Blumea balsamifera nano-particle hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to blumea balsamifera nano-particle hydrogel as well as a preparation method and application thereof. The blumea balsamifera nano-particle hydrogel is prepared from blumea balsamifera nano-particle dispersion liquid and a hydrogel matrix, wherein the blumea balsamifera nano-particle dispersion liquid is prepared from L-borneol, tween-80, glycerol and ultrapure water, and the hydrogel matrix is prepared from sapindoside, radix puerariae polysaccharide, konjac glucomannan and ultrapure water. The blumea balsamifera nano-particle hydrogel with good physicochemical properties, biological safety and multifunctional activity is successfully constructed, the hydrogel can significantly improve the healing disorder of diabetic wounds through a multi-target and multi-path synergistic effect, and the curative effect of the blumea balsamifera nano-particle hydrogel is significantly superior to that of a clinical common control drug. The blumea balsamifera nano-particle hydrogel provided by the invention has a good transformation application prospect, and provides a new strategy and an experimental basis for clinical treatment of chronic wounds of diabetes mellitus.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an artemisia nanoparticle hydrogel, its preparation method, and its application. Background Technology

[0002] Diabetic wound healing disorders pose a significant challenge to global healthcare systems. With the continued rise in diabetes prevalence, its incidence is increasing annually, becoming the leading cause of non-traumatic amputations. Unlike acute wounds, diabetic wounds possess unique pathophysiological characteristics: driven by persistent hyperglycemia, a series of pathological cascades are triggered, including chronic inflammation, excessive accumulation of reactive oxygen species (ROS), impaired angiogenesis, neuropathy, and abnormal extracellular matrix (ECM) remodeling. These pathogenic factors intertwine, forming a self-reinforcing vicious cycle that stalls the wound healing process at the inflammatory stage, ultimately leading to chronic, refractory wounds.

[0003] Currently, the clinical management of diabetic wounds mainly relies on multidisciplinary comprehensive treatment, including strict glycemic control, regular surgical debridement, revascularization, and the application of functional dressings. However, these traditional treatments primarily focus on symptom relief and supportive care, failing to fundamentally reverse the imbalance in the wound microenvironment. In recent years, with in-depth research in regenerative medicine and immunology, novel treatment methods have emerged. Among them, immunomodulation therapy shows great potential, such as reshaping the wound immune microenvironment through strategies like regulating macrophage polarization and promoting regulatory T cell (Treg) recruitment. However, these cutting-edge therapies still face key bottlenecks such as low delivery efficiency, insufficient bioavailability, and potential off-target toxicity, severely hindering their clinical translation.

[0004] Among various innovative dressing designs, hydrogels, with their unique physicochemical properties and biological functions, have become an ideal platform for the treatment of diabetic wounds. High-performance hydrogel dressings can not only maintain a moist wound environment and regulate exudate, but also serve as multifunctional carriers to achieve controlled release of therapeutic molecules. Of particular note is the ability of stimulus-responsive "smart" hydrogels to dynamically respond to specific biomarkers (such as ROS and matrix metalloproteinases) in the microenvironment of diabetic wounds, enabling on-demand drug release and significantly improving treatment precision. However, existing hydrogel systems still have inherent limitations: synthetic polymer-based hydrogels may raise biocompatibility and degradation product safety issues; natural polymer hydrogels often suffer from weak mechanical properties and limited drug loading efficiency, making it difficult to synergistically address the multiple pathological characteristics of diabetic wounds.

[0005] Based on this, the present invention has developed an innovative acetylcholine nanoparticle-hydrogel integrated system through rational molecular design and material assembly strategies. It aims to solve the problems of existing technologies, provide a novel strategy with great translational potential for the treatment of diabetic wounds, and provide an innovative paradigm for the efficient delivery and functional integration of natural bioactive ingredients. Summary of the Invention

[0006] The purpose of this invention is to provide an acetylene nanoparticle hydrogel and its preparation method.

[0007] Another object of the present invention is to provide the application of the above-mentioned acetylcholine nanoparticle hydrogel in the preparation of a drug for promoting the healing of diabetic wounds.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The Artemisia annua nanoparticle hydrogel of the present invention is made of Artemisia annua nanoparticle dispersion and hydrogel matrix, wherein: the Artemisia annua nanoparticle dispersion is composed of 0.83-5 mg / mL of L-borneol and 1%-10% of Tween-80 and 1% of glycerol by mass percentage, and the solvent is ultrapure water; the hydrogel matrix is ​​composed of 1%-2% of Sapindus mukorossi saponins, 2.5%-10% of Pueraria lobata polysaccharide and 1%-2% of konjac glucomannan by mass percentage, and the solvent is ultrapure water.

[0009] Preferably, in the Artemisia annua nanoparticle hydrogel of the present invention, the Artemisia annua nanoparticle dispersion is composed of 2.50-3.33 mg / mL of L-borneol, 1%-3% of Tween-80 and 1% of glycerol by mass, and the solvent is ultrapure water; the hydrogel matrix is ​​composed of 1.5%-2% of Sapindus mukorossi saponins, 2.5%-5% of Pueraria lobata polysaccharide and 1.5-2% of konjac glucomannan by mass, and the solvent is ultrapure water.

[0010] In a further preferred embodiment, the moxa nanoparticle hydrogel of the present invention comprises a dispersion of 3.33 mg / mL of L-borneol, 1% Tween-80, and 1% glycerol by mass percentage, with ultrapure water as the solvent; and a hydrogel matrix comprising 2% Sapindus mukorossi saponins, 2.5% Pueraria lobata polysaccharides, and 2% konjac glucomannan by mass percentage, with ultrapure water as the solvent.

[0011] The preparation method of the artemisia nanoparticle hydrogel of the present invention includes the following steps: S1. Preparation of Ai Pian nanoparticle dispersion: Oil phase preparation: Accurately weigh L-carnitine, dissolve it in dichloromethane, place it in a water bath at 25±2℃, and sonicate it to obtain a clear and transparent oil phase solution; Aqueous phase preparation: Ultrapure water, Tween-80 and glycerol were added sequentially to a sterile container and stirred thoroughly to obtain a homogeneous aqueous solution; Emulsification and evaporation: The above oil phase solution is slowly and constantly added dropwise to the continuously stirred aqueous phase solution. After the addition is completed, the system is kept at 25±2℃ and stirred to allow the dichloromethane to evaporate completely, thus obtaining the Ai Pian nanoparticle dispersion. Preparation of S2, Ai Pian nanoparticle hydrogel: Take the Artemisia annua nanoparticle dispersion prepared in the above steps and mix it with ultrapure water. Add Sapindus mukorossi saponins and stir at room temperature until the Sapindus mukorossi saponins are completely dissolved. Add Pueraria lobata polysaccharide and continue stirring at the same speed until the Pueraria lobata polysaccharide is fully dissolved, the system is uniform and there are no visible particles. Slowly and evenly sprinkle in konjac glucomannan while stirring continuously, and continue stirring to obtain Artemisia annua nanoparticle hydrogel.

[0012] Preferably, in the preparation method of the Ai Pian nanoparticle hydrogel of the present invention, the stirring speed in step S1 or step S2 is 300-500 rpm.

[0013] Preferably, in the preparation method of the Ai Pian nanoparticle hydrogel of the present invention, the ultrasonic power in the oil phase preparation in step S1 is 300W and the ultrasonic time is 5min.

[0014] Preferably, in the preparation method of the Ai Pian nanoparticle hydrogel of the present invention, the stirring time in the aqueous phase preparation in step S1 is 5-10 min.

[0015] Preferably, in the preparation method of the Ai Pian nanoparticle hydrogel of the present invention, in the emulsification and evaporation step S1: the dropping rate of the oil phase solution is 1-2 drops / second; after the dropping is completed, the stirring time is continued for 1 hour.

[0016] Preferably, in the preparation method of the Artemisia annua nanoparticle hydrogel of the present invention, in step S2: the mixing volume ratio of the Artemisia annua nanoparticle dispersion to ultrapure water is 1:4; the stirring time after adding Sapindus mukorossi saponin is 15-20 min; the stirring time after adding Pueraria lobata polysaccharide is 10-15 min; and the stirring time after adding konjac glucomannan is 20-30 min.

