Dual-response nano hydrogel for promoting healing of chronic wound of diabetes mellitus, preparation method and application

Abstract

The invention relates to a biomedical functional material and nano-drug delivery technology, in particular to dual-response nano-hydrogel for promoting healing of chronic wound surfaces of diabetes mellitus, a preparation method and application. According to the hydrogel, chitosan / sodium tripolyphosphate / hyaluronic acid nanoparticles are constructed to stably deliver miR-144-5p, oxymatrine is synergistically entrapped in a gelatin-TG enzyme cross-linked hydrogel matrix, in-situ gelling under the body temperature condition is achieved, and difunctional drugs are sequentially released. Animal experiments prove that the composition can significantly accelerate wound closure, improve VEGF and CD31 expression and reconstruct a dermal vascular network, has good biocompatibility and controlled release performance, and has wide clinical application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to biomedical functional materials and nanomedicine delivery technology, and particularly relates to a dual-response nanohydrogel for promoting chronic wound healing of diabetes, a preparation method and application. BACKGROUND

[0002] The global number of diabetes patients continues to rise, and chronic foot ulcers, pretibial ulcers and other refractory wounds have become an important inducement for hospitalization, amputation and death. The mutual superposition of sustained oxidative stress induced by hyperglycemia and microangiopathy causes the local ROS level to rise, the endothelial cell function to be impaired, and the blood perfusion to be insufficient, and the wound stays in the inflammation phase for a long time and is difficult to enter the proliferation-remodeling stage. Conventional dressing change, negative pressure treatment, growth factor spraying and other means can only partially improve the inflammatory environment, but it is difficult to simultaneously solve the core pathological contradiction of "excessive ROS + insufficient angiogenesis", and the healing period is long and the recurrence rate is high.

[0003] To reduce ROS, Yuanmeng He et al. have studied the incorporation of natural or inorganic antioxidants (such as dopamine, CeO2, sulfide nanoszyme, etc.) into intelligent hydrogel to achieve pH or glucose-triggered slow release, and obtain certain healing promotion effect in animal models. However, the long-term retention of inorganic nanoszyme poses a potential metabolic / accumulation risk, and simply removing ROS still cannot reverse the nutrient deficiency caused by insufficient angiogenesis.

[0004] The literature "Oxymatrine Hydrogel Promotes Wound Healing by Activating the Nrf2 / HO-1 Pathway in Keratinocytes" (Chinese Journal of Tissue Engineering Research, 2024, Vol. 28, Issue (29): 4620-4627) discloses that the plant small molecule oxymatrine (OX) has been proven to reduce oxidative damage and accelerate epidermal repair by activating the Nrf2 / HO-1 pathway, but its water solubility is generally poor, it is prone to initial burst release and insufficient dosage later, and it cannot directly promote the formation of new blood vessels, resulting in limited comprehensive efficacy on refractory ulcers.

[0005] In recent years, the use of recombinant VEGF protein, exosomes or miRNA to regulate angiogenesis has become a research hotspot. Among them, miRNA-144-5p has been reported to significantly improve the migration and tube formation capacity of vascular endothelial cells by targeting RICTOR, regulating Akt / mTOR and eNOS signals, and is a potential pro-angiogenic candidate nucleic acid drug. However, naked miRNA is easily degraded by nucleases in body fluids, and it is difficult to cross the membrane into cells due to its negative charge, so finding a safe and efficient non-viral delivery system is a prerequisite for its clinical application.

[0006] Chitosan (CS) / triple sodium phosphate (TPP) ion cross-linked nanoparticles have been used as non-viral carriers of miRNA and siRNA in many works due to their positive charge and easy biodegradation. The introduction of an outer layer of hyaluronic acid (HA) can mediate endocytosis through CD44, further improving targeting and blood compatibility. However, the current public literature mainly focuses on tumor suppression or bone defect repair, and there is no report on the use of this system in combination with antioxidant small molecules for diabetic wounds.

[0007] In the face of complex diabetic microenvironment, more and more studies attempt to develop multi-responsive hydrogels, while integrating antibacterial, antioxidant, pro-vascular and visual monitoring functions. For example, Zhendong Li et al. recently reported a glucose-pH dual-responsive QO / PV / AB7 hydrogel that can scavenge ROS and induce angiogenesis, but 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 solved. SUMMARY

[0008] To solve the above technical problems, the purpose of the present application is to provide a dual-responsive nanohydrogel hydrogel for promoting the healing of diabetic chronic wounds, which uniformly disperses positively charged nanoparticles loaded with miR-144-5p in a mild cross-linked gelatin hydrogel three-dimensional network, and simultaneously embeds oxymatrine, achieving antioxidant, pro-vascular dual pathway synergy, in-situ gelation, sequential controlled release, aiming to break through the key technical bottleneck of long-term non-healing of diabetic chronic wounds.

