Temperature-sensitive hydrogel loaded with ginger outer vesicles and tannic acid, preparation method thereof and application thereof in preparing anti-infection and healing-promoting drugs

Abstract

This research relates to the biomedical field and describes a thermosensitive hydrogel loaded with ginger extracellular vesicles and tannic acid, its preparation method, and its application in the preparation of anti-infective drugs. The process includes extracting ginger-derived extracellular vesicles; loading tannic acid onto these vesicles to obtain TA@GDEVs; preparing SP hydrogels; and slowly adding TA@GDEVs to the SP hydrogel to form a GTSP hydrogel system. GDEVs exhibit anti-inflammatory and pro-repair activities; TA provides broad-spectrum antibacterial, anti-inflammatory, and antioxidant functions; the SP hydrogel endows the thermosensitive hydrogel composite system with injectable and in-situ gelation properties; the SP hydrogel not only provides in-situ gelation and a protective barrier for GDEVs and TA but also enables controlled release of the loaded material; through synergistic effects, the three components enable the thermosensitive hydrogel composite system to possess responsiveness to the infection microenvironment, enhanced antibacterial efficacy, immune remodeling, and tissue repair functions, demonstrating its potential as a multifunctional anti-infective wound repair material.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a thermosensitive hydrogel loaded with ginger vesicles and tannic acid, its preparation method, and its application in the preparation of anti-infective drugs. Background Technology

[0002] As the body's largest defense organ, the skin is easily affected by external stimuli such as strong impacts, intense friction, and sharp cuts, which often lead to skin trauma. If the trauma is severe and not treated promptly and effectively, it may lead to wound infection and even tissue necrosis due to microbial invasion and inflammatory infiltration.

[0003] Currently, the treatment of bacterial infections primarily relies on antibiotics. Since their advent in the 20th century, antibiotics have saved countless lives, marking a significant turning point in the long-term struggle between humanity and pathogenic bacteria. However, due to the overuse of antibiotics, bacterial resistance has become a major global public health threat. Studies have shown that the mortality rate of those infected with drug-resistant bacteria is 0.7 to 12 times higher than that of those infected with susceptible bacteria, meaning that skin infections caused by drug-resistant bacteria can lead to more serious consequences, including death. As resistance increases, the number of effective antibiotics is decreasing. Simultaneously, drug-resistant bacteria exacerbate the socioeconomic burden, leading to continuously rising healthcare costs. This not only places a financial burden on families but also strains the social healthcare system. Therefore, the search for and development of anti-infective drugs with excellent antibacterial effects that do not induce resistance is urgently needed.

[0004] Plant-derived substances have long been a focus of attention, as they hold promise as a potential alternative to antibiotics. Ginger-derived extracellular vesicles (GDEVs) are lipid bilayer vesicles with a diameter of approximately 100-300 nanometers, secreted by ginger cells. They carry various bioactive molecules, including proteins, lipids, and miRNAs, and possess negative surface potentials and good stability. Studies have shown that GDEVs can interact with bacterial surface proteins through their unique phosphatidic acid (PA), leading to selective endocytosis by pathogenic bacteria in a lipid-dependent manner. After uptake, GDEVs can disrupt bacterial cell membrane integrity, induce membrane depolarization, and inhibit pathogen growth. Unlike traditional antibiotics that rely on passive diffusion into cells, GDEVs, as natural nanodelivery carriers, can directly deliver multiple bioactive molecules into bacterial cells via membrane fusion or endocytosis, exerting a multi-target synergistic inhibitory effect. Furthermore, GDEVs also possess immunomodulatory functions, regulating macrophage polarization towards the repair-oriented M2 phenotype, providing a favorable microenvironment for tissue repair. Therefore, GDEVs possess antibacterial effects, drug delivery capabilities, and immunomodulatory functions.