[0017] The application of the Ai Pian nanoparticle hydrogel described in this invention in the preparation of drugs that promote the healing of diabetic wounds.

[0018] The beneficial effects of this invention are: 1. This invention successfully constructed an arbutin nanoparticle hydrogel with excellent physicochemical properties, biosafety, and multifunctional activity. Characterization methods, including scanning electron microscopy, swelling and degradation experiments, and drug release kinetics, confirmed that the hydrogel possesses a porous, interconnected three-dimensional network structure, good water absorption, and controllable degradation behavior, and can achieve sustained release of L-borneol. Biocompatibility evaluation confirmed that the hydrogel has no significant toxicity to L929 cells, HUVEC cells, and erythrocytes, exhibiting good cell and blood compatibility. Furthermore, the hydrogel demonstrates significant antioxidant activity, effectively scavenging DPPH, ABTS, and hydroxyl radicals, and its protective effect against oxidative stress was demonstrated at the cellular level.

[0019] 2. This invention optimized the preparation process of the Artemisia annua nanoparticle dispersion through single-factor experiments, determining the optimal dosage of Tween-80 to be 1% and the optimal dosage of L-borneol to be 3.33 mg / mL. Nanoparticles prepared under these conditions exhibited uniform particle size and good stability. Orthogonal experimental design determined the optimal mass ratio of SPK hydrogel to be: Sapindus mukorossi saponins 2%, Pueraria lobata polysaccharides 2.5%, and konjac glucomannan 2%. The hydrogel prepared under these conditions possessed suitable mechanical properties, adhesion, and workability.

[0020] 3. In vivo animal experiments confirmed that the Artemisia annua nanoparticle hydrogel provided by this invention has significant healing-promoting efficacy. Macroscopic healing observation showed that compared with the model group, the positive drug Centella asiatica cream group, and the blank hydrogel group, the drug-loaded hydrogel group had the fastest wound healing speed, with a healing rate of 86.9±4.5% at 10 days post-operation, significantly better than other groups. Histopathological analysis further confirmed that the Artemisia annua nanoparticle hydrogel provided by this invention can significantly promote re-epithelialization, increase granulation tissue thickness, guide orderly deposition of collagen fibers, and significantly improve the structural repair quality of diabetic wounds. Molecular mechanism studies confirmed that this hydrogel can effectively regulate the inflammatory microenvironment of the wound, inhibit the expression of pro-inflammatory factor TNF-α, increase the levels of anti-inflammatory factor IL-10 and pro-angiogenic factor VEGF, and promote macrophage polarization from M1 type to M2 type, thereby effectively breaking the vicious cycle of inflammation in diabetic wounds and significantly promoting the transformation of the healing process from the inflammatory phase to the proliferative repair phase.

[0021] 4. The acetylcholine nanoparticle hydrogel provided by this invention can significantly improve the healing obstacles of diabetic wounds through multi-target and multi-pathway synergistic effects, and has good prospects for translational application, providing new strategies and experimental evidence for the clinical treatment of chronic diabetic wounds. Attached Figure Description

[0022] Figure 1The effect of Tween-80 dosage on the particle size and dispersibility of BONPs (in the figure: a is the effect of Tween-80 dosage on the average particle size and PDI of nanoparticles; b is a macroscopic view of different Tween-80 dosages). Figure 2 The effect of levorotatory borneol dosage on the particle size, dispersibility and stability of BO-NPs (in the figure: a) effect of levorotatory borneol dosage on the average particle size and PDI of nanoparticles; b) effect of levorotatory borneol dosage on the zeta potential of nanoparticles; c) macroscopic diagram of different concentrations of levorotatory borneol). Figure 3 The storage stability of BONPs at 25℃ is shown in the figure (a is the change in average particle size and PDI of nanoparticles at different time points; b is the macroscopic view of stability at different time points). Figure 4 The impact of various factors (SA, PPS, KGM concentration) on liquidity score; Figure 5 The effects of various factors (SA, PPS, KGM concentration) on adhesion time; Figure 6 The effects of various factors (SA, PPS, KGM concentration) on the flow area; Figure 7 Electron microscopy morphology of freeze-dried SPKgel and SPK@BONPsgel; Figure 8 The in vitro swelling properties of SPKgel and SPK@BONPsgel; Figure 9 The in vitro degradation behavior of SPKgel and SPK@BONPsgel; Figure 10 The in vitro release curve of L-borne borneol in SPK@BONPsgel; Figure 11 The effects of hydrogel extracts on the proliferation of L929 and HUVEC cells are shown in the figure (a: effect of SPKgel extract on L929 cells; b: effect of SPK@BONPsgel extract on L929 cells; c: effect of SPKgel extract on HUVEC cells; d: effect of SPK@BONPsgel extract on HUVEC cells). Figure 12 The results of live / dead staining of L929 cells after co-culturing with hydrogel extract; Figure 13 The results are for the blood compatibility (hemolysis test) of the hydrogel extract; Figure 14 Effects of SPK@BONPsgel extract on the proliferation of L929 and HUVEC cells; Figure 15The effect of hydrogel extract on the migration of L929 and HUVEC cells (in the figure: a is a microscopic image of L929 scratch test; b is a microscopic image of HUVEC scratch test). Figure 16 The free radical scavenging ability of the hydrogel extract is shown in the figure (a and d are DPPH free radical scavenging rates; b and e are ABTS free radical scavenging rates; c and f are •OH free radical scavenging rates; a-1 and d-1 are DPPH free radical UV spectra; b-1 and e-1 are ABTS free radical UV spectra; c-1 and f-1 are •OH free radical UV spectra). Figure 17 The effect of hydrogel extract on intracellular ROS scavenging (in the figure: a is the LPS-stimulated group; b is the high glucose-stimulated group). Figure 18 The protective effect of hydrogel extract on RAW264.7 cells under oxidative stress (Figure: a is the LPS stimulation group; b is the high glucose stimulation group). Figure 19 The changes in blood glucose and body weight in STZ-induced diabetic rats (in the figure: a is the trend of body weight change; b is the trend of blood glucose change). Figure 20 Macroscopic manifestations of wound healing in diabetic rats from 0 to 10 days post-surgery; Figure 21 A quantitative analysis curve of wound healing rate in diabetic rats; Figure 22 The H&E staining of wound tissues in each group on postoperative day 5 and day 10; Figure 23 Masson staining (collagen deposition) of wound tissues in each group on postoperative day 5 and day 10; Figure 24 The results show the expression of inflammatory factors in the wound (in the figure: a is IL-4, 5 days post-operation; b is IL-10, 5 days post-operation; c is TNF-α, 5 days post-operation; d is VEGF, 5 days post-operation; e is IL-4, 10 days post-operation; f is IL-10, 10 days post-operation; g is TNF-α, 10 days post-operation; h is VEGF, 10 days post-operation). Figure 25 Immunohistochemical expression results of inflammatory factors in wounds (in the figure: a is IL-10; b is TNF-α; c is VEGF; df is the quantitative expression of each group at different time points). Figure 26 Immunofluorescence results of M1 / M2 polarization of macrophages in wounds (in the figure: a is the fluorescence staining pattern; b is the quantification of CD86 (M1) cells; c is the quantification of CD206 (M2) cells). Figure 27Immunofluorescence results of α-SMA in wound angiogenesis (Figure: a is the fluorescence staining image; b is the quantitative data of α-SMA vessels). Detailed Implementation

[0023] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are for explanation and illustration only, and do not constitute a limitation on the technical solution of the present invention.

[0024] Example 1 Artemisia annua nanoparticle hydrogel is made of an artemisia annua nanoparticle dispersion and a hydrogel matrix. The artemisia annua nanoparticle dispersion consists of 3.33 mg / mL of L-borneol, 1% Tween-80 and 1% glycerol by mass percentage, and ultrapure water as the solvent. The hydrogel matrix consists of 2% Sapindus mukorossi saponins, 2.5% Pueraria lobata polysaccharide and 2% konjac glucomannan by mass percentage, and ultrapure water as the solvent.

