Mitochondrial targeting nano-enzyme based on dendrobium officinale nano-vesicles as well as preparation method and application of mitochondrial targeting nano-enzyme

Abstract

The invention discloses a mitochondrial targeting nano-enzyme based on dendrobium officinale nano-vesicles as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials and tissue engineering. The mitochondrial targeting nano-enzyme provided by the invention has the functions of enhancing ROS (reactive oxygen species) removal capability and mitochondrial targeting. The mitochondrial targeting nano-enzyme is combined with dendrobium officinale nano-vesicles to form a complex with a core-shell structure, so that the biocompatibility is remarkably improved. The formed complex is further fused in hydrogel, so that the composite hydrogel dressing for promoting healing of refractory wounds of diabetes mellitus can be obtained.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and tissue engineering technology, specifically relating to a mitochondrial-targeting nanozyme based on Dendrobium officinale nanovesicles, its preparation method, and its application. Background Technology

[0002] Diabetic refractory wounds are a serious complication of diabetes, and their slow healing mechanism is closely related to a vicious cycle of oxidative stress triggered by hyperglycemia. Sustained hyperglycemia leads to abnormal cellular metabolism, which in turn overloads the mitochondrial electron transport chain, generating excessive reactive oxygen species (ROS). Excessive ROS directly damages mitochondrial components, causing dysfunction, and disrupts autophagy, ultimately leading to the accumulation of damaged mitochondria. These abnormal mitochondria, in turn, continue to produce ROS, forming a vicious cycle that amplifies oxidative stress. Therefore, developing a novel wound dressing that can precisely target and repair mitochondrial dysfunction has significant clinical value and application prospects for breaking the vicious cycle of oxidative stress in diabetic wounds and achieving fundamental treatment.

[0003] In recent years, nanozymes with enzyme-like catalytic activity have attracted widespread attention due to their great potential in regulating oxidative stress. They exert catalytic activity on ROS scavenging through various antioxidant enzymes, thus providing precise intervention to regulate intracellular redox homeostasis. Among them, cerium vanadate (CeVO4) nanozymes have attracted attention due to their good stability and tunable catalytic activity. Studies have shown that CeVO4 nanozymes possess superoxide dismutase (SOD)-like activity, which helps maintain mitochondrial function. Their performance can be further optimized through elemental doping strategies (such as vanadium and titanium), and the oxygen vacancies and variable valence states introduced by doping can effectively improve their electron transfer and catalytic efficiency. However, to realize the effective application of nanozymes in diabetic wound treatment, it is necessary to systematically address the key issues of their targeting and biosafety. Traditional nanozymes lack active targeting capabilities and have low enrichment efficiency in mitochondrial sites, limiting their therapeutic effects. Triphenylphosphine (TPP), a classic mitochondrial-targeting molecule, can achieve mitochondrial enrichment of nanoparticles driven by mitochondrial membrane potential. However, how to stably and efficiently modify the surface of nanozymes with TPP while maintaining its high catalytic activity after modification remains a key technical challenge. Furthermore, the potential immunogenicity and biocompatibility of nanomaterials remain limiting factors for clinical application. In addition, simple nanoparticle formulations are difficult to release stably into wounds, thus affecting their sustained therapeutic efficacy.

[0004] To address the above issues, plant-derived exosomes (DOEVs) from *Dendrobium officinale* have been explored as delivery carriers for bioactive ingredients or drugs due to their naturally low immunogenicity, good biocompatibility, and inherent delivery potential. Using biocompatible hydrogels such as hyaluronic acid as dressing matrices can enhance the efficacy of nanozymes at the wound site. However, these methods often only address single problems and lack a comprehensive treatment approach that can synergistically achieve precise mitochondrial targeting and enhanced biocompatibility. Therefore, constructing an integrated multifunctional dressing by combining targeted modification, biocompatible encapsulation, and gelation loading strategies holds promise for solving these key problems and providing a novel solution for diabetic wound treatment. Summary of the Invention

[0005] The present invention aims to provide a mitochondrial-targeting nanozyme based on Dendrobium officinale nanovesicles, its preparation method, and its application. The provided mitochondrial-targeting nanozyme possesses both enhanced ROS scavenging ability and mitochondrial targeting function. This mitochondrial-targeting nanozyme combines with Dendrobium officinale nanovesicles to form a core-shell structured composite, thereby significantly improving its biocompatibility. The formed composite is further fused into a hydrogel to obtain a composite hydrogel dressing for promoting the healing of refractory diabetic wounds.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] One of the technical solutions of this invention: a method for preparing mitochondrial-targeted nanozymes, comprising the following steps:

[0008] (1) Soluble cerium salt, ethylenediaminetetraacetic acid (EDTA), soluble vanadate and titanium source are mixed in water, the pH value is adjusted to alkaline, and CeVO4-Ti nanozyme is obtained after hydrothermal reaction.

[0009] (2) The CeVO4-Ti nanozyme was dispersed in PBS buffer, and ethylenediaminetetraacetic acid was added to activate the surface of the CeVO4-Ti nanozyme. Then, the unreacted ethylenediaminetetraacetic acid was removed and the nanozyme was resuspended in PBS buffer. Triphenylphosphine (TPP) was added and the pH was adjusted to 6.5-7.5. After the reaction was carried out in the dark, CeVO4-Ti-TPP nanozyme, i.e., the mitochondrial-targeting nanozyme, was obtained.

[0010] Optionally, in step (1), the soluble cerium salt is Ce(NO3)3; the soluble vanadate is Na3VO4; and the titanium source is Ti(SO4)2.

[0011] Preferably, in step (1), the soluble cerium salt contains Ce 3+ The ethylenediaminetetraacetic acid and the VO4 in the soluble vanadate 3-The molar ratio of Ti in the titanium source is 1:0.8~1.2:0.8~1.2:0.05~0.3; more preferably 1:1:1:0.1.

[0012] Preferably, in step (1), the temperature of the hydrothermal reaction is 140-220°C and the time is 12-24h; more preferably, the temperature is 180°C and the time is 18h.

[0013] Preferably, in step (1), the substance used to adjust the pH value is ammonia water, and the adjustment endpoint is pH==8.0~11.0; more preferably, the endpoint pH=9.0.

[0014] Preferably, in step (2), the ratio of the CeVO4-Ti nanozyme, the ethylenediaminetetraacetic acid, and the triphenylphosphine is 1g:1mol:0.5g.

[0015] Preferably, in step (2), the activation condition is incubation at 37°C for 1 hour.

[0016] Preferably, in step (2), the light-avoidance reaction time is 2 hours.

[0017] The second technical solution of the present invention provides a mitochondrial-targeting nanozyme prepared according to the above-mentioned preparation method of mitochondrial-targeting nanozyme based on Dendrobium officinale nanovesicles.