[0005] Tannic acid (TA) is a naturally occurring hydrolyzable tannin primarily derived from plants in the Anacardiaceae family (such as Rhus chinensis). Its chemical structure consists of a glucose core linked by multiple gallic acids via ester bonds, and the molecule is rich in catechol and pyrogallol groups. This unique polyphenolic structure allows it to interact with biomolecules such as proteins and polysaccharides through various mechanisms, including hydrogen bonding, hydrophobic interactions, and metal coordination. Based on this structural foundation, tannic acid exhibits broad-spectrum and multi-target antibacterial activity: it can disrupt bacterial cell membrane integrity, inhibit biofilm formation, interfere with quorum sensing systems, and chelate essential metal ions. This multi-modal attack makes it difficult to induce bacterial resistance. Studies have shown that the introduction of tannic acid not only endows hydrogels with excellent antibacterial properties but also enables them to self-repair within minutes of damage, while also possessing anti-inflammatory and antioxidant functions. However, plant-derived substances generally suffer from low bioavailability, difficulty in storage, and easy degradation when used alone, thus requiring the involvement of drug delivery platforms.

[0006] Poloxamer 407 (P407) is a PEO-PPO-PEO triblock copolymer composed of polyethylene oxide (PEO) and polypropylene oxide (PPO). Its thermosensitive behavior in aqueous solution stems from its unique amphiphilic molecular structure. At low temperatures, the hydrated hydrophilic PEO blocks interact strongly with water molecules through hydrogen bonds, allowing the polymer to exist in the form of single chains or loose micelles, exhibiting a free-flowing sol. As the temperature rises, the hydrophobic PPO blocks dehydrate and shrink, and the enhanced hydrophobic interaction drives micelle aggregation, forming a three-dimensional physically cross-linked network, achieving a sol-gel phase transition. Based on this property, P407 is widely used in the construction of injectable hydrogels, showing promising applications in drug delivery and tissue engineering. However, single P407 hydrogels have significant limitations in practical applications: their gel network relies entirely on dynamic physical entanglement, resulting in typically low mechanical strength; they are prone to rapid dissolution under physiological conditions, leading to poor structural stability, difficulty in long-term maintenance, and significant drug burst release phenomena. Summary of the Invention

[0007] The purpose of this invention is to provide a thermosensitive hydrogel loaded with ginger vesicles and tannic acid, its preparation method, and its application in the preparation of anti-infective and healing-promoting drugs. This invention aims to address the problem of antibiotic overuse, improve the bioavailability of GDEVs and TA, reduce the frequency of medication, and achieve sustained drug release.

[0008] The technical solution adopted by this invention to solve the technical problem is as follows:

[0009] In a first aspect, the present invention provides a method for preparing a thermosensitive hydrogel loaded with ginger vesicles and tannic acid.

[0010] The present invention provides a method for preparing a thermosensitive hydrogel loaded with ginger vesicles and tannic acid, comprising the following steps:

[0011] Step 1: Extract extracellular vesicles from ginger cells;

[0012] Step 2: Tannins were encapsulated in ginger-derived extracellular vesicles to obtain TA@GDEVs;

[0013] Step 3: Prepare SP hydrogel;

[0014] Step 4: Slowly add TA@GDEVs into the SP hydrogel to prepare a GTSP hydrogel system.

[0015] In a preferred embodiment, in step one, fresh ginger is juiced and then subjected to low-speed centrifugation, high-speed centrifugation, and ultracentrifugation to obtain ginger-derived extracellular vesicles. The low-speed centrifugation is performed at a speed of 4500-5500 rpm for 25-35 min; the high-speed centrifugation is performed at a speed of 8000-12000 rpm for 0.5-1.5 h; and the ultracentrifugation is performed at a speed of 22500-27500 rpm for 1.5-2.5 h.

[0016] In a preferred embodiment, in step two, ginger-derived extracellular vesicles and tannic acid are prepared at a concentration of 4.5-5.5 mg / mL, subjected to ultrasonic treatment, and centrifuged to obtain TA@GDEVs.

[0017] As a preferred embodiment, the parameters of the ultrasonic treatment are: amplitude 15-25%, oscillation 25-35s, cooling 25-35s, duration 2-4min, cooling 1-3min and cycled 5-7 times.

[0018] In a preferred embodiment, in step three, sodium alginate powder is added to pre-cooled pure water at 4°C in a refrigerator or ice bath, and a clear solution is obtained after stirring. At the same time, P407 powder is added to pre-cooled pure water at 4°C, and a viscous liquid is obtained after stirring. The clear solution is slowly poured into the viscous liquid, and SP hydrogel is obtained after stirring under ice bath conditions.