[0025] Example 2 Artemisia annua nanoparticle hydrogel is made of an Artemisia annua nanoparticle dispersion and a hydrogel matrix. The Artemisia annua nanoparticle dispersion consists of 0.83 mg / mL of L-borneol, 3% Tween-80, and 1% glycerol by mass percentage. The hydrogel matrix consists of 1.5% Sapindus mukorossi saponins, 5% Pueraria lobata polysaccharide, and 1.5% konjac glucomannan by mass percentage.

[0026] Example 3 Artemisia annua nanoparticle hydrogel is made of an Artemisia annua nanoparticle dispersion and a hydrogel matrix. The Artemisia annua nanoparticle dispersion consists of 5 mg / mL of L-borneol, 10% Tween-80, and 1% glycerol by mass percentage. The hydrogel matrix consists of 1% Sapindus mukorossi saponins, 10% Pueraria lobata polysaccharides, and 1% konjac glucomannan by mass percentage.

[0027] Example 4 The preparation of the Ai Pian nanoparticle hydrogel using the formulation of Example 1 is as follows: (1) Preparation of Artemisia annua nanoparticle dispersion Oil phase preparation: Accurately weigh 100 mg of L-borneol, dissolve it in 1 mL of dichloromethane, place it in a water bath at 25±2℃, and sonicate for 5 min to obtain a clear and transparent oil phase solution.

[0028] Aqueous phase preparation: In a 10 mL sterile vial, add 2940 μL of ultrapure water, 30 μL of Tween-80, and 30 μL of glycerol in sequence; fix the vial on a magnetic stirrer and stir at 300-500 rpm for 5-10 min to obtain a homogeneous aqueous phase system.

[0029] Emulsification and evaporation: Using a micro-injection pump, 100 μL of the above oil phase solution was added dropwise to the continuously stirred aqueous phase at a rate of 1-2 drops / second. After the addition was completed, the system was continuously stirred at 25±2℃ and 300-500 rpm for 1 h to allow the dichloromethane to evaporate completely, resulting in a dispersion of 3.33 mg / mL of borneol nanoparticles.

[0030] (2) Preparation of Artemisia nanoparticle hydrogel Take 2 mL of the prepared Artemisia annua nanoparticle dispersion and 8 mL of ultrapure water, and mix them evenly in a 50 mL beaker; add 0.2 g of Sapindus mukorossi saponin to the mixture, and stir at room temperature and 300-500 rpm for 15-20 min until the Sapindus mukorossi saponin is completely dissolved and the solution is clear; add 0.25 g of Pueraria lobata polysaccharide, and continue stirring at the same speed for 10-15 min until the Pueraria lobata polysaccharide is fully dissolved, the system is uniform and there are no visible particles; under continuous stirring, slowly and evenly sprinkle in 0.2 g of konjac glucomannan, and stir at 300-500 rpm for 20-30 min to obtain Artemisia annua nanoparticle hydrogel.

[0031] Example 5 The acetylcholine nanoparticle hydrogel prepared in Example 4 was applied to the treatment of diabetic wounds.

[0032] To further verify the reliability of the present invention and select the optimal solution, the inventors conducted a series of experiments, as follows: I. Preparation and Characterization of Artemisia Nanoparticle Hydrogel 1. Experimental Methods 1.1 Preparation of Artemisia annua nanoparticle dispersion The dispersion of Ai Pian nanoparticles was prepared using a modified emulsification-solvent evaporation method, and the steps are as follows: Oil phase preparation: Accurately weigh 100 mg of purified L-carnitine, dissolve it in 1 mL of dichloromethane, place it in a water bath at 25±2℃, and sonicate it for 5 min until a clear and transparent oil phase solution is obtained, resulting in a L-carnitine stock solution with a concentration of 100 mg / mL. Seal and store at room temperature for later use.

[0033] Aqueous phase preparation: In a 10 mL sterile vial, add 2940 μL of ultrapure water, 30 μL of Tween 80, and 30 μL of glycerol sequentially. Fix the vial on a magnetic stirrer and stir at 300-500 rpm for 5-10 min to fully disperse the surfactant and co-surfactant in the water and form a homogeneous aqueous phase system.

[0034] Emulsification and evaporation: Using a micro-injection pump, the above oil phase solution was slowly and constantly added to the continuously stirred aqueous phase at a constant rate (approximately 1-2 drops / second). After the addition was complete, the system was continuously stirred at 25±2℃ and 300-500 rpm for 1 h to ensure complete evaporation of the organic solvent dichloromethane. Finally, a uniform, semi-transparent or opalescent dispersion of Ai Pian nanoparticles was obtained. This dispersion was then stored in a sealed container at 4℃ for later use.

[0035] To optimize the stability, particle size distribution, and drug loading of the acetaminophen nanoparticles, the research team conducted the following single-factor and stability studies: (1) Investigation on the dosage of Tween-80 To optimize the stability and particle size distribution of the nanoparticles, a fixed amount of 100 mg / mL loratadine stock solution (50 μL) and glycerol (1%) was used, and the dosage gradient of Tween-80 was set as follows: 1%, 3%, 5%, 7%, and 10%. Three batches of nanoparticle dispersions were prepared in parallel according to the above method. After preparation, the average hydrodynamic particle size (Z-Average) and polydispersity index (PDI) were immediately determined using a dynamic light scattering particle size analyzer. The formulation with smaller and more uniform particle size distribution (PDI < 0.3) was selected as the optimal dosage of Tween-80.

[0036] (2) Drug loading investigation To investigate the effect of drug loading on nanoparticle properties, the optimized amounts of Tween-80 and glycerol were fixed, and the feed amounts of 100 mg / mL levonorgestrel stock solution were set at 25 μL, 50 μL, 75 μL, 100 μL, and 150 μL. Nanoparticle dispersions were prepared in parallel, and changes in particle size, PDI, and Zeta potential were measured.

[0037] (3) Stability test The nanoparticle dispersion prepared according to the optimal formulation was dispensed into transparent glass bottles and stored at 4°C (refrigerated) and 25°C (room temperature), respectively. Samples were taken on days 0, 7, 14, 21, and 28 of storage to observe changes in appearance (e.g., precipitation, flocculation, or stratification), and changes in particle size and PDI were measured. The short- and medium-term storage stability of the nanoparticle dispersion was evaluated by monitoring the trends of these key physical parameters over time.

[0038] 1.2 Preparation of SPK hydrogel SPK composite hydrogels, composed of Sapindus mukorossi saponins (SA), Pueraria lobata polysaccharides (PPS), and konjac glucomannan (KGM), were constructed based on all-natural components using a sequential mixing and stirring method. During the matrix solution premixing stage, when preparing SPK@BO NPsgel, 2 mL of the optimized BO NPs dispersion was mixed thoroughly with 8 mL of ultrapure water in a 50 mL beaker; when preparing the blank hydrogel (SPK gel), 10 mL of ultrapure water was used directly. During the sequential addition of polymer components, 0.2 g of Sapindus mukorossi saponin (SA) was first added to the mixture. The beaker was placed on a magnetic stirrer and stirred at 300-500 rpm for 15-20 min at room temperature until the SA was completely dissolved and the solution was clear. Then, 0.25 g of Pueraria lobata polysaccharide (PPS) was added, and stirring was continued at the same speed for 10-15 min until the PPS was fully dissolved and the system was homogeneous with no visible particles. Finally, 0.2 g of konjac glucomannan (KGM) was slowly and evenly sprinkled in while stirring continuously. After the addition was complete, stirring was continued at 300-500 rpm for 20-30 min. The viscosity of the system was observed to gradually increase, eventually forming a homogeneous, transparent gel with a three-dimensional network structure, namely SPK@BO NPs gel. The prepared hydrogel was transferred to a sample bottle and sealed and stored at 4℃ for later use.

[0039] To obtain a hydrogel formulation that combines suitable mechanical properties, adhesion, and spreadability, this study used L9(3) 4 Orthogonal experimental design was used to systematically optimize the composition of the composite hydrogel. The concentrations of Sapindus mukorossi saponins, Pueraria lobata polysaccharides, and konjac glucomannan were used as three evaluation factors, with three levels for each factor, as shown in Table 1. Flowability score, adhesion time, and flow area were selected as key evaluation indicators to comprehensively reflect the hydrogel's operability, adhesion properties, and mechanical strength.

