of Lut@CELNs prepared in Example 2 was added, wherein the mass ratio of methacrylamide gelatin to Lut@CELNs was 50:3. The mixture was magnetically stirred for 30 min until uniformly dispersed. S3, same as Example 4, yields a hydrogel composite material, denoted as Lut@C@GelMA.

[0077] Comparative Example 4 The only difference between this comparative example and Example 4 is that the hydrogel composite material does not contain Lut@CELNs. The specific steps are as follows: S1, same as Example 4; S2, Irgacure 2959 was added to the methacrylamide gelatin prepolymer solution and dispersed with PBS buffer. The mixture was vortexed to achieve a final concentration of 0.3% (w / v) of Irgacure 2959 in the prepolymer solution system. Subsequently, 0.5 g of UIO-66-NHCl prepared in Example 3 was added, wherein the mass ratio of methacrylamide gelatin to UIO-66-NHCl was 50:5. The mixture was magnetically stirred for 30 min until uniformly dispersed. S3, same as in Example 4, yields UIO-66-NHCl@GelMA.

[0078] Comparative Example 5 The only difference between this comparative example and Example 4 is that UIO-66-NHCl in the hydrogel composite material is replaced with ZIF-8. The specific steps are as follows: S1, same as Example 4; S2, Irgacure 2959 was added to the methacrylamide gelatin prepolymer solution and dispersed with PBS buffer. The mixture was vortexed to achieve a final concentration of 0.3% (w / v) of Irgacure 2959 in the prepolymer solution system. Subsequently, Lut@CELNs and ZIF-8 prepared in Example 2 were added, wherein the mass ratio of methacrylamide gelatin:Lut@CELNs:ZIF-8 was 50:3:5, and the final concentration of ZIF-8 in the system was 0.5% (w / v). The mixture was magnetically stirred for 30 min until uniformly dispersed. S3, same as Example 4, yields Lut@C / ZIF-8@GelMA.

[0079] ZIF-8 preparation (Jiang J, Sun J, Zhu L, et al. Macrophage membrane encapsulated with manganese-doped ZIF-8 loaded quercetin alleviates acute lugin jury[J]. Materials & Design, 2026: 1155-18.).

[0080] Comparative Example 6 The only difference between this comparative example and Example 4 is that UIO-66-NHCl in the hydrogel composite material is replaced with hexadecyltrimethylammonium bromide (CTAB). To ensure the consistency of the antibacterial component concentration and the comparability of the experiments, the final concentration of CTAB is kept the same as the final concentration of UIO-66-NHCl in Example 4, which is 0.5% (w / v). Based on the amount of methacryloyl gelatin prepolymer solution added and the system volume in Example 4, the mass ratio of each component was calculated to be methacryloyl gelatin:Lut@CELNs:CTAB = 50:3:5. The specific steps are as follows: S1, same as Example 4; S2, Irgacure 2959 was added to the methacrylamide gelatin prepolymer solution and dispersed with PBS buffer. The mixture was vortexed to achieve a final concentration of 0.3% (w / v) of Irgacure 2959 in the prepolymer solution system. Subsequently, 0.3 g of Lut@CELNs and 0.5 g of hexadecyltrimethylammonium bromide (CTAB) prepared in Example 2 were added. The mass ratio of methacrylamide gelatin, Lut@CELNs and CTAB was 50:3:5. The final concentration of CTAB in the system was 0.5% (w / v). The mixture was magnetically stirred for 30 min until uniformly dispersed.

[0081] S3, same as Example 4, yields Lut@C / CTAB@GelMA.

[0082] Comparative Example 7 The only difference between this comparative example and comparative example 4 is that UIO-66-NHCl in the hydrogel composite material is replaced with vancomycin (Wan). The specific steps are as follows: S1, same as Example 4; S2, Irgacure 2959 was added to the methacrylamide gelatin prepolymer solution and dispersed with PBS buffer. The mixture was vortexed to achieve a final concentration of 0.3% (w / v) of Irgacure 2959 in the prepolymer solution system. Subsequently, 0.3g of Lut@CELNs and 0.5g of vancomycin (Wan) prepared in Example 2 were added, with a final concentration of vancomycin of 0.5% (w / v). The mass ratio of methacrylamide gelatin:Lut@CELNs:vancomycin was 50:3:5. The mixture was magnetically stirred for 30 minutes until uniformly dispersed. S3, same as Example 4, yields Lut@C / Wan@GelMA.

