Colored rice exosome hydrogel and application thereof in preparation of wound dressing

Abstract

The colored rice exosome hydrogel is prepared by the following steps: uniformly mixing bletilla striata polysaccharide and konjac polysaccharide, adding distilled water into the polysaccharide mixture, uniformly stirring, adding a sodium hydroxide solution, continuously stirring until the solution is uniform, standing at room temperature, filtering, washing, and drying to obtain the colored rice exosome hydrogel. The preparation method comprises the following steps: preparing a hydrogel precursor solution, putting the hydrogel precursor solution into a hot water bath, heating to prepare the hydrogel precursor solution, adding a colored rice exosome into the hydrogel precursor solution, placing at room temperature, and co-incubating to form gel, thereby obtaining the colored rice exosome hydrogel. The colored rice exosome hydrogel is applied to an animal experiment for promoting wound healing, an experiment result shows that the colored rice exosome hydrogel can promote wound healing, and the effect of the colored rice exosome hydrogel is better than that of hydrogel purchased in the market and a common wound dressing band-aid; the colored rice exosome hydrogel disclosed by the invention is simple to prepare, rapid in gelling and high in reproducibility, can be used for large-scale industrial production, has the advantages of high biological safety, remarkable anti-inflammatory, antibacterial and antioxidant effects and the like, and also has the characteristics of good air permeability, swelling property and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine and medical devices, and particularly relates to a colored rice exosome hydrogel with antioxidant, anti-inflammatory and wound healing promotion and application thereof in preparation of wound dressings. BACKGROUND

[0002] Exosomes are currently a hot research topic, but there are few reports on gel products using plant-derived exosomes for promoting wound healing. The colored rice exosome is an exosome extracted from natural plants, which has the advantages of wide source, high biological safety and low immunogenicity. In addition, the colored rice in this study is a poor quality rice that is difficult to sell and is about to be discarded, which is purchased from the hands of rice farmers in Yunnan. The exosomes are extracted from it to make full use of the resources and avoid waste.

[0003] Gels have been widely used as drug delivery platforms in drug release and tissue engineering fields. The hydrogel has high biological safety, good biocompatibility and biodegradability, and can be combined with exosomes in a physical or chemical adsorption manner to prepare the corresponding dosage form. Through diffusion, penetration and other ways, it enters the human body, so that the active ingredients are continuously and slowly released in the human body at a stable and controllable rate and appropriate concentration. Its advantage is that it can effectively solve the problems of low bioavailability and short half-life of exosomes. And the colored rice exosome hydrogel prepared in this study has antioxidant, anti-inflammatory and wound healing promotion biological activities, which provides a new development idea for the research and development of new type of wound dressings. SUMMARY

[0004] To solve the above technical problems, the present application provides a colored rice exosome hydrogel with antioxidant, anti-inflammatory and wound healing promotion, which is prepared as follows: (1) The colored rice exosome is prepared according to the method in patent application 202511343447.3, that is, the washed and crushed colored rice powder is added to PBS buffer with pH 6-7.4 and homogenized at high speed, and then α - amylase is added to the rice slurry, and enzyme hydrolysis is carried out at pH 6-7.4 and 90-97℃ for 1-2h, filtration, and the filtrate is left overnight; then gradient differential centrifugation is carried out at 4℃, the supernatant is collected, filtered, and the filtrate is ultracentrifuged at 4℃ and 12000-15000g for 1.5-3h, and the precipitate is collected; the precipitate is resuspended and enriched with MWCO ultrafiltration tube with a molecular weight cut-off of 100kDa, and the precipitate at the bottom of the ultrafiltration tube is collected to prepare the colored rice exosome; (2) Pretreatment of polysaccharides: mix small white yam polysaccharide and konjac polysaccharide, and then add distilled water to the polysaccharide mixture and stir uniformly; The mass ratio of Bletilla striata polysaccharide to konjac polysaccharide is 1:2-4, and the mass-volume ratio of the polysaccharide mixture to distilled water is 1:8-12 (g:mL). (3) Add sodium hydroxide solution to the mixture in step (2), continue stirring until the solution is uniform, let it stand at room temperature for 0.5-1.5 hours, and then heat it in a hot water bath at 65-75℃ for 25-35 minutes to obtain the hydrogel precursor solution; The sodium hydroxide has a mass concentration of 0.1-0.3%, and the mass-to-volume ratio (g:mL) of the polysaccharide mixture to the sodium hydroxide solution is 1:4-6. (4) Add colored rice exosomes to the hydrogel precursor solution, and then incubate at room temperature for 1-3 hours to form a gel, thus obtaining colored rice exosome hydrogel; The mass-to-volume ratio (mg:mL) of the colored rice exosomes to the hydrogel precursor solution is 1:2-3.

