A dual-responsive nanohydrogel for promoting chronic wound healing in diabetes, preparation method and application

Abstract

The present application relates to biomedical functional materials and nano drug delivery technology, and particularly relates to a double-response nano hydrogel for promoting chronic wound healing of diabetes, a preparation method and application. The hydrogel is stable in delivery of miR-144-5p by constructing chitosan / sodium tripolyphosphate / hyaluronic acid nanoparticles, and is synergistically loaded with oxymatrine in a gelatin-TG enzyme crosslinking hydrogel matrix, so that in-situ gelation and sequential release of double functional drugs are realized under body temperature. Animal experiments prove that the composition can significantly accelerate wound closure, improve VEGF and CD31 expression and reconstruct dermal vascular network, and has good biocompatibility and controlled release performance, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention relates to biomedical functional materials and nanomedicine delivery technology, and more particularly to a dual-response nanohydrogel that promotes the healing of chronic diabetic wounds, its preparation method, and its application. Background Technology

[0002] The global number of people with diabetes continues to rise, and chronic foot ulcers, anterior tibial ulcers, and other refractory wounds have become significant causes of hospitalization, amputation, and death. The cumulative effect of persistent oxidative stress induced by hyperglycemia and microvascular complications leads to elevated local ROS levels, impaired endothelial cell function, and insufficient blood perfusion, causing the wound to remain in the inflammatory phase and hindering its progression to the proliferative-remodeling stage. Conventional dressing changes, negative pressure therapy, and growth factor spraying can only partially improve the inflammatory environment but cannot simultaneously address the core pathological contradiction of "excessive ROS + insufficient angiogenesis," resulting in a prolonged healing period and a high recurrence rate.

[0003] To reduce ROS, Yuanmeng He et al. have incorporated natural or inorganic antioxidants (such as dopamine, CeO2, and sulfide nanozymes) into smart hydrogels to achieve pH- or glucose-triggered sustained release, demonstrating a certain healing-promoting effect in animal models. However, the long-term retention of inorganic nanozymes poses potential metabolic / accumulation risks, and simply clearing ROS is insufficient to reverse nutritional deficiencies caused by inadequate angiogenesis.

[0004] The literature “Oxymatrine hydrogel promotes wound healing by activating the Nrf2 / HO-1 pathway in keratinocytes” in Chinese Journal of Tissue Engineering Research, 2024, Vol. 28, Issue (29): 4620-4627. has shown that the plant small molecule oxymatrine (OX) can reduce oxidative damage and accelerate epidermal repair by activating the Nrf2 / HO-1 pathway. However, its water solubility is generally poor, and it is easy to release in the early stage but the dose is insufficient in the later stage. At the same time, it cannot directly promote angiogenesis, resulting in limited comprehensive efficacy for refractory ulcers.

[0005] In recent years, the regulation of angiogenesis using recombinant VEGF protein, exosomes, or miRNAs has become a research hotspot. Among them, miRNA-144-5p has been reported to significantly improve the migration and tube-forming ability of vascular endothelial cells by targeting RICTOR and regulating Akt / mTOR and eNOS signaling, making it a potential candidate nucleic acid drug for promoting angiogenesis. However, naked miRNAs are easily degraded by nucleases in body fluids, and due to their negative charge, they are not easily able to cross the membrane and enter cells. Therefore, finding a safe and efficient non-viral delivery system is a prerequisite for their clinical application.

[0006] Chitosan (CS) / sodium tripolyphosphate (TPP) ion-crosslinked nanoparticles, due to their positive charge and easy biodegradability, have been used as non-viral carriers for miRNA and siRNA in various studies. Introducing an outer layer of hyaluronic acid (HA) can further improve targeting and blood compatibility through CD44-mediated endocytosis. However, current published literature mainly focuses on tumor suppression or bone defect repair, and has not reported the synergistic application of this system with antioxidant small molecules in diabetic wounds.

[0007] In response to the complex diabetic microenvironment, an increasing number of studies are attempting to develop multi-responsive hydrogels that integrate antibacterial, antioxidant, pro-angiogenic, and visual monitoring functions. For example, Zhendong Li et al. recently reported that the glucose-pH dual-responsive QO / @PV@AB7 hydrogel can scavenge ROS and induce angiogenesis, but it still relies on inorganic quantum dots and metal ions and does not integrate gene regulation modules. Long-term safety and cost issues remain to be resolved. Summary of the Invention

[0008] To address the aforementioned technical problems, the present invention aims to provide a dual-response nano-hydrogel for promoting the healing of chronic diabetic wounds. This hydrogel uniformly disperses positively charged miR-144-5p nanoparticles within a mildly cross-linked gelatin hydrogel three-dimensional network and simultaneously embeds oxymatrine, achieving synergistic effects of anti-oxidation and angiogenesis, in-situ gelation, and sequential controlled release. This invention aims to overcome the key technical bottleneck of the long-term difficulty in healing chronic diabetic wounds.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A dual-responsive nanohydrogel for promoting the healing of chronic diabetic wounds, the hydrogel comprising:

[0011] 1) Chitosan / sodium tripolyphosphate / hyaluronic acid ion-crosslinked nanoparticles loaded with microRNA-144-5p, wherein the concentration in the hydrogel is 0.5-10.0 mg / mL; the volume average particle size of the nanoparticles is 200-800 nm (preferably 200-300 nm), the zeta potential is +25 mV to +35 mV, and the mass ratio of chitosan:sodium tripolyphosphate:hyaluronic acid is 1:0.05-0.25:0.05-0.20;

[0012] 2) The antioxidant oxymatrine, in which the concentration in the hydrogel is 0.05-0.50 mg / mL;

[0013] 3) A gelatin hydrogel matrix cross-linked by transglutaminase at 30-40℃, wherein the concentration of gelatin in the hydrogel is 20-150 mg / mL and the concentration of cross-linking enzyme is 3.0-15.0 mg / mL;

[0014] The nanoparticles are uniformly dispersed within the three-dimensional network of the hydrogel, enabling the hydrogel to continuously release microRNA-144-5p to promote angiogenesis and release oxymatrine to scavenge reactive oxygen free radicals in the wound microenvironment, thereby synergistically accelerating the healing of diabetic wounds.

[0015] Preferably, the mass ratio of chitosan:sodium tripolyphosphate:hyaluronic acid is 1:0.10-0.20:0.08-0.15.

[0016] Preferably, the loading of microRNA-144-5p is 10-200 pmol / mg nanoparticles.