[0083] In vitro drug release evaluation Take 2 mL of each of the drug-loaded exosomes (Cur@CELNs, Que@CELNs, Lut@CELNs) prepared in Example 2 and Comparative Examples 1-2 (containing 2 mg of dry weight of exosomes), put them into a dialysis bag with a molecular weight cutoff of 14 kDa and seal it. Immerse it in 50 mL of PBS buffer and shake at 37 °C and 100 rpm in the dark. Take 2 mL of samples at 0 h, 6 h, 12 h, 24 h, 48 h and 72 h for detection, and add an equal amount of fresh medium at the same time.

[0084] Luteolin: Detection wavelength 350 nm, column temperature 30 ℃, injection volume 10 μL, flow rate 1.0 mL / min, mobile phase methanol-0.2% phosphoric acid aqueous solution (58:42).

[0085] Curcumin: Detection wavelength 428 nm, column temperature 30 ℃, injection volume 10 μL, flow rate 1.0 mL / min, mobile phase methanol-water (80:20).

[0086] Quercetin: Detection wavelength 374 nm, column temperature 30 ℃, injection volume 10 μL, flow rate 1.0 mL / min, mobile phase methanol-0.1% phosphoric acid aqueous solution (60:40).

[0087] Calculate the cumulative release rate. Set up 3 parallel samples in each group. The experiment was independently repeated 3 times. The results were taken as mean ± standard deviation.

[0088] Encapsulation efficiency EE% = (M1 / M) × 100% In the formula, M1 is the mass of Lut, Cur, and Que in Lut@CELNs, Cur@CELNs, and Que@CELNs determined by HPLC; M is the total mass of luteolin, curcumin, and quercetin added.

[0089] The results are shown in Table 1.

[0090] Table 1

[0091] Experimental results showed that cucumber exosomes loaded with luteolin (Lut@CELNs) had the highest encapsulation efficiency (78.2%), indicating good compatibility and encapsulation stability between luteolin and cucumber exosomes. The cumulative drug release rate of Lut@CELNs reached 80.0% ± 2.5% at 72 h, and the overall release curve was relatively smooth with no obvious burst release, indicating good sustained-release performance. In contrast, cucumber exosomes loaded with curcumin (Cur@CELNs) showed relatively slower initial release, with a cumulative drug release rate of 32.1% ± 1.7% at 24 h; cucumber exosomes loaded with quercetin (Que@CELNs) showed a cumulative drug release rate of 45.7% ± 2.2% at 24 h, with relatively faster initial release followed by a slower release. Considering the encapsulation efficiency, release rate, and release curve stability, Lut@CELNs exhibited superior overall drug release performance under the experimental conditions.

[0092] Physicochemical property testing of hydrogel composites Scanning electron microscope (SEM) images of the hydrogel composite materials prepared in Examples 4 and Comparative Examples 5-7 are shown below. Figure 7 As shown in the figure, it can be clearly observed that the antibacterial components (ZIF-8 and CTAB) in Comparative Examples 5 and 6 exhibit significant aggregation, forming localized "hard spots" and disrupting the continuity of the gel matrix; the surface of the hydrogel composite material in Comparative Example 7 shows cracking, indicating poor structural integrity. In the hydrogel composite material prepared in Example 4, UIO-66-NHCl particles are uniformly dispersed in the GelMA matrix without aggregation or cracking, and the three-dimensional gel network structure remains intact. This result confirms that the compatibility of UIO-66-NHCl with the gel matrix is ​​significantly better than that of other types of antibacterial drugs, and its regular morphology and dispersion characteristics can avoid damaging the dressing structure.

[0093] The hydrogel composite materials prepared in Examples 4 and Comparative Examples 1-7, as well as blank methacrylamide gelatin (GelMA, without UIO-66-NHCl and Lut@CELNs), were tested for their physicochemical properties according to the following methods: Each hydrogel composite material was cut into standard rectangular strips of 20mm (effective length) × 5mm (width) × 2mm (thickness), with 5 parallel samples per group. The strips were equilibrated for 2 hours at 25℃ and 50% relative humidity. Three tests were then performed using a universal testing machine (equipped with a 50N tensile sensor) at a tensile rate of 10mm / min and a clamping distance of 10mm at 25±1℃: ① Fracture strain test: Stretch the sample until it fractures, record the force-displacement curve, calculate the fracture strain, and the stress-strain curve is shown below. Figure 8 As shown: Fracture strain = (fracture elongation / initial clamping distance) × 100%.