[0009] To achieve the above purpose, the technical scheme adopted by the present application is as follows: A dual-responsive nanohydrogel hydrogel for promoting the healing of diabetic chronic wounds, comprising: 1) chitosan / triple sodium phosphate / hyaluronic acid ion cross-linked nanoparticles loaded with microRNA-144-5p, 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 (preferably 200-300 nm), the zeta potential is +25 mV to +35 mV, and the mass ratio of chitosan:triple sodium phosphate:hyaluronic acid is 1:0.05-0.25:0.05-0.20; 2) 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 at 30-40°C, 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; The nanoparticles are uniformly dispersed in the three-dimensional network of the hydrogel, so that the hydrogel can continuously release microRNA-144-5p to promote angiogenesis and release oxymatrine to remove active oxygen free radicals in the microenvironment of the wound surface, thereby synergistically accelerating the healing of diabetic wounds.

[0010] Preferably, the mass ratio of chitosan: sodium tripolyphosphate: hyaluronic acid is 1:0.10-0.20:0.08-0.15.

[0011] Preferably, the loading amount of microRNA-144-5p is 10-200 pmol / mg of nanoparticles.

[0012] 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.

[0013] Preferably, the nanoparticles are subjected to ultrafiltration treatment with a high molecular weight of 100 kDa, and the residual amount of free nucleic acids or small molecules is ≤5 wt%.

[0014] Preferably, 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.

[0015] Further, the present application also provides a preparation method of the hydrogel, which 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 into the chitosan solution in the 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 at 35-40℃ in a mold and post-curing at 2-5℃.

[0016] Further, the present application also provides a sterile syringe filling or a paste-type wound dressing for improving chronic or difficult-to-heal diabetic wounds, wherein the sterile syringe filling or the paste-type wound dressing comprises the hydrogel.

[0017] Further, the present application also provides the use of the hydrogel in the preparation of a medicament for improving the chronic or refractory diabetic wound surface.

[0018] Further, the present application also provides the use of the hydrogel in the preparation of a skin external preparation for simultaneously increasing the density of new blood vessels and reducing the level of active oxygen.

[0019] The present application, due to the adoption of the above technical solutions, forms a synergy in the aspects of material science, pharmacokinetic control release and biology: the nanoparticles present a light blue opalescence, the particle size is mainly distributed in 200-300 nm, the PDI is <0.4, the zeta potential is about +25 / +35 mV, the free nucleic acid and small molecules are removed after 100 kDa ultrafiltration, the stable loading of nucleic acid and cell uptake are ensured; the hydrogel is rapidly in-situ gelled at 37°C in 8-9 min, the equilibrium swelling rate is 4-7%, the mass loss is ≤15% in 24 h, the nanoparticles are uniformly dispersed in the three-dimensional porous network, forming the sequential and sustained release of miRNA and small molecule drugs. In terms of drug efficacy, the system significantly improves the healing rate in the full-thickness wound model of STZ diabetic rats: compared with the model group (healing rate of 34.72% on the 9th day), the miR group, the miN group, the Ox group and the miN+Ox group reach 86.84%, 90.60%, 78.75% and 85.54% respectively; the wound is significantly reduced (29.75% / 44.41% / 31.16% / 45.61%) in the early stage (3rd day), and the average temperature of the wound is increased by about 1.5-2.0°C in 3-9 days after drug administration, indicating that the local perfusion is improved. Histology and molecular indicators further confirm the vascular reconstruction and microenvironment repair: HE shows that the epidermis / dermis structure is reconstructed, and the appendages are restored, and the number of CD31 positive blood vessels is significantly increased; the expression of VEGF is up-regulated (miR, miN, miN+Ox are better than the model / blank carrier), and the ITGB8 protein is restored to close to or higher than the normal level, revealing the synergistic mechanism of "pro-angiogenic + antioxidant". At the same time, the system has good safety, and there is no significant difference in BUN, CREA and TCH between the normal group and the percutaneous administration group, and there is no local toxicity or abnormal inflammatory reaction; the whole aqueous phase formula, mild cross-linking and injectable / pasting integration are integrated, and have the advantages of bedside operation friendliness and scalability feasibility. In summary, the angiogenesis-antioxidant dual-responsive nanohydrogel of the present application is significantly superior to the existing single-function dressing in terms of controlled release performance, wound healing speed, vascular reconstruction depth and biological safety, and provides an efficient and industrialized new strategy for the treatment of diabetic and other refractory wounds. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1Figure 1 is a figure of the morphology and characterization of the nanoparticles; wherein: A represents the figure of the morphology characteristics of the nanoparticles; B represents the particle size distribution of the blank nanoparticles; C represents the particle size distribution of the miR-144-5p-loaded nanoparticles; D represents the electric potential distribution of the blank nanoparticles; and E represents the electric potential distribution of the miR-144-5p-loaded nanoparticles.

