Application of hydrogel preparation loaded with quercetin nanoparticles in preparation of chronic difficult-to-heal wound treatment material

Abstract

According to the application of the hydrogel preparation loaded with the quercetin nanoparticles to preparation of a chronic difficult-to-heal wound surface treatment material, Que-coated HZ hydrogel is obtained through physical loading of QueNPs and crosslinking of modified hyaluronic acid and Zn < 2 + >, full-stage management of wound healing can be achieved, rapid healing and tissue regeneration of the wound surface are promoted through the synergistic effect of all the components, and the healing effect of the wound surface is improved. A comprehensive treatment strategy is provided for chronic wound surfaces difficult to heal, and the Que-coated HZ hydrogel promotes transformation of macrophages from an M1 phenotype to an M2 phenotype and shows a good anti-inflammatory effect. In the early stage, the hydrogel preferentially releases Zn < 2 + > to achieve the antibacterial effect and relieve local inflammation, in the later stage, QueNps gradually releases Que to reduce ROS generation, so that the anti-oxidation and anti-inflammatory effects are achieved, wound repair is accelerated, and meanwhile the released Que can resist mitochondrial damage and improve the mitochondrial function.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to the application of hydrogel formulations loaded with quercetin nanoparticles in the preparation of therapeutic materials for chronic, difficult-to-heal wounds. Background Technology

[0002] Chronic, non-healing wounds are a significant challenge in global public health today. With the increasing prominence of the global aging population, the number of patients with chronic, non-healing wounds such as diabetic ulcers and bedsores is rising, placing a huge socioeconomic burden on the population. Chronic wounds are mainly characterized by persistent bacterial infection, continuous oxidative stress, and inflammatory dysregulation. There are many causes of chronic, non-healing wounds, including underlying diseases (diabetes, vascular diseases, immune diseases, etc.), recurrent bacterial infections, persistent damage, and radiation therapy. Taking diabetic non-healing wounds as an example, excessive infiltration of innate immune cells such as macrophages and neutrophils releases large amounts of pro-inflammatory cytokines (IL-6, TNF-α, etc.), resulting in a persistent inflammatory response. In addition, the long-term hyperglycemic microenvironment leads to impaired or delayed function of wound repair-related cells (endothelial cells, fibroblasts, keratinocytes) and dysregulation of related growth factors (TGF-β, VEGF, etc.), hindering angiogenesis and tissue regeneration, ultimately resulting in difficulty in wound repair. Furthermore, most chronic wounds are also accompanied by bacterial infection. Therefore, the key to promoting chronic wound healing lies in reducing bacterial infection, reducing oxidative stress, suppressing inflammation, and promoting the formation of new blood vessels.

[0003] Clinical treatments for chronic, non-healing wounds include surgical debridement, skin grafting, and wound dressings. Wound dressings, as temporary substitutes for damaged skin, can isolate bacteria and protect the wound to some extent. Hydrogel dressings, a three-dimensional network structure formed by hydrophilic polymers, are similar to the extracellular matrix (EMC), thus providing a temporary matrix for cell migration, EMC deposition, and angiogenesis, while also keeping the wound site moist. To date, hydrogel dressings containing natural polysaccharides such as chitosan, gelatin, hyaluronic acid, and sodium alginate have been developed for chronic wounds. These hydrogel dressings have promoted the transition from the inflammatory phase to the proliferative phase to some extent. However, most of these dressings focus on a single point of action and lack a holistic approach to the entire healing process, thus limiting their potential for clinical application. Summary of the Invention

[0004] To address the technical deficiencies of existing technologies, this invention provides an application of a hydrogel formulation loaded with quercetin nanoparticles in the preparation of materials for treating chronic, difficult-to-heal wounds. This is achieved through physical loading of Quercetin nanoparticles and crosslinking of hyaluronic acid with Zn. 2+The Que@HZ hydrogel was developed. This hydrogel not only enables full-stage management of wound healing, but also promotes rapid wound healing and tissue regeneration through the synergistic effect of its components. It provides a comprehensive treatment strategy for chronic, difficult-to-heal wounds.

[0005] The technical solution adopted in this invention is the application of hydrogel formulations loaded with quercetin nanoparticles in the preparation of therapeutic materials for chronic, difficult-to-heal wounds.