[0005] Another object of the present invention is to provide the use of the above-mentioned colored rice exosome hydrogel, namely, its use in the preparation of wound dressings.

[0006] Advantages and technical effects of this invention: 1. The colored rice exosome hydrogel provided by this invention has antioxidant, anti-inflammatory and wound healing functions, as well as high biocompatibility, good air permeability and swelling properties, providing a new approach for the treatment of wounds; 2. This invention employs methods including physical cross-linking and co-incubation, combined with a natural gel base, to effectively encapsulate active ingredients for sustained release and responsive drug delivery. It also forms a physical barrier on the wound skin, promoting skin tissue repair and regeneration. Furthermore, it significantly accelerates wound healing and reduces scar formation, improving the biosafety of the hydrogel, simplifying the preparation process, and reducing production costs, thus facilitating large-scale production and application. It has enormous application potential in the field of biomedical devices such as wound dressings. 3. Plant-derived colored rice exosomes, as natural products, have advantages such as high biocompatibility and low immunogenicity; In summary, this invention broadens the types of drug active ingredients that can be loaded onto hydrogels, especially by utilizing the unique characteristics of colored rice, providing more options for the source of drug exosomes and enhancing resource availability. The colored rice exosome hydrogel of this invention has broad application prospects in the fields of anti-oxidation, anti-inflammation, and wound healing promotion, providing a new direction for the development of biomedical devices, and is expected to be used to treat a variety of complex wounds and related diseases. Attached Figure Description

[0007] Figure 1 Morphological images of purple rice exosome hydrogels or red rice exosome hydrogels; Figure 2This is a schematic diagram showing that the hydrogel prepared in control group 1 was difficult to shape or had a soft texture. Figure 3 Fourier transform infrared (FTIR) spectra of colored rice exosome hydrogel and other control samples; Figure 4 The images show the scanning electron microscope (SEM) results of the hydrogels. The top left image is the pore size diagram of the blank hydrogel, the top right image is the blank hydrogel, the bottom left image is the purple rice exosome hydrogel, and the bottom right image is the red rice exosome hydrogel. Figure 5 The figure shows the rheological test results of the blank hydrogel. Figure 6 The figure shows the rheological test results of the red rice exosome hydrogel. Figure 7 The figure shows the rheological and mechanical test results of the purple rice exosome hydrogel. Figure 8 The graph shows the water vapor transmission rate test results for the hydrogel. Figure 9 The swelling rates of purple rice and red rice exosome hydrogels at different pH values ​​are shown. Figure 10 The swelling rates of purple rice and red rice exosome hydrogels under different concentrations of ROS are shown. Figure 11 The image shows the results of the blood compatibility test for the hydrogel. Figure 12 The graph shows the antioxidant test results of the hydrogel. The left graph shows the ABTS scavenging rate, and the right graph shows the DPPH scavenging rate. Figure 13 The results of CCK-8 assay for the effect of hydrogel extract on cell viability are shown. The left image is purple rice hydrogel; the right image is red rice hydrogel. Figure 14 The results show the experimental findings of nitric oxide measurement (top left) and ELISA detection of IL-6 (top right), IL-1β (bottom left), and TNF-α (bottom right) levels in cells. Figure 15 The images show the results of testing the effect of hydrogel on wound healing using an animal model method. The top image shows the macroscopic situation of wound healing, and the bottom image shows the wound area healing rate analysis. Figure 16 Figure showing the experimental results of H&E section staining method for detecting the wound healing effect of hydrogel; Figure 17 Figure showing the experimental results of using the Masson section staining method to detect the wound-healing effect of hydrogels. Detailed Implementation