[0094] ② Toughness test: quantified by the area under the stress-strain curve (unit: kJ / m). 3 This reflects the dressing's ability to absorb energy and resist breakage.

[0095] ③ Elastic recovery rate test: Stretch the sample to 100% strain and hold for 5 minutes, then restore it to its initial length at the same rate. Repeat this cycle 5 times and calculate the elastic recovery rate in the 5th cycle. Elastic recovery rate = (recovery length / stretching length) × 100%.

[0096] The results are shown in Table 2.

[0097] Table 2

[0098] The results showed that: Example 4 had the best mechanical properties; Comparative Examples 1, 3, and 4 had moderate properties, Comparative Example 2 had slightly lower properties, all of which were better than the blank group; Comparative Examples 5-7 had properties similar to the blank GelMA, but their performance was significantly degraded; The blank GelMA had no functional components and the weakest mechanical properties.

[0099] The above experimental results demonstrate that the combination of methacrylamide gelatin, cucumber-derived exosomes loaded with luteolin, and UIO-66-NHCl has a synergistic effect, resulting in a complete three-dimensional network structure of the hydrogel with excellent extensibility, fracture resistance, and elastic recovery, adaptable to dynamic deformation of the wound. Adding or replacing a single functional component leads to a decrease in the mechanical properties of the hydrogel, proving that the combination of luteolin and cucumber-derived exosomes is more advantageous in improving mechanical properties.

[0100] Replacing UIO-66-NHCl with ZIF-8, CTAB, or vancomycin easily leads to gel aggregation, cracking, and disruption of the three-dimensional network, resulting in mechanical properties deteriorating to near the control group. This confirms that UIO-66-NHCl is the optimal antibacterial component in this system. The rational formulation of functional components can effectively improve the mechanical properties of pure methacryloyl gelatin hydrogel, providing excellent mechanical support for its use as a wound dressing.

[0101] Fit test: The hydrogel composite dressing prepared in Example 4 was cut into circular dressings with a diameter of 2 cm and applied to the skin surface on the dorsal side of the finger joints of healthy volunteers. During the test, the volunteers sequentially performed the following actions: fingers in a naturally extended state (0°), slowly bent to 15°, 45°, and 90°, and performed free fingertip movements and multiple rapid flicks to simulate the dynamic deformation process of the joints during daily activities. The adhesion between the dressing and the skin was recorded using a digital camera during each action state. The results are as follows: Figure 9 As shown.

[0102] Figure 9 The results showed that under static and dynamic deformation conditions, including finger joint flexion of 0°, 15°, 45°, and 90°, as well as fingertip movement and repeated shaking, the dressing remained intact without any peeling, cracking, lifting, or displacement, demonstrating its ability to adhere closely to the irregular skin surface of the joint area. This result indicates that the composite hydrogel material prepared in this invention possesses excellent flexibility, adhesion, and mechanical adaptability, enabling it to adapt to the dynamic deformation requirements of joints and other areas. In clinical applications, it exhibits significant advantages in adaptability to irregular wounds and ease of use.

[0103] swelling rate The hydrogel composite material prepared in Example 4 was cut into circular samples with a diameter of 10 mm and a thickness of 2 mm. The samples were placed in PBS buffer (pH 7.2~7.4), and the samples were taken out every 2 hours until the testing time was 24 hours. The surface moisture was blotted with filter paper, and the wet weight (W) was accurately measured. t ); Calculate the swelling ratio (SR) according to the following formula, plot the swelling curve, and set up 3 parallel samples for each group.

[0104] SR(%)=[(W t -W0) / W0]×100% In the formula: W t Wt is the wet weight (g) of the sample at time t; W0 is the initial dry weight (g) of the sample.

[0105] The swelling curve of the composite hydrogel is as follows Figure 10 As shown in the figure, the swelling rate gradually increases with time and tends to stabilize after 24 hours, which is consistent with the typical swelling characteristics of hydrogels. The Lut@C / UIO-66-NHCl@GelMA composite hydrogel has a swelling rate of 59.1% after 24 hours, indicating that it can effectively absorb wound exudate, avoid the risk of infection caused by exudate accumulation, and maintain the stability of the gel structure, preventing rupture due to excessive swelling. It provides a continuously moist repair microenvironment for the wound, meeting the swelling performance requirements of wound dressings.