[0021] Figure 2 Figure 2 is a figure of the morphology and characterization of the hydrogel; wherein: A represents the gelation time determined by the inverted test tube method; B represents the morphology characteristics of the hydrogel; C represents the swelling property of the hydrogel; and D represents the gelation time of the hydrogel.

[0022] Figure 3 Figure 3 is a figure of the micro-morphology (SEM) of the hydrogel.

[0023] Figure 4 Figure 4 is a figure of the rat signs and blood biochemistry; wherein: A represents the diabetic rats (left) and normal rats (right); B represents the rats with the hydrogel wrapped and fixed; C represents the blood glucose change during the administration; D represents the blood glucose after the last administration; E represents the urea nitrogen (BUN) after the last administration; F represents the creatinine (CREA) after the last administration; and G represents the 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.

[0024] Figure 5 Figure 5 is a figure of the wound healing rate; wherein: 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.

[0025] Figure 6 Figure 6 is a figure of the wound thermal imaging; wherein: A represents the back wound picture taken by the infrared camera; and B represents the wound temperature thermal map. Compared with the normal group: #P<0.05, ##P<0.01; compared with the model group: *P<0.05, **P<0.01.

[0026] Figure 7 Figure 7 is a figure of the pathological tissue; wherein: A represents the HE staining picture (100x, 200x); B represents the epidermis thickness; C represents the dermis thickness; and D represents the 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.

[0027] Figure 8 Figure 8 is a figure of the CD31 immunohistochemical staining; wherein: A represents the CD31 immunohistochemical representative figure of the skin cross-section (400x); and B represents the CD31 immunohistochemical representative figure of the skin longitudinal section (400x).

[0028] Figure 9Figure A-VEGF immunohistochemical staining representative graph (400x); Figure B-WB picture; Figure C-VEGF statistical result; Figure D-WB statistical result. 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.

[0029] Figure 10 Figure ROS staining graph; wherein: skin ROS staining representative graph (400x), blue is DAPI-stained nucleus, and red is DHE-stained ROS. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the protection scope of the present application.

[0031] 1.1 Materials 1.1.1 Experimental animals SPF level male SD rats, 21, 8-10 weeks old, provided by Hangzhou Hengsi Biological Technology Co., Ltd. Ventilated well, room temperature 20℃, humidity 55%, standard light-dark rhythm feeding. Rat feed was provided by Beijing Huafukang Biological Technology Co., Ltd. Ethical review number: KTSC2024406.

[0032] 1.1.2 Experimental reagents The batch numbers and manufacturers of the main experimental reagents are shown in Table 1.

[0033] Table 1 Main experimental reagents 1.1.3 Main experimental instruments and equipment The model and manufacturer of the main experimental instruments and equipment are shown in Table 2.

[0034] Table 2 Main experimental instruments and equipment 1.2 Methods 1.2.1 Modeling and grouping After 7 days of adaptive feeding, the SD rats were injected intraperitoneally with streptozotocin (STZ, 45mg / kg) to establish a diabetic model. After 3 days of STZ injection, the random blood glucose of rats was measured by tail vein, and the blood glucose value was ≥16.7mmol / L, which was maintained for one week to be considered as a successful modeling.

[0035] Back full-thickness skin defect surgery to construct a diabetic wound rat model: using a respiratory anesthetic machine to make rats inhale isopentane anesthesia, place a 1.0 cm diameter perforating biopsy instrument in the center of the back of the rat, and generate appropriate pressure downward, when a circular mark appears. Then use scissors to cut off the middle part of the skin along the circular mark. The depth of the cut tissue is full-thickness skin, leaving the subcutaneous dorsal muscle. Two diabetic wounds are prepared for each rat.

[0036] 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), oxymatrine + miR-144-5p nanoparticle hydrogel group (miN+Ox group).

[0037] 1.2.2 Preparation of nanoparticles and characterization determination (1) Nanoparticle preparation: ion crosslinking method was used to prepare nanoparticles

[40] ① A certain amount of CS was dissolved in 0.1 mol / L acetic acid solution to prepare a 1 mg / ml CS acetic acid solution. A certain amount of 0.1 mol / L NaOH was added to adjust the pH to 5.5.

[0038] ② A certain amount of TPP and HA was dissolved in deionized water to obtain 1 mg / ml TPP and HA solutions, and filtered through a 0.22 μm pore size filter membrane.

[0039] ③ The volume ratio of CS:TPP:HA was 1:0.15:0.1. Under magnetic stirring, TPP, HA and drug solution were added dropwise to a fixed volume of CS solution at a magnetic stirrer speed of 1200 r / min. After dropping, the speed was continued at 1200 r / min for 10 min to form a milky colored nanoparticle suspension.

[0040] ④ The milky colored nanoparticle suspension was placed on the upper end of the Nanosep® 100KDa ultrafiltration centrifuge tube and centrifuged at 3600 r / min for 10 min.