[0006] The hydrogel formulation loaded with quercetin nanoparticles consists of quercetin nanoparticles (QueNPs) loaded on cross-linked Zn. 2+ Que@HZ hydrogel was obtained by modifying hyaluronic acid.

[0007] The modified hyaluronic acid mentioned is norbornene-modified hyaluronic acid (HA-Nor).

[0008] The cross-linked Zn 2+ The concentration is 0.05M.

[0009] The concentration of quercetin nanoparticles (QueNPs) in the Que@HZ hydrogel is 2%wt.

[0010] The quercetin nanoparticles (QueNPs) were prepared via the following steps: quercetin (Que) was dissolved in anhydrous ethanol and sonicated; gelatin was dissolved in deionized water and heated with stirring in a constant-temperature water bath; the anhydrous ethanol solution of quercetin was slowly and uniformly added dropwise; after the reaction was complete, the pH of the system was controlled to 7, and the mixture was stirred until completely homogeneous and cooled to room temperature. Formaldehyde was added under ice bath conditions, and stirring was continued. The precipitate was collected by centrifugation, washed with deionized water and ethanol respectively, and then freeze-dried to obtain the QueNPs precipitate.

[0011] The concentration of quercetin is 4 mg / mL.

[0012] The anhydrous quercetin ethanol solution was added slowly and uniformly at a rate of 3 mL / h.

[0013] The hydrogel formulation loaded with quercetin nanoparticles was prepared by the following steps: HA-Nor was dissolved in phosphate buffer containing photoinitiator PI2959 at room temperature; then, dithiol-functionalized polyethylene glycol HS-PEG-SH was added to the HA-Nor solution until completely dissolved; QueNPs were dispersed in the above solution at a concentration of 2%; the mixture was stirred at 1500 rpm to obtain a homogeneous solution system, which was then exposed to 15 mW·cm⁻¹. -2 Under ultraviolet light, the obtained hydrogel was immersed in a 0.05M ZnCl2 solution to obtain the hydrogel formulation Que@HZ loaded with quercetin nanoparticles.

[0014] The beneficial effects of this invention are: This invention provides an application of a hydrogel formulation loaded with quercetin nanoparticles in the preparation of therapeutic materials for chronic, difficult-to-heal wounds, through physical loading of QueNPs and modification of hyaluronic acid crosslinking with Zn. 2+ The resulting Que@HZ hydrogel not only enables comprehensive wound healing management throughout its entire lifecycle, but also promotes rapid wound healing and tissue regeneration through the synergistic effect of its components. It provides a comprehensive treatment strategy for chronic, difficult-to-heal wounds. Hyaluronic acid (HA), with its superior moisturizing properties, maintains a moist wound environment and can also mimic the extracellular matrix to promote cell migration and proliferation. Zn 2+ Zn is cross-linked to the hydrogel via metallic bonds (HZ), and the HZ hydrogel achieves Zn 2+ The sustained release of quercetin inhibits the growth of bacteria and fungi, reducing delayed healing caused by infection. Then, by physically loading gelatin-encapsulated quercetin nanoparticles (QueNPs) onto the hydrogel (Que@HZ), the hydrogel was endowed with significant antioxidant and anti-inflammatory functions. Que can alleviate oxidative stress, particularly mitochondrial damage, by eliminating mitochondrial reactive oxygen species (mtROS) and alleviating membrane potential damage, thereby protecting mitochondrial function. The Que@HZ hydrogel promotes the transformation of macrophages from the M1 phenotype to the M2 phenotype, exhibiting good anti-inflammatory effects. In the early stages, the hydrogel preferentially releases Zn. 2+ This process achieves antibacterial effects and alleviates local inflammation. In the later stages, QueNps gradually releases Que, reducing ROS production and thus achieving antioxidant and anti-inflammatory effects, accelerating wound repair. Simultaneously, the released Que can also combat mitochondrial damage and improve mitochondrial function. The Que@HZ hydrogel synergistically releases Que and Zn. 2+ It can programmatically regulate each stage of wound healing to achieve anti-infection, anti-oxidation, anti-inflammation and healing-promoting treatment. Attached Figure Description

[0015] Figure 1 Characterization of the properties of HA-Nor; where (a) HA-Nor's 1 (a) HA-NMR spectrum; (b) HA-Nor FT-IR spectrum.