[0106] Degradation performance The hydrogel composite material prepared in Example 4 was cut into circular samples with a diameter of 10 mm and a thickness of 2 mm and dried to constant weight (W0).

[0107] Three degradation media were prepared: (1) 0.01 mol / L PBS (pH 7.4) containing 1 mg / mL collagenase type I (from bovine pancreas, activity ≥ 125 U / mg), filtered and sterilized; (2) 0.01 mol / L PBS (pH 7.4), autoclaved; (3)10 3 A bacterial culture of CFU / mL Escherichia coli and Staphylococcus aureus mixed at a volume ratio of 1:1.

[0108] The cut hydrogel samples were immersed in 50 mL of each of the three degradation media at a material-to-liquid ratio of 1:50 (w / v), and cultured at 37°C with shaking at 100 rpm. Samples were taken every 2 days, rinsed, dried, and weighed (w / v). t The degradation rate was calculated, and the results are as follows: Figure 11 As shown.

[0109] Degradation rate = [(W0-W)] t ) / W0]×100%.

[0110] The results showed that the dressing was completely degraded within 8 days in a collagenase environment and stably degraded within 25 days in a PBS and bacterial culture environment, indicating good biodegradability.

[0111] Antibacterial properties The antibacterial properties of the hydrogel composite materials prepared in Example 4 and Comparative Examples 1-7 were tested according to the following method: (1) Preparation of sterile liquid LB medium: Dissolve 10g of tryptone, 5g of yeast extract and 10g of NaCl in 950mL of deionized water, stir until completely dissolved, adjust the pH to 7.0 with 5mol / L NaOH, bring the volume to 1L, autoclave at 121℃ for 20min, and cool to room temperature for later use.

[0112] (2) LB solid plates: Add 15g of agar powder to the liquid LB medium, sterilize and wait for the temperature to drop to 55℃, then pour into sterile petri dishes (about 15mL per dish), let it solidify naturally and then seal and store at 4℃. Use within 1 week.

[0113] (3) Preparation of extract: Take 1.0g of each hydrogel composite material, aseptically cut it into 1mm×1mm pieces, add 10mL of sterile liquid LB medium, extract at 37℃ and 100rpm for 24h, sterilize with 0.22μm filter membrane to obtain 0.1g / mL extract.

[0114] (4) Simultaneously prepare a single GelMA extract (blank control 1) and a blank LB medium (blank control 2). Escherichia coli and Staphylococcus aureus were cultured to the logarithmic growth phase and adjusted to 10⁻⁶. 3 CFU / mL bacterial suspension. Take 1 mL of bacterial suspension (10... 3 Mix (CFU / mL) with 9 mL of extract (final concentration 10). 2 After incubating at 37°C for 24 hours (CFU / mL), take 0.1 mL of the mixture and serially dilute it (10 CFU / mL). 0 10 1 10 2 (Multiple dilutions), inoculated onto LB plates (3 replicates per dilution), incubated at 37°C for 16 hours, and then counted. Sterilization rate = [(Number of colonies in the control group - Number of colonies in the experimental group) / Number of colonies in the control group] × 100%.

[0115] Table 3

[0116] The results showed that the hydrogel composite material prepared in Example 4 had a significantly higher bactericidal rate against the two bacteria than the control materials, and exhibited significant antibacterial activity.

[0117] Figure 13 The images show scanning electron microscope (SEM) images of the bacterial morphology in Example 4 and Comparative Examples 3-4. As can be seen from the images, the bacteria in the single GelMA group have intact morphology; the bacterial cell membranes in the other groups show varying degrees of shrinkage and rupture. Among them, the bacterial morphology in Example 4 group is the most obviously ruptured, and the contents flow out.

[0118] In vitro bioactivity test In this experiment, different cell types were cultured and used in subsequent experiments using their respective dedicated culture media: L929 mouse fibroblasts and RAW264.7 mouse macrophages were cultured in DMEM high-glucose complete medium, and human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell-specific medium (ECM). The hydrogel extracts for each cell type were prepared using the cell's basal culture medium and diluted according to experimental requirements before use.

[0119] 1. Biocompatibility: L929 mouse fibroblasts were seeded in DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. The basic components of DMEM high glucose medium contained glucose (4.5 g / L), L-glutamine (0.584 g / L), sodium pyruvate (110 mg / L), and NaCl (6400 mg / L).