[0019] In a preferred embodiment, the final concentration of the sodium alginate powder is 12%-20% W / V; and the final concentration of the P407 powder is 0.5%-1.5% W / V.

[0020] In a preferred embodiment, in step four, the GTSP hydrogel system is allowed to stand at 4°C for 2-4 hours to remove air bubbles from the system.

[0021] In a preferred embodiment, the volume ratio of TA@GDEVs to SP hydrogel is (1-3):(7-9).

[0022] Secondly, the present invention provides a thermosensitive hydrogel loaded with ginger vesicles and tannic acid obtained by the above preparation method.

[0023] Thirdly, the present invention provides the application of a thermosensitive hydrogel loaded with ginger vesicles and tannic acid in the preparation of an anti-infective and healing-promoting drug.

[0024] The beneficial effects of this invention are:

[0025] 1. This invention constructs a thermosensitive hydrogel composite system loaded with ginger vesicles and tannic acid by combining GDEVs, TA and SP hydrogel. This thermosensitive hydrogel composite system constructs a spatiotemporally ordered and functionally synergistic dynamic treatment platform through multiple non-covalent interactions, realizing the integration of multi-component functions.

[0026] GDEVs have anti-inflammatory and repair-promoting activities, and their natural lipid structure endows the thermosensitive hydrogel composite system with bacterial targeted endocytosis and immune regulation capabilities.

[0027] TA, with its polyphenol structure, enhances the mechanical properties of temperature-sensitive hydrogel composite systems while providing broad-spectrum antibacterial, anti-inflammatory, and antioxidant functions;

[0028] SP hydrogel, as a temperature-responsive framework, endows thermosensitive hydrogel composite systems with injectable and in-situ gelation properties; SP hydrogel not only provides in-situ gelation and protective barriers for GDEVs and TA, but also enables the controlled release of the loads;

[0029] Through a reasonable combination and synergistic effect, the three components enable this thermosensitive hydrogel composite system to possess functions such as infection microenvironment responsiveness, antibacterial enhancement, immune remodeling, and tissue repair. It can work together on multiple aspects of the healing of infected wounds, exerting a complementary effect and demonstrating its potential as a multifunctional anti-infective wound repair material.

[0030] 2. This invention employs a hierarchical structure of "external vesicles encapsulating polyphenols and hydrogel-loaded vesicles," which is beneficial for maintaining the stability of the active ingredients. The preparation process of this invention uses an ultrasonic method to encapsulate TA within GDEVs to form TA@GDEVs, which are then dispersed in SP hydrogel. This hierarchical structure design utilizes the phospholipid membrane of GDEVs to encapsulate and protect TA, reducing its oxidation or enzymatic degradation during storage and application; simultaneously, the SP hydrogel matrix, acting as an outer carrier, can delay the release of TA@GDEVs, prolonging its retention time on the wound surface.

[0031] In preparing SP hydrogels, this invention introduces the natural polysaccharide sodium alginate (SA) and poloxamer 407 (P407). The long molecular chains of SA can permeate and entangle within the P407 micelle network, forming an interpenetrating polymer network structure, which significantly enhances the rigidity and mechanical strength of the thermosensitive hydrogel. More importantly, as an anionic polysaccharide, the carboxylate groups on SA chains can form hydrogen bonds with the ether bonds of P407, and influence the assembly behavior of the P407 micelle network by regulating the ionic strength of the local microenvironment, thereby synergistically enhancing network stability, delaying the degradation of the thermosensitive hydrogel, and improving drug release behavior.

[0032] 3. This invention employs a preparation process combining gradient centrifugation and low-temperature ultrasound to reduce potential damage to the active ingredients. The preparation process is carried out under low-temperature conditions (4°C or ice bath), and impurities are removed through optimized gradient centrifugation to obtain GDEVs. The TA encapsulation process uses intermittent ultrasound to avoid damage to the GDEV membrane structure caused by prolonged continuous ultrasound. This process condition is relatively mild, which helps maintain the integrity of GDEVs and the activity of TA.