[0008] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Example 1: Preparation of colored rice exosome hydrogels and control samples 1. Preparation of colored rice exosome hydrogels (1) Rinse the purple rice with deionized water and drain. Grind it into ultrafine powder to 110-180 mm using an air jet mill. Add PBS with pH 6.0 to 100 g of ultrafinely ground purple rice powder and homogenize it 8 times (1 min / time) at 1500 rpm / min using a vortex mixer. Add PBS at a ratio of 200 μg / 100 mL. α- Amylase was added to rice liquid and enzymatically hydrolyzed at pH 6.0 and 97℃ for 2 hours. The mixture was filtered through eight layers of gauze, and the filtrate was allowed to stand overnight. The enzymatically hydrolyzed and stood rice liquid was then centrifuged at low temperature and high speed using a centrifuge, specifically at 4℃, 1000g for 10 min, 2000g for 20 min, 4000g for 40 min, and 10000g for 60 min. The supernatant was collected, filtered through a 0.22 μm filter, and then centrifuged at 4℃ and 12000g for 2 hours. The precipitate was collected. The precipitate from step 4 was resuspended and enriched using an MWCO ultrafiltration tube with a molecular weight cutoff of 100 kDa. The precipitate at the bottom of the ultrafiltration tube was collected to obtain purple rice exosomes. Red rice exosomes were prepared using the same method. (2) Weigh and mix the powders of Bletilla striata polysaccharide and konjac polysaccharide in a mass ratio of 1:3 to prepare a polysaccharide mixture; add distilled water to the polysaccharide mixture in a mass-volume ratio of g:mL of 1:10 and stir evenly. (3) In the solution after stirring, add sodium hydroxide solution with a concentration of 0.2% according to the mass-volume ratio of polysaccharide mixture to sodium hydroxide solution of 1:5 g:mL, continue stirring until the solution is uniform, let it stand at room temperature for 1 hour, and then heat it in a 70℃ hot water bath for 30 minutes to obtain the hydrogel precursor solution. (4) Add purple rice exosomes or red rice exosomes from step 1 to the hydrogel precursor solution, and then incubate at room temperature for 2 hours to form a gel, thus obtaining purple rice exosome hydrogel or red rice exosome hydrogel. Figure 1 ); 2. Preparation of blank hydrogel: Refer to steps 1 (2)-(4), except that exosomes are not added in step (4); Control group 1 followed the same method as step 1, except that hydrogels were prepared using *Bletilla striata* polysaccharide, konjac polysaccharide, and *Bletilla striata* polysaccharide + chitosan, respectively. During the preparation process, it was found that hydrogels prepared using a single polysaccharide or a combination of *Bletilla striata* polysaccharide and chitosan were difficult to form or had a soft texture. Figure 2 The existing hydrogels cannot meet the requirements for medical hydrogels; only the hydrogels prepared by combining Bletilla striata polysaccharide and konjac polysaccharide have stable molding properties. 3. Fourier transform infrared spectroscopy (FTIR) was used to analyze the spectra of Bletilla striata polysaccharides (BSPs), konjac polysaccharides (KGM), blank hydrogel (FEG), purple rice exosome hydrogel (PEG), and red rice exosome hydrogel (REG). First, dried potassium bromide and the treated samples were mixed at a mass ratio of 100:1 and ground. The mixed sample powder was placed in a mold and compressed into tablets using a tablet press. The mold loaded with potassium bromide tablets was placed in the slot of the infrared spectrometer, ensuring stable placement. The infrared spectrometer parameters were set, and the test was started. After the test was completed, the result images were saved.

[0009] Infrared spectral results as follows Figure 3 As shown, the results indicate that at 3423cm -1 Characteristic peaks of polysaccharides can be observed at locations such as 1587 cm⁻¹. -1 The peak revealed the amide I band (C=O stretching) of the protein at 2107 cm⁻¹. -1 At this point, the peaks for Bletilla striata polysaccharides (BSPs) and konjac polysaccharides (KGM) were not obvious, but the peaks in the other three experimental groups were significantly broadened. However, the peaks in this range were not characteristic absorption peaks of the two polysaccharides, but rather characteristic bands of terminal alkynes. This may be due to the formation of alkyne groups at the ends after the two polysaccharides were combined. This result indicates that the prepared hydrogel was successfully cross-linked and loaded with colored rice exosomes.

[0010] 4. The morphology of blank hydrogel (FEG), purple rice exosome hydrogel (PEG), and red rice exosome hydrogel (REG) was examined using a scanning electron microscope (SEM) (Hitachi, SU8100). First, the hydrogel samples were sputter-coated with gold and then scanned with an SEM. The experimental conditions were: accelerating voltage 10 kV, adaptive magnification adjustment, fixed-point imaging, and observation of the surface structure of the hydrogel. The morphology of the samples was observed and photographed under the SEM. The results are as follows Figure 4As shown, the blank hydrogel (FEG) exhibits a dense and relatively complete mesh-like structure with most pores having an average diameter between 160-200 μm, which is larger than the maximum diameter of exosomes, allowing for effective loading of exosomes. The electron micrograph of the hydrogel loaded with colored rice exosomes clearly shows that some pores are not permeable, possibly because the exosomes are loaded and enter the pores. Although no direct observation of exosomes is possible, the mesh-like and pore structure of the hydrogel is still preserved, indicating that this hydrogel has good air permeability and water absorption properties.