[0120] Incubate the cells in a constant temperature cell culture incubator at 37℃, 5% CO2, and saturated humidity until the cell confluence reaches 80-90%. Discard the old culture medium, gently wash once with 5 mL of sterile PBS (pH 7.4), add 2 mL of 0.25% trypsin-EDTA solution and digest at 37℃ for 2-3 min. Observe under a microscope that the cells become rounded and the gaps between them increase. Immediately add 8 mL of complete culture medium to stop the digestion, mix well by pipetting, and then mix 10 μL of cell suspension with 10 μL of 0.4% trypan blue staining solution. Use an automated cell counter to count the viable cells to ensure a viability of ≥95%.

[0121] The cell suspension was adjusted to a density of 5.0 × 10⁻⁶ using complete culture medium. 4 Cells / mL, add 100 μL of cell suspension to each well of a 96-well plate, and place the cell plate in a 37℃, 5% CO2 incubator for 24 h to allow the cells to adhere fully and enter the logarithmic growth phase.

[0122] Discard the original culture medium in the 96-well plate. Add 100 μL of fresh 0.05 g / mL complete DMEM medium (blank control group), the extract of Lut@C@GelMA prepared in Comparative Example 3, the extract of UIO-66-NHCl@GelMA prepared in Comparative Example 4 (negative control group), and the extract of Lut@C / UIO-66-NHCl@GelMA prepared in Example 4 (experimental group) to each well, with 3 replicates per group. At 24 h and 48 h of co-incubation, remove the 96-well plate, add 10 μL of CCK-8 reagent to each well, and continue incubation for 2 h. After incubation, measure the absorbance (OD) of each well using a multi-mode microplate reader at a detection wavelength of 450 nm. 450 ).

[0123] Preparation of extract: Take 1.0 g of the hydrogel composite material prepared in Example 4, cut it into 1 mm × 1 mm pieces under aseptic conditions, add 10 mL of complete DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, and extract at 37 °C and 100 pm for 24 h under constant temperature shaking conditions; after extraction, take the supernatant and filter it through a 0.22 μm sterile filter membrane to obtain an extract with a concentration of 0.1 g / mL.

[0124] Cell viability (%) = (Experimental group OD) 450 Mean / Blank Control Group OD 450 (Average) × 100%. Results are shown in Table 4 and... Figure 14 As shown.

[0125] Table 4

[0126] The experimental results show that all three types of dressings in this experiment have good in vitro biocompatibility, no obvious cytotoxicity, and meet the cell compatibility safety requirements for biomedical materials.

[0127] 2. Cell migration ability: L929 cells in the logarithmic growth phase were harvested and their density adjusted to 2 × 10⁻⁶ cells using the complete DMEM medium described above. 5 Add 2 mL of cell suspension to each well of a 6-well cell culture plate at 37°C and 5% CO2 for 24–36 h until the cell confluence reaches 90% or more and a dense monolayer is formed. Use a 200 μL sterile pipette tip to draw a straight line, discard the old culture medium, and gently rinse the bottom of the well three times with pre-warmed PBS (pH 7.4) to thoroughly remove detached cell debris.

[0128] Add 2 mL of complete DMEM medium containing 0.05 g / mL hydrogel composite material extract to each well. Set up a blank control group (with only complete medium) and a negative control group (containing 0.05 g / mL GelMA dressing extract), with 3 replicate wells in each group. Return the 6-well plate to the incubator for further incubation, and take fixed-point photographs of the same scratch area at three time points: 0 h, 24 h, and 48 h after scratching, using an inverted phase contrast microscope.

[0129] The method for preparing a complete DMEM culture medium containing 0.05 g / mL hydrogel composite material extract is as follows: Take the hydrogel composite material extract with a concentration of 0.1 g / mL and mix it with a complete DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics at a volume ratio of 1:1 to obtain a complete culture medium with a concentration of 0.05 g / mL hydrogel composite material extract.

[0130] ImageJ software was used to analyze the image, and the scratch width was measured. The average value was taken as the scratch width (W) at that time point. t Using the 0h scratch width (W0) as a baseline, the wound healing rate is calculated as follows: Healing rate (%) = [1 - (W0) / (W0)] t / W0)]×100%.

[0131] Experimental results showed that after treatment with the extract of the hydrogel composite material prepared in Example 4 for 48 hours, the scratch healing rate of L929 cells was 86.84%, which was significantly higher than that of the blank control group (56.32%) and the negative control group (65.24%). This indicates that the hydrogel composite material can effectively promote fibroblast migration and accelerate the re-epithelialization process of the wound.