[0033] 4. The thermosensitive hydrogel composite system loaded with ginger vesicles and tannic acid constructed in this invention possesses injectability, thermosensitive gel properties, and anti-infection and repair-promoting functions, demonstrating potential for further clinical translation. The thermosensitive hydrogel loaded with GDEVs and TA constructed in this invention balances convenience and effectiveness in clinical use in its dosage form design: its injectability allows for minimally invasive application to irregular or deep wounds, while its thermosensitive properties ensure rapid in-situ gelation in vivo, avoiding secondary damage caused by frequent dressing changes. Furthermore, the combination of GDEVs and TA exhibits good anti-infection and healing-promoting effects in both in vitro and in vivo models, and all components used are derived from natural products or pharmaceutical excipients, demonstrating high biocompatibility.

[0034] The above-mentioned characteristics make the thermosensitive hydrogel composite system loaded with ginger vesicles and tannic acid constructed in this invention potentially valuable for the development of novel drugs or medical devices for the treatment of infected wounds. Attached Figure Description

[0035] Figure 1 TEM image of GDEVs.

[0036] Figure 2 Images for GDEVs NTA detection.

[0037] Figure 3 This is a gelation diagram of SP hydrogel.

[0038] Figure 4 Images of SP hydrogel flowability and injectability.

[0039] Figure 5This is a SEM image of the SP hydrogel.

[0040] Figure 6 XRD image of GTSP hydrogel.

[0041] Figure 7 The in vitro drug release curve of GTSP hydrogel is shown.

[0042] Figure 8 The in vitro antibacterial rate of GTSP hydrogel.

[0043] Figure 9 The wound healing rate of mice in each treatment group with Escherichia coli-infected wounds was measured.

[0044] Figure 10 The wound healing rate of mice in each treatment group with Staphylococcus aureus-infected wounds was measured.

[0045] Figure 11 The wound healing rate of mice in different treatment groups infected with methicillin-resistant Staphylococcus aureus was measured.

[0046] Figure 12 The results are from the biosafety experiments of the GTSP hydrogel.

[0047] Figure 13 The results of experiments on the effect of GTSP hydrogel on promoting HDF cell proliferation. Detailed Implementation

[0048] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0049] Example 1: Extraction of ginger-derived extracellular vesicles (GDEVs)

[0050] This embodiment provides an extraction process for ginger-derived extracellular vesicles (GDEVs), which are prepared by gradient centrifugation. The specific implementation process is as follows:

[0051] (1) Juice fresh ginger and water at a ratio of 1:6;

[0052] (2) Use a low-speed centrifuge for initial centrifugation, with a speed of 5000 rpm and a time of 30 min;

[0053] (3) Further centrifuge using a high-speed centrifuge at a speed of 10,000 rpm for 1 hour;

[0054] (4) Use an ultra-high speed centrifuge to perform ultra-high speed centrifugation at a speed of 25,000 rpm for 2 hours to obtain ginger-derived extracellular vesicles (GDEVs).

[0055] The TEM images of GDEVs extracted in this embodiment are as follows: Figure 1 As shown, transmission electron microscopy reveals that GDEVs exhibit a classic circular vesicle shape.

[0056] The NTA detection images of GDEVs extracted in this embodiment are as follows: Figure 2 As shown, the analysis images from the nanoparticle size analyzer show that the average diameter of GDEVs is approximately 132.80 nm, which is consistent with the typical size range of exovesicles.

[0057] Example 2: Tannins (TA) encapsulated in ginger-derived extracellular vesicles (GDEVs)

[0058] The ginger-derived extracellular vesicles (GDEVs) prepared in Example 1 were mixed with tannic acid (TA) to form a 5 mg / mL solution. The solution was then subjected to an ultrasonic method with the following parameters: amplitude 20%, oscillation for 30 s, cooling for 30 s, continuous for 3 min, cooling for 2 min, and repeated 6 times. The solution was then centrifuged at 1000 rpm for 30 min to obtain TA@GDEVs.