[0041] (2) Nanoparticle characterization determination: Malvern laser particle size analyzer was used to determine the particle size, PDI and potential of the nanoparticles.

[0042] ① Particle size, PDI: first rinse the Malvern plastic sample cell with ultrapure water 3 times, then rinse the sample solution 1-2 times, add the sample solution to the Malvern plastic sample cell to about one-third, and wipe off the surface moisture. Put it into the Malvern laser particle size analyzer to measure the particle size and PDI for 3 times.

[0043] ②Zeta potential: The Malvern Zeta potential cell was rinsed with ultrapure water for 3 times, and then rinsed with sample solution for 1-2 times. The sample solution was added into the Malvern Zeta potential cell, which was filled with no bubbles and wiped clean. The Zeta potential was measured for 3 times by Malvern laser particle size analyzer.

[0044] 1.2.3 Preparation of hydrogel and characterization determination (1) Hydrogel preparation: The hydrogel was prepared using gelatin, sterile PBS and TG enzyme crosslinking as the matrix. The drug proportion was shown in Table 3.

[0045] ① Gelatin melting: The appropriate amount of gelatin was weighed and sterilized under ultraviolet light for 30 min. Then, the appropriate amount of sterile PBS was added, and the solution was heated to complete dissolution in a water bath at 55°C.

[0046] ② Drug addition: After the water bath, the solution was cooled to room temperature, and the drug solution and TG enzyme solution (0.1 g / ml) were added and stirred uniformly. A polytetrafluoroethylene mold with a diameter of 1.5 cm was sterilized with 75% alcohol, and 500 μL of the gel solution was added to each well. The crosslinking was performed at 37°C for 2 h.

[0047] ③ Crosslinking and solidification: After crosslinking at 37°C for 2 h, the sample was placed at 4°C overnight.

[0048] Table 3 Drug dosage of hydrogel (2) Hydrogel characterization determination: The morphological characteristics of the hydrogel were observed, and the gelation time, swelling property, pore size and microstructure of the hydrogel were determined.

[0049] ① Swelling property: The prepared hydrogel was weighed (Wa). Then, 1000 μL of sterile PBS solution was added to the top of the hydrogel, which was then incubated at 37±0.5°C. The sample was taken out every certain time, and the excess water on the surface of the hydrogel was carefully absorbed with filter paper, and then it was placed on the balance for weighing (Wb). Each experiment was repeated 3 times. The following formula was used to calculate the swelling rate of the hydrogel.

[0050] ② Gelation time: The inverted test tube method was used. 2 ml of gelatin solution was injected into an 8 ml test tube, which was placed at a set temperature (e.g. 37°C). The test tube was tilted every 30 seconds, and the liquid flow was observed; when the liquid did not flow, the gelation time was recorded. To ensure the reliability and accuracy of the experimental data, the principle of repeated experiments was strictly followed, and three parallel experiments were performed for each group of samples.

[0051] ③ Pore size and microstructure: The pore size and microstructure (SEM) test was used. The hydrogel was rapidly frozen with liquid nitrogen and freeze-dried for 24 h. The dried sample was fixed on a conductive glue, and gold was sprayed (thickness 5-10 nm). The acceleration voltage was selected as 20 kV, and the images were taken at different magnifications.

[0052] 1.2.4 Animal experiments The rats were allowed to eat and drink freely during the experiment. Each group had 3 rats, and the rats were shaved on the back and then depilated with depilatory cream. Two skin wounds were made on the back of each rat. Under anesthesia, a small amount of iodophor was applied to prevent infection, and the prepared hydrogel was gently applied to the skin wound site. The hydrogel was fixed with an elastic self-adhesive bandage and medical tape, and the wound was wrapped neatly.

[0053] 1.3 Detection indicators 1.3.1 Animal signs During the experiment, the rats' food and water intake were measured daily, and the rats' random blood glucose was measured before and after modeling by tail measurement to evaluate diabetes-related signs.

[0054] 1.3.2 Wound healing rate On the basis of the diabetic model, a full-thickness skin excision model was established, and the hydrogel was given to the wound every other day. From the day of modeling, standardized photographs of the wound were taken at a fixed time point 21 cm vertically away from the wound on days 3, 5, 7, and 9. Then, ImageJ image analysis software was used to accurately measure the wound area. The wound healing rate was calculated as follows: wound healing rate = (1 - residual wound area / original wound area) x 100%.

[0055] 1.3.3 Infrared imaging Every other day, the wound blood flow was measured using an infrared thermal imager, and standardized photographs of the wound were taken at a fixed time point 21 cm vertically away from the wound. The wound healing rate was measured before the experiment on days 0, 3, 5, 7, and 9.