[0132] 3. Cell proliferation capacity: The cell proliferation experiment used the CCK-8 assay, and the specific steps are as follows: L929 mouse fibroblasts were used at a rate of 5 × 10⁻⁶ 3 Cells were seeded at a density of 100 cells / well in 96-well plates using DMEM high-glucose complete medium (containing 4.5 g / L glucose, 0.584 g / L L-glutamine, 110 mg / L sodium pyruvate, and phenol red-free) containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin antibiotics. Cells were incubated at 37°C and 5% CO2 for 24 h to allow cell adhesion. The original medium in the 96-well plates was discarded, and 100 μL of fresh 0.05 g / mL complete DMEM medium (blank control), extract of single GelMA dressing (negative control), or extract of the hydrogel composite material prepared in Example 4 (experimental group) were added to each well. Each group had 3 replicates. After co-culturing for 24 h, 48 h, and 72 h, the chromogenic agent was added according to the CCK-8 reagent instructions. After incubation for 2 h, the absorbance (OD) was measured at 450 nm. 450 The results are shown in Table 5.

[0133] Preparation method of extract: Take the hydrogel composite material extract with a concentration of 0.1 g / mL and mix it with complete DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at a volume ratio of 1:1 to obtain the complete culture medium of hydrogel composite material extract with a concentration of 0.05 g / mL.

[0134] Proliferation rate (%) = (Experimental group OD) 450 Mean / Blank Control Group OD 450 (Average) × 100%.

[0135] Table 5

[0136] The results showed that the hydrogel composite material prepared in Example 4 could significantly promote the proliferation of L929 cells at 48h and 72h time points, and the proliferation rate was significantly higher than that of the negative control group and the blank control group.

[0137] 4. Angiogenesis Promoting Experiment (1) Human umbilical vein endothelial cells (HUVECs) were obtained and cultured using endothelial cell culture medium (ECM). The ECM culture medium consisted of basal medium, 5% (v / v) fetal bovine serum, 1% (v / v) endothelial cell growth supplement, and 1% (v / v) penicillin-streptomycin solution. The basal ECM culture medium (1L formulation) contained: glucose 5.5g, L-glutamine 0.584g, L-arginine hydrochloride 0.147g, L-lysine hydrochloride 0.073g, L-isoleucine 0.052g, L-leucine 0.052g, L-phenylalanine 0.032g, L-threonine 0.048g, L-tryptophan 0.009g, L-valine 0.046g, L-histidine hydrochloride monohydrate 0.042g, L-methionine 0.015g, and glycine 0. 0.015g, L-alanine 0.025g, L-asparagine monohydrate 0.015g, L-aspartic acid 0.02g, L-cysteine ​​hydrochloride monohydrate 0.035g, L-glutamic acid 0.02g, L-proline 0.028g, L-serine 0.023g, L-tyrosine disodium salt dihydrate 0.046g, sodium chloride 6.8g, potassium chloride 0.4g, calcium chloride 0.02g, magnesium sulfate 0.2g, anhydrous sodium dihydrogen phosphate 0.12g, sodium bicarbonate 2.2g, HEPES 2.383g of vitamin B1, 0.001g of vitamin B2, 0.001g of vitamin B6, 0.001g of nicotinamide, 0.002g of folic acid, 0.0002g of biotin, and 0.01g of phenol red are added to a final volume of 1L with deionized water, and the pH is adjusted to 7.2-7.4.

[0138] (2) Before the experiment, the Matrigel matrix was melted overnight in a 4°C refrigerator and operated on ice throughout to prevent premature gelation. 50 μL of pre-cooled Matrigel was added to the center of the bottom of each well of a 96-well plate to avoid the formation of air bubbles. The plate was then placed in a 37°C, 5% CO2 incubator and allowed to stand for 45 min to allow it to fully polymerize and form a gel layer.

[0139] (3) Adjust the cell density of HUVECs in the logarithmic growth phase to 2×10⁻⁶ cells using ECM medium. 4 Add 100 μL of HUVEC cell suspension (i.e., 2 × 10³ cells / well) to the solidified Matrigel wells, and immediately add 100 μL of the extract of the hydrogel composite material prepared in Example 4 (final concentration 0.05 g / mL, diluted with ECM medium). Set up a blank control group (add only 200 μL of ECM medium), with 5 replicates per group.