[0059] Example 3 Preparation of SP hydrogel

[0060] This embodiment provides a method for preparing SP hydrogel, the specific implementation process of which is as follows:

[0061] (1) The entire preparation process was carried out in a 4°C refrigerator or ice bath to prevent poloxamer 407 (P407) from forming micelles prematurely;

[0062] (2) Preparation of SA matrix solution (solution A): Accurately weigh sodium alginate powder (16% W / V), put the sodium alginate powder into a small beaker, add 2-3 mL of ice-pre-cooled pure water, place it on a magnetic stirrer in a 4℃ refrigerator, and stir at low speed for 2-4 h until it is completely dissolved into a uniform, slightly viscous clear solution.

[0063] (3) Preparation of P407 solution (solution B): Accurately weigh P407 powder (1.0% W / V); put P407 powder into a small beaker, very slowly sprinkle in 4-5 mL of ice-precooled pure water, place it on a magnetic stirrer in a 4℃ refrigerator, and stir at low speed for 2-4 h to obtain a clear, transparent viscous liquid.

[0064] (4) While solution B is completely dissolved and kept cold, slowly pour solution A into solution B, rinse the beaker containing solution A with a small amount (about 1 mL) of ice water, and pour the rinsing solution into the mixture to ensure that all materials are transferred.

[0065] (5) Under ice bath conditions, stir at low speed for 4 h to obtain SP hydrogel, and store at 4℃ overnight.

[0066] SP hydrogel temperature response image as follows Figure 3 As shown, at 25°C, SP hydrogel has good fluidity and can flow with the tilt of the centrifuge tube. When the temperature rises to 35°C, SP hydrogel becomes a semi-solid gel.

[0067] like Figure 4 As shown, the top image is a flowability image of the SP hydrogel, and the bottom image is a printability image of the SP hydrogel. At room temperature, the SP hydrogel exhibits excellent flowability, passing smoothly through a syringe and allowing for writing, indicating that the SP hydrogel meets the requirements for injectable gels.

[0068] like Figure 5 As shown, the microstructure of SP hydrogel was observed by scanning electron microscopy (SEM). The SEM images revealed a unique porous structure with pore sizes mainly between 21.5 µm and 33.7 µm. The dense hydrogel network enables drug encapsulation and achieves sustained drug release.

[0069] Example 4: Preparation of GTSP hydrogel

[0070] Slowly add 1-3 mL of TA@GDEVs prepared in Example 2 to 7-9 mL of SP hydrogel prepared in Example 3 to finally prepare a 10 mL GTSP hydrogel system. Stir gently and let the prepared final solution stand at 4°C for 2-4 h to remove air bubbles and allow the system to fully equilibrate.

[0071] XRD images of GTSP hydrogel are as follows Figure 6As shown, compared with SA and P407, the diffraction pattern of SP hydrogel shows a new broadened peak at 2θ≈18.0°, and the intensity of the strong peak at 24.0° in solution B is significantly reduced and the peak shape is broadened. This indicates that sodium alginate and poloxamer 407 form a semi-crystalline network structure through intermolecular interactions during gelation, and the orderliness of the crystalline regions decreases, confirming the successful construction of SP hydrogel. After encapsulating ginger-derived extracellular vesicles (GDEVs), the diffraction peak positions of GTSP hydrogel are basically consistent with those of SP hydrogel, and the main structure of hydrogel is preserved. At the same time, the scattering intensity of GTSP hydrogel is enhanced in the low-angle region (2θ≈5.0°), which may indicate that nanoscale ginger-derived extracellular vesicles (GDEVs) are uniformly distributed in the gel matrix, confirming the successful encapsulation of ginger-derived extracellular vesicles (GDEVs).

[0072] Results of GTSP hydrogel in vitro drug release: Figure 7 As shown, GDEVs were continuously released within 96 hours, reaching a peak of 62.83% at 72 hours and 62.07% at 96 hours. It is speculated that the excessively long degradation time led to the large-scale breakage of GDEVs.