[0011] 5. Rheological properties were tested using a rotational oscilloscope. First, hydrogel samples of the appropriate size were prepared in a cylindrical mold. Oscillatory scanning was performed within a gap distance of 8 mm, 1% strain, and an angular frequency range of 0.1-100 rad / s. The results are as follows: Figures 5-7 As shown, the three groups of rheological tests were blank hydrogel (FEG), purple rice exosome hydrogel (PEG), and red rice exosome hydrogel (REG). The results showed that the G' of all three groups was much larger than G'', indicating that the hydrogel has excellent elastic properties. A higher G' usually also means that the material has a more compact and ordered structure, and better mechanical strength. Furthermore, it remained stable throughout the entire test range, confirming that it formed a stable three-dimensional network structure with high elasticity.

[0012] 6. Water vapor transmission rate test Cut the hydrogel sample into circular slices and place them on the mouth of a glass bottle with a corresponding diameter. Add an appropriate amount of pure water to the glass bottle. To fix the hydrogel on the bottle mouth, apply a layer of Vaseline to the edge of the mouth and use sealing film to secure it. Then place the glass bottle containing the hydrogel in a 37°C incubator for 24 hours. After removing it, weigh it again. Perform three parallel replicates for each hydrogel sample and take the average value. The results are as follows Figure 8 As shown, the blank hydrogel group had FEG (1145±18.46 g / m³). 2 • 24h)> Purple rice exosome hydrogel group PEG (1065±7.45g / m 2 • 24h)> Red rice exosome hydrogel group REG (1006±2.65g / m 2 (24h) Due to the addition of exosomes, a small number of pores were blocked, but the water vapor permeability (i.e., air permeability) of all three groups of hydrogels met the national requirements for medical hydrogels (500~2000 g / m³). 2 The 24-hour timeframe indicates that this hydrogel has the potential to be used as a medical hydrogel dressing.

[0013] 7. Swelling rate detection of hydrogels under different pH and ROS conditions Hydrogels (5 mm high × 10 mm in diameter) were placed in 6-well plates containing PBS buffer solution (pH 1.2–11) or 0.01 mM–10 mM H₂O₂ aqueous solution, respectively. At specific time points (1–24 h), the swollen hydrogels were removed, and residual moisture was blotted off with absorbent paper. The weights of the samples at each time point were compared with the initial weights. The swelling ratio was calculated using the following formula: Swelling ratio = (W / W) * ... t -W0) / W0×100%. Where, W t W represents the weight of the hydrogel after t hours; W0 represents the initial weight of the hydrogel. The results are as follows Figures 9-10 As shown, in the pH test, the swelling rate of the hydrogel reached its peak at 4 hours and then gradually decreased. In the ROS test, the swelling rate reached its maximum at 2 hours and then began to decline. Between pH 1.2 and pH 4, the swelling rate of the hydrogel decreased and reached its minimum. This is likely because at this point, the positive charge in the system gradually decreased, and the hydrogen bonds between hydroxyl groups and the attraction between positive and negative charges became dominant. These forces shortened the distance between molecular chains, reducing the porosity of the hydrogel network, decreasing its water absorption capacity, and thus lowering the swelling rate to its lowest point. At pH 7.4, the system contained a large number of negative charges, increasing the repulsive forces between molecules and breaking hydrogen bonds, thus increasing the porosity of the gel network and improving the swelling capacity of the hydrogel. When the pH continued to increase to 9, the swelling rate slowly decreased again, as intramolecular and intermolecular hydrogen bonds formed, shortening the distance between molecular chains. Between pH 9 and 11, with the increase in hydroxyl concentration, the degree of ionization may further increase, leading to an increase in the swelling rate. This demonstrates excellent pH response performance and a high swelling rate even under strong acid or strong alkali conditions, reflecting the stability of its gel properties.