[0140] Preparation method of extract: Take 1.0g of the hydrogel composite material prepared in Example 4, cut it into 1mm×1mm pieces under aseptic conditions, add 10mL of ECM basic culture medium, and extract under constant temperature shaking conditions of 37℃ and 100rpm for 24h; after extraction, take the supernatant and filter it through a 0.22μm sterile filter membrane to remove bacteria, and obtain an ECM extract with a concentration of 0.1g / mL. Mix it with ECM complete culture medium at a volume ratio of 1:1 to obtain an extract complete culture medium with a final concentration of 0.05g / mL.

[0141] (4) The 96-well plate was placed in a 37℃, 5% CO2 incubator and cultured for another 6 hours. After the culture was completed, five fields of view were randomly selected and photographed under an inverted phase contrast microscope. ImageJ software was used to perform quantitative analysis on the images, and the number of lumen branch nodes and the total lumen length in each field of view were counted. The results are shown in Table 6.

[0142] Table 6

[0143] Experimental results show that, compared with the blank control group, the number of branch nodes in the lumen of the hydrogel composite material prepared in Example 4 of this invention increased by 75%, and the total lumen length reached 1.72 times that of the control group, showing significant pro-angiogenic activity.

[0144] 5. Evaluation of Inflammation Regulation (1) Take mouse macrophages RAW264.7 and culture them in DMEM high glucose medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin double antibiotics (basic components are the same as the biocompatibility test section).

[0145] (2) RAW264.7 cells were fed with 1×10 5 Inoculate the cells at a density of 1 cell / well in a 24-well plate, add 1 mL of complete culture medium to each well, and incubate at 37°C and 5% CO2 for 24 h. Discard the original culture medium and replace it with fresh complete culture medium containing 1 μg / mL lipopolysaccharide (LPS). Continue stimulation for 6 h to establish an in vitro inflammation model.

[0146] (3) After LPS stimulation, the LPS-containing medium was discarded, and 1 mL of fresh medium (experimental group) containing 0.05 g / mL of the extract of the hydrogel composite material prepared in Example 4 (diluted with the above-mentioned complete DMEM medium) was added. At the same time, an LPS model control group (only complete medium was added) was set up. After culturing for another 24 h, the cell culture supernatant was collected, centrifuged at 1000×g for 5 min at 4 °C to remove cell debris, aliquoted and stored at -80 °C for testing.

[0147] Preparation method of extract: Take the hydrogel composite material extract with a concentration of 0.1 g / mL and mix it with complete DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at a volume ratio of 1:1 to obtain the complete culture medium of hydrogel composite material extract with a concentration of 0.05 g / mL.

[0148] (4) The concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) in the supernatant were detected using an ELISA kit. The specific procedures were strictly performed according to the kit instructions: including the preparation of standard gradients (usually 0–1000 pg / mL), sample loading (with replicates), incubation at 37°C, plate washing, enzyme-labeled antibody reaction, TMB color development, and absorbance measurement at 450 nm. The actual concentrations (pg / mL) of each cytokine were calculated based on the standard curve. The results are shown in Table 7.

[0149] Table 7

[0150] Experimental results showed that, compared with the LPS model control group, the secretion levels of pro-inflammatory factors TNF-α and IL-6 in the hydrogel composite material extract treatment group prepared in Example 4 of this invention decreased by 52.1% and 48.3%, respectively, while the secretion level of anti-inflammatory factor IL-10 increased by 65.2%. This indicates that the dressing can effectively inhibit excessive inflammatory response, promote the transformation of inflammation to the repair stage, and thus optimize the inflammatory microenvironment of the wound.

[0151] Internal healing ability: (1) Thirty SPF-grade male rats aged 8 weeks and weighing 200-250g were selected. The hair on the back of the rats was removed, and the area was about 5cm×8cm. The rats were disinfected and defatted with 75% ethanol. They were anesthetized by intraperitoneal injection of 10% chloral hydrate solution (0.3mL / 100g body weight). About 1cm below the scapula on both sides of the spine, a sterile skin punch with a diameter of 1.5cm was used to press vertically and rotate to remove the full thickness of the skin (including the epidermis, dermis and subcutaneous fascia) in one go, forming two symmetrical full-thickness skin defects. The wound edges were kept neat and free from burns or tears.