[0016] Figure 2 Synthesis and property characterization of QueNPs; (a) SEM image of QueNPs; (b) particle size distribution map of QueNPs. Figure 3Synthesis and property characterization of Que@HZ hydrogel; (a) schematic diagram of Que@HZ hydrogel synthesis; (b) schematic diagram of Que@HZ hydrogel gelation; (c) SEM image of Que@HZ hydrogel; (d) compression properties of Que@HZ hydrogel; (e) rheological properties of Que@HZ hydrogel; (f) swelling properties of Que@HZ hydrogel; (g) Que drug release from Que@HZ hydrogel at different pH values.

[0017] Figure 4 Evaluation of the in vitro antibacterial activity of Que@HZ hydrogel; (a) agar plate image after co-culturing hydrogel with bacteria; (b) colony count on agar plate.

[0018] Figure 5 The biocompatibility of Que@HZ hydrogel was evaluated; (a) images and hemolysis rate of the hydrogel hemolysis test; (b) L929 cytotoxicity test.

[0019] Figure 6 Evaluation of Que@HZ hydrogel's in vitro free radical scavenging capacity; including (a) DPPH free radical scavenging capacity; (b) ABTS free radical scavenging capacity; (c) ·OH free radical scavenging capacity.

[0020] Figure 7 The in vitro anti-inflammatory effects of Que@HZ hydrogel were evaluated; (a) NO expression level in different experimental groups; (b) IL-6 expression level in different experimental groups; and (c) TNF-α expression level in different experimental groups.

[0021] Figure 8 The therapeutic effect of Que@HZ hydrogel on chronic wounds in diabetic rats; (a) images of the wound healing process; (b) a quantitative map of the wound healing area. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: Synthesis of norbornene-modified hyaluronic acid (HA-Nor) 2g of sodium hyaluronate was dissolved in 100mL of deionized water. 6g of Dowex 50W proton exchange resin was added to the solution. After 5 hours, the resin was filtered off, and the filtrate was titrated to pH 7 with tetrabutylammonium hydroxide. HA-TBA was obtained after freeze-drying.

[0024] Dissolve 0.5g of HA-TBA in 40mL of anhydrous DMSO. Heat the solution to 90℃ until completely dissolved. After returning to room temperature, add 100μL of triethylamine. React at room temperature for 24h, then add 4-(dimethylamino)pyridine and norbornene anhydride. React again for 24h. Dialyze in pure water for 3 days using a dialysis bag with a molecular weight of 3500kDa, and then freeze-dry for further use.

[0025] Dissolve the above-mentioned lyophilized material in deionized water at 2% wt, add NaCl (1 g NaCl per 100 mL of solution), and after 2 hours, precipitate the solution in 10 times the excess of cold isopropanol. Dissolve the precipitate in deionized water and lyophilize HA-Nor using a lyophilizer.

[0026] Example 2 Synthesis of QueNPs nanoparticles Quercetin (Que, 4 mg / mL) was dissolved in 10 mL of anhydrous ethanol and sonicated until completely dissolved. Gelatin (0.2 g) was dissolved in 10 mL of deionized water and heated in a 40°C constant temperature water bath with stirring. The anhydrous ethanol solution of quercetin was slowly and uniformly added dropwise (3 mL / h). After the reaction was complete, the pH of the system was controlled at 7, and the mixture was stirred until completely mixed and cooled to room temperature. 200 μL of formaldehyde was added under ice bath conditions, and stirring was continued for 30 min. The mixture was centrifuged at 12000 rpm for 20 min at 4°C, and the precipitate was collected. The precipitate was washed three times with deionized water and ethanol, respectively, and then lyophilized using a freeze dryer.

[0027] Example 3: Synthesis of HZ and Que@HZ hydrogels At room temperature, 50 mg of HA-Nor was dissolved in phosphate buffer containing 1% photoinitiator PI2959. Then, 50 mg of dithiol-functionalized polyethylene glycol HS-PEG-SH was added to the HA-Nor solution until completely dissolved. QueNPs were dispersed in the above solution at a concentration of 2%, and the mixture was stirred at 1500 rpm for 3 min to obtain a homogeneous solution system. This system was then exposed to 15 mW·cm⁻¹. -2 The obtained hydrogels were immersed in ZnCl2 solutions of concentrations of 0, 0.01M, 0.05M, and 0.1M under ultraviolet light for 300 s. They were named HZ-0, HZ-1, HZ-5, and HZ-10, respectively.