[0056] 1.3.4 Detection of blood biochemical indicators At the end of the experiment, blood was taken from the abdominal aorta of the rats, and after 0.5 h of warm bath at 37℃, the supernatant was obtained by centrifugation twice (1500 r / min, 10 min). The blood glucose (GLU), renal function (urea nitrogen BUN, creatinine CREA), and total cholesterol (TCH) were detected by automatic blood biochemistry instrument.

[0057] 1.3.5 Histological examination On the 10th day of the experiment, the diabetic rats were euthanized with an overdose of anesthetic. The skin wound tissue was taken, and part of it was placed in formalin. After 72 h, it was embedded in paraffin. Finally, the tissue sections were treated with hematoxylin-eosin staining for morphological evaluation. The specific steps are as follows: (1) Weighing and fixation After the skin was washed with physiological saline and dried, the tissue at the same site was taken, the blood and other tissues of the tissue were washed away with physiological saline, and the tissue was fixed with 4% neutral formalin buffer for 72 h and washed with tap water for 0.5-1 h. The tissue at the same site of the skin was taken and stored at -80°C.

[0058] (2) Dehydration The program is shown in Table 4.

[0059] Table 4 Dehydration program table of tissue (1) Tissue embedding After dehydration, the tissue was immersed in liquid paraffin heated to a liquid, and a suitable mold was selected, the skin was placed in an embedding box, liquid paraffin was poured, and the ice platform was cooled and solidified.

[0060] (2) Sectioning, unfolding, adhering, and baking The solidified paraffin was fixed on a microtome, and 4-5 μm thick paraffin sections were cut, unfolded in a 37°C water bath, adhered to the wax section with a glass slide water bath, and dried and baked in a 37°C oven for 12 h. Before staining, the section was baked in a 60°C oven for 20 min.

[0061] (3) H&E staining, mounting, and reading The H&E staining steps are shown in Table 5. After 1 h in a ventilated place, the xylene was evaporated, the cover glass was sealed with neutral gum, and the histopathological changes were observed under a light microscope.

[0062] Table 5 H&E staining steps 1.3.6 Immunohistochemistry The prepared paraffin sections were incubated with VEGF and CD31 antibodies after antigen repair and hydrogen peroxide blocking at 4°C overnight. Then, the secondary antibody was incubated at room temperature for 1 h, the cell nucleus was stained with DAPI, and the image was observed and collected under an inverted fluorescence microscope. After the section was transparent in xylene, it was placed in a ventilated place for 1 h, the xylene was evaporated, the cover glass was sealed with neutral gum, and the histopathological changes were observed under a light microscope. The immunohistochemical DAB staining steps are shown in Table 6.

[0063] Table 6 Immunohistochemical DAB staining steps 1.3.7 Western-blot detection Skin tissue protein concentration was measured using a BCA kit. Equal amounts of protein were subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. After 1.5 hours, the membrane was blocked with a rapid blocking buffer for 2 hours at room temperature. Primary antibody against ITGB8 protein was added and incubated overnight at 4°C. Secondary antibody blocking was performed for 2 hours at room temperature, followed by ECL development and image acquisition using an imaging analysis system. The images were analyzed and the relative expression levels of each protein were determined.

[0064] 1.3.8 ROS Detection Prepared paraffin sections were rehydrated, antigen retrieved, and incubated with BSA before staining with DHE staining solution. Cell nuclei were then counterstained with DAPI staining solution, and the sections were mounted with anti-fluorescence quenching mounting media. The sections were then observed and images were collected under an inverted fluorescence microscope. Specific steps are shown in Table 7. Table 7 ROS staining steps 1.3.9 Statistical analysis Excel software was used for data processing in this experiment. Measurement data were expressed as mean ± standard deviation (x¯±s). The t-test was used for comparison between groups. P < 0.05 was considered statistically significant.

[0065] 2 Results and Analysis 2.1 Nanoparticle morphology and characterization 2.1.1 Morphological characteristics of nanoparticle solutions The prepared blank nanoparticles and the miR-144-5p loaded nanoparticle solution exhibited a significant and pure light blue opalescence under natural light. This unique optical phenomenon is caused by the Rayleigh scattering effect of nanoparticles on light: when the light beam penetrates the nanoparticle solution, the nanometer-sized particles interact with the incident light, causing the short-wavelength blue light to be strongly scattered, thereby giving the solution a unique light blue opalescence appearance. Figure 1 As shown in A.

[0066] 2.1.2 Particle Size Distribution and Polydispersity Index (PDI) A Malvern laser particle size analyzer was used to systematically characterize the key physical parameters of the blank and miR-144-5p-loaded nanoparticles, accurately measuring their particle size distribution and polydispersity index (PDI). Experimental data showed that the particle sizes of both the blank and drug-loaded nanoparticles were primarily concentrated in the 200-300 nm range, which meets the size definition of nanomaterials.