[0017] Preferably, the concentration of cross-linked nanoparticles in the hydrogel is 1.0-5.0 mg / mL; the concentration of oxymatrine is 0.10-0.30 mg / mL; the concentration of gelatin is 50-100 mg / mL; and the concentration of cross-linking enzyme is 5.0-10.0 mg / mL.

[0018] Preferably, the nanoparticles are treated with high molecular weight 100kDa ultrafiltration, and the amount of free nucleic acid or small molecules remaining is ≤5wt%.

[0019] Preferably, the hydrogel has a gelation time of 8-10 min at 37°C, an equilibrium swelling rate of 4-7%, and a cumulative mass loss of ≤15% within 24 h.

[0020] Furthermore, the present invention also provides a method for preparing the hydrogel, the method comprising the following steps:

[0021] a) Dissolve chitosan in a 0.05-0.2 mol / L aqueous acetic acid solution and adjust the pH to 5.0-6.0;

[0022] b) Prepare sodium tripolyphosphate solution and hyaluronic acid solution with a concentration of 0.5-2 mg / mL respectively;

[0023] c) Add sodium tripolyphosphate solution, hyaluronic acid solution and microRNA-144-5p solution dropwise to chitosan solution under stirring at 800-1500 r / min according to the volume ratio to form nanoparticles;

[0024] d) Nanoparticles obtained by ultrafiltration purification;

[0025] e) Dissolve gelatin in sterile PBS, and after complete dissolution at 50-60℃, cool to 30-37℃. Then add the nanoparticle suspension from step d), oxymatrine solution and transglutaminase in sequence. After mixing, crosslink the mixture in a mold at 35-40℃ and allow it to mature at 2-5℃.

[0026] Furthermore, the present invention also provides a sterile syringe filler or patch-type wound dressing for improving chronic or refractory diabetic wounds, the sterile syringe filler or patch-type wound dressing comprising the hydrogel.

[0027] Furthermore, the present invention also provides the use of the hydrogel in the preparation of a medicament for improving chronic or refractory diabetic wounds.

[0028] Furthermore, the present invention also provides the use of the hydrogel in the preparation of topical skin formulations for simultaneously increasing angiogenesis density and reducing reactive oxygen species levels.

[0029] This invention, by employing the above-mentioned technical solutions, achieves synergy in materials science, pharmacokinetics, controlled release, and biology: the nanoparticles exhibit a pale blue opalescent luster, with a particle size mainly distributed between 200–300 nm, a PDI < 0.4, and a zeta potential of approximately +25 / +35 mV. After 100 kDa ultrafiltration, free nucleic acids and small molecules are removed, ensuring stable nucleic acid loading and cellular uptake; the hydrogel rapidly forms in situ gels at 37°C for 8–9 min, with an equilibrium swelling rate of 4–7% and a mass loss of ≤15% over 24 h. The nanoparticles are uniformly dispersed in the three-dimensional porous network, enabling the sequential and sustained release of miRNA and small molecule drugs. In terms of efficacy, this system significantly improved the healing rate in the STZ diabetic rat full-thickness wound model: compared with the model group (healing rate of 34.72% on day 9), the miR group, miN group, Ox group, and miN+Ox group achieved healing rates of 86.84%, 90.60%, 78.75%, and 85.54%, respectively; the wound area was significantly reduced in the early stage (day 3) (29.75% / 44.41% / 31.16% / 45.61%), and the average wound temperature was increased by about 1.5–2.0℃ from day 3 to day 9 after administration, indicating improved local perfusion. Histological and molecular indicators further confirmed vascular remodeling and microenvironment repair: HE showed epidermal / dermal structure reconstruction and appendage recovery, with a significant increase in the number of CD31-positive vessels; VEGF expression was upregulated (miR, miN, and miN+Ox were all superior to the model / blank vector), and ITGB8 protein recovered to near or above normal levels, revealing a synergistic mechanism of "pro-angiogenesis + anti-oxidation". Meanwhile, the system exhibits good safety; after transdermal administration, BUN, CREA, and TCH levels showed no significant differences compared to the normal group, and no local toxicity or abnormal inflammatory reactions were observed. The formulation is entirely aqueous, mildly cross-linked, and can be integrated for injection / application, combining bedside operation friendliness with the feasibility of large-scale production. In summary, the angiogenesis-antioxidant dual-response nanohydrogel of this invention significantly outperforms existing single-function dressings in terms of controlled-release performance, wound healing speed, vascular reconstruction depth, and biocompatibility, providing a highly efficient and industrially scalable new strategy for the treatment of diabetic and other refractory wounds. Attached Figure Description

[0030] Figure 1The figures show the morphology and characterization of nanoparticles; where: A - representative morphological characteristics of nanoparticles; B - particle size distribution of blank nanoparticles; C - particle size distribution of loaded miR-144-5p nanoparticles; D - potential distribution of blank nanoparticles; E - potential distribution of loaded miR-144-5p nanoparticles.

[0031] Figure 2 Figure 1 shows the morphology and characterization of the hydrogel; where: A - gelation time determined by the inverted test tube method; B - morphological characteristics of the hydrogel; C - swelling properties of the hydrogel; D - gelation time of the hydrogel.

[0032] Figure 3 The microstructure of the hydrogel is shown in the image (SEM).

[0033] Figure 4 Images show the physical signs and blood biochemistry of rats; where: A - diabetic rats (left) and normal rats (right); B - rats bandaged and fixed with hydrogel; C - blood glucose changes during drug administration; D - blood glucose after the last administration; E - blood urea nitrogen (BUN) after the last administration; F - creatinine (CREA) after the last administration; G - total cholesterol (TCH) after the last administration. Compared with the normal group: #P<0.05, ##P<0.01; Compared with the model group: *P<0.05, **P<0.01.

[0034] Figure 5 The figure shows the wound healing rate. The differences are as follows: Compared with the normal group: #P<0.05, ##P<0.01; Compared with the model group: *P<0.05, **P<0.01; Compared with the NN group: *P<0.05, **P<0.01.

[0035] Figure 6 The images show thermal images of the wound; where: A - image of the back wound taken by an infrared camera; B - wound temperature thermal image. Compared with the normal group: #P<0.05, ##P<0.01; Compared with the model group: *P<0.05, **P<0.01.

[0036] Figure 7 The images show pathological tissue images; where: A - HE stained images (100×, 200×); B - epidermal thickness; C - dermal thickness; D - total skin thickness. Compared with the normal group: #P<0.05, ##P<0.01; Compared with the model group: *P<0.05, **P<0.01; Compared with the NN group: *P<0.05, **P<0.01.