[0018] The third technical solution of the present invention provides a method for preparing a mitochondrial targeted drug delivery system, comprising the following steps:

[0019] The above-mentioned mitochondrial-targeting nanozyme was dispersed in PBS buffer, then mixed with a solution containing Dendrobium officinale nanovesicles (DOEVs), and after reaction, squeezed through a membrane with a pore size of 200 nm to obtain the DOEVs@CTT complex; that is, the mitochondrial-targeting drug delivery system.

[0020] Preferably, the mass ratio of the mitochondrial-targeting nanozyme to the Dendrobium officinale nanovesicles is 1:2.

[0021] Preferably, the reaction is carried out at a temperature of 4°C for 60 minutes.

[0022] The fourth technical solution of the present invention provides a mitochondrial targeted drug delivery system prepared according to the above-described method for preparing a mitochondrial targeted drug delivery system.

[0023] The fifth technical solution of the present invention provides a composite hydrogel dressing for promoting healing of refractory diabetic wounds, wherein the active ingredient includes the above-mentioned mitochondrial targeted drug delivery system.

[0024] The sixth technical solution of this invention provides a method for preparing the above-mentioned composite hydrogel dressing for promoting healing of refractory diabetic wounds, comprising the following steps:

[0025] The above-mentioned mitochondrial targeted drug delivery system (i.e., DOEVs@CTT complex) was mixed with an aqueous solution containing dopamine-methacrylated hyaluronic acid (DHM) and crosslinked to obtain DOEVs@CTT / DHM composite hydrogel, which is the composite hydrogel dressing for promoting healing of refractory diabetic wounds.

[0026] Preferably, the crosslinking method is photocrosslinking, and a photoinitiator is added before crosslinking.

[0027] The beneficial technical effects of the present invention are as follows:

[0028] The mitochondrial-targeted nanozyme provided by this invention has the following characteristics:

[0029] 1. Excellent antioxidant and enzyme-like activities: CeVO4-Ti-TPP nanozymes exhibit significantly superior superoxide dismutase and catalase-like activities compared to unmodified CeVO4. In standard antioxidant tests such as DPPH, ABTS, and salicylic acid colorimetric methods, this material demonstrates strong free radical scavenging capabilities.

[0030] 2. Highly effective antibacterial properties: Antibacterial experiments on Staphylococcus aureus and Escherichia coli showed that the composite hydrogel prepared with the mitochondrial targeted drug delivery system as the active ingredient can effectively inhibit the growth of common wound infection bacteria and reduce the risk of infection.

[0031] 3. Excellent biocompatibility and cell protection: CCK-8 and live / dead staining experiments confirmed that CeVO4-Ti-TPP nanozymes had no significant toxicity to HUVECs. In a high glucose-induced oxidative stress model, the composite hydrogel prepared with CeVO4-Ti-TPP nanozymes as the active ingredient effectively reduced intracellular reactive oxygen species levels, increased endogenous antioxidants such as glutathione, and protected cells from oxidative damage.

[0032] 4. Significant mitochondrial function repair effect: MitoSOX and JC-1 staining and transmission electron microscopy confirmed that the composite hydrogel prepared with CeVO4-Ti-TPP nanozyme as active ingredient can target and reduce mitochondrial oxidative stress, improve mitochondrial membrane potential, and maintain normal mitochondrial morphology and structure.

[0033] 5. Significant angiogenesis-promoting ability: Scratch assay, Transwell migration assay and in vitro tube formation assay all showed that the composite hydrogel prepared with CeVO4-Ti-TPP nanozyme as the active ingredient can significantly promote the migration and tube formation of endothelial cells, providing the necessary blood supply for wound healing.

[0034] 6. Significant in vivo healing-promoting effect: In a full-thickness skin defect model in db / db diabetic mice, topical application of a composite hydrogel prepared with CeVO4-Ti-TPP nanozyme as the active ingredient significantly accelerated wound contraction and closure. Histological (H&E and Masson staining) analysis showed that the treatment group had more intact new epithelium, richer collagen deposition, and more orderly arrangement, demonstrating its comprehensive tissue regeneration-promoting capacity. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 Characterization spectra of raw materials and products during the preparation of CTT nanozymes are shown below. A is the SEM image of CeVO4; B is the XRD spectrum of CeVO4 and CeVO4-Ti; C is the SEM image of CeVO4, CeVO4-Ti, and CTT nanozymes; DF represent the particle size distributions of CeVO4, CeVO4-Ti, and CTT nanozymes, respectively; G is the XPS spectrum of CeVO4-Ti and CTT nanozymes; H is the FTIR spectrum of CeVO4-Ti and CTT nanozymes; I is the UV-Vis spectrum of CTT nanozymes and other samples; J is the Zeta potential value of CTT nanozymes and other samples; K is the GSH depletion capacity of CTT nanozymes at different concentrations; L is the GSH enzyme activity of CTT nanozymes under different pH treatments; and M is the CAT enzyme activity of CTT nanozymes under different pH treatments.

[0037] Figure 2 Characterization spectra of raw materials and products during the preparation of DOEVs@CTT complex; where A is TEM image of DOEVs and DOEVs@CTT complex; B is particle size distribution of DOEVs (top) and DOEVs@CTT complex (bottom); C is elemental mapping image of DOEVs@CTT complex; D is absorption spectrum of ABTS measured in DOEVs@CTT complex and other samples; E is ABTS scavenging rate of DOEVs@CTT complex and other samples; F is absorption spectrum of DPPH measured in DOEVs@CTT complex and other samples; G is DPPH scavenging rate of DOEVs@CTT complex and other samples; H is absorption spectrum of salicylic acid colorimetric reaction of DOEVs@CTT complex and other samples; I is absorbance value of salicylic acid colorimetric reaction of DOEVs@CTT complex and other samples at 510 nm.

[0038] Figure 3 Characterization spectra of raw materials and products during the preparation of DOEVs@CTT / DHM composite hydrogels are shown below. A represents the FTIR spectra of hyaluronic acid (HA), hyaluronic acid methacrylate (HAMe), and DHM; B represents the 1H NMR spectra of HA, HAMe, and DHM; C represents the macroscopic images of DHM hydrogel and DOEVs@CTT / DHM composite hydrogel formation under UV light; D represents the release of CTT nanozymes from the DOEVs@CTT / DHM composite hydrogel; E represents the compressive stress-strain curves of the DOEVs@CTT / DHM composite hydrogel and DHM hydrogel; F represents the compressive elastic modulus of the DOEVs@CTT / DHM composite hydrogel and DHM hydrogel; G represents the compressive elastic modulus of samples A and B of the DOEVs@CTT / DHM composite hydrogel. Absorption spectra of TS determination; H represents the ABTS scavenging rate of DOEVs@CTT / DHM composite hydrogel and other samples; I represents the absorption spectrum of DPPH determination of DOEVs@CTT / DHM composite hydrogel and other samples; J represents the DPPH scavenging rate of DOEVs@CTT / DHM composite hydrogel and other samples; K represents the absorption spectrum of salicylic acid colorimetric reaction of DOEVs@CTT / DHM composite hydrogel and other samples; L represents the absorbance value of salicylic acid colorimetric reaction of DOEVs@CTT / DHM composite hydrogel and other samples at 510 nm; M represents the bacterial colony image of DOEVs@CTT / DHM composite hydrogel and other samples; NO represents the growth curves of DOEVs@CTT / DHM composite hydrogel and other samples treated with Staphylococcus aureus and Escherichia coli, respectively.