[0040] .

[0041] 1.3 Characterization of hydrogels (1) Observation by scanning electron microscope (SEM) Scanning electron microscopy (SEM) was used to observe the cross-sectional micromorphology of SPK gel and SPK@BO NPs gel: Fresh samples were quick-frozen in liquid nitrogen for 10 min and then freeze-dried at -50℃ and <10 Pa for 48 h to remove moisture. Fresh cross-sections of the freeze-dried samples were broken off and attached to conductive adhesive. After sputtering a ~10 nm gold film, the morphology was observed and recorded by SEM under an accelerating voltage of 5 kV.

[0042] (2) Swelling performance test The water absorption properties of hydrogels were systematically evaluated using a gravimetric method. Specifically, three sets of hydrogel samples of uniform size were prepared in parallel, and their initial weights were weighed and recorded as W0. Subsequently, the samples were completely immersed in phosphate-buffered saline (PBS) at a constant temperature of 37°C, ensuring sufficient solution volume. At predetermined time points, the hydrogel samples were carefully removed using sterile forceps, and residual liquid on the surface was gently blotted with absorbent paper. Their weights were immediately weighed and recorded as W0. t Repeat the above steps until the hydrogel reaches water absorption equilibrium. Calculate the equilibrium swelling ratio (ESR) of the hydrogel using the following formula: ; In the formula: W0 is the initial weight of the hydrogel, W t This represents the weight of the hydrogel after expansion equilibrium.

[0043] (3) Degradation performance test The degradation performance was evaluated using an in vitro immersion method. Lyophilized hydrogel cylinder samples with a known dry weight (W0) were immersed in centrifuge tubes containing PBS (pH=7.4) and incubated in a constant temperature shaker at 37℃ and 100 rpm. Fresh PBS was replaced every 24 h to maintain the medium conditions. At specified time points, the samples were removed, the surface was gently rinsed with ultrapure water, and after re-lyophilization, the remaining dry weight (W0) was measured. t Three parallel samples were set up at each time point. The degradation rate (DR) was calculated using the following formula: ; In the formula: W0 is the initial weight of the hydrogel, W t This represents the weight of the hydrogel after degradation equilibrium.

[0044] (4) Drug release kinetics detection Drug release kinetics were assessed using the dialysis bag method. First, a standard curve for levonorgestrel was established via GC. A 10 mg / mL levonorgestrel stock solution was serially diluted with ethyl acetate, and a linear regression of peak area against concentration was performed to obtain the standard curve equation. In the release study, 1.0 g of hydrogel was accurately weighed and placed in a pre-treated dialysis bag, then immersed in 50 mL of PBS release medium containing 0.5% Tween-80, and stirred at a constant speed of 100 rpm at 37°C. At predetermined time intervals, 1 mL of release medium was aspirated and replenished with an equal volume of fresh medium. The collected samples were filtered through a 0.22 μm filter membrane, and the levonorgestrel concentration was determined by GC. The cumulative release percentage was calculated using the formula: ; In the formula: C n and C xThis represents the drug concentration in the supernatant obtained from time n and x.

[0045] 1.4 Biocompatibility assessment of hydrogels (1) Cell culture and treatment RAW 264.7 and HUVEC cells were cultured in high-glucose DMEM medium containing 10% FBS and 1% penicillin-streptomycin, while L929 cells were cultured in RPMI-1640 medium containing the same proportion of FBS and antibiotics. All cells were routinely cultured in a 37°C, 5% CO2 saturated humidity incubator, and passaged periodically, with logarithmic growth phase cells used for experiments. SPK gel and SPK@BO NPs gel were dispensed at 1 cm intervals. 2 Immerse the extract in serum-free culture medium at a ratio of / mL, and extract in a shaker at 37℃ for 24 h. After sterilization by filtration through a 0.22 μm filter membrane, the extract is diluted with complete culture medium to concentrations of 25%, 50%, 75%, and 100% for later use.

[0046] (2) Cell compatibility test Cell compatibility was evaluated using the CCK-8 assay and live / dead staining. For the CCK-8 assay, L929 and HUVEC cells were prepared at a ratio of 5 × 10⁶ cells / mL. 3 Cells were seeded per well in 96-well plates and pre-cultured for 24 h until adherence. The medium was then changed, and complete culture medium containing 25%, 50%, 75%, and 100% hydrogel extract was added to each well. Cell-free wells served as blank controls. After culturing for 24, 48, and 72 h, 10 μL CCK-8 solution was added to each well and incubated for 1–2 h. Absorbance was measured at 450 nm. Relative Growth Rate (RGR) was calculated using the following formula: ; In the live / dead staining method, L929 cells were seeded at an appropriate density in 24-well plates and cultured for 24 h. The culture medium was then replaced with a medium containing 50% hydrogel extract and cultured for another 48 h. The medium was then discarded, and the cells were gently washed once with PBS. A PBS staining working solution containing 2 μM calcein AM and 4 μM propidium iodide was added, and the cells were incubated at 37°C in the dark for 30 min. After washing with PBS, the cells were immediately observed and photographed under an inverted fluorescence microscope.

[0047] (3) Blood compatibility test (hemolysis test) Hemolysis assay to evaluate blood compatibility: Fresh blood from healthy SD rats was anticoagulated with heparin sodium and centrifuged at 1500 rpm for 10 min. The supernatant and pale yellow layer were discarded, and red blood cells were collected and washed three times with PBS. The cells were resuspended to prepare a 5% (v / v) red blood cell suspension. 800 μL of hydrogel extract at different concentrations (diluted with PBS) was added to a centrifuge tube, and 200 μL of the red blood cell suspension was added and mixed well. PBS was used as a negative control, and deionized water was used as a positive control. The mixture was incubated at 37℃ for 1 h. After incubation, the cells were centrifuged at 3000 rpm for 5 min, and 200 μL of the supernatant was transferred to a 96-well plate. The absorbance was measured at 540 nm. The hemolysis rate (HR) was calculated using the following formula: ; In the formula: A b A represents the absorbance value of the supernatant of the hydrogel-treated solution. c A represents the absorbance value of the hydrogel extract in PBS solution (0.01M, pH 7.4). w A is the absorbance value of the supernatant of a deionized water treatment solution. r The absorbance value is the supernatant of the solution treated with PBS.

[0048] 1.5 Assessment of HUVEC cell proliferation and migration capacity (1) Cell proliferation assay (CCK-8 assay) The effect of hydrogel on the proliferation of L929 and HUVEC cells was detected by the CCK-8 assay, and the procedure was the same as the cytotoxicity assay described above.

[0049] (2) Cell migration test (scratch test) Scratch assay for cell migration: HUVEC cells were prepared at 8 × 10⁻⁶ cm⁻¹. 4 Cells were seeded per well in 24-well plates and cultured until confluence >90% to form a dense monolayer. A sterile 200 μL pipette tip was used to make a straight incision in the center of the monolayer, perpendicular to the bottom of each well. Cells were washed twice with PBS to remove detached cells. The medium was changed to a medium containing 2% FBS and supplemented with 50% SPK gel or SPK@BO NPs gel extract; the control group received medium containing only 2% FBS. Images were taken at the same location under an inverted microscope at 0 h, 24 h, and 36 h post-incision. The incision area (A) was measured using ImageJ software. t The migration rate was calculated to assess the cell migration-promoting effect of the hydrogel. Cell migration rate (MR) was calculated using the following formula: ; In the formula: A0 represents the initial scratch area, A t This indicates the area of ​​the scratch at the corresponding time point.