[0014] When the H2O2 solution concentration is 10 mM, the hydrogel may loosen due to oxidation of the network structure, resulting in enhanced water absorption and a larger swelling ratio. At a concentration of 5 mM, some structures remain largely intact due to cross-linking, and while the swelling ratio increases, it is still less than at 10 mM. Similarly, the swelling degree of the hydrogel is even lower at a concentration of 0.1 mM. During the inflammatory phase of wound healing, immune cells (mainly neutrophils and macrophages) produce explosive amounts of ROS, including H2O2, to clear pathogens. The responsiveness and efficiency of the hydrogel in this experiment increase with increasing H2O2 concentration. Utilizing this property to encapsulate beneficial components and apply them to the wound site enables on-demand, precise release and treatment, demonstrating the therapeutic potential of this hydrogel in promoting wound healing.

[0015] 8. Blood compatibility test (1) Add whole blood from mice with EDTA as an anticoagulant to a centrifuge tube, centrifuge at 3000 rpm for 15 minutes, take out the supernatant, and then dilute the lower layer of red blood cells with physiological saline to 5%; (2) Trim the hydrogel into small rectangles and place them in centrifuge tubes as experimental groups; add 10 mL of physiological saline (NS) to the centrifuge tubes of the negative control and add purified water to the centrifuge tubes of the positive control; then add 200 μL of diluted red blood cells to each centrifuge tube, incubate in a 37℃ incubator for 2 hours, remove all centrifuge tubes, centrifuge at 1200 rpm for 5 minutes, use a pipette to take out 200 μL of supernatant and aspirate it into a 96-well cell culture plate, and measure its absorbance value at 540 nm; The results are as follows Figure 11 As shown, the hemolysis rates of the blank hydrogel (FEG), purple rice exosome hydrogel (PEG), and red rice exosome hydrogel (REG) were 4.240±0.20%, 4.067±0.07%, and 3.923±0.24%, respectively, all below 5%, meeting the safety standards for hemolysis rates of drugs or medical devices. This indicates that the hydrogels in this experiment have the potential to become biomedical materials (wound dressings) and can be applied to medical treatment.

[0016] 9. Antioxidant capacity test (1) Antioxidant analysis by ABTS method Take (20- x Add 10 μL of PBS (pH=6.0) to a 96-well plate, then add the sample solution. x μL ( x The sample solution volume was determined based on the preliminary test results (i.e., 0.5, 1, 2, 4 μL), then 80 μL of ABTS solution was added, mixed, and reacted at 37℃ in the dark for 6 min. The absorbance was measured at 734 nm. The ABTS scavenging rate was calculated using the following formula: ABTS clearance rate = ( A 0- A ) / A 0×100% In the formula: A 0 represents the absorbance of 20 μL PBS (pH=6.0) and 80 μL ABTS; A The absorbance of 20 μL of samples at different dilutions and 80 μL of ABTS.

[0017] (2) Antioxidant analysis by DPPH method Take (20- x Add 10 μL of PBS (pH=6.0) to a 96-well plate, then add the sample solution. x μL ( xThe sample solution volume was determined based on the preliminary test results (i.e., 0.5, 1, 2, 4 μL), then 80 μL of DPPH solution was added, mixed, and reacted at 37℃ in the dark for 30 min. The absorbance was measured at 519 nm. The DPPH scavenging rate was calculated using the following formula: DPPH removal rate = ( A 0- A ) / A 0×100% In the formula: A 0 represents the absorbance of 20 μL PBS (pH=6.0) and 80 μL DPPH; A The absorbance of 20 μL of samples at different dilutions and 80 μL of DPPH; Meanwhile, an experimental group supplemented with vitamin C was set up as a control group; See results Figure 12 The results of ABTS antioxidant scavenging rate were as follows: Vitamin C group (99.86±0.16%) > shellac group (91.63±0.58%) > red gum group (87.09±1.63%) > empty gum group (77.00±1.49%).

[0018] The results of DPPH antioxidant scavenging rate were as follows: Vitamin C group (99.36±0.52%) > shellac group (90.30±0.76%) > red gum group (83.76±1.39%) > empty gum group (69.67±1.06%).

[0019] The results showed that colored rice exosome hydrogels had better antioxidant effects than blank hydrogels, with the shellac group showing the best results. This is likely because purple rice contains a large amount of anthocyanins, more than red rice, and anthocyanins themselves have strong antioxidant capabilities. Encapsulation in the hydrogel allows for better release of these anthocyanins, resulting in superior antioxidant effects.