[0037] Figure 8 The images show CD31 immunohistochemical staining patterns; where: A - representative image of CD31 immunohistochemistry in a cross section of the skin (400×); B - representative image of CD31 immunohistochemistry in a longitudinal section of the skin (400×).

[0038] Figure 9This is a diagram showing VEGF protein expression.

[0039] Figure 10 The image shows ROS staining. The image represents skin ROS staining (400×). Blue represents DAPI-stained cell nuclei, and red represents DHE-stained ROS. Detailed Implementation

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present invention.

[0041] 1.1 Materials

[0042] 1.1.1 Laboratory Animals

[0043] Twenty-one SPF-grade male SD rats, aged 8-10 weeks, were provided by Hangzhou Hangsi Biotechnology Co., Ltd. They were housed in well-ventilated conditions at a room temperature of 20°C and humidity of 55%, under standard light-dark rhythm conditions. Rats were fed a diet provided by Beijing Huafukang Biotechnology Co., Ltd. The ethics review number is KTSC2024406.

[0044] 1.1.2 Experimental Reagents

[0045] The batch numbers and manufacturers of the main experimental reagents are shown in Table 1.

[0046] Table 1 Main experimental reagents

[0047] name batch number Manufacturers Streptozotocin (STZ) JS312051 Shanghai Yuanye Biotechnology Co., Ltd. Citric acid, sodium citrate 20200615 Beijing Boaigang Biotechnology Co., Ltd. Chitosan (CS) 16389032 Shanghai McLean Biotechnology Co., Ltd. Hyaluronic acid (HA) KA741129 Shanghai Jieshikai Biotechnology Co., Ltd. Sodium tripolyphosphate (TPP) 20240702 Sinopharm Chemical Reagent Co., Ltd. Oxymatrine 211011 Beijing Zhongke Quality Inspection Biotechnology Co., Ltd. Glutamine transferase JS31205 Shanghai Jieshikai Biotechnology Co., Ltd. miRNA inhibitor (miR-144-5p) LSS0601 Beixin Biotechnology Co., Ltd. sterile PBS 19HO2A21 Bosch Biotechnology Co., Ltd. ITGB8 antibody 6100008357 Wuhan Aiboteke Biotechnology Co., Ltd. CD31 antibody H651213025 Hangzhou Huaan Biotechnology Co., Ltd. VEGF antibody H650661037 Hangzhou Huaan Biotechnology Co., Ltd. Total cholesterol test kit 240918101 Meikang Biotechnology Co., Ltd. Urea test kit 240906201 Meikang Biotechnology Co., Ltd. Glucose test kit 230804101 Meikang Biotechnology Co., Ltd. Creatinine test kit 240524101 Meikang Biotechnology Co., Ltd. DAPI staining solution 17J12B77 Bosch Biotechnology Co., Ltd. Superoxide anion reactive oxygen species detection kit (DHE) A264250505 Biyuntian Biotechnology Co., Ltd. PAGE Gel Rapid Preparation Kit (10%) 039211800 Shanghai Yamei Biomedical Technology Co., Ltd.

[0048] 1.1.3 Main experimental instruments and equipment

[0049] The models and manufacturers of the main experimental instruments and equipment are shown in Table 2.

[0050] Table 2 Main Experimental Instruments and Equipment

[0051] name model Manufacturer Electronic analytical balance PMK223ZH / E Ohaus Instruments (Shanghai) Co., Ltd. pH meter pH 100 Shanghai Lichen Bangxi Instrument Technology Co., Ltd. Magnetic stirrer SCI280-Pro Scilogex, Inc. Malvern laser particle size analyzer KA741129 Malvern Panalytical (UK) Malvern potential sample cell DTS1070 Malvern Panalytical (UK) Malvern Plastic Sample Cell DTS0012 Malvern Panalytical (UK) ELISA reader Epoch2c Berton Instruments, Inc. low-speed centrifuge TLXJ-IIB Shanghai Anting Scientific Instrument Factory Refrigerated centrifuge VELOCITY18R Beijing Dameco Biotechnology Co., Ltd. Fully automated biochemical analyzer HITACHI70 Hitachi Manufacturing Co., Ltd. Automatic dehydrator KH-TO Hubei Xiaogan Kuohai Medical Technology Co., Ltd. Paraffin embedding machine JB-L7 Wuhan Junjie Electronics Co., Ltd. High-throughput tissue homogenizer SCIENTZ-48 Ningbo Xinzhi Biotechnology Co., Ltd. Electric thermostatic incubator DHP-9162 Shanghai Heheng Instrument Equipment Co., Ltd. OLYMPUS microscope BX43 Olympus Corporation Fluorescence Imaging System Upgrade Module BX3-URA Olympus Corporation

[0052] 1.2 Methods

[0053] 1.2.1 Modeling and Grouping

[0054] After 7 days of acclimatization feeding, SD rats were intraperitoneally injected with streptozotocin (STZ, 45 mg / kg) to establish a diabetic model. Three days after STZ injection, random blood glucose levels were measured in the tail vein. A blood glucose level ≥16.7 mmol / L, maintained for one week, was considered a successful model establishment.

[0055] A rat model of diabetic wounds was established using dorsal full-thickness skin defect surgery: Rats were anesthetized with isopentane via inhalation using a respiratory anesthesia machine. A 1.0 cm diameter perforation biopsy instrument was placed in the center of the rat's back, and appropriate downward pressure was applied until a circular imprint appeared. Then, the skin in the middle section was excised along the circular imprint. The excised tissue was full-thickness skin, leaving the subcutaneous back muscle intact. Two diabetic wounds were prepared for each rat.

[0056] The samples were randomly divided into 7 groups: normal group (blank hydrogel), model group (blank hydrogel), blank nanoparticle hydrogel group (NN group), oxymatrine hydrogel group (Ox group), miR-144-5p hydrogel group (miR group), miR-144-5p nanoparticle hydrogel group (miN group), and oxymatrine + miR-144-5p nanoparticle hydrogel group (miN+Ox group).

[0057] 1.2.2 Preparation and Characterization of Nanoparticles

[0058] (1) Nanoparticle preparation: Nanoparticles were prepared by ion crosslinking

[40]

[0059] ① Dissolve an appropriate amount of CS in a 0.1 mol / L acetic acid solution to prepare a 1 mg / ml CS acetic acid solution. Add an appropriate amount of 0.1 mol / L NaOH to adjust the pH to 5.5.