[0067] By repeating the measurement three times for each group of samples, the PDI values ​​obtained were all stable and less than 0.4. According to the nanoparticle dispersibility evaluation standard, this result fully demonstrates that the particle size distribution uniformity of blank nanoparticles and drug-loaded nanoparticles in the system is good, and there is no obvious abnormal particle size aggregation phenomenon. Figure 1As shown in B and C.

[0068] 2.1.3 Surface potential The key physical parameters of blank nanoparticles and nanoparticles loaded with miR-144-5p were systematically characterized using a Malvern laser particle size analyzer. The average potential of blank nanoparticles was accurately determined to be 28.9±1.77mV, and the average potential of miR-144-5p-loaded nanoparticles was 33.1±1.57mV. 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, is positively charged due to its molecular structure characteristics; hyaluronic acid (HA) and sodium tripolyphosphate (TPP) are negatively charged. Since CS is in an excess state during the preparation process, its positive charge effect dominates, making the surface of the nanoparticles finally prepared positively charged as a whole. Figure 1 As shown in D and E.

[0069] 2.2 Hydrogel preparation and characterization 2.2.1 Hydrogel morphology characteristics The hydrogel prepared by cross-linking gelatin and transglutaminase (TG enzyme) is pure, colorless and transparent, and exhibits 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.

[0070] 2.2.2 Gelation time The gelation time of the blank hydrogel was about 12.5 min. The gelation time of the hydrogel in the experimental group with the addition of oxymatrine, miR-144-5p, and miR-144-5p nanoparticles was concentrated between 8 and 9 min. The gelation time of the hydrogel in the experimental group with the addition of blank nanoparticles was about 14 min. Figure 2 As shown in A and D.

[0071] Within the observed range of this experiment, the gelation times of several hydrogels exhibited relatively stable characteristics, demonstrating high consistency and controllability in the gelation kinetics of each hydrogel formulation. This stability provides an important process foundation for the standardized application of hydrogels in animal experiments.

[0072] 2.2.3 Swelling In the study of hydrogel swelling properties, sterile phosphate buffered saline (PBS) was selected as the swelling medium, and a standardized process of three repeated experiments was also used to reduce experimental errors. The 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 C. This result reflects that the hydrogel of this system has relatively stable and controllable swelling properties, providing a key parameter basis for its application in drug sustained release.

[0073] 2.2.4 Microstructure The microstructure of the hydrogel after lyophilization was analyzed in depth by scanning electron microscopy (SEM). At 100 times magnification, the cross-section of the hydrogel showed an irregular porous morphology, and these pore structures were interconnected, forming a complex and unique three-dimensional network architecture, which laid a structural foundation for the swelling and mass transfer performance of the hydrogel. When the magnification was increased to 500 times, the uniformly loaded nanoparticles in the hydrogel matrix could be directly observed, which were closely combined with the hydrogel network, further confirming the effective loading and stable distribution of the nanoparticles in the hydrogel system. As shown in Figure 3 .

[0074] 2.3 Pharmacodynamic results 2.2.1 Influence of hydrogel on the signs of diabetic rats After a single intraperitoneal injection of streptozotocin (STZ) to construct a diabetic rat model, the tail tip blood samples of the rats were regularly tested for blood glucose by a blood glucose meter every few days. Compared with the normal control group (blood glucose value range 3.9-6.1 mmol / L), the random blood glucose level of the model rats showed a significant increase, and the blood glucose value was stably higher than 16.7 mmol / L, as shown in Figure 4 .

[0075] In addition, it was found through continuous observation that the successfully modeled diabetic rats showed typical symptoms of diabetes: body weight loss and a significant decrease in skin luster; the daily water consumption of each rat increased from 15-20 ml before modeling to 80-100 ml, accompanied by a significant increase in urination frequency, and the cage bedding had to be replaced daily. This suggests that the diabetic rat model was successfully constructed.

[0076] 2.2.2 Influence of hydrogel on blood biochemical indicators of diabetic rats Blood urea nitrogen (BUN) is a key indicator reflecting glomerular filtration function. When the kidney function is decompensated, BUN will increase, so BUN is an important indicator for evaluating kidney damage. Creatinine (CREA) in the blood is mainly filtered by the glomerulus, but not absorbed by the renal tubule, and its value can reflect the damage to the glomerular filtration function; if it is significantly increased, it indicates that drugs and their metabolites have toxic effects on the kidney parenchyma or cause inflammatory damage, etc. Total cholesterol (TCH) refers to the cholesterol contained in various lipoproteins in the serum, i.e., the sum of bound cholesterol and free cholesterol, and the size of TCH can reflect the blood lipid metabolism.

[0077] After the end of the experiment, the blood glucose and kidney function (CREA, BUN), TCH, and other blood biochemical indicators were detected by a fully automatic blood biochemical analyzer. The results are shown in Figure 4As shown in D~G, compared with the model group, the blood glucose and serum urea nitrogen (BUN), blood creatinine (CREA), and total cholesterol (TCH) indicators of each group of rats showed no significant difference after hydrogel transdermal administration. It is suggested that the drug absorbed through the skin into the circulatory system does not cause damage to the kidney function and blood lipid metabolism, and has high safety.