[0050] 1.6 Antioxidant Performance Evaluation (1) DPPH free radical scavenging ability test DPPH free radical scavenging capacity assay: A 0.2 mM DPPH working solution was prepared with anhydrous ethanol. 100 μL of hydrogel extracts (prepared with PBS) of different concentrations (25%, 50%, 75%, 100%) were mixed with an equal volume of DPPH solution in a 96-well plate. The mixture was incubated at room temperature in the dark for 30 min, centrifuged at 8000 r / min for 5 min, and the supernatant was measured at 517 nm (A1). Anhydrous ethanol was used as the blank group (A2) instead of the DPPH solution, and distilled water was used as the control group (A0) instead of the sample solution. Free radical scavenging rate R0 DPPH The calculation is as follows: ; (2) ABTS free radical scavenging ability test ABTS free radical scavenging capacity assay: A stock solution was prepared by mixing 7 mmol / L ABTS reagent with an equal volume of 2.45 mmol / L potassium persulfate solution and reacting at 4℃ in the dark for 12–16 h. The stock solution was diluted to an absorbance of 0.7 ± 0.02 at 734 nm to obtain the working solution. 1 mL of sample was mixed with 3 mL of working solution and reacted at 37℃ in the dark for 6 min. The absorbance at 734 nm was measured (A1). Distilled water was used as a blank group (A2) to replace the sample. Free radical scavenging rate R ABTS The calculation is as follows: ; (3) Hydroxyl radical scavenging ability test The hydroxyl radical scavenging capacity was tested using the salicylic acid capture method. 1 mL of sample was added to 0.5 mL of 10 mmol / L salicylic acid solution, 0.5 mL of 10 mmol / L ferrous sulfate solution, and 0.5 mL of 8.8 mmol / L hydrogen peroxide solution. The absorbance was measured at 510 nm (A1). Distilled water was used instead of salicylic acid solution as the blank group (A2), and an equal volume of distilled water was used instead of the sample solution as the control group (A0). The free radical scavenging rate R... •OH The calculation is as follows: ; (4) Intracellular ROS scavenging capacity test (DCFH-DA probe) DCFH-DA probe detection of intracellular ROS scavenging ability: RAW264.7 cells were seeded in 96-well plates, and after adhesion, the medium was changed to serum-free medium, 10 μM DCFH-DA was added, and the cells were incubated at 37℃ in the dark for 30 min, followed by washing three times with PBS. Cells were divided into a normal control group, a model group (LPS / high glucose), and a treatment group (LPS / high glucose + 50% SPK@BO NPs gel extract) and cultured for 6 h. Fluorescence intensity was measured in 96-well plates using a fluorescence microplate reader (485 nm excitation / 525 nm emission); images were taken in 24-well plates using a fluorescence microscope, and quantification was performed using ImageJ. Differences between groups were compared to assess ROS scavenging effect.

[0051] (5) Cell protection experiment under oxidative stress Oxidative stress cell protection experiment: RAW 264.7 cells were injected at a dose of 1×10⁻⁶. 4 Cells were seeded per well in 96-well plates and cultured for 24 h, with the medium changed afterward. Five groups were established: normal control, LPS stimulation (1 μg / mL), high glucose stimulation (50 mM), LPS + 50% extract, and high glucose + 50% extract, with six replicates per group. Cell viability was measured using the CCK-8 assay after 12 h of culture. The differences between the stimulation groups and the co-treatment groups were compared to evaluate the cell-protective effect of the hydrogel extract.

[0052] 2. Results and Discussion 2.1 Preparation and Characterization of BO NPs The effect of Tween-80 dosage on the particle size and dispersibility of BO NPs is as follows: Figure 1 As shown in the figure. Based on the single-factor optimization experiment, the amount of Tween-80 in the range of 1% to 10% had no significant effect on the particle size of BO-NPs (particle size fluctuated between 9.40 and 12.64 nm), while the PDI gradually decreased with increasing amount (dispersion uniformity gradually improved). Although a high amount of Tween-80 can improve dispersibility, based on the toxicity of Tween-80 and the formulation principle of "appropriate amount and as low as possible", and the small difference in particle size under different amounts, 1% Tween-80 was finally selected as the optimal preparation condition.

[0053] This study investigated the effects of levorotatory borneol dosage (corresponding to theoretical concentrations of 0.83-5 mg / mL) on the particle size, dispersibility, and stability of BO-NPs. The results are as follows: Figure 2As shown in the results, within the range of 0.83-3.33 mg / mL, the average particle size of BO-NPs remained stable, with polydispersity index (PDI) below 0.3, indicating uniform particle distribution. When the feed concentration increased to 5 mg / mL, the particle size significantly increased, the system became turbid, and the dispersion uniformity decreased. Actual experimental data showed no significant difference in the absolute value of the Zeta potential among the different feed concentration groups, indicating no significant difference in stability. Considering the overall particle size performance (suitable and stable particle size within the 0.83-3.33 mg / mL range, with an abnormal increase in particle size at 5 mg / mL), the optimal concentration of levonorgestrel was ultimately determined to be 3.33 mg / mL.

[0054] Storage stability of BO NPs at 25°C as follows Figure 3 As shown in the figure, BO-NPs exhibited good physical stability during a 28-day stability assessment. Throughout this period, the average particle size remained within the range of 10.2-11.0 nm, and the polydispersity index (PDI) was consistently below 0.3, indicating uniform particle dispersion and no significant aggregation. This confirms that the system possesses sufficient electrostatic repulsion to maintain colloidal stability. Visually, the samples remained translucent and opalescent, without precipitation, stratification, or flocculation. This stability is primarily attributed to the small size effect of the nanoparticles, their high surface charge, and the effective encapsulation and protection of the drug by the mixed micelles formed by Tween-80 and glycerol. In conclusion, BO-NPs demonstrate good short-term storage and transport stability at room temperature, meeting the basic requirements for subsequent formulation processing and application.

[0055] 2.2 Preparation and Characterization of SPK@BO NPs Hydrogel (1) Optimization of hydrogel preparation process and gelation conditions The results of the orthogonal experiment for hydrogel optimization are shown in Table 2. The results of the key performance analysis and optimal formulation screening of hydrogel are shown in Table 3. The effects of each factor on flowability score, adhesion time and flow area are shown in Table 3. Figure 4-6 As shown.

[0056] SPK composite hydrogels were successfully prepared by sequentially mixing three natural polysaccharides: SA, PPS, and KGM. During stirring, the viscosity of the system gradually increased, eventually forming a self-supporting transparent gel. To obtain an optimal formulation with suitable mechanical properties, adhesion, and flowability, this study used L9(3) 4An orthogonal experimental design was used, with the concentrations of SA, PPS, and KGM as three factors, each with three levels. Flowability score, adhesion time, and flow area were used as key evaluation indicators. Higher adhesion scores and adhesion times indicated stronger adhesion of the hydrogel, while lower flow areas indicated better mechanical properties. Intuitive analysis and analysis of variance showed that the order of influence of each factor on the hydrogel's performance was: KGM > PPS > SA. Considering all factors, to obtain strong adhesion (high adhesion score and time) and moderate mechanical properties (lower flow area), the optimal mass percentage composition of the SPK hydrogel was determined to be: SA 2%, PPS 2.5%, and KGM 2% (i.e., the SAL3 / PPSL1 / KGML3 combination). The hydrogel prepared by this formulation is uniform and transparent in appearance, possesses suitable spreadability and in vivo retention capacity, and meets the basic application requirements for wound dressings.

[0057] ; .

[0058] (2) Microscopic morphology analysis Figure 7 The electron microscopy images show the morphology of the lyophilized SPK gel and SPK@BO NPs gel. The results indicate that both exhibit a porous, interconnected three-dimensional network structure, facilitating oxygen and nutrient exchange, cell migration, and blood vessel ingrowth. Compared to the blank hydrogel, the drug-loaded hydrogel has a denser framework and slightly smaller pores, possibly due to the influence of the nitrocellulose nanoparticles on polymer chain interactions. No obvious nanoparticle aggregation was observed under high magnification, suggesting uniform dispersion. This structure provides a good foundation for hydrogel swelling, drug release, and cell infiltration.

[0059] (3) Swelling and Degradability Analysis Figure 8 The in vitro swelling properties of SPK gel and SPK@BO NPs gel were compared. Results showed that SPK@BO NPs gel exhibited superior swelling performance compared to the blank gel. Within 24 hours, the swelling ratio of both groups increased over time, with the drug-loaded group showing a higher swelling rate at all time points, reaching an equilibrium swelling ratio of 48.3% (compared to 43.0% in the blank group). BO-NPs did not inhibit the hydrophilicity of the gel; instead, they enhanced its water absorption capacity, which is beneficial for wound exudate management.