[0060] ② Dissolve appropriate amounts of TPP and HA in deionized water to obtain a 1 mg / ml TPP and HA solution, and filter it through a 0.22 μm pore size filter membrane.

[0061] ③The volume ratio of CS:TPP:HA is 1:0.15:0.1. Under magnetic stirring, TPP, HA and drug solution are added dropwise to a fixed volume of CS solution at a magnetic stirrer speed of 1200 r / min. After the addition is complete, stirring is continued at 1200 r / min for 10 min to form a milky white nanoparticle suspension.

[0062] ④ Place the opalescent nanoparticle suspension into the top of a Nanosep® 100 kDa ultrafiltration centrifuge tube and centrifuge at 3600 r / min for 10 min.

[0063] (2) Nanoparticle characterization: The particle size, PDI and potential of the nanoparticles were determined using a Malvern laser particle size analyzer.

[0064] ① Particle size and PDI: First, rinse the Malvern plastic sample cell three times with ultrapure water, then rinse it 1-2 times with the sample solution. Add the sample solution to the Malvern plastic sample cell to about one-third full, and wipe off the surface moisture. Place the sample cell into the Malvern laser particle size analyzer and measure the particle size and PDI three times.

[0065] ② Potential: First, rinse the Malvern potential sample cell three times with ultrapure water, then rinse it 1-2 times with the sample solution. Add the sample solution to the Malvern potential sample cell, filling it completely without air bubbles, and wipe off any surface moisture. Place it in a Malvern laser particle size analyzer and measure the potential three times.

[0066] 1.2.3 Preparation and Characterization of Hydrogels

[0067] (1) Hydrogel preparation: Hydrogels were prepared using gelatin, sterile PBS, and TG enzyme crosslinking as the matrix. The drug proportions are shown in Table 3.

[0068] ① Melting gelatin: Weigh an appropriate amount of gelatin and sterilize it by irradiating it with a UV lamp for 30 minutes. Then add an appropriate amount of sterile PBS and heat it in a water bath at 55°C until it is completely dissolved.

[0069] ② Adding the drug: After the water bath, cool to room temperature, add the drug solution and TG enzyme solution (0.1g / ml), and stir well. Use a 1.5cm diameter PTFE mold, sterilize the mold with 75% alcohol, and add 500μL of adhesive solution to each hole. Crosslink at 37℃ for 2 hours.

[0070] ③ Crosslinking and curing: Crosslink at 37℃ for 2 hours, then incubate at 4℃ overnight.

[0071] Table 3. Dosage of Hydrogel Drugs

[0072] blank Ox miR NN miN miN+Ox Gelatin (g) 0.35 0.35 0.35 0.35 0.35 0.35 Sterile PBS (ml) 5 5 5 5 5 5 TG enzyme solution (0.1 g / ml) (μL) 350 350 350 350 350 350 Ox solution (0.1 g / ml) (μL) 8 8 NN solution (μL) 250 miR solution (μL) 40 miN solution (μL) 250 250

[0073] (2) Hydrogel characterization and determination: Observe the morphological characteristics of the hydrogel and determine the gelation time, swelling, pore size and microstructure of the hydrogel.

[0074] ① Swelling property: After preparation, the hydrogel was weighed (Wa). Then, 1000 μL of sterile PBS solution was added above the hydrogel, followed by incubation at 37 ± 0.5 °C. Samples were removed at regular intervals, excess water was carefully blotted off the surface of the hydrogel with filter paper, and then weighed again on a balance (Wb). Each experiment was repeated three times. The swelling ratio of the hydrogel was calculated using the following formula.

[0075]

[0076] ② Gel formation time: Use the inverted test tube method. Pour 2 ml of gelatin solution into an 8 ml vial and place it at the set temperature (e.g., 37°C). Tilt the vial every 30 seconds and observe the liquid flow; record the gel formation time when the liquid stops flowing. To ensure the reliability and accuracy of the experimental data, strictly follow the principle of repeated experiments and perform three parallel experiments for each group of samples.

[0077] ③ Pore size and microstructure: Pore size and microstructure (SEM) were measured. The hydrogel was rapidly frozen with liquid nitrogen and freeze-dried for 24 hours. The dried sample was fixed on a conductive adhesive and sputtered with gold (5-10 nm thickness). An accelerating voltage of 20 kV was selected, and images were taken at different magnifications.

[0078] 1.2.4 Animal Experiments

[0079] During the experiment, rats had free access to food and water. Each group consisted of 3 rats. After shaving the backs of the rats, depilatory cream was used to remove the downy hair, leaving 2 skin wounds on the backs of each rat. Under anesthesia, a small amount of iodine was applied to prevent infection. The prepared hydrogel was then gently applied to the skin wounds of the rats, and the hydrogel was secured with elastic self-adhesive bandages and medical tape. The wounds were then neatly bandaged.

[0080] 1.3 Detection Indicators

[0081] 1.3.1 Animal physical signs

[0082] During the experiment, the rats' diet and water intake were measured daily, and random blood glucose levels were measured in the tails of the rats before and after modeling to evaluate diabetes-related signs.

[0083] 1.3.2 Wound healing rate

[0084] A full-thickness skin resection model was established based on a diabetic model, and hydrogel wound dressing was applied every other day. From the day of model establishment, standardized photographs of the wound were taken at fixed time points (21 cm vertically above the wound) on days 3, 5, 7, and 9. The wound area was then precisely measured using ImageJ image analysis software. The wound healing rate was calculated as (1 - residual wound area / original wound area) × 100%.

[0085] 1.3.3 Infrared Imaging

[0086] Infrared thermal imaging was used to measure wound blood flow every other day. Standardized photos of the wound were taken at a fixed time point at a vertical height of 21 cm from the wound. The experiment was conducted before measuring the wound healing rate on days 0, 3, 5, 7, and 9.

[0087] 1.3.4 Detection of blood biochemical indicators

[0088] At the end of the experiment, blood was collected from the abdominal aorta of rats. After being incubated at 37°C for 0.5 h, the blood was centrifuged twice (1500 r / min, 10 min) and the supernatant was collected. The blood glucose (GLU), renal function (BUN, creatinine, CREA), total cholesterol (TCH) and other indicators were detected by a fully automated blood biochemistry analyzer.