[0152] (2) Thirty SPF-grade male rats were randomly divided into three groups of 10 each (n=10 / group): Experimental group: The hydrogel composite material Lut@C / UIO-66-NHCl@GelMA prepared in Example 4 was cut into circular pieces with a diameter of 1.8 cm for wound coverage. Blank control group: covered with a single GelMA (same size); Lut@C@GelMA group: Lut@C@GelMA (same size) prepared in Comparative Example 3.

[0153] (3) Change the dressing every 48 hours: Take photos before changing the dressing. Standardized photos of the wound were taken under the same lighting conditions on postoperative days 0, 3, 5, 7, and 12. The photos were imported into ImageJ software to calculate the remaining wound area (mm²). 2 The initial wound area (A0) was measured on postoperative day 0. The wound healing rate at each time point was calculated using the following formula: Healing rate (%) = [(A0) A t ) / A0]×100%, where A t The remaining wound area (mm²) on postoperative day t. 2 ).

[0154] The results are as follows Figure 15 As shown.

[0155] The results showed that by the 12th day after the operation, the average healing rate of the experimental group reached 89.6%, which was significantly higher than that of the blank control group (single GelMA dressing, 52.3%) and the Lut@C@GelMA group (65.1%), indicating that the hydrogel composite material prepared in Example 4 of the present invention can significantly accelerate the repair process of full-thickness skin defects.

[0156] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A hydrogel composite material, characterized in that, include: A three-dimensional network matrix formed by methacrylamide gelatin; as well as Plant-derived exosomes carrying luteolin and N-chloroamino-functionalized metal-organic framework materials are uniformly dispersed in the methacrylamide gelatin matrix.

2. The hydrogel composite material as described in claim 1, characterized in that, The swelling rate of the hydrogel composite material in PBS buffer for 24 hours was 55%~60%, and the fracture strain was 230%~280%.

3. The hydrogel composite material as described in claim 1, characterized in that, The mass ratio of the methacrylamide gelatin matrix, the plant-derived exosomes carrying luteolin, and the N-chloroaminofunctionalized metal-organic framework material is 50:(1~5):(1~10).

4. The hydrogel composite material as described in claim 1, characterized in that, The plant-derived exosomes are exosomes derived from cucumber fruit; and / or The N-chloroaminofunctionalized metal-organic framework material is UIO-66-NHCl.

5. The hydrogel composite material as described in claim 4, characterized in that, The method for preparing plant-derived exosomes loaded with luteolin includes the following steps: The cucumber fruit was juiced, and after differential centrifugation to remove cell debris and large particulate impurities, the juice was filtered through a microporous membrane. The filtrate was then subjected to ultracentrifugation, and the precipitate was collected to obtain cucumber-derived exosomes. The cucumber-derived exosomes were dispersed in a buffer solution to obtain a cucumber-derived exosome suspension; Luteolin was dissolved in an alcohol solvent and added to the cucumber-derived exosome suspension. The mixture was incubated to allow luteolin to enter the exosomes. Then, the unloaded luteolin was removed to obtain the plant-derived exosomes loaded with luteolin.

6. The hydrogel composite material as described in claim 4, characterized in that, The preparation method of the N-chloroamino-functionalized metal-organic framework material includes the following steps: The N-chloroaminofunctionalized metal-organic framework material was obtained by reacting UIO-66-NH2 and sodium p-toluenesulfonyl chloride with N-chloro.

7. The method for preparing the hydrogel composite material according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1, dissolve methacrylamide gelatin in buffer solution, add photoinitiator to obtain prepolymer solution; S2, plant-derived exosomes loaded with luteolin and N-chloroamino-functionalized metal-organic framework materials are added to the prepolymer liquid, mixed evenly, and photocrosslinked and cured to obtain a hydrogel composite material.

8. The method for preparing the hydrogel composite material as described in claim 7, characterized in that, In S1, the mass-to-volume ratio of the methacrylamide gelatin to the buffer solution is (5~15) g:100 mL; and / or In S1, the mass ratio of the photoinitiator to methacrylamide gelatin is 1:(15~30); and / or In S2, the light wavelength for photocrosslinking curing is 320nm~400nm, and the light intensity is 5mW / cm². 2 ~15mW / cm 2 The illumination time is 8 to 15 minutes.

9. The use of the hydrogel composite material according to any one of claims 1 to 6 in the preparation of medical dressings.

10. A medical dressing, characterized in that, Including the hydrogel composite material according to any one of claims 1 to 6.

Images