[0060] Figure 9The in vitro degradation behavior of SPK gel and SPK@BO NPs gel was investigated. Results showed that both gels gradually degraded over time in PBS; the degradation rate of the blank gel was 139.4% on day 1 and decreased to 15.2% on day 7, while the degradation rates of the drug-loaded gel were 135.8% and 4.8% during the same period. The degradation originated from polymer chain hydrolysis and physical cross-linking relaxation. Its controllable and slow degradation characteristics are well-suited to a 1-2 week wound healing cycle, providing a stable barrier and drug release platform, and eliminating the need for secondary removal, thus meeting the requirements of an ideal dressing.

[0061] (4) Drug release kinetics The in vitro release curve of L-borneol in SPK@BO NPs gel is as follows: Figure 10 As shown, its release exhibits a typical two-phase characteristic: a burst release of the drug from 0 to 12 hours, with a cumulative release rate of 40.1% (originating from rapid release from the gel surface / near-surface nanoparticles); and a transition to sustained release from 12 to 48 hours, with a cumulative release rate reaching 82.6% after 48 hours. The release curve conforms to the Higuchi model (R²>0.98), with Fickian diffusion being the dominant mode. This "rapid-then-slow" pattern can quickly establish an effective drug concentration and maintain a therapeutic level, meeting the long-term healing needs of diabetic wounds.

[0062] 2.3 Biocompatibility assessment (1) Cell compatibility results Effects of hydrogel extract on the proliferation of L929 and HUVEC cells, such as Figure 11 As shown in the figure. The results showed that, according to the CCK-8 assay, after L929 fibroblasts and HUVEC endothelial cells were co-cultured with different concentrations (25%-100%) of SPK gel and SPK@BONPs hydrogel extracts for 1-3 days, the relative cell growth rate (RGR) was higher than 80%, and some groups (especially the low concentration group) even exceeded 100%, showing a certain proliferative trend. According to the ISO 10993-5 standard, it can be considered that the two hydrogel extracts have no cytotoxicity to these two cell types.

[0063] The results of live / dead staining of L929 cells after co-culturing with hydrogel extract are as follows: Figure 12 As shown, the results of double staining of live / dead cells provide direct evidence: after co-culturing with 50% extract for 48 h, the vast majority of L929 cells in the field of view showed bright green fluorescence (live cells), with only a very few scattered red fluorescent spots (dead cells), showing no significant difference from the negative control group. This indicates that the hydrogel and its extract have no adverse effects on cell membrane integrity and cell metabolic activity, and have good cell compatibility, which is closely related to the fact that the hydrogel is composed entirely of natural polysaccharides.

[0064] (2) Blood compatibility results The results of the hemocompatibility (hemolysis test) of the hydrogel extract are as follows: Figure 13 As shown in the figure. The hemolysis test is a key indicator for evaluating the safety of biomaterials in contact with blood. The results showed that the color of the red blood cell supernatant after treatment with different concentrations of SPK hydrogel extract was similar to that of the negative control (PBS) group, appearing pale yellow, while the positive control (deionized water) group appeared dark red. Quantitative determination showed that the hemolysis rate of all extract groups was below the 5% biomaterial safety threshold (ASTM F756), indicating that SPK hydrogel extract does not cause significant red blood cell rupture and has good blood compatibility, providing a safety guarantee for its use in wound dressings and avoiding the risk of bleeding or thrombosis.

[0065] 2.4 HUVEC cell proliferation and migration ability (1) Cell proliferation promotion effect The effects of SPK@BO NPs gel extract on the proliferation of HUVEC and L929 cells are as follows: Figure 14 As shown in the figure. The results showed that the 50% concentration of the extract had a statistically significant regulatory effect on the proliferation activity of both cell types. Compared with the control group, the cell proliferation activity of the gel group was significantly different after 48 h and 72 h of culture. This phenomenon may be related to the regulation of cell signaling pathways by the sustained-release polysaccharides and active ingredients such as L-carnitine released by the gel. These results indicate that SPK@BO NPs gel is a non-biologically inert material, and its local application can actively regulate cell behavior, providing in vitro experimental evidence for elucidating the mechanism of promoting healing and optimizing biocompatibility.

[0066] (2) Cell migration promotion effect Effects of hydrogel extract on the migration of L929 and HUVEC cells, such as Figure 15 As shown in the figure, both 50% SPK gel and SPK@BO NPs gel extract significantly promoted HUVEC migration, with the latter exhibiting a more pronounced migration-promoting effect and a larger scratch closure area. This indicates that the hydrogel matrix possesses migration-promoting capabilities, and loading SPK@BO NPs nanoparticles can enhance this effect. Endothelial cell migration is a key step in angiogenesis, suggesting that an appropriate concentration of SPK@BO NPs gel has the potential to promote angiogenesis, providing experimental evidence for its application in diabetic wound repair.

[0067] 2.5 Antioxidant properties (1) Scavenging ability of DPPH, ABTS, and •OH free radicals The scavenging ability of hydrogel extracts on free radicals, such as Figure 16As shown in the figure. Evaluations using DPPH, ABTS, and •OH scavenging experiments revealed that the SPK@BO NPs gel extract exhibited concentration-dependent scavenging activity against all three free radicals, with scavenging rates of 78.1±1.5%, 81.1±0.2%, and 81.8±1.1% at 100% concentration, respectively. The blank SPK gel extract, due to the inherent scavenging ability of its natural components, showed significantly enhanced scavenging ability after loading with L-borneol (p<0.01). The two components exhibited a synergistic antioxidant effect. This composite system provides a more reliable molecular basis for neutralizing excessive reactive oxygen species (ROS) accumulated in diabetic wounds through a dual antioxidant mechanism.

[0068] (2) Intracellular ROS scavenging effect The scavenging effect of hydrogel extract on intracellular ROS is as follows: Figure 17 As shown, a RAW264.7 macrophage oxidative stress model was constructed using LPS / high glucose. DCFH-DA probe detection revealed a significant upregulation of green fluorescence intensity in the model group cells, indicating a dramatic increase in ROS levels. After co-treatment with SPK@BO NPs gel extract, the fluorescence signal was strongly suppressed (p<0.001), approaching normal levels. These results confirm that this hydrogel extract can penetrate the cell membrane, efficiently remove excess ROS induced by exogenous stimuli, and exert a protective effect against oxidative damage to cells.

[0069] (3) Cell protection under oxidative stress The protective effect of hydrogel extract on RAW264.7 cells under oxidative stress, such as Figure 18 As shown in the figure, a cellular inflammation / metabolic oxidative stress model was constructed using LPS (1 μg / mL) and high glucose (50 mM). CCK-8 assay showed that the extract significantly improved cell viability within 12 h (p<0.001), and there was no statistically significant difference in cell viability recovery compared to the blank SPK gel group. These results confirm that the antioxidant stress protective effect of SPK@BO-NPs gel stems from the synergistic effect of matrix polysaccharides and saponins, providing experimental support for its application in pathological microenvironments such as diabetic wounds.

[0070] II. Application of hydrogels in wound healing in diabetic rats 1. Materials and Methods 1.1 Laboratory Animals Healthy female SD rats (180-220 g) were provided by the Animal Experiment Center of Guizhou University of Traditional Chinese Medicine. They were housed separately in SPF-grade animal rooms with free access to food and water. The environment was maintained at 25±2℃ and 50±10% humidity, with a 12-hour light-dark cycle. They were acclimatized for one week. The experimental procedures strictly followed the protocols of the university's Animal Experiment Ethics Committee and the "3R" principle.