[0078] 2.2.3 Effect of hydrogel on wound healing rate The 3 days after modeling and administration is the acute phase of wound healing, the 5~7 days is the healing medium-term, and the 9 days is the healing end-term, as shown in the wound pictures. Figure 5 Compared with the normal group, the wound healing process of the model group rats was significantly delayed; the wound healing rate of the normal group rats reached 39.67% on the 3rd day, while that of the model group rats was only 9.08% at the same period; the wound healing rate of the normal group rats reached 92.86% on the 9th day, while that of the model group rats was only 35.43% at the same period, and the difference between the groups was statistically significant (P<0.01). It can be seen that the model group rats showed typical characteristics of wound healing disorders in a diabetic environment.

[0079] Compared with the model group, the wound healing rates of the Ox group, miR group, miN group, and miN+Ox group rats on the 3rd day were 26.38%, 29.75%, 45.59%, and 53.05%, respectively; and on the 9th day, the wound healing rates were 78.35%, 86.84%, 85.94%, and 87.55%, respectively, and the wounds were close to complete healing. The wound healing rates of the four administration groups were significantly faster than those of the model group (P<0.01). P <0.01).

[0080] Compared with the model group, the wound healing rate of the NN group rats on the 9th day reached 69.77%, which may be due to the good biocompatibility and antibacterial properties of chitosan and hyaluronic acid, which can effectively maintain a moist wound environment and promote wound healing. Compared with the NN group, the wound healing rates of the miN group and miN+Ox group rats on the 3rd, 5th, 7th, and 9th days were also significantly increased (P<0.05, 0.01). It can be seen that miR, miN, and miN+Ox effectively promote wound healing. P

[0081] Further comparison of the wound healing rates of miR, miN, and Ox with miN+Ox showed that the wound healing rate of the miN+Ox group was significantly higher than that of the miR, miN, and Ox groups on the 3rd, 5th, 7th, and 9th days (P<0.05, 0.01). P <0.05, 0.01).

[0082] 2.2.4 Effect of hydrogel on wound microcirculation and temperature ​On the 3rd, 5th, 7th and 9th days of administration, the back skin was photographed and measured using an infrared camera (21 cm vertically from the wound) at fixed time points to measure microcirculation and thermal imaging. Figure 6 As shown in middle A, compared with the normal group, the wound microcirculation of the rats in the model group was significantly decreased; while compared with the model group, the wound microcirculation of the rats in the Ox group, miR group, miN group and miN+Ox group was significantly increased.

[0083] The infrared images were processed uniformly using FlirTools software, and the image temperature range was standardized to 20°C (lowest temperature) to 35°C (highest temperature). A thermal map was drawn based on the temperature values ​​obtained at the wound. Figure 6 As shown in B, compared with the normal group, the wound temperature of the rats in the model group was significantly reduced, especially in the acute phase ( P <0.01); compared with the model group, the wound temperature of rats in the Ox group, miR group, miN group, and miN+Ox group increased to a certain extent, and there were significant differences on the third day ( P <0.01). It can be seen that miR, miN and miN+Ox can significantly improve the microcirculation and back temperature of animals.

[0084] 2.2.5 Effects of hydrogel on skin pathological tissue After H&E staining, the skin pathological tissue was observed under a microscope. Figure 7 As shown in Figure A. Observation of the healed skin under a 100x microscope clearly reveals the layered structure of the skin: epidermis, dermis, and subcutaneous tissue. The epidermis, the outermost layer of the skin, is composed of keratinized stratified squamous epithelium and appears purple-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, appearing light pink on H&E-stained sections. Compared with the normal group, blue-purple non-nuclear material was observed in the wound site of the rats in the model group, indicating the infiltration of a large number of inflammatory cells. Compared with the model group, significant recovery of capillary growth and skin appendages in the wound site was observed in the rats in the Ox, miR, miN, and miN+Ox groups on day 9.

[0085] OPLENIC software was used to measure the thickness of the epidermis, dermis, and total skin. Compared with the normal group, the thickness of the epidermis and dermis of the wound skin regeneration in the model group rats were significantly reduced ( P <0.05, 0.01), indicating that the skin of diabetic wounds is difficult to regenerate. Compared with the model group, the thickness of the epidermal layer of the wound skin of rats in the miN group and miN+Ox group increased significantly ( P <0.01); compared with the NN group, the thickness of the epidermal layer of the wound surface in the miN+Ox group was significantly increased ( P<0.05). Compared with the model group, the dermal thickness of the wound surface skin of 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 thickness of the dermis layer of the wound surface of rats 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.