[0020] Example 2: Cell proliferation, toxicity, and inflammation experiments of colored rice exosome hydrogels from Example 1 1. CCK-8 assay to detect the effect of hydrogel extract on cell viability RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-dextrin. The cells were incubated at 37°C in a 5% CO2 incubator. The hydrogel was cut into small pieces and then cell culture medium was added as the extraction medium. The pieces were placed in centrifuge tubes and incubated at 37°C with shaking for 24 hours. After centrifugation at 3000 rpm, the supernatant was filtered through a sterile filter membrane to obtain the exosome hydrogel extract. When the cell culture reaches 80%–90% confluence, passage the cells, discard the culture medium, wash the cells with PBS to remove residual culture medium and serum, add an appropriate amount of PBS (pH=7.4) to the wash dish, gently pipette to remove all cells, and continue pipetting until a single-cell suspension is formed. Count the cells using a cell counter and adjust the cell density to 1×10⁻⁶. 5 Cells were seeded at 100 μL / well in 96-well plates (to eliminate edge effects, 100 μL of PBS (pH=7.4) was added to the outermost ring of the 96-well plate). After gentle mixing, the plates were incubated at 37°C and 5% CO2 for 24 h. The culture medium was then removed, and the cells were treated with different concentrations of hydrogel extract (0~1500 μg / mL). After 6 h, 100 μL of medium containing 10% CCK8 solution was added to each well, and the plates were incubated at 37°C and 5% CO2 for another 1 h. A blank group (no cells were seeded, only DMEM complete medium) and a control group (cells were seeded without exosome hydrogel) were also set up. The absorbance was measured at 450 nm, and the cell viability was calculated based on the absorbance using the following formula: Cell viability (%) = (OD1 - OD0) / (OD2 - OD0) In the formula: OD0 represents the absorbance value of the blank group, OD1 represents the absorbance value of the sample group, and OD2 represents the absorbance value of the control group; See results Figure 13 As shown in the figure, cell viability significantly increased with decreasing concentration. At a concentration of PEG or REG of 1500 μg / mL, cell viability was only 19.37±0.84% ​​and 18.40±0.28%, respectively, exhibiting strong cytotoxicity. When the concentration was reduced to 750 μg / mL, cell viability increased to 30.00±0.27% and 27.33±1.79%, respectively, but cytotoxicity remained significant. Further reducing the concentration to 500 μg / mL significantly increased cell viability to 88.81±1.45% and 87.47±1.38%, respectively, indicating that the cytotoxicity of the compound was greatly reduced at this concentration. At concentrations of 300 μg / mL and 100 μg / mL, cell viability reached 98.73±0.92% and 94.06±1.59%, 106.40±0.68% and 102.40±0.98%, respectively, which were close to or even slightly higher than the untreated control group. This indicates that these concentrations may not be toxic to cells and may even have a certain promoting effect on cell proliferation. Therefore, 100 μg / mL was selected as a safe and effective concentration for subsequent applications.

[0021] 2. Determination of nitric oxide (NO) RAW264.7 was cultured using the method described above and then inoculated with 1×10⁻⁶. 52 mL of cells were seeded per well into 6-well plates. After 24 h of complete cell adhesion, the culture medium was removed, and hydrogel extract of the prepared concentration was added to the test wells. Dexamethasone (DXM) at 1 μg / mL was selected as the positive control. The culture medium was replaced in the blank and model wells, and the cells were pretreated for 6 h. Then, the cell supernatant was removed, and the cells were carefully washed with PBS. After that, except for the blank wells, 2 mL of 1 μg / mL LPS was added to each of the other wells, and the cells were incubated for another 24 h. After the culture was completed, the culture medium was collected, and the cells and debris were removed by centrifugation at 1000 rpm to obtain the supernatant. The inhibition of NO production in LPS-induced RAW264.7 cells by the hydrogel extract was determined according to the NO assay kit instructions. 3. ELISA method to determine the expression level of inflammatory factors The initial culture and hydrogel extraction treatment of RAW264.7 cells were performed using the same method as in step 2. After cell culture, the supernatant was collected, centrifuged at 5500 rpm to remove cell debris, and then tested. The effect of the hydrogel extraction on LPS-induced inflammatory factors produced by RAW264.7 cells was determined according to the ELISA kit instructions. The specific measurement method is as follows: (1) Double antibody sandwich: Take out the ELISA kit stored at 4℃ and equilibrate to room temperature (the well plate used has been coated and blocked with primary antibody); set blank wells (without adding sample and enzyme labeling reagent, the other steps are the same), standard wells, and sample wells respectively; add 50 μL of different concentrations of standard to each well containing primary antibody, and add 40 μL of sample diluent (provided with the kit) to the sample wells first, and then add 10 μL of sample to be tested (the final sample dilution is 5 times); shake slowly to mix well, seal all reaction wells with sealing film, and incubate at 37℃ in the dark for 30 min; (2) Washing the plate: After incubation, gently shake out the reaction solution in the well and pat dry on a thick stack of absorbent paper; add 350 μL of the prepared washing solution to each well, let stand for 30 seconds, then gently shake out the liquid and blot it with absorbent paper. Repeat 5 times. (3) Enzyme conjugation: Add 50 μL of enzyme conjugation working solution to each well, gently shake left and right to mix evenly; then seal all reaction wells and incubate at 37°C in the dark for 30 min. (4) Washing the plate: After incubation, wash the plate 5 times according to step (3); (5) Color development: Add 50 μL of color developer A to each well, then add 50 μL of color developer B, incubate at 37°C in the dark for 10 min, and observe the color change. (6) Termination of reaction: Add 50 μL of reaction termination solution to each well, mix well and observe the color reaction. When the color of the reaction solution in the well changes from blue to yellow, the reaction is terminated. Then measure the absorbance at a wavelength of 450 nm. The content of inflammatory factors in cells of each group of samples is calculated based on the standard curve plotted with the standard. Experimental data are expressed as mean ± standard deviation (Mean ± SD) (sample size ≥ 3) and analyzed using one-way ANOVA. A statistically significant result was considered to be p < 0.05. All analyses were performed using GraphPadprism 9.0.0.