[0039] Figure 4 The effects of DOEVs@CTT / DHM composite hydrogel and other samples on cellular oxidative stress are shown in the following figures: A represents the quantitative analysis results of HUVECs cell viability after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; B represents HUVECs cell staining images after treatment with DOEVs@CTT / DHM composite hydrogel and other samples for different time periods; C represents live / dead HUVECs cell staining images after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress; D represents DCF-DA staining images of HUVECs cells after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress; E represents SOD activity in HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress; F represents GSH level in HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress; and G represents MDA level in HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress.

[0040] Figure 5This study illustrates the effects of DOEVs@CTT / DHM composite hydrogels and other samples on mitochondrial function. A shows the mitochondrial red staining images of HUVECs after treatment with DOEVs@CTT / DHM composite hydrogels and other samples under oxidative stress; B shows the quantitative analysis results of the Mito-tracker red fluorescence intensity of HUVECs after treatment with DOEVs@CTT / DHM composite hydrogels and other samples under oxidative stress; C shows the MPTP detection images of HUVECs after treatment with DOEVs@CTT / DHM composite hydrogels and other samples under oxidative stress; D shows the quantitative analysis results of the MPTP fluorescence intensity of HUVECs after treatment with DOEVs@CTT / DHM composite hydrogels and other samples under oxidative stress; and E shows the DOEVs@CTT / DHM composite hydrogels and other samples under oxidative stress. JC-1 staining images of HUVECs after treatment with Vs@CTT / DHM composite hydrogel and other samples; F shows the quantitative analysis results of the JC-1 fluorescence ratio of HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress; G shows electron microscopy images of mitochondrial structures of HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress (yellow arrows indicate mitophagy); H shows the quantitative analysis results of mitochondrial damage in HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress; I shows representative WB bands of the Nrf2 / Parkin / Pink1 pathway in HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress.

[0041] Figure 6 The effects of DOEVs@CTT / DHM composite hydrogel and other samples on in vitro angiogenesis are shown in the following figures: A: Scratch migration experiment image of DOEVs@CTT / DHM composite hydrogel and other samples; B: Quantitative analysis results of scratch area in scratch migration experiment of DOEVs@CTT / DHM composite hydrogel and other samples; C: Transfer well experiment image of DOEVs@CTT / DHM composite hydrogel and other samples; D: Quantitative analysis results of number of migrated cells in transfer well experiment of DOEVs@CTT / DHM composite hydrogel and other samples; E: Channel formation experiment image of DOEVs@CTT / DHM composite hydrogel and other samples; F: Quantitative analysis results of vascular connection in channel formation experiment of DOEVs@CTT / DHM composite hydrogel and other samples; G: Immunofluorescence staining image of VEGFA in HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; H: Quantitative analysis results of VEGFA expression in HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples.

[0042] Figure 7Images show the therapeutic effects of diabetic wounds on mice in each group. A represents a representative image of the wound within 14 days; B represents the wound healing rate within 14 days; CD represents the quantitative reepithelialization rate on days 7 and 14, respectively; E represents H&E staining of the wound on days 7 and 14; F represents Masson staining of the wound on days 7 and 14; and GH represents the quantitative collagen deposition on days 7 and 14, respectively.

[0043] Figure 8 The oxidative stress resistance of DOEVs@CTT / DHM composite hydrogel and other samples in mice is shown in Figure 1. A represents the superoxide dismutase (SOD) activity in mouse serum after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; B represents the catalase (CAT) activity in mouse serum after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; C represents the glutathione (GSH) level in mouse serum after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; D represents the malondialdehyde (MDA) level in mouse serum after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; E represents the immunohistochemical staining images of TNF-α, IL-6, and IL-10 in mice after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; F and H represent the quantitative analysis results of IL-6, TNF-α, and IL-10, respectively.

[0044] Figure 9 The effects of DOEVs@CTT / DHM composite hydrogel and other samples on angiogenesis in mice are shown. A shows immunohistochemical staining of MMP9 at mouse wound sites after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; B shows immunofluorescence staining of CD31 and α-SMA at mouse wound sites after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; CD shows the quantitative analysis results of α-SMA and CD31 in mice after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; E shows HE staining images of various organs in mice after treatment with DOEVs@CTT / DHM composite hydrogel and other samples. Detailed Implementation

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0046] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0047] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0048] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0049] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0050] In this invention, room temperature refers to a temperature of 20±10℃.

[0051] The preparation steps of DHM in this invention are as follows:

[0052] (1) Synthesis of methacrylated hyaluronic acid (HAMe): 1.0 g of hyaluronic acid (HA) was added to 100 mL of deionized water, and the pH of the HA solution was adjusted to 8.0 with 1.0 mM NaOH. 4 mL of methacrylic anhydride was added to the above solution, and the mixture was magnetically stirred for 4 hours. After the reaction was completed, the product was dialyzed in deionized water and lyophilized to obtain HAMe.

[0053] (2) Synthesis of DHM: 1.0 g HAMe was dissolved in 100 mL of deionized water, and the pH of the solution was adjusted to 5.5. 506.0 mg EDC and 303.7 mg NHS were added to the solution. After reacting for 0.5 hours, 0.5 g dopamine hydrochloride was added, and the mixture was incubated at 25 °C for 12 hours. Finally, the product was dialyzed in acidified deionized water and lyophilized to obtain DHM.

[0054] Example 1

[0055] Preparation, performance characterization, and biological evaluation of multifunctional composite wound dressings:

[0056] 1.1 Synthesis of CeVO4-Ti nanozymes

[0057] Ce(NO3)3·6H2O and EDTA (molar ratio 1:1) were weighed and dissolved in deionized water. After complete dissolution, an aqueous solution containing Na3VO4 and Ti(SO4)2 (molar ratio of Ce(NO3)3·6H2O, Na3VO4, and Ti(SO4)2 was added, and the pH of the mixed solution was adjusted to 9.0 with ammonia. The mixture was transferred to a high-pressure reactor and reacted at 180℃ for 18 hours. After the reaction, the product was washed successively with deionized water and ethanol, and dried to obtain CeVO4-Ti nanozyme. Its morphology and elemental composition were characterized by transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDS).