[0086] 2.2.6 Effect of hydrogel on CD31 and VEGF protein expression CD31, as a highly specific endothelial cell differentiation marker, can specifically bind to surface antigens on vascular endothelial cells. Under the action of the DAB colorimetric system, positively expressed vascular endothelial cells exhibit typical brown-yellow granular staining, thereby accurately presenting the spatial distribution and expression abundance of blood vessels in the dermal tissue.

[0087] like Figure 8 As shown in the results, compared with the normal group, CD31 expression in the wound skin of rats in the model and NN groups was significantly reduced, indicating that the dermal vascular network in diabetic wounds was significantly atrophied and reduced. Compared with the model and NN groups, CD31 expression and the number of blood vessels in the wound skin of rats in the miR, miN, and miN+Ox groups were significantly increased, especially in the miN+Ox group. This suggests that the miR-144 inhibitor and oxymatrine hydrogel can synergistically promote dermal angiogenesis.

[0088] Vascular endothelial growth factor (VEGF), a core cytokine that regulates angiogenesis, can significantly enhance the permeability of venules and venules by specifically activating surface receptors on endothelial cells, driving endothelial cell proliferation, migration, and lumen formation. It is currently the most active and specific angiogenic factor known. In normal skin tissue, VEGF is primarily expressed in epidermal basal cells, hair follicle stem cells, and areas of wound repair.

[0089] like Figure 9 As shown in A and C, 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), indicating that VEGF-mediated angiogenesis in diabetic wound skin was weakened. Compared with the model group, the expression of VEGF in the wound skin of rats in the miR group, miN group and miN+Ox group 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 showed that miN and miN+Ox could effectively promote dermal angiogenesis and vascular network reconstruction by upregulating VEGF expression.

[0090] 2.2.7 Effect of hydrogel on ROS content Reactive oxygen species (ROS) is a general term for a class of widely existing oxygen-derived free radicals and non-free radical substances, including superoxide anions (O2 - ), hydrogen peroxide (H2O2), hydroxyl radical (OH - ), ozone (O3) and singlet oxygen ( 1 O2), due to the presence of unpaired electrons in these substances, is highly chemically reactive. Abnormally elevated levels can cause significant damage to cellular structures. Dihydroethidium (DHE) is a commonly used fluorescent probe for detecting intracellular superoxide anion levels. DHE undergoes a dehydrogenation reaction in response to intracellular superoxide anions, producing ethidium. This product can bind to RNA or DNA, generating a red fluorescent signal.

[0091] like Figure 10 As shown in the figure, compared with the normal group, ROS expression in the wound skin of rats in the model and NN groups was significantly increased, indicating increased oxidative damage in diabetic wounds. Compared with the model and NN groups, ROS expression in the wound skin of rats in the Ox and miN+Ox groups was significantly reduced. These results suggest that miR-144 inhibitors and oxymatrine hydrogel can synergistically reduce oxidative damage in diabetic wounds.

[0092] The above is a description of the embodiments of the present invention. The above description of the disclosed embodiments will enable professionals in the field to implement or use the present invention. Various modifications to these embodiments will be apparent to professionals in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

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

2. The hydrogel according to 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 according to claim 1, wherein The loading amount of microRNA-144-5p was 10-200 pmol / mg nanoparticles.

4. The hydrogel according to 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 according to claim 1, wherein The nanoparticles are treated by high molecular weight 100 kDa ultrafiltration, and the residual amount of free nucleic acid or small molecules is ≤5 wt %.

6. The hydrogel according to claim 1, wherein The hydrogel has a gelation time of 8-10 minutes at 37° C., an equilibrium swelling rate of 4-7%, and a cumulative mass loss of ≤15% within 24 hours.

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 acetic acid aqueous solution and adjusting the pH to 5.0-6.0; b) preparing 0.5-2 mg / mL sodium tripolyphosphate solution and hyaluronic acid solution respectively; c) adding the sodium tripolyphosphate solution, the hyaluronic acid solution, and the microRNA-144-5p solution dropwise to the chitosan solution at the volume ratio while stirring at 800-1500 rpm to form nanoparticles; d) purifying the obtained nanoparticles by ultrafiltration; e) dissolving gelatin in sterile PBS, cooling to 30-37°C after complete dissolution at 50-60°C, and sequentially adding the nanoparticle suspension from step d), oxymatrine solution, and transglutaminase. After mixing, cross-linking into gel in a mold at 35-40°C and post-cooking at 2-5°C.

8. A sterile syringe filler or patch-type wound dressing for improving chronic or refractory diabetic wounds, characterized in that: The sterile syringe filler or patch-type wound dressing comprises the hydrogel according to any one of claims 1 to 6.

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

10. Use of the hydrogel according to any one of claims 1 to 6 in the preparation of a skin external preparation for simultaneously increasing the density of new blood vessels and reducing the level of reactive oxygen species.

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