[0071] 1.2 Establishment of a Diabetic Animal Model STZ intraperitoneal injection to induce a type 1 diabetes model: Rats were fasted for 12 h and weighed. STZ was dissolved in pre-cooled 0.1M sodium citrate buffer (pH 4.5) in a light-protected ice bath to prepare a 2% solution (20 mg / mL), which was then injected intraperitoneally at a dose of 60 mg / kg. After injection, the rats were fed a full diet, and fasting blood glucose was measured via the tail vein 72 h later. Rats with two consecutive FBG ≥16.7 mmol / L and exhibiting the "three highs and one low" symptoms (polyuria, polydipsia, polyphagia, and weight loss) were considered to have successfully established the model. After one week of stable rearing, the model rats were used for wound modeling.

[0072] 1.3 Skin wound modeling and treatment plan Diabetic rats were randomly divided into four groups (n=6 per group): model group (wound covered with PBS gauze), positive control group (Centella asiatica extract cream), blank hydrogel group (SPK gel), and drug-loaded hydrogel group (SPK@BO NPs gel). After anesthesia, the rats' back hair was shaved, and the wounds were disinfected three times alternately with iodine and ethanol. Four 10 mm full-thickness skin defects (deep to the fascia) were created on both sides of the spine on the back, and hemostasis was achieved. 0.5 mL of the drug / hydrogel was applied to each group to cover the wound and its edges, and the wounds were secured with Vaseline gauze and adhesive tape. Rats were housed individually post-surgery, with dressings changed every two days. Post-surgery, photos were taken at a fixed distance under light on days 0, 2, 4, 6, 8, and 10, with the wound cleaned before each photo.

[0073] 1.4 Skin Sample Collection At predetermined time points, rats were euthanized and dissected to obtain regenerated dorsal skin tissue for pathological evaluation. The obtained tissue samples were immediately fixed in 4% paraformaldehyde solution for 24 hours. After fixation, tissue sections were prepared, and histological analysis was performed using various staining methods: hematoxylin-eosin (H&E) staining was used to assess tissue morphology, and Masson staining was used to observe collagen deposition; immunohistochemical staining was used to detect platelet endothelial cell adhesion molecule (VEGF) to assess angiogenesis, and tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were detected to analyze inflammatory responses; simultaneously, CD206 (a marker for M2 macrophages) and CD86 (a marker for M1 macrophages) staining were used to characterize macrophage polarization.

[0074] 2. Results and Discussion 2.1 Establishment of a diabetic rat model Figure 19This study demonstrates the changes in blood glucose and body weight in STZ-induced diabetic rats. 72 h after modeling, fasting blood glucose levels in rats increased from 5.0 ± 0.5 mmol / L to 24.1 ± 3.7 mmol / L (p < 0.001), and remained above 16.7 mmol / L; this was accompanied by typical symptoms of polydipsia, polyuria, polyphagia, and stunted / decreased weight gain. This model successfully simulates the pathological state of impaired wound healing in diabetic rats, providing a reliable zoological basis for subsequent experiments.

[0075] 2.2 Macroscopic assessment of wound healing in diabetic rats Macroscopic manifestations of wound healing in diabetic rats 0-10 days post-surgery are as follows: Figure 20 As shown in the figure, the healing efficacy varied significantly among the groups. The Control group exhibited severely delayed healing, with slow wound contraction and abnormal scab formation, consistent with the pathological characteristics of chronic inflammation and stagnant healing in diabetic wounds. The CTC group showed better healing than the model group, but the improvement was limited, suggesting that Centella asiatica cream ointment is ill-suited to the complex pathological microenvironment of diabetic wounds. The SPK gel group showed better healing than the positive control group; its matrix-constructed moist barrier reduced wound dehydration, inhibited bacterial growth, and promoted granulation tissue and epithelial migration. The SPK@BO NPs gel group showed the best healing efficacy, with the wound essentially closed 10 days post-surgery and exhibiting vigorous granulation tissue growth, directly demonstrating the synergistic therapeutic value of the acetic acid nanoparticles and the natural polysaccharide matrix.

[0076] Quantitative analysis curve of wound healing rate in diabetic rats as shown in the figure Figure 21 As shown in the figure, at 10 days post-surgery, the healing rate was only 51.9±4.7% in the Control group, 67.4±3.9% in the CTC group, 80.2±3.5% in the SPK gel group, and 86.9±4.5% in the SPK@BO NPs gel group (p<0.001 vs Control group), showing a statistically significant difference compared to the other three groups. Post-operative measurements at time points 2-8 days showed that the healing rate in the SPK@BO NPs gel group was significantly higher than that in other groups (p<0.05 or p<0.01), with the difference becoming more pronounced from day 4 onwards. These results confirm that SPK@BO NPs gel is significantly more effective than Centella Asiatica extract cream and blank hydrogel in promoting the healing of diabetic wounds, providing quantitative support for its clinical translation.

[0077] 2.3 H&E staining to evaluate wound reepithelialization in rats Figure 22The H&E staining results of wound tissues in each group at 5 and 10 days postoperatively are shown. At 5 days postoperatively, the Control group showed significant wound defects with a large amount of inflammatory exudate and necrotic tissue, weak re-epithelialization, sparse granulation tissue, and healing stalled in the inflammatory phase. The CTC group showed slightly improved re-epithelialization and granulation tissue thickness, reduced inflammatory infiltration, but still significant exudate, and limited repair benefit from positive drugs. The SPK gel group showed significantly prolonged re-epithelialization length, thickened new epithelium, and denser granulation tissue. Its moist barrier could drive keratinocyte migration and proliferation, accelerating epithelial reconstruction and matrix synthesis. The SPK@BO NPs gel group showed the best performance, with the fastest re-epithelialization process, intact new epithelial structure, regular granulation tissue arrangement, and almost disappearance of inflammatory cell infiltration. The difference from the other groups was statistically significant (p<0.01). The synergistic effect of AiPian nanoparticles can effectively inhibit chronic inflammation and optimize the repair microenvironment.

[0078] During the 10-day remodeling period post-surgery, the Control group still had significant epithelial gaps and inflammation, with low maturity of granulation tissue and severely delayed healing. Although the wounds in the CTC and SPK gel groups were basically covered by epithelium, the granulation tissue was loose and the collagen fibers were disordered. The wounds in the SPK@BO NPs gel group were completely covered by new epithelium, with high maturity of granulation tissue, gradually orderly arrangement of collagen fibers, and basic regression of inflammatory cells, thus providing a solid histological basis for wound healing.

[0079] 2.4 Masson staining to evaluate collagen deposition in rat wounds Figure 23 Masson staining results of wound tissues in each group at 5 and 10 days post-surgery reveal key characteristics of extracellular matrix remodeling. At 10 days post-surgery, the Control group showed light and sparse collagen staining and disordered fiber arrangement, suggesting impaired collagen synthesis and remodeling function of fibroblasts in diabetic conditions, making it difficult to form a stable tissue scaffold. The CTC group showed deeper collagen staining and slightly improved arrangement, but still disordered, indicating that the positive drug had limited effect on promoting collagen synthesis and was unable to regulate collagen arrangement. The SPK gel group showed deeper collagen staining and more regular fiber arrangement, confirming that the blank hydrogel could optimize the wound microenvironment, activate fibroblast activity, and enhance collagen synthesis and remodeling processes. The SPK@BO NPs gel group showed deep and dense blue collagen staining and neat and dense fiber arrangement, forming a solid tissue scaffold, with a highly statistically significant difference from the other three groups. Collagen is a core component of granulation tissue and scar tissue. Its sufficient and orderly deposition is a key marker of improved wound tensile strength and normal remodeling. This result confirms that SPK@BO NPs gel can significantly improve the damaged collagen synthesis and arrangement in diabetic wounds, promote structural repair of wounds, enhance their mechanical strength, and provide a solid material basis for healing.