[0089] 1.3.5 Histological examination

[0090] On day 10 of the experiment, diabetic rats were euthanized using an overdose of anesthetic. Skin wound tissue was collected, and a portion was placed in formalin. After 72 hours, it was embedded in paraffin. Finally, the tissue sections were stained with hematoxylin and eosin for morphological evaluation. The specific steps are as follows:

[0091] (1) Weighing and fixing

[0092] After rinsing the skin with physiological saline, blot dry, and weigh, collect tissue from the same skin site. Wash away blood and other tissues with physiological saline, then fix with 4% neutral formalin buffer for 72 hours, followed by rinsing with tap water for 0.5-1 hour. Store the tissue from the same skin site at -80℃.

[0093] (2) Dehydration

[0094] The procedure is shown in Table 4.

[0095] Table 4. Dehydration Procedure

[0096] Serial Number step time Serial Number step time 1 50% ethanol 30min 7 Anhydrous ethanol II 30min 2 75% ethanol 30min 8 Anhydrous ethanol xylene (1:1) mixture 10min 3 85% ethanol 30min 9 Xylene I 5min 4 95% Ethanol I 30min 10 Xylene II 5min 5 95% ethanol 30min 11 Paraffin I 50min 6 Anhydrous ethanol 30min 12 Paraffin II 50min

[0097] (1) Tissue embedding

[0098] The dehydrated tissue is immersed in heated paraffin wax, a mold of appropriate size is selected, the skin is placed upright in the embedding cassette, liquid paraffin wax is poured in, and the mixture is cooled and solidified on an ice plate.

[0099] (2) Slicing, unfolding, adhering, baking slices

[0100] The fully solidified paraffin was fixed in a microtome and cut into 4-5 μm thick sections. The sections were then developed in a 37°C water bath, attached to glass slides in a water bath, and air-dried before being baked in a 37°C oven for 12 hours. Before staining, the sections were baked in a 60°C oven for 20 minutes.

[0101] (3) H&E staining, mounting, and reading of slides

[0102] The H&E staining procedure is shown in Table 5. After staining, place the tissue in a ventilated area for 1 hour to allow the xylene to evaporate. Seal the tissue with a coverslip using neutral resin and observe the histopathological changes under a light microscope.

[0103] Table 5 H&E staining steps

[0104] Serial Number step Time required Serial Number step Time required 1 Xylene dewaxing I 10min 13 Wash with tap water 1min 2 Xylene Dewaxing II 10min 14 1% dilute ammonia solution turns blue 30s 3 Anhydrous ethanol I 2min 15 Wash with tap water 1min 4 Anhydrous ethanol II 2min 16 Eosin staining 5s 5 95% Ethanol I 2min 17 Wash with tap water 1min 6 95% Ethanol II 2min 18 80% ethanol 20s 7 70% ethanol 2min 19 95% ethanol 30s 8 50% ethanol 2min 20 Anhydrous ethanol I 2min 9 Wash with tap water 2min 21 Anhydrous ethanol II 2min 10 Hematoxylin staining 7min 22 air dry 1h 11 Wash with tap water 2min 23 Xylene Clear I 10min 12 1% hydrochloric acid and 75% ethanol differentiation 20s 24 Xylene Clear II 10min

[0105] 1.3.6 Immunohistochemistry

[0106] Prepared paraffin sections were incubated with VEGF and CD31 antibodies after antigen retrieval and hydrogen peroxide blockade at 4°C overnight. Then, they were incubated with secondary antibody at room temperature for 1 hour, and the cell nuclei were counterstained with DAPI. The sections were observed and images were acquired under an inverted fluorescence microscope. After clearing with xylene, the sections were placed in a ventilated area for 1 hour to allow the xylene to evaporate. The sections were then mounted with neutral resin on coverslips and observed under a light microscope for histopathological changes. The immunohistochemical DAB staining procedure is shown in Table 6.

[0107] Table 6 Immunohistochemical DAB staining steps

[0108] Serial Number step Time required Serial Number step Time required 1 Xylene dewaxing 1 10min 20 DAB colorimetric assay Microscopic control 2 Xylene dewaxing 2 10min 21 Wash with tap water 2min 3 Anhydrous ethanol 1 2min 22 Hematoxylin staining 7min 4 Anhydrous ethanol 2 2min 23 Wash with tap water 1min 5 95% ethanol 2min 24 1% hydrochloric acid 75% ethanol 20s 6 80% ethanol 2min 25 Wash with tap water 1min 7 50% ethanol 2min 26 1% dilute ammonia 30s 8 PBS wash ×3 3min×3 27 Wash with tap water 1min 9 Antigen repair Microwave for 2 minutes, then bake in a 100℃ oven for 40 minutes. 28 70% ethanol 30s 10 Cool to room temperature 29 80% ethanol 30s 11 PBS wash ×3 3min×3 30 95% ethanol 1 30s 12 Immunohistochemistry circle 31 95% ethanol 2 30s 13 3% H2O2, room temperature, protected from light 15min 32 Anhydrous ethanol 1 2min 14 PBS wash ×3 5min×3 33 Anhydrous ethanol 2 2min 15 5%BSA, room temperature 2h 34 air dry 2h 16 Primary antibody (1:200), 4℃ 12h 35 Xylene transparent 1 10min 17 PBS wash ×3 5min×3 36 Xylene transparent 2 10min 18 Secondary antibody, room temperature 2h 37 air dry 2h 19 PBS wash ×3 5min×3 38 Cover

[0109] 1.3.7 Western blot detection

[0110] The protein concentration in skin tissue was detected using a BCA kit. Equal volumes of protein were subjected to SDS-PAGE electrophoresis; the membrane was transferred to a PVDF membrane; after 1.5 h, it was blocked for 2 h at room temperature with a rapid blocking buffer; ITGB8 protein primary antibody was added, and the membrane was incubated overnight at 4°C; secondary antibody was added, and the membrane was blocked for 2 h at room temperature, followed by ECL imaging; images were acquired using an imaging analysis system; the images were analyzed, and the relative expression levels of each protein were determined.

[0111] 1.3.8 ROS Detection

[0112] The prepared paraffin sections were rehydrated, subjected to antigen retrieval, and incubated with BSA before being stained with DHE staining solution. The cell nuclei were then counterstained with DAPI staining solution, mounted with anti-fluorescence quenching mounting medium, and observed and images acquired under an inverted fluorescence microscope. Specific steps are shown in Table 7.