[0058] 1.2 Preparation of CTT nanozymes

[0059] 10 mg of the CeVO4-Ti nanozyme synthesized in step 1.1 was dispersed in 10 mL of PBS buffer (0.01 M, pH 7.4). 1 mL of EDTA solution (10 mM) was added, and the mixture was incubated in a shaker at 37 °C for 1 hour. Subsequently, the supernatant was removed by centrifugation at 10,000 rpm for 10 minutes, and the mixture was resuspended in PBS and washed twice. 1 mL of ethanol solution of TPP (5 mg / mL) was added dropwise to the activated nanozyme dispersion, and the pH of the mixture was adjusted to 7.2 with 0.1 M NaOH solution. The reaction was carried out at room temperature with shaking in the dark for 2 hours. After the reaction, the precipitate was collected by centrifugation, washed three times with deionized water, and lyophilized to obtain CeVO4-Ti-TPP (CTT) nanozyme powder. The modification effect was characterized by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), and Zeta potential analysis.

[0060] 1.3 Preparation of DOEVs@CTT complex

[0061] 10 mg of CTT nanozyme powder was dispersed in 10 mL of PBS and sonicated on ice for 5 minutes. DOEVs extracted by ultracentrifugation were prepared into a 1 mg / mL solution with PBS. The CTT nanozyme dispersion and DOEVs solution were mixed at a volume ratio of 1:2 and incubated at 4°C for 60 minutes. The mixture was transferred to a liposome extruder and repeatedly extruded 18 times using a 200 nm polycarbonate membrane under ice bath conditions to obtain a homogeneous DOEVs@CTT complex suspension.

[0062] 1.4 Preparation of DOEVs@CTT / DHM composite hydrogel

[0063] Weigh 2.0 mg (calculated as CTT nanozyme) of the DOEVs@CTT composite prepared in step 1.3 and add it to 10 mL of DHM aqueous solution (5 w / v%). Stir magnetically for 10 minutes to ensure thorough dispersion. Then, add the photoinitiator I2959 stock solution (final concentration 0.1 w / v%), stir evenly, pour into a mold, and irradiate under a UV lamp for 10 minutes to crosslink and form a DOEVs@CTT / DHM composite hydrogel.

[0064] 1.5 Evaluation parameters for enzyme catalytic activity and antioxidant capacity: Enzyme activity detection wavelength:

[0065] Catalase (CAT)-like activity was monitored at 240 nm using the characteristic peak of H2O2; glutathione peroxidase (GPx)-like activity was measured at 412 nm using absorbance changes.

[0066] DPPH method: The sample was mixed with 2 mL of DPPH solution and reacted at room temperature in the dark for 30 min; after centrifugation (5000 r / min, 10 min), the absorbance at 517 nm was measured.

[0067] ABTS method: The sample is mixed with ABTS solution and reacted in the dark for 10 min; the detection wavelength is 734 nm.

[0068] Salicylic acid colorimetric method: Mix salicylic acid, ferrous sulfate, and hydrogen peroxide solution and heat in a water bath; detection wavelength is 510 nm. Standard curve: The regression equation for the DOEVs@CTT complex is y = 0.00857x + 0.02862 (R²). 2 =0.99787).

[0069] 1.6 Antimicrobial performance evaluation parameter model strains:

[0070] Staphylococcus aureus and Escherichia coli. Culture volume and conditions: The material was introduced into 5 mL of bacterial suspension and incubated at 37°C for 8 h. Evaluation indicators: Optical density (OD value) was continuously measured; serial dilutions were performed and colonies were counted on agar plates.

[0071] 1.7 Biocompatibility Evaluation Parameters: Cell Lines

[0072] Human umbilical vein endothelial cells (HUVECs). Dosage parameters: 5% DHM solution (w / v).

[0073] Detection time points: Cytotoxicity was assessed at 24 hours and 48 hours, respectively.

[0074] Analytical methods: Calcein-AM / PI staining kit and CCK-8 kit were used.

[0075] 1.8 Establishment of in vitro antioxidant and mitochondrial function evaluation parameter models:

[0076] HUVECs were treated with 5 mg / mL glucose to simulate a high glucose-induced oxidative stress environment.

[0077] Processing time: The material processing time is 24 hours.

[0078] Molecular probes: Cellular ROS levels were detected using the DCFH-DA probe; mitochondrial superoxide dismutase was detected using MitoSOX fluorescent dye; mitochondrial membrane potential was measured using JC-1 dye. Mitochondrial permeability: Assessed using a mitochondrial permeability transition pore (MPTP) kit. Western blot parameters: Electrophoresis was performed using a 10% separating gel; membranes were transferred to 0.22 μm PVDF membranes; and blocked with 5% skim milk.

[0079] 1.9 Experimental methods for evaluating in vitro angiogenesis-promoting capacity

[0080] To evaluate the repair effects of DOEVs@CTT / DHM composite hydrogel and related materials on endothelial cell function, this study systematically evaluated its influence on the migration and tube-forming ability of HUVECs through cell scratch assay, Transwell migration assay and Matrigel tube formation assay.

[0081] (1). Cell Scratch Experiment

[0082] Cell seeding and pretreatment: HUVECs were seeded in plates and treated with 5 mg / mL glucose to simulate the high glucose-induced oxidative stress environment in vitro.

[0083] Scratching technique: After the cells have grown into a dense monolayer, use a pipette tip to make uniform scratches vertically on the cell surface.

[0084] Drug administration: The original culture medium was discarded, and culture media containing CTT nanozyme, DOEVs@CTT complex and DOEVs@CTT / DHM composite hydrogel were added for intervention. The control group was given normal culture treatment.

[0085] Image acquisition and analysis: Images of the scratched area were captured at 0 and 24 hours post-scratching. ImageJ software was used to measure and calculate changes in scratch area to assess cell migration.

[0086] (2). Transwell Migration Assay

[0087] Cell seeding: HUVECs treated with different materials were seeded into the upper chamber of the Transwell chamber.

[0088] Migration induction: Add culture medium containing chemokines to the lower cavity and incubate for further culture.

[0089] Staining and counting: After culture, remove unmigrated cells from the upper surface of the chamber membrane by wiping with a cotton swab. Migrated cells on the lower surface are fixed and stained with **crystal violet**.

[0090] Quantitative analysis: Randomly selected fields of view were photographed under a microscope, and the migration ability of cells was quantitatively assessed by counting the number of stained cells.

[0091] (3). Matrigel Tube Formation Assay

[0092] Matrigel application: Matrigel is added to the 24-well plate beforehand and incubated at 37°C to cure and set.

[0093] Cell seeding: HUVECs treated with different groups of materials were resuspended and seeded into well plates that had been laid with matrix gel.

[0094] Observation of the tube: Place the well plate in an incubator and incubate for 6 hours.

[0095] Angiogenesis index assessment: Images of the vascular network structure were captured under a microscope. ImageJ software was used to quantitatively analyze angiogenesis indicators such as the number of junctions and vessel length.