[0073] Example 5: In vitro antibacterial activity of GTSP hydrogel

[0074] To evaluate the in vitro antibacterial effect of GTSP hydrogel, 1×10 8 CFU / mL E. coli, 1×10 8 Staphylococcus aureus (S. aureus), methicillin-resistant Staphylococcus aureus (MRSA), and GTSP hydrogel were co-cultured, and OD values ​​were measured at 0 h, 6 h, 12 h, 18 h, and 24 h to calculate the inhibition rate.

[0075] The in vitro antibacterial rate of GTSP hydrogel is as follows: Figure 8 As shown, after 24 hours of co-culture, the GTSP hydrogel exhibited in vitro inhibition rates of 91.80%, 75.95%, and 72.37% against Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus, respectively.

[0076] Example 6: Effects of GTSP hydrogel on anti-wound infection and wound healing in mice

[0077] Mice were acclimatized for one week, then anesthetized and had their backs shaved. A full-skin wound model was prepared using surgical scissors. Escherichia coli (50 μL, 1×10⁻⁶) was then introduced. 8 CFU / mL), Staphylococcus aureus (50 μL, 1×10⁻⁶ ... 8 CFU / mL) and methicillin-resistant Staphylococcus aureus (50 μL, 1×10⁻⁶ CFU / mL)8 GTSP hydrogel (CFU / mL) was applied to the wound site of mice for infection for 48 h. When redness and swelling and a large amount of tissue exudate appeared at the wound site, GTSP hydrogel was applied to the wound and the wound healing was observed and compared with the control group, the free GDEVs group, and the SP hydrogel group. The wound healing rate of mice in each treatment group was calculated at 0, 4, 7, 10, and 14 days.

[0078] Wound healing rates of various bacterial species in mice, as follows: Figures 9 to 11 As shown, the GTSP hydrogel group had better wound healing than the other groups throughout the treatment process, indicating that GTSP hydrogel has a positive effect on wounds and can promote wound healing in a short period of time.

[0079] Example 7: Biocompatibility of GTSP Hydrogel

[0080] The biocompatibility of GTSP hydrogels was evaluated using the MTT assay. L929 mouse fibroblasts were injected at a concentration of 1×10⁻⁶ cells / mL. 4 GTSP hydrogels were seeded at a density of 100 μg / well in 96-well plates and cultured in DMEM medium containing 10% fetal bovine serum for 24 h. The GTSP hydrogels were then immersed in PBS and incubated at 37 °C with constant shaking at 100 rpm for 48 h. The extract was collected, filtered through a 0.22 μm filter for sterilization, and stored at 4 °C for later use. The original medium was discarded, and 45 μg / mL (based on GDEV content) GTSP hydrogel extract prepared in serum-free DMEM medium and a blank control (serum-free DMEM medium) were added. 20 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated at 37 °C for 4 h. The supernatant was discarded, and 150 μL of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader. Figure 12 As shown, the experimental results indicated that the cell viability was above 85% within 48 hours, confirming that the GTSP hydrogel extract has no toxic effect on mammalian cells and has excellent in vitro cell compatibility.

[0081] Example 8: Experiment on the effect of GTSP hydrogel on promoting HDF cell proliferation

[0082] The effect of this treatment on the proliferation of HDF cells was evaluated using a cell scratch assay. Human skin fibroblasts (HDFs) were injected at a rate of 2 × 10⁻⁶ cells / year. 5 Seed cells at a density of 1 cell / well in 6-well plates and cultured to 90-95% confluence. Using a sterile 200 μL pipette tip, create linear scratches along a ruler on the cell monolayer. Gently wash three times with PBS to remove detached cells. Add serum-free DMEM medium containing the following treatments:

[0083] (1) Blank control group: only serum-free DMEM medium was added, without any hydrogels, exovesicles or other additives;

[0084] (2) SP hydrogel group: SP hydrogel extract was added (prepared according to Example 7, i.e., SP hydrogel was immersed in PBS, incubated at 37 °C and 100 rpm for 48 h, the extract was collected, filtered through a 0.22 μm filter membrane for sterilization, and stored at 4 °C for later use).

[0085] (3) Free GDEVs group: GDEVs prepared in Example 1 were added (diluted to 30 μg / mL with serum-free DMEM (based on GDEV content));

[0086] (4) GTSP hydrogel group: Add the GTSP hydrogel extract prepared in Example 7 (diluted to 30 μg / mL with serum-free DMEM (based on GDEVs content)).