[0113] Table 7 ROS staining steps

[0114] Serial Number step Time required Serial Number step Time required 1 Xylene dewaxing I 10min 11 PBS wash 3min×3 2 Xylene Dewaxing II 10min 12 5%BSA room temperature 2h 3 Anhydrous ethanol I 2min 13 PBS wash 3min×3 4 Anhydrous ethanol II 2min 14 DHE staining Incubate at 37°C in the dark for 20 minutes. 5 95% ethanol 2min 15 Wash with PBS (protect from light) 5min×3 6 80% ethanol 2min 16 DAPI staining (avoid light) 15min 7 50% ethanol 2min 17 Wash with PBS (protect from light) 5min×3 8 PBS wash 3min×3 18 Anti-fluorescence quenching mounting medium (protected from light) - 9 Antigen retrieval (50x) Microwave boiling 19 Observation under a fluorescence microscope - 10 Cool to room temperature

[0115] 1.3.9 Statistical Analysis

[0116] Data processing was performed using Excel software. Quantitative data were expressed as mean ± standard deviation (x̄ ± s). The t-test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0117] 2 Results and Analysis

[0118] 2.1 Nanoparticle morphology and characterization

[0119] 2.1.1 Nanoparticle solution morphology characteristics

[0120] The prepared blank nanoparticles and the solution of nanoparticles loaded with miR-144-5p exhibit a significant and pure pale blue opalescence under natural light. This unique optical phenomenon arises from the Rayleigh scattering effect of the nanoparticles: when a beam of light penetrates the nanoparticle solution, the nanoparticles, with their nanoscale size, interact with the incident light, causing strong scattering of short-wavelength blue light, thus giving the solution its characteristic pale blue opalescent appearance. Figure 1 As shown in Figure A.

[0121] 2.1.2 Particle size distribution and polydispersity index (PDI)

[0122] The key physical parameters of blank nanoparticles and drug-loaded nanoparticles were systematically characterized using a Malvern laser particle size analyzer, and their particle size distribution and polydispersity index (PDI) were accurately measured. Experimental data showed that the particle size of both blank and drug-loaded nanoparticles was mainly concentrated in the 200-300 nm range, which conforms to the size definition of nanomaterials.

[0123] By performing three repeated measurements on each group of samples, the obtained PDI values ​​were consistently less than 0.4. Based on the nanoparticle dispersibility evaluation standard, this result fully demonstrates that the particle size distribution of blank nanoparticles and drug-loaded nanoparticles in the system is uniform, with no obvious abnormal particle size aggregation. Figure 1 As shown in B and C.

[0124] 2.1.3 Surface Potential

[0125] The key physical parameters of blank nanoparticles and miR-144-5p-loaded nanoparticles were systematically characterized using a Malvern laser particle size analyzer. The average potential of the blank nanoparticles was accurately measured to be 28.9 ± 1.77 mV, while that of the miR-144-5p-loaded nanoparticles was 33.1 ± 1.57 mV. During the preparation of nanoparticles, the formation of their surface potential characteristics is closely related to the raw materials used. Chitosan (CS), as the main material, exhibits positive charge due to its molecular structure; hyaluronic acid (HA) and sodium tripolyphosphate (TPP) carry negative charges. Because CS is in an excess state during preparation, its positive charge effect dominates, resulting in an overall positive charge on the surface of the final nanoparticles. Figure 1 As shown in D and E.

[0126] 2.2 Hydrogel Preparation and Characterization

[0127] 2.2.1 Morphological characteristics of hydrogels

[0128] The hydrogel prepared by crosslinking gelatin with transglutaminase (TG enzyme) exhibits a pure, colorless, and transparent appearance, displaying uniform and transparent optical properties under natural light. Its clear texture provides ideal matrix conditions for subsequent performance studies. Figure 2 As shown in B.

[0129] 2.2.2 Gel formation time

[0130] The gelation time of the blank hydrogel was approximately 12.5 min. The gelation times of the experimental group hydrogels with added matrine, miR-144-5p, and miR-144-5p nanoparticles were concentrated between 8 and 9 min. The gelation time of the experimental group hydrogel with added blank nanoparticles was approximately 14 min. Figure 2 As shown in A and D.

[0131] Within the scope of this experiment, the gelation times of several hydrogels exhibited relatively stable characteristics. This indicates that the various hydrogel formulations demonstrate high consistency and controllability in their gelation kinetics. This stability provides an important technological basis for the standardized application of hydrogels in animal experiments.

[0132] 2.2.3 Swelling property

[0133] In the study of hydrogel swelling properties, sterile phosphate-buffered saline (PBS) was selected as the swelling medium, and a standardized procedure of three replicate experiments was employed to reduce experimental error. Experimental data showed that the swelling rates of the hydrogels in each experimental group were concentrated in the range of 4%–7%. Figure 2 As shown in Figure C, this result reflects that the hydrogel of this system possesses relatively stable and controllable swelling properties, providing key parameter basis for its application in drug sustained release.

[0134] 2.2.4 Microscopic characteristics

[0135] The microstructure of the lyophilized hydrogel was analyzed in depth using scanning electron microscopy (SEM). At 100x magnification, the irregular porous morphology of the hydrogel cross-section was clearly visible. These interconnected pores form a complex and unique three-dimensional network architecture, laying the structural foundation for the hydrogel's swelling and mass transport properties. Increasing the magnification to 500x, uniformly loaded nanoparticles within the hydrogel matrix were directly observed, tightly bound to the hydrogel network, further confirming the effective loading and stable distribution of nanoparticles in the hydrogel system. Figure 3 As shown.

[0136] 2.3 Efficacy Results

[0137] 2.2.1 Effects of hydrogels on the physical signs of diabetic rats

[0138] After establishing a diabetic rat model through a single intraperitoneal injection of streptozotocin (STZ), blood glucose levels were measured every few days using a glucometer on tail blood samples. Compared to the normal control group (blood glucose range 3.9–6.1 mmol / L), the random blood glucose levels of the model rats showed a significant increase, consistently exceeding 16.7 mmol / L. Figure 4 As shown in C.

[0139] Furthermore, continuous observation revealed that the successfully modeled diabetic rats exhibited typical symptoms of diabetes: weight loss and significantly reduced coat luster; daily water intake surged from 15-20 ml per rat before modeling to 80-100 ml per rat, accompanied by a significant increase in urination frequency, requiring frequent daily changes of cage bedding. This indicates that the diabetic rat model was successfully established.