[0096] Experimental results:

[0097] 1. Structural characterization and enzyme-like catalytic performance of CTT nanozymes

[0098] Figure 1Characterization spectra of raw materials and products during the preparation of CTT nanozymes are shown below. A is the SEM image of CeVO4; B is the XRD spectrum of CeVO4 and CeVO4-Ti; C is the SEM image of CeVO4, CeVO4-titanium, and CTT nanozymes; DF represent the particle size distributions of CeVO4, CeVO4-Ti, and CTT nanozymes, respectively; G is the XPS spectrum of CeVO4-Ti and CTT nanozymes; H is the FTIR spectrum of CeVO4-Ti and CTT nanozymes; I is the UV-Vis spectrum of CTT nanozymes and other samples; J is the Zeta potential value of CTT nanozymes and other samples; K is the GSH depletion capacity of CTT nanozymes at different concentrations; L is the GSH enzyme activity of CTT nanozymes under different pH treatments; and M is the CAT enzyme activity of CTT nanozymes under different pH treatments.

[0099] 1.1 Morphology and Size Analysis: Cerium vanadate (CeVO4) nanorods were successfully synthesized via a hydrothermal method. Figure 1 Based on this, CTT nanozymes were constructed through titanium doping and electrostatic modification with triphenylphosphine (TPP). Transmission electron microscopy (TEM) showed that CeVO4, CeVO4-Ti, and CTT nanozymes all maintained typical rod-shaped structures. Figure 1 Dynamic light scattering particle size analysis showed that the average hydrated particle size of CTT nanozymes was 134.34 ± 3.13 nm, which was an increase compared to CeVO4 (116.27 ± 2.52 nm). This was attributed to the successful surface modification of TPP. Figure 1 (DF).

[0100] 1.2 Crystal Structure and Chemical Composition Analysis: X-ray diffraction (XRD) patterns showed that both CeVO4 and CeVO4-Ti exhibited clear diffraction peaks on the (200) crystal plane, confirming the preservation of the crystal structure. Figure 1 (B) The characteristic peaks of Ce, V, Ti, and P elements are clearly visible in the full X-ray photoelectron spectroscopy (XPS) spectrum, proving the presence of all target elements in the CTT nanozyme. Figure 1 (G). High-resolution spectral analysis of Ce 3d shows that its binding energy (Ce 3d) 3 / 2 900 eV and 903 eV; Ce 3d 5 / 2 The values ​​of 881 eV and 885 eV are consistent with literature reports. In Fourier transform infrared (FTIR) spectroscopy, the CTT nanozyme showed a value at 1458 cm⁻¹. -1 and 1504cm -1 The characteristic absorption peak of the TPP benzene ring appeared at the 260-280 nm wavelength range, and the UV-Vis spectrum also showed the characteristic absorption of TPP in the 260-280 nm wavelength range. Both of these findings confirm that TPP has been successfully grafted onto the CeVO4-Ti surface. Figure 1 (H, I).

[0101] 1.3 Surface Potential Analysis: Zeta potential measurements revealed charge changes during the modification process. The surface potential of CeVO4 was +9.28 mV, which changed to -14.26 mV after Ti doping. The introduction of ethylenediaminetetraacetic acid (EDTA) during synthesis aimed to increase the surface negative charge (-23.31 mV) to promote its electrostatic binding with the positively charged TPP. The final potential of the CTT nanozyme was -8.27 mV. This series of changes confirmed the successful modification based on electrostatic interactions. Figure 1 (J).

[0102] 1.4 Enzyme-like Catalytic Activity Analysis: CTT nanozymes exhibited significant enzyme-like catalytic activity. Their dose-dependent glutathione peroxidase (GPx)-like activity could be assessed by consuming glutathione (GSH), and this activity showed a clear pH dependence. Figure 1 (K, L). More importantly, CTT nanozymes exhibit highly efficient catalase-like (CAT) activity in a weakly acidic environment (pH 6-7). Figure 1 (M).

[0103] 2. Preparation and Antioxidant Capacity Analysis of DOEVs@CTT Complex

[0104] Figure 2 Characterization spectra of raw materials and products during the preparation of DOEVs@CTT complex; where A is TEM image of DOEVs and DOEVs@CTT complex; B is particle size distribution of DOEVs (top) and DOEVs@CTT complex (bottom); C is elemental mapping image of DOEVs@CTT complex; D is absorption spectrum of ABTS measured in DOEVs@CTT complex and other samples; E is ABTS scavenging rate of DOEVs@CTT complex and other samples; F is absorption spectrum of DPPH measured in DOEVs@CTT complex and other samples; G is DPPH scavenging rate of DOEVs@CTT complex and other samples; H is absorption spectrum of salicylic acid colorimetric reaction of DOEVs@CTT complex and other samples; I is absorbance value of salicylic acid colorimetric reaction of DOEVs@CTT complex and other samples at 510 nm.

[0105] 2.1 Preparation and characterization of DOEVs@CTT complex: The Dendrobium officinale exosomes (DOEVs) obtained by gradient centrifugation were elliptical in shape, with an average diameter of approximately 382.25 ± 1.73 nm. Figure 2(AB). A DOEVs@CTT composite was successfully prepared by encapsulating CTT nanozymes within DOEVs using a thin-film extrusion method. The composite was nearly spherical with an average particle size reduced to 85.55 ± 0.43 nm. Energy-dispersive X-ray spectroscopy (EDS) surface scanning analysis showed that P, Ti, V, and Ce elements were uniformly distributed within the composite structure. The strong P signal originated from phospholipids, the main component of the DOEVs membrane, which confirms from an elemental distribution perspective that the CTT nanozyme was successfully encapsulated within DOEVs. Figure 2 (C)

[0106] 2.2 In vitro antioxidant capacity analysis: The DOEVs@CTT complex exhibited significantly enhanced broad-spectrum antioxidant capacity. Its antioxidant capacity against ABTS was significantly enhanced. + The free radical scavenging rate was higher than that of pure CeVO4-Ti, indicating that the synergistic effect of Ti doping and DOEVs encapsulation improved the overall antioxidant capacity. Figure 2 (DE). DPPH radical scavenging experiments showed that the DOEVs@CTT complex could change the solution color from purple to light purple, and significantly decrease the absorbance at 517 nm ( Figure 2 (FG). In the salicylic acid colorimetric reaction, the DOEVs@CTT complex can effectively and competitively scavenge hydroxyl radicals (·OH), leading to a decrease in the absorbance of the reaction system, demonstrating its highly efficient ·OH scavenging ability (FG). Figure 2 (China HI).