[0022] The results are as follows Figure 14 As shown, compared with the untreated blank control group, the levels of inflammatory factors NO, IL-6, TNF-α, and IL-1β in the LPS-induced RAW264.7 cell model group were significantly increased, confirming the successful establishment of the LPS-induced in vitro anti-inflammatory model of RAW264.7 cells. Furthermore, the addition of positive control drug (DXM), purple rice exosome extract (PEG), and red rice exosome extract (REG) resulted in a significant decrease in the levels of inflammatory factors NO, IL-6, TNF-α, and IL-1β compared to the model group. This indicates that the PEG and REG groups can promote inflammation recovery by reducing the concentration of pro-inflammatory cytokines and have a good anti-inflammatory effect.

[0023] Example 3: Animal experiment on the effect of colored rice exosome hydrogel on wound healing as described in Example 1 1. Macroscopic wound healing analysis The experimental mice were healthy 4-week-old 30g Kunming mice. A 10mm circular superficial skin wound model was constructed, and six groups were set up: a control group (Con), a shellac group (PEG), a red glue group (REG), an empty glue group (FEG), a commercially available hydrogel group (MSG), and a band-aid group. The mice were fed normally and had their dressings changed for 14 days, and wound changes were photographed and recorded. Finally, ImageJ software was used to simulate and analyze the wound area. Experimental results are as follows Figure 15 As shown, on days 3 and 6, each group exhibited varying degrees of normal wound healing, including scab formation and inflammation. By day 9, except for the control group, all other groups showed good wound healing. By day 14, most wounds had healed, but the control group, the gelatin group, and the bandage group still showed some inflammation or incomplete scab formation, and the gelatin group also had some scarring. In contrast, the hair on the back wounds of the lac and red lac groups had regrowth, and there were no obvious scars. Analysis of wound area using ImageJ software showed the wound healing rate in mice to be: lac group > red lac group > gelatin group > gelatin group > bandage group > control group.

[0024] Throughout the experimental period, all three groups of experimental adhesives demonstrated superior healing effects compared to commercially available hydrogels and ordinary wound dressings. Furthermore, the wound healing rates of the shellac and red adhesive groups were consistently higher than those of the empty adhesive group, indicating that the addition of exosomes further enhanced the therapeutic effect on wound healing.

[0025] 2. H&E wound section staining Fourteen days after dissection, fresh wound skin was collected from experimental mice and fixed in tissue fixative for 24 hours. After fixation, the tissue was embedded in paraffin, and the paraffin block was cut into 5 μm thick sections. These sections were then stained with hematoxylin and eosin (H&E) to prepare H&E sections. The sections and images were observed using an Olympus IX83 microscope for histopathological evaluation of the mouse wound tissue.