[0140] 2.2.2 Effects of hydrogels on blood biochemical parameters in diabetic rats

[0141] Blood urea nitrogen (BUN) is a key indicator reflecting glomerular filtration function. In cases of renal insufficiency and decompensation, BUN levels will rise, making it an important indicator for assessing renal function impairment. Serum creatinine (CREA) is primarily filtered by the glomeruli but not absorbed by the renal tubules. Its value reflects the extent of glomerular filtration function impairment; a significant increase suggests toxic effects of drugs and their metabolites on the renal parenchyma or inflammatory damage. Total cholesterol (TCH) refers to the cholesterol contained in various lipoproteins in serum, namely the sum of bound cholesterol and free cholesterol. The level of TCH reflects the status of lipid metabolism.

[0142] After the experiment, a fully automated blood biochemistry analyzer was used to measure blood glucose, renal function (CREA, BUN), TCH, and other blood biochemical indicators. The results are as follows: Figure 4 As shown in Figures D-G, compared with the model group, there were no significant differences in blood glucose, serum urea nitrogen (BUN), serum creatinine (CREA), and total cholesterol (TCH) levels among the rat groups after transdermal administration of the hydrogel. This suggests that after the drug is absorbed through the skin and enters the circulatory system, it does not cause damage to renal function or lipid metabolism, indicating a high safety profile.

[0143] 2.2.3 Effect of hydrogel on wound healing rate

[0144] The acute phase of wound healing occurs within 3 days of drug administration during the modeling process, the intermediate phase within 5-7 days, and the final phase within 9 days. Figure 5The wound images shown are illustrated. Compared to the normal group, the wound healing process in the model group rats was significantly delayed; the wound healing rate in the normal group rats reached 39.67% on day 3, while the healing rate in the model group rats was only 9.08% at the same time; by day 9, the healing rate in the normal group rats was as high as 92.86%, while the healing rate in the model group rats was only 35.43% at the same time, and the difference between the groups was statistically significant (P<0.01). It is evident that the model group rats exhibited typical wound healing impairment characteristics under diabetic conditions.

[0145] Compared with the model group, the wound healing rates of rats in the Ox group, miR group, miN group, and miN+Ox group reached 26.38%, 29.75%, 45.59%, and 53.05% on day 3, respectively; and reached 78.35%, 86.84%, 85.94%, and 87.55% on day 9, respectively, indicating that the wounds were almost completely healed. The wound healing rate of rats in all four drug-treated groups was significantly faster. P <0.01).

[0146] Compared with the model group, the wound healing rate of rats in the NN group reached 69.77% on day 9. This may be because chitosan and hyaluronic acid have good biocompatibility and antibacterial properties, effectively maintaining a moist wound environment and promoting wound healing. Compared with the NN group, the healing rates of rats in the miN and miN+Ox groups were also significantly increased on days 3, 5, 7, and 9. P <0.05, 0.01). It can be seen that miR, miN, and miN+Ox effectively promote wound healing.

[0147] Further comparisons were made between the wound healing rates of miR, miN, and Ox groups and the miN+Ox group. Results showed that on days 3, 5, 7, and 9 after intervention, the healing rate of the miN+Ox group was significantly higher than that of the miR, miN, and Ox group. P <0.05, 0.01).

[0148] 2.2.4 Effects of hydrogels on wound microcirculation and temperature

[0149] On days 3, 5, 7, and 9 after drug administration, standardized photographic measurements of microcirculation and thermal images of the back skin were taken using an infrared camera (21 cm vertically above the wound) at fixed time points. The microcirculation imaging results are shown below. Figure 6 As shown in Figure A, compared with the normal group, the wound microcirculation of rats in the model group was significantly reduced; while compared with the model group, the wound microcirculation of rats in the Ox group, miR group, miN group and miN+Ox group was significantly increased.

[0150] The acquired infrared images were processed using FlirTools software, and the image temperature range was standardized to 20℃ (minimum temperature) to 35℃ (maximum temperature). A thermogram was then created based on the temperature values ​​at the wound site. The thermogram results are shown below. Figure 6 As shown in Figure B, compared with the normal group, the wound temperature of the rats in the model group was significantly lower, especially during the acute phase. P <0.01); and compared with the model group, the wound temperature of rats in the Ox group, miR group, miN group and miN+Ox group was increased to a certain extent, and the differences were significant on day 3. P <0.01). This indicates that miR, miN, and miN+Ox can significantly improve animal microcirculation and back temperature.

[0151] 2.2.5 Effects of hydrogels on skin pathological tissues

[0152] After H&E staining, skin pathological tissue was observed under a microscope, such as... Figure 7 As shown in Figure A, under a 100x microscope, the healed skin clearly reveals its layered structure: epidermis, dermis, and subcutaneous tissue. The epidermis, the outermost layer of the skin, is composed of keratinized stratified squamous epithelium and appears purplish-blue on H&E-stained sections. The dermis, located beneath the epidermis, is composed of dense connective tissue and contains numerous skin appendages such as hair follicles, sebaceous glands, and sweat glands; it appears light pink on H&E-stained sections. Compared to the normal group, the model group rats showed accumulations of bluish-purple non-nuclear material at the wound site, indicating extensive inflammatory cell infiltration. Compared to the model group, rats in the Ox, miR, miN, and miN+Ox groups showed significant growth of capillaries and recovery of skin appendages at day 9.

[0153] The thickness of the epidermis, dermis, and total skin was measured using OPLENIC software. Compared with the normal group, the thickness of the epidermis and dermis in the wound regeneration area of ​​rats in the model group was significantly reduced. P <0.05, 0.01), indicating that skin damage in diabetic wounds is difficult to regenerate. Compared with the model group, the epidermal layer thickness of the wound skin in rats in the miN group and miN+Ox group was significantly increased ( P <0.01); compared with the NN group, the thickness of the epidermal layer of the wound skin in the miN+Ox group was significantly increased ( P <0.05. Compared with the model group, the dermal thickness of the wound skin in rats in the Ox group, miR group, miN group, and miN+Ox group was significantly increased ( P <0.05, 0.01); Compared with the NN group, the dermal layer thickness of the skin wound in the miN group and miN+Ox group was significantly increased ( P <0.01). This indicates that Ox, miR, miN, and miN+Ox can effectively promote skin wound repair.

[0154] 2.2.6 Effects of hydrogel on the expression of CD31 and VEGF proteins

[0155] CD31, as a highly specific marker of endothelial cell differentiation, can specifically bind to vascular endothelial cell surface antigens. Under the action of the DAB staining system, positively expressed vascular endothelial cells exhibit typical brownish-yellow granular staining, thereby accurately revealing the spatial distribution and expression abundance of blood vessels in dermal tissue.