[0107] 3. Preparation and physicochemical properties of DOEVs@CTT / DHM composite hydrogels

[0108] Figure 3Characterization spectra of raw materials and products during the preparation of DOEVs@CTT / DHM composite hydrogels are shown below. A represents the FTIR spectra of hyaluronic acid (HA), hyaluronic acid methacrylate (HAMe), and DHM; B represents the 1H NMR spectra of HA, HAMe, and DHM; C represents the macroscopic images of DHM hydrogel and DOEVs@CTT / DHM composite hydrogel formation under UV light; D represents the release of CTT nanozymes from the DOEVs@CTT / DHM composite hydrogel; E represents the compressive stress-strain curves of the DOEVs@CTT / DHM composite hydrogel and DHM hydrogel; F represents the compressive elastic modulus of the DOEVs@CTT / DHM composite hydrogel and DHM hydrogel; G represents the compressive elastic modulus of samples A and B of the DOEVs@CTT / DHM composite hydrogel. Absorption spectra of TS determination; H represents the ABTS scavenging rate of DOEVs@CTT / DHM composite hydrogel and other samples; I represents the absorption spectrum of DPPH determination of DOEVs@CTT / DHM composite hydrogel and other samples; J represents the DPPH scavenging rate of DOEVs@CTT / DHM composite hydrogel and other samples; K represents the absorption spectrum of salicylic acid colorimetric reaction of DOEVs@CTT / DHM composite hydrogel and other samples; L represents the absorbance value of salicylic acid colorimetric reaction of DOEVs@CTT / DHM composite hydrogel and other samples at 510 nm; M represents the bacterial colony image of DOEVs@CTT / DHM composite hydrogel and other samples; NO represents the growth curves of DOEVs@CTT / DHM composite hydrogel and other samples treated with Staphylococcus aureus and Escherichia coli, respectively.

[0109] 3.1 Chemical Structure and Formation Analysis of Hydrogels: Infrared Spectroscopy and Hydrogen Nuclear Magnetic Resonance Spectroscopy (H NMR) 1 1H NMR analysis confirmed that DHM was successfully synthesized. Figure 3 (AB). After adding the DOEVs@CTT composite to the DHM precursor solution, free radical polymerization was successfully initiated under UV irradiation in the presence of photoinitiator (I2959), forming a cross-linked network structure. This indicates that the addition of the DOEVs@CTT composite does not affect the photocuring of the hydrogel. Figure 3 (C)

[0110] 3.2 pH-responsive release behavior analysis: Under the simulated weakly acidic environment of a diabetic wound (pH 6.0), the cumulative release rate of the DOEVs@CTT composite in the DOEVs@CTT / DHM composite hydrogel reached 79.46% within 36 hours, significantly higher than the release rate under neutral conditions (pH 7.4) (43.64%), demonstrating good pH-responsive release characteristics. Figure 3 (D).

[0111] 3.3 Mechanical property testing: Compression tests showed that after loading the DOEVs@CTT composite, the hydrogel had an elastic modulus of 8.61 kPa, possessing the flexibility and mechanical support suitable for wound dressing applications. Figure 3 Medium EF).

[0112] 3.4 Antioxidant and Antibacterial Performance Analysis: After encapsulation into a hydrogel, the DOEVs@CTT / DHM composite hydrogel maintained the high antioxidant activity of the composite, with a total antioxidant capacity of up to 96.4 ± 1.3%. Figure 3 (Glucose content). Furthermore, this hydrogel exhibits strong antibacterial activity against both Staphylococcus aureus and Escherichia coli. Plate counts showed that the bacterial survival rate after treatment with the DOEVs@CTT / DHM composite hydrogel was less than 1%, meeting the antibacterial standards for medical dressings; absorbance monitoring of the bacterial suspension also confirmed its effective inhibition of bacterial proliferation. Figure 3 middle MO).

[0113] 4. In vitro cell and molecular biological efficacy analysis

[0114] Figure 4 The effects of DOEVs@CTT / DHM composite hydrogel and other samples on cellular oxidative stress are shown in the following figures: A represents the quantitative analysis results of HUVECs cell viability after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; B represents HUVECs cell staining images after treatment with DOEVs@CTT / DHM composite hydrogel and other samples for different time periods; C represents live / dead HUVECs cell staining images after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress; D represents DCF-DA staining images of HUVECs cells after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress; E represents SOD activity in HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress; F represents GSH level in HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress; and G represents MDA level in HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress.

[0115] Figure 5This study illustrates the effects of DOEVs@CTT / DHM composite hydrogels and other samples on mitochondrial function. A shows the mitochondrial red staining images of HUVECs after treatment with DOEVs@CTT / DHM composite hydrogels and other samples under oxidative stress; B shows the quantitative analysis results of the Mito-tracker red fluorescence intensity of HUVECs after treatment with DOEVs@CTT / DHM composite hydrogels and other samples under oxidative stress; C shows the MPTP detection images of HUVECs after treatment with DOEVs@CTT / DHM composite hydrogels and other samples under oxidative stress; D shows the quantitative analysis results of the MPTP fluorescence intensity of HUVECs after treatment with DOEVs@CTT / DHM composite hydrogels and other samples under oxidative stress; and E shows the DOEVs@CTT / DHM composite hydrogels and other samples under oxidative stress. JC-1 staining images of HUVECs after treatment with Vs@CTT / DHM composite hydrogel and other samples; F shows the quantitative analysis results of the JC-1 fluorescence ratio of HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress; G shows electron microscopy images of mitochondrial structures of HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress (yellow arrows indicate mitophagy); H shows the quantitative analysis results of mitochondrial damage in HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress; I shows representative WB bands of the Nrf2 / Parkin / Pink1 pathway in HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples under oxidative stress.

[0116] Figure 6 The effects of DOEVs@CTT / DHM composite hydrogel and other samples on in vitro angiogenesis are shown in the following figures: A: Scratch migration experiment image of DOEVs@CTT / DHM composite hydrogel and other samples; B: Quantitative analysis results of scratch area in scratch migration experiment of DOEVs@CTT / DHM composite hydrogel and other samples; C: Transfer well experiment image of DOEVs@CTT / DHM composite hydrogel and other samples; D: Quantitative analysis results of number of migrated cells in transfer well experiment of DOEVs@CTT / DHM composite hydrogel and other samples; E: Channel formation experiment image of DOEVs@CTT / DHM composite hydrogel and other samples; F: Quantitative analysis results of vascular connection in channel formation experiment of DOEVs@CTT / DHM composite hydrogel and other samples; G: Immunofluorescence staining image of VEGFA in HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; H: Quantitative analysis results of VEGFA expression in HUVECs after treatment with DOEVs@CTT / DHM composite hydrogel and other samples.

[0117] 4.1 Biocompatibility Analysis: CCK-8 assay and live / dead cell staining results showed that the DOEVs@CTT / DHM composite hydrogel had good biocompatibility, and its cell viability was similar to that of the control group. Figure 4 (AB). In the H2O2-induced oxidative stress model, the DOEVs@CTT / DHM composite hydrogel significantly improved cell survival ( ). Figure 4 (C)

[0118] 4.2 Oxidative Stress Results: DCFH-DA fluorescent probe detection showed that treatment with DOEVs@CTT / DHM composite hydrogel significantly reduced intracellular reactive oxygen species (ROS) levels. Figure 4 (D). Simultaneously, it can restore the activity of intracellular superoxide dismutase (SOD) and the content of glutathione (GSH), and effectively reduce the level of malondialdehyde (MDA), a lipid peroxidation product, indicating that it can comprehensively protect cells from oxidative damage. Figure 4 (Zhong EG).