[0080] 2.5 Analysis of Key Inflammatory Factor Expression and Immune Microenvironment This study used enzyme-linked immunosorbent assay (ELISA) to systematically detect the dynamic changes in the concentrations of key cytokines—pro-inflammatory TNF-α, anti-inflammatory IL-4 and IL-10, and pro-angiogenic VEGF—in wound tissue of diabetic rats on postoperative days 5 (inflammation / proliferation phase) and 10 (remodeling phase). The aim was to elucidate the deep regulatory role of SPK@BO NPs gel composite hydrogel on the wound inflammatory microenvironment. The results of wound inflammatory factor expression are shown below. Figure 24 As shown in the figure, the model group wounds exhibited characteristics of chronic inflammatory imbalance, with significantly high expression of the pro-inflammatory factor TNF-α and suppressed expression of anti-inflammatory factors IL-4 / IL-10 and the pro-angiogenic factor VEGF. In contrast, the SPK@BO NPs gel group demonstrated precise and comprehensive regulatory efficacy, significantly downregulating TNF-α to curb excessive inflammation, and simultaneously upregulating IL-4 / IL-10 and VEGF to activate anti-inflammatory repair processes, constructing a microenvironment conducive to healing. Its multi-target synergistic effect of "anti-inflammatory and repair-promoting" drives the transformation of the wound microenvironment from a pro-inflammatory state to a repair state, and the regulatory effect is stably maintained during the 10-day remodeling period postoperatively. This hydrogel breaks the vicious cycle of inflammation in diabetic wounds by precisely remodeling the cytokine network, laying a key molecular foundation for tissue regeneration.

[0081] Immunohistochemical expression results of inflammatory factors in the wound, as follows Figure 25 As shown in the figure. Immunohistochemical results elucidated the role of hydrogels in regulating the inflammatory microenvironment at the molecular level. Five days post-surgery, the Control group showed high expression of the pro-inflammatory factor TNF-α and low expression of the anti-inflammatory factor IL-10 and the pro-angiogenic factor VEGF, indicating a severe inflammatory imbalance. The CTC group showed slight improvement in factor expression, but no significant difference, suggesting that the regulatory effect of positive drugs was limited. The SPK gel group showed significantly suppressed TNF-α expression and upregulated IL-10 and VEGF expression (p<0.05), confirming that its matrix can regulate factor balance by optimizing the local microenvironment. The SPK@BO NPs gel group showed the best regulatory efficacy, with significantly reduced TNF-α expression (p<0.01) and significantly increased IL-10 and VEGF expression (p<0.01), successfully driving the wound microenvironment from a pro-inflammatory state to a repair state.

[0082] Ten days post-surgery, the inflammatory factor network in the Control group remained in an imbalanced state; factor expression in the CTC group and SPK gel group was further improved, but both were not as good as in the SPK@BO NPs gel group; TNF-α expression in this group was reduced to the lowest level, while IL-10 and VEGF maintained high expression levels, confirming that it can effectively regulate the inflammatory factor network and provide a stable molecular microenvironment for wound healing.

[0083] 2.6 Macrophage polarization at the site of a diabetic wound Immunofluorescence results of M1 / M2 polarization of macrophages in wounds are as follows: Figure 26 As shown, the polarization of macrophages from the pro-inflammatory M1 phenotype to the reparative M2 phenotype is a key event in the resolution of inflammation and the initiation of tissue repair. M2 macrophages can secrete factors such as IL-10 and TGF-β, promoting fibroblast proliferation, collagen synthesis, and angiogenesis, while inhibiting the secretion of pro-inflammatory factors, thus coordinating anti-inflammatory and pro-repair effects. This study shows that SPK@BO NPs gel can effectively drive the transformation of macrophages in diabetic wounds to the M2 phenotype, which may be the central link in its coordination of anti-inflammatory and pro-repair effects. By regulating the innate immune response, it breaks the vicious cycle of chronic inflammation and lays the cellular basis for wound healing.

[0084] Immunofluorescence results of α-SMA in wound angiogenesis are as follows: Figure 27 As shown in the figure. The results showed that the drug-loaded hydrogel group had more abundant α-SMA cells covering the neovascularization in the wound, indicating that the neovascularization structure was more mature and stable, which helps to improve the blood supply to the wound and provide sufficient oxygen and nutrients for tissue repair.

[0085] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A type of moxa nanoparticle hydrogel, characterized in that, The moxa nanoparticle hydrogel is made of moxa nanoparticle dispersion and hydrogel matrix; wherein: The dispersion of the acetic acid nanoparticles consists of 0.83-5 mg / mL of levorotatory borneol, 1%-10% by mass of Tween-80, and 1% by mass of glycerol, with ultrapure water as the solvent. The hydrogel matrix is ​​composed of 1%-2% by mass of Sapindus mukorossi saponins, 2.5%-10% by mass of Pueraria lobata polysaccharides, and 1%-2% by mass of konjac glucomannan, with ultrapure water as the solvent.

2. The acetaminophen nanoparticle hydrogel according to claim 1, characterized in that: The dispersion of the acetic acid nanoparticles consists of 2.50-3.33 mg / mL of loratadine, 1%-3% by mass of Tween-80, and 1% by mass of glycerol, with ultrapure water as the solvent. The hydrogel matrix is ​​composed of 1.5%-2% by weight of Sapindus mukorossi saponins, 2.5%-5% by weight of Pueraria lobata polysaccharides, and 1.5%-2% by weight of konjac glucomannan, with ultrapure water as the solvent.

3. The acetaminophen nanoparticle hydrogel according to claim 2, characterized in that: The acetylene nanoparticle dispersion is composed of 3.33 mg / mL of L-borneol, 1% by mass of Tween-80, and 1% by mass of glycerol, with ultrapure water as the solvent. The hydrogel matrix is ​​composed of 2% by weight of Sapindus mukorossi saponins, 2.5% by weight of Pueraria lobata polysaccharides, and 2% by weight of konjac glucomannan, with ultrapure water as the solvent.

4. The method for preparing the acetylene nanoparticle hydrogel as described in claim 1, characterized in that, The steps are as follows: S1. Preparation of Ai Pian nanoparticle dispersion: Oil phase preparation: Accurately weigh L-carnitine, dissolve it in dichloromethane, place it in a water bath at 25±2℃, and sonicate it to obtain a clear and transparent oil phase solution; Aqueous phase preparation: Ultrapure water, Tween-80 and glycerol were added sequentially to a sterile container and stirred thoroughly to obtain a homogeneous aqueous solution; Emulsification and evaporation: The above oil phase solution is slowly and constantly added dropwise to the continuously stirred aqueous phase solution. After the addition is completed, the system is kept at 25±2℃ and stirred to allow the dichloromethane to evaporate completely, thus obtaining the Ai Pian nanoparticle dispersion. Preparation of S2, Ai Pian nanoparticle hydrogel: Take the Artemisia annua nanoparticle dispersion prepared in the above steps and mix it with ultrapure water. Add Sapindus mukorossi saponins and stir at room temperature until the Sapindus mukorossi saponins are completely dissolved. Add Pueraria lobata polysaccharide and continue stirring at the same speed until the Pueraria lobata polysaccharide is fully dissolved, the system is uniform and there are no visible particles. Slowly and evenly sprinkle in konjac glucomannan while stirring continuously and continue stirring to obtain Artemisia annua nanoparticle hydrogel.

5. The method for preparing the acetylene nanoparticle hydrogel according to claim 4, characterized in that, The stirring speed in step S1 or step S2 is 300-500 rpm.

6. The method for preparing the acetylene nanoparticle hydrogel according to claim 4, characterized in that, The ultrasonic power in the oil phase preparation in step S1 is 300 W and the ultrasonic time is 5 min.

7. The method for preparing the acetylene nanoparticle hydrogel according to claim 4, characterized in that, The stirring time in the aqueous phase preparation in step S1 is 5-10 min.

8. The method for preparing the acetylene nanoparticle hydrogel according to claim 4, characterized in that, In step S1, during the emulsification and evaporation process: the dropping rate of the oil phase solution is 1-2 drops / second; after the dropping is completed, the stirring time is 1 hour.

9. The method for preparing the acetylene nanoparticle hydrogel according to claim 4, characterized in that, In step S2: the volume ratio of the Artemisia annua nanoparticle dispersion to ultrapure water is 1:4; the stirring time after adding Sapindus mukorossi saponins is 15-20 min; the stirring time after adding Pueraria lobata polysaccharide is 10-15 min; and the stirring time after adding konjac glucomannan is 20-30 min.

10. The use of the acetylcholine nanoparticle hydrogel as described in claim 1 in the preparation of a drug to promote the healing of diabetic wounds.

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