[0156] like Figure 8 As shown, compared with the normal group, the expression of CD31 in the wound skin of rats in the model group and NN group was significantly reduced, indicating that the dermal vascular network in diabetic wounds was significantly atrophied and reduced. Compared with the model group and NN group, the expression of CD31 in the wound skin of rats in the miR group, miN group, and miN+Ox group was significantly increased, and the number of blood vessels was increased, especially in the miN+Ox group. This suggests that miR-144 inhibitors and oxymatrine hydrogel can synergistically promote dermal angiogenesis.

[0157] Vascular endothelial growth factor (VEGF), a core cytokine regulating angiogenesis, can significantly enhance the permeability of microveins and small veins by specifically activating receptors on the surface of vascular endothelial cells, driving endothelial cell proliferation, migration, and luminal structure formation. It is currently the most potent and specific pro-angiogenic factor known. In normal skin tissue, VEGF is mainly positively expressed in basal cells of the epidermis, hair follicle stem cells, and wound repair areas.

[0158] Compared with the normal group, the expression of VEGF in the wound skin of rats in the model group was significantly downregulated. P <0.05 indicates weakened VEGF-mediated angiogenesis in diabetic wounds. Compared with the model group, VEGF expression in the wound skin of rats in the miR, miN, and miN+Ox groups was upregulated ( P <0.05, 0.01). Compared with the NN group, the expression of VEGF in the wound skin of rats in the miN group and miN+Ox group was upregulated ( P <0.05, 0.01). The results indicate that miN and miN+Ox can effectively promote dermal angiogenesis and vascular network reconstruction by upregulating VEGF expression.

[0159] 2.2.7 Effect of hydrogel on ROS content

[0160] Reactive oxygen species (ROS) are a collective term for a wide range of oxygen-based free radicals and non-free radical substances, including superoxide anion (O2). - Hydrogen peroxide (H2O2), hydroxyl radicals (OH-) -), ozone (O3) and singlet oxygen ( 1 O2, due to the presence of unpaired electrons in its molecules, exhibits extremely high chemical reactivity. Abnormally elevated levels can cause significant damage to cell structures. Dihydroethidium (DHE) is a commonly used fluorescent probe for detecting intracellular superoxide anion levels. DHE undergoes a dehydrogenation reaction under the influence of intracellular superoxide anions, generating ethidium. This product can bind to RNA or DNA, thereby producing a red fluorescent signal.

[0161] like Figure 10 As shown, compared with the normal group, the ROS-positive expression in the wound skin of rats in the model group and NN group was significantly increased, indicating increased oxidative damage in diabetic wounds. Compared with the model group and NN group, the ROS-positive expression in the wound skin of rats in the Ox group and miN+Ox group was significantly reduced. These results indicate that miR-144 inhibitors and oxymatrine hydrogel can synergistically reduce oxidative damage in diabetic wounds.

[0162] The foregoing description of embodiments of the present invention, through which those skilled in the art are able to implement or use the present invention, will be readily apparent to those skilled in the art. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novelty disclosed herein.

Claims

1. A dual-responsive nanohydrogel for promoting chronic wound healing in diabetes, characterized in that, The hydrogel comprises: 1) microRNA-144-5p-loaded chitosan / sodium tripolyphosphate / hyaluronic acid ionically cross-linked nanoparticles, the concentration of which in the hydrogel is 0.5-10.0 mg / mL; the volume average particle size of the nanoparticles is 200-800 nm, the zeta potential is +25 mV to +35 mV, and the mass ratio of chitosan:sodium tripolyphosphate:hyaluronic acid is 1:0.05-0.25:0.05-0.20; 2) an antioxidant oxymatrine, the concentration of which in the hydrogel is 0.05-0.50 mg / mL; 3) a gelatin hydrogel matrix cross-linked and shaped by transglutaminase under the condition of 30-40℃, the concentration of gelatin in the hydrogel is 20-150 mg / mL, and the concentration of cross-linking enzyme is 3.0-15.0 mg / mL; wherein the nanoparticles are uniformly dispersed in the three-dimensional network of the hydrogel, enabling the hydrogel to continuously release microRNA-144-5p to promote angiogenesis and release oxymatrine to scavenge reactive oxygen free radicals in the microenvironment of a wound surface, thereby synergistically accelerating the healing of a diabetic wound.

2. The hydrogel of claim 1, wherein, The mass ratio of chitosan:sodium tripolyphosphate:hyaluronic acid is 1:0.10-0.20:0.08-0.

15.

3. The hydrogel of claim 1, wherein, The loading amount of microRNA-144-5p is 10-200 pmol / mg of nanoparticles.

4. The hydrogel of claim 1, wherein, The concentration of cross-linked nanoparticles in the hydrogel is 1.0-5.0 mg / mL; the concentration of oxymatrine is 0.10-0.30 mg / mL; the concentration of gelatin is 50-100 mg / mL; and the concentration of cross-linking enzyme is 5.0-10.0 mg / mL.

5. The hydrogel of claim 1, wherein, The nanoparticles are subjected to ultrafiltration treatment with high molecular weight 100 kDa, and the residual amount of free nucleic acids or small molecules is ≤5 wt%.

6. The hydrogel of claim 1, wherein, The hydrogel has a gelation time of 8-10 min at 37℃, an equilibrium swelling rate of 4-7%, and a cumulative mass loss of ≤15% within 24 h.

7. A method for preparing the hydrogel according to any one of claims 1 to 6, characterized in that, The method comprises the following steps: a) dissolving chitosan in 0.05-0.2 mol / L aqueous acetic acid solution, and adjusting the pH to 5.0-6.0; b) respectively preparing 0.5-2 mg / mL sodium tripolyphosphate solution and hyaluronic acid solution; c) adding the sodium tripolyphosphate solution, the hyaluronic acid solution and the microRNA-144-5p solution to the chitosan solution in a volume ratio under stirring at 800-1500 r / min to form nanoparticles; d) ultrafiltration purification of the obtained nanoparticles; e) dissolving gelatin in sterile PBS, completely dissolving at 50-60℃, and then cooling to 30-37℃, and then sequentially adding the nanoparticle suspension of step d), the oxymatrine solution and the transglutaminase, mixing uniformly, and then cross-linking and gelling in a mold at 35-40℃ and overnight at 2-5℃.

8. A sterile syringe filling or a patch-type wound dressing for improving chronic or non-healing diabetic wounds, characterized in that, The sterile syringe filling or the paste-type wound dressing comprises the hydrogel of any one of claims 1-6.

9. Use of the hydrogel of any one of claims 1-6 in the preparation of a medicament for improving chronic or refractory diabetic wounds.

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