[0119] 4.3 Analysis of mitochondrial morphology and membrane integrity: Mito-Tracker staining and transmission electron microscopy (TEM) showed that treatment with DOEVs@CTT / DHM composite hydrogel could improve abnormal morphology of mitochondria caused by oxidative stress, such as fragmentation, swelling, and cristae structure damage, and restore the normal elliptical structure of mitochondria. Figure 5 (AB, GH).

[0120] 4.4 Mitochondrial membrane potential and permeability analysis: JC-1 staining results showed that the DOEVs@CTT / DHM composite hydrogel could stabilize the mitochondrial membrane potential and prevent its depolarization. Figure 5 (Middle EF). MPTP assay showed that it can reduce the abnormal opening of the mitochondrial permeability transition pore and maintain the integrity of the mitochondrial membrane. Figure 5 Medium CD).

[0121] 4.5 Western Blot Results: Western blot analysis further revealed that the DOEVs@CTT / DHM composite hydrogel upregulated the expression of nuclear factor E2-related factor 2 (Nrf2) and key mitophagy proteins Pink1 and Parkin, indicating that it enhances mitophagy and clears damaged mitochondria by activating the Nrf2 / Pink1 / Parkin signaling pathway. Figure 5 Middle I).

[0122] 4.6 Results of experiments promoting angiogenesis:

[0123] 4.6.1 Cell migration results analysis: Both scratch assay and Transwell migration assay confirmed that the DOEVs@CTT / DHM composite hydrogel significantly promoted the migration ability of human umbilical vein endothelial cells (HUVECs). Figure 6 (Chinese AD).

[0124] 4.6.2 Tube-forming ability analysis: In vitro Matrigel tube-forming experiments showed that DOEVs@CTT / DHM composite hydrogel can effectively enhance the ability of endothelial cells to form vascular-like network structures. Figure 6 Medium EF).

[0125] 4.6.3 Detection of vascular factor expression: Immunofluorescence staining showed that treatment with DOEVs@CTT / DHM composite hydrogel significantly upregulated the expression of vascular endothelial growth factor A (VEGFA) in vascular endothelial cells. Figure 6 (Zhong GH).

[0126] Example 2

[0127] Evaluation of the therapeutic effect of intra-arterial diabetic wound treatment

[0128] 2.1 Animal Model Establishment and Drug Administration

[0129] All animal experiments were approved by the Animal Ethics Committee of Shenzhen Bao'an Pure Traditional Chinese Medicine Treatment Hospital (TOPGM-IACUC-2025-0159). db / db diabetic model mice were selected and, after one week of acclimatization, a 1.5 cm diameter full-thickness skin defect was created on their backs. The mice were randomly divided into four groups: a blank control group (Control), a simple hydrogel group (DHM), a DOEVs@CTT complex group, and a DOEVs@CTT / DHM composite hydrogel group. The corresponding material was administered topically every other day for 14 days, as shown in Table 1.

[0130] Table 1

[0131]

[0132] 2.2 Wound healing assessment and histological analysis

[0133] Wound area was recorded regularly and healing rate was calculated. Wound tissue was harvested on day 14 for histological analysis. Hematoxylin and eosin (H&E) staining was used to observe skin tissue regeneration, inflammatory cell infiltration, and epithelialization; Masson staining was used to observe collagen fiber deposition, arrangement, and maturity. Results showed that, compared with the control group, the DOEVs@CTT / DHM composite hydrogel group exhibited the fastest wound healing speed and the highest healing rate; tissue sections showed the most complete newly formed epithelium, the mildest inflammatory infiltration, and the largest amount of collagen deposition with a more orderly arrangement, most closely resembling the structure of normal skin.

[0134] Experimental results:

[0135] 1. Promotes wound healing in diabetic patients

[0136] Figure 7 Images show the therapeutic effects of diabetic wounds on mice in each group. A represents a representative image of the wound within 14 days; B represents the wound healing rate within 14 days; CD represents the quantitative reepithelialization rate on days 7 and 14, respectively; E represents H&E staining of the wound on days 7 and 14; F represents Masson staining of the wound on days 7 and 14; and GH represents the quantitative collagen deposition on days 7 and 14, respectively.

[0137] 1.1 Healing Observation: In the untreated control group, wound healing was slow; by day 14, the wound remained dark red with depressed edges. In contrast, the wound in the DOEVs@CTT / DHM composite hydrogel treatment group showed a light pink color by day 14, indicating that it had entered the epithelialization stage. Quantitative analysis showed that the wound healing rate in the DOEVs@CTT / DHM composite hydrogel group was close to 100%, significantly better than the control group. Figure 7 (AB).

[0138] 1.2 H&E staining analysis: On day 7 post-treatment, epithelialization was delayed and the newly formed epithelial layer was thinner in the control group; while the epidermis in the DOEVs@CTT / DHM composite hydrogel treatment group was significantly thickened, and the epithelialization rate was significantly improved. By day 14, abundant hair follicle formation and a large number of new blood vessels were observed in the wound tissue of the DOEVs@CTT / DHM composite hydrogel treatment group, indicating that it can effectively promote the regeneration of skin appendages and functional tissue reconstruction. Figure 7 (CE).

[0139] 1.3 Masson staining analysis: In the control group, the blue staining areas were sparse and light, indicating severe under-synthesis of collagen fibers and impaired extracellular matrix synthesis. The DOEVs@CTT / DHM composite hydrogel treatment group, however, exhibited continuously optimized collagen structure. From day 7 to day 14, collagen fibers gradually transformed from an initial disordered arrangement to an ordered, dense, mature structure, demonstrating its effective promotion of continuous collagen synthesis, maturation, and remodeling. Figure 7 (FH).

[0140] 2. Regulatory effects on oxidative stress and inflammatory responses in the body

[0141] Figure 8The oxidative stress resistance of DOEVs@CTT / DHM composite hydrogel and other samples in mice is shown in Figure 1. A represents the superoxide dismutase (SOD) activity in mouse serum after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; B represents the catalase (CAT) activity in mouse serum after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; C represents the glutathione (GSH) level in mouse serum after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; D represents the malondialdehyde (MDA) level in mouse serum after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; E represents the immunohistochemical staining images of TNF-α, IL-6, and IL-10 in mice after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; F and H represent the quantitative analysis results of IL-6, TNF-α, and IL-10, respectively.