[0026] The results are as follows Figure 16 As shown, at 7 days, significant inflammatory cell aggregation and severe dermal damage were observed in the control group. Although inflammation was also evident in the shellac and bandage groups, some angiogenesis was also observed. The shellac, red glue, and empty glue groups all generated a large number of angiogenesis cells, with the shellac group showing the best results, although mild inflammation was still present. At 14 days, inflammation in the control, shellac, and bandage groups decreased, and the number of angiogenesis cells increased and gradually formed. The number of angiogenesis cells further increased and formed in the shellac, red glue, and empty glue groups, dermal cells returned to normal, and inflammation subsided.

[0027] 3. Masson wound section staining Wound sections from mice collected 14 days after dissection were dewaxed and rehydrated, then stained and treated with hematoxylin, Ponceau S, phosphomolybdic acid, and aniline blue. After treatment, the sections were differentiated, dehydrated, and mounted. Finally, the sections and images were observed and acquired using an Olympus IX83 microscope, and the mouse wound tissues were evaluated histopathologically.

[0028] The results are as follows Figure 17 As shown, at 7 days, compared to the control group, bandage group, and commercially available hydrogel group, the empty gel group, shellac group (PEG), and red gel group (REG) showed more collagen fiber formation (blue network), and the network was denser, indicating that the density of newly formed collagen fibers at the wound site in the experimental gel groups was significantly higher than that in the control group, bandage group, and commercially available gel group. In addition, significant generation of hair follicle cells and vascular cells was observed in all three experimental gel groups. In contrast, only a small amount of vascular cell generation was observed in the control group, bandage group, and commercially available gel group on day 7, and the dermis was clearly in an unrepaired state.

[0029] On day 14, follicle-like shapes were clearly observed in the shellac and red gel groups, indicating the formation of hair follicle tissue at the wound site, with significantly more hair follicle cells than in other groups. This suggests that the exosome-loaded hydrogel group had a superior therapeutic effect and good wound healing. However, no mature hair follicle structures were observed in the blank group, bandage group, and gel group. Considering collagen deposition, dermal repair, and angiogenesis, the degree of wound recovery was: shellac group > red gel group > empty gel group > gel group > bandage group > blank group. This is consistent with the macroscopic wound healing results, possibly because the *Bletilla striata* polysaccharide in the hydrogel itself has hemostatic and healing-promoting effects, and it is loaded with anthocyanin-rich colored rice with excellent antioxidant and anti-inflammatory properties. The hydrogel encapsulation and slow release prolong the action time of exosomes at the wound site, thereby enhancing collagen deposition and synergistically promoting wound healing.

[0030] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. Colored rice exosome hydrogel, characterized by: The Bletilla striata polysaccharide and konjac polysaccharide are mixed, then distilled water is added to the polysaccharide mixture, stirred uniformly, then sodium hydroxide solution is added, and stirring is continued until the solution is uniform, and the solution is left to stand at room temperature for 0.5-1.5 hours, then heated in a 65-75℃ hot water bath for 25-35 minutes to obtain a hydrogel precursor solution, then the colored rice exosome is added to the hydrogel precursor solution, and then left to stand at room temperature for 1-3 hours for incubation and gelation to obtain the colored rice exosome hydrogel.

2. Colored rice exosome hydrogel according to claim 1, characterized by: The mass ratio of the Bletilla striata polysaccharide and the konjac polysaccharide is 1:2-4, and the mass-volume ratio of the polysaccharide mixture to distilled water is 1:8-12.

3. Colored rice exosome hydrogel according to claim 1, characterized by: The mass concentration of sodium hydroxide is 0.1-0.3%, and the mass-volume ratio of the polysaccharide mixture to the sodium hydroxide solution is 1:4-6.

4. The colored rice exosome hydrogel of claim 1, wherein: The mass-volume ratio of the colored rice exosome to the hydrogel precursor solution is 1:2-3.

5. The colored rice exosome hydrogel of claim 1, wherein: The color rice powder after cleaning and crushing was added to PBS buffer with pH 6-7.4 and homogenized at high speed, and then α - amylase was added to the rice slurry, and enzymolysis was carried out at pH 6-7.4 and 90-97℃ for 1-2h, filtration was performed, and the filtrate was left overnight; then gradient differential centrifugation was carried out at 4℃, the supernatant was collected, filtration was performed, the filtrate was ultracentrifuged at 4℃ and 12000-15000g for 1.5-3h, and the precipitate was collected; the precipitate was resuspended and enriched by using a MWCO ultrafiltration tube with a molecular weight cutoff of 100kDa, the precipitate at the bottom of the ultrafiltration tube was collected, and the color rice exosome was prepared.

6. Use of the colored rice exosome hydrogel of any one of claims 1-5 in the preparation of a wound dressing.

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