[0087] The healing process of the scratches was recorded by taking photos of the same location under an inverted microscope at 0 h, 6 h, 12 h and 24 h.

[0088] like Figure 13 As shown in the results, the scratch healing in the blank control group was slow, with a relatively wide exposed area remaining after 24 hours. The SP hydrogel group showed slight improvement compared to the control group, with a reduction in scratch width. The free GDEVs group exhibited an increasing trend in promoting migration with increasing concentration, and the scratch closure was significantly better than that of the control group and the SP hydrogel group. The GTSP hydrogel group showed the best scratch healing effect at each corresponding concentration, with a concentration-dependent increase, and the high-dose group showed almost complete scratch closure. These results confirm that GTSP, through continuous local release from the hydrogel matrix, effectively enhances GDEVs-mediated fibroblast recruitment and migration, and has significant wound repair activity.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a thermosensitive hydrogel loaded with ginger vesicles and tannic acid, characterized in that, Includes the following steps: Step 1: Extract extracellular vesicles from ginger cells; Step 2: Tannins were encapsulated in ginger-derived extracellular vesicles to obtain TA@GDEVs; Step 3: Prepare SP hydrogel; Step 4: Slowly add TA@GDEVs into the SP hydrogel to prepare a GTSP hydrogel system.

2. The method for preparing the thermosensitive hydrogel loaded with ginger vesicles and tannic acid according to claim 1, characterized in that, In step one, fresh ginger is juiced and then subjected to low-speed centrifugation, high-speed centrifugation, and ultracentrifugation to obtain ginger-derived extracellular vesicles. The low-speed centrifugation is performed at a speed of 4500-5500 rpm for 25-35 min; the high-speed centrifugation is performed at a speed of 8000-12000 rpm for 0.5-1.5 h; and the ultracentrifugation is performed at a speed of 22500-27500 rpm for 1.5-2.5 h.

3. The method for preparing the thermosensitive hydrogel loaded with ginger vesicles and tannic acid according to claim 1, characterized in that, In step two, ginger-derived extracellular vesicles and tannic acid were prepared into a solution with a concentration of 4.5-5.5 mg / mL. After mixing, the solution was sonicated and centrifuged to obtain TA@GDEVs.

4. The method for preparing the thermosensitive hydrogel loaded with ginger vesicles and tannic acid according to claim 3, characterized in that, The parameters for the ultrasonic treatment are: amplitude 15-25%, oscillation 25-35s, cooling 25-35s, continuous for 2-4min, cooling for 1-3min, and repeated 5-7 times.

5. The method for preparing the thermosensitive hydrogel loaded with ginger vesicles and tannic acid according to claim 1, characterized in that, In step three, sodium alginate powder is added to ice-precooled pure water under 4°C refrigerator or ice bath conditions, and a clear solution is obtained after stirring. At the same time, P407 powder is added to ice-precooled pure water, and a viscous liquid is obtained after stirring. The clear solution was slowly poured into the viscous liquid, and the SP hydrogel was obtained after stirring under ice bath conditions.

6. The method for preparing the thermosensitive hydrogel loaded with ginger vesicles and tannic acid according to claim 5, characterized in that, The final concentration of the sodium alginate powder is 12%-20% W / V; the final concentration of the P407 powder is 0.5%-1.5% W / V.

7. The method for preparing the thermosensitive hydrogel loaded with ginger vesicles and tannic acid according to claim 1, characterized in that, In step four, the GTSP hydrogel system is allowed to stand at 4°C for 2-4 hours to remove air bubbles from the system.

8. The method for preparing the thermosensitive hydrogel loaded with ginger vesicles and tannic acid according to claim 1, characterized in that, The volume ratio of TA@GDEVs to SP hydrogel is (1-3):(7-9).

9. The thermosensitive hydrogel loaded with ginger vesicles and tannic acid obtained by the preparation method according to any one of claims 1-8.

10. The application of the thermosensitive hydrogel loaded with ginger vesicles and tannic acid as described in claim 9 in the preparation of an anti-infective and healing-promoting drug.

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