[0142] 2.1 Antioxidant Analysis: Wound tissue homogenate was analyzed using an ELISA kit. Compared with the control group, the activities of superoxide dismutase (SOD) and catalase (CAT), as well as the content of glutathione (GSH), were significantly increased in the DOEVs@CTT / DHM composite hydrogel treatment group. Figure 8 (AC). This indicates that the dressing can effectively activate the local cascade antioxidant defense system in the wound, enhancing the ability to scavenge excess free radicals. Simultaneously, the level of malondialdehyde (MDA), an end product of lipid peroxidation, was significantly reduced in the treatment group ( Figure 8 The results (D) indicate that lipid peroxidation damage induced by oxidative stress has been effectively improved.

[0143] 2.2 Effects on inflammatory factors: The control group showed a large number of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) positive cells in the wound tissue. After treatment with DOEVs@CTT / DHM composite hydrogel, the number of positive cells for these two pro-inflammatory factors was significantly reduced. Figure 8 In untreated diabetic wounds, the expression of the anti-inflammatory factor IL-10 is weak. After treatment with the prepared material, the levels of anti-inflammatory factors were significantly increased, with the DOEVs@CTT / DHM composite hydrogel group showing the best anti-inflammatory and repairing effect. Figure 8 (H). The above results indicate that the DOEVs@CTT / DHM composite hydrogel can effectively break the vicious cycle of oxidative stress and inflammatory response, transforming the wound microenvironment from a pro-inflammatory state to a pro-repair state.

[0144] 3. Impact on angiogenesis and safety evaluation

[0145] Figure 9The effects of DOEVs@CTT / DHM composite hydrogel and other samples on angiogenesis in mice are shown. A shows immunohistochemical staining of MMP9 at mouse wound sites after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; B shows immunofluorescence staining of CD31 and α-SMA at mouse wound sites after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; CD shows the quantitative analysis results of α-SMA and CD31 in mice after treatment with DOEVs@CTT / DHM composite hydrogel and other samples; E shows HE staining images of various organs in mice after treatment with DOEVs@CTT / DHM composite hydrogel and other samples.

[0146] 3.1 Inhibition of excessive matrix degradation: Immunohistochemistry showed that, compared with the control group, the number and staining intensity of matrix metalloproteinase-9 (MMP9) positive cells were significantly reduced in the DOEVs@CTT / DHM composite hydrogel treatment group. Figure 9 (A). This indicates that the dressing can inhibit the overexpression of MMP9, thereby protecting the newly formed extracellular matrix from damage and providing a stable scaffold for tissue regeneration.

[0147] 3.2 Promoting Angiogenesis: Immunofluorescence co-staining was performed on vascular markers CD31 (endothelial cell marker) and α-smooth muscle actin (α-SMA, perivascular cell marker). The control group showed only weak CD31 and α-SMA fluorescence signals. In contrast, the DOEVs@CTT / DHM composite hydrogel treatment group exhibited strong co-localization signals of CD31 and α-SMA, indicating the formation of more structurally stable and functionally mature neovascularization. Figure 9 (BD).

[0148] 3.3 Safety Assessment: At the study endpoint, H&E staining histological analysis was performed on the major organs (heart, liver, spleen, lung, and kidney). Results showed that no significant pathological abnormalities were observed in any of the major organs of mice treated with the DOEVs@CTT / DHM composite hydrogel. Figure 9 The presence of E indicates that the topical application of this dressing did not cause significant systemic toxicity and has good in vivo safety.

[0149] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing mitochondrial-targeted nanozymes based on Dendrobium officinale nanovesicles, characterized in that, Includes the following steps: (1) Soluble cerium salt, ethylenediaminetetraacetic acid, soluble vanadate and titanium source are mixed in water, the pH value is adjusted to alkaline, and CeVO4-Ti nanozyme is obtained after hydrothermal reaction; (2) The CeVO4-Ti nanozyme was dispersed in PBS buffer, and ethylenediaminetetraacetic acid was added to activate the surface of the CeVO4-Ti nanozyme. Then, the unreacted ethylenediaminetetraacetic acid was removed and the nanozyme was resuspended in PBS buffer. Triphenylphosphine was added and the pH was adjusted to 6.5-7.

5. After the reaction was carried out in the dark, CeVO4-Ti-TPP nanozyme, i.e., the mitochondrial-targeting nanozyme, was obtained.

2. The method for preparing mitochondrial-targeted nanozymes based on Dendrobium officinale nanovesicles according to claim 1, characterized in that, In step (1), the Ce in the soluble cerium salt 3+ The ethylenediaminetetraacetic acid and the VO4 in the soluble vanadate 3- The molar ratio of Ti in the titanium source is 1:0.8~1.2:0.8~1.2:0.05~0.

3.

3. The method for preparing mitochondrial-targeted nanozymes based on Dendrobium officinale nanovesicles according to claim 1, characterized in that, In step (1), the temperature of the hydrothermal reaction is 140-220℃ and the time is 12-24h; and / or, in step (1), the substance used to adjust the pH value is ammonia water, and the adjustment endpoint is pH=8.0-11.

0.

4. The method for preparing mitochondrial-targeted nanozymes according to claim 1, characterized in that, In step (2), the ratio of the CeVO4-Ti nanozyme, the ethylenediaminetetraacetic acid and the triphenylphosphine is 1g:1mol:0.5g; and / or, in step (2), the activation condition is incubation at 37°C for 1h; and / or, in step (2), the light-protected reaction time is 2h.

5. A mitochondrial-targeting nanozyme prepared by the method for preparing mitochondrial-targeting nanozymes based on Dendrobium officinale nanovesicles according to any one of claims 1 to 4.

6. A method for preparing a mitochondrial-targeted drug delivery system, characterized in that, Includes the following steps: The mitochondrial-targeting nanozyme described in claim 5 was dispersed in PBS buffer, then mixed with a solution containing Dendrobium officinale nanovesicles, and after reaction, squeezed through a membrane with a pore size of 200 nm to obtain the DOEVs@CTT complex; that is, the mitochondrial-targeting drug delivery system.

7. The method for preparing the mitochondrial targeted drug delivery system according to claim 6, characterized in that, The mass ratio of the CeVO4-Ti-TPP nanozyme to the Dendrobium officinale nanovesicles is 1:2; and / or the reaction temperature is 4°C and the time is 60 min.

8. A mitochondrial targeted drug delivery system prepared by the method of preparation of the mitochondrial targeted drug delivery system according to claim 6 or 7.

9. A composite hydrogel dressing for promoting healing of refractory diabetic wounds, characterized in that, The active ingredient includes the mitochondrial targeted drug delivery system of claim 8.

10. A method for preparing the composite hydrogel dressing for promoting healing of refractory diabetic wounds as described in claim 9, characterized in that, Includes the following steps: The mitochondrial targeted drug delivery system of claim 8 is mixed with an aqueous solution containing dopamine-methacrylated hyaluronic acid, and crosslinked to obtain DOEVs@CTT / DHM composite hydrogel, which is the composite hydrogel dressing for promoting healing of refractory diabetic wounds.

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