[0028] Similarly, drug-loaded hydrogels Que@HZ can be obtained.

[0029] Example 4 Physical characterization of Que@HZ hydrogel The microstructure of HZ and Que@HA hydrogels was observed using scanning electron microscopy. The compressibility of the hydrogels was tested using a universal testing machine (UTM2102, SUNS, China). Each group of hydrogels (8 mm in diameter, 10 mm in height) was placed in a six-well plate, and PBS was added to each well. The plates were incubated at 37°C for 24 h to allow the hydrogels to fully swell. The samples were then placed on the universal testing machine, and the upper and lower plates were adjusted to ensure no stress was applied. A 1 mm / min compression test was then performed. -1 The hydrogel was compressed to 60% at a certain speed.

[0030] The rheological properties of the hydrogel were tested using a hybrid rheometer (DHR-2, USA). An 8 mm diameter, 2 mm high hydrogel was placed on a TADiscovery hybrid rheometer with a parallel plate clamp of 8 mm diameter. The oscillation frequency was 0.1–100 Hz, the constant strain was 1%, and the temperature was 37 °C.

[0031] To determine the water content of the hydrogel, the initial weight was measured, followed by the weight of the freeze-dried hydrogel. To determine the swelling ratio (SR) of the hydrogel at different pH values, the freeze-dried hydrogel samples were weighed (W0) and then immersed in PBS at different pH values ​​(ph5, 7, and 9) and weighed at predetermined time points (W). t ), through SR = w n / w0 calculates the swelling ratio.

[0032] In vitro release of quercetin was calculated using a UV spectrophotometer (TU-1901, China). Quercetin hydrogels (1 cm in diameter, 0.6 cm in height) were immersed in 10 mL of PBS buffer solutions at pH 5, 7, and 9 (containing 1% Tween 80 and 5 μg / mL). -1 Type I collagenase was placed in a shaker at 37°C and 100 rpm. 5 mL of buffer was removed at predetermined time intervals, and an equal volume of buffer was replenished. The concentration of Que in the release medium was determined by UV absorbance at 374 nm, and quantitative analysis was performed based on the Que standard curve.

[0033] Example 5: Biocompatibility evaluation of Que@HZ hydrogel The in vitro cytotoxicity of the materials to L929 was determined using the CCK-8 assay. Sample groups (Gelatin NPs, Que NPs, HZ, and QueHZ) were immersed in DMEM solution for 24 h, and all obtained DMEM extracts were filtered through a 0.22 μm filter. L929 cells were seeded in 96-well plates (5000 cells / well) and statically cultured in an incubator to ensure full cell adhesion. The prepared sample extracts were then added to the 96-well plates and incubated. At corresponding time points, 10 μL of CCK-8 assay reagent was added, and incubation continued for 4 h. The absorbance was measured at 450 nm.

[0034] Fresh rabbit blood anticoagulated with sodium citrate was diluted 10-fold with physiological saline. Samples from each group were mixed with the diluted blood and incubated at 37°C for 6 hours. The mixture was centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected. The absorbance at 540 nm was measured. The positive control group used whole blood diluted 10-fold with deionized water, and the negative control group used whole blood diluted 10-fold with physiological saline. The hemolysis rate H (%) was calculated using the following formula: H (%) = (OD) sample -OD negativecontrol ) / (OD positivecontrol -OD negativecontrol ) × 100%; Example 6: Antibacterial performance test of Que@HZ hydrogel To assess antibacterial activity, suspensions of Staphylococcus aureus and Escherichia coli (1×10⁻⁶) were prepared. 6 The bacterial suspension (CFU / mL) was co-cultured with LB broth (control group) or sample groups (GelatinNPs, QueNPs, HZ, and QueHZ) at 37°C for 24 h, and then diluted to 10⁻⁶ CFU / mL with LB broth. 4 CFU / mL, inoculate 100 μL of bacterial suspension evenly onto LB agar plates. After incubation at 37°C for 24 hours, record and count the bacterial colonies.

[0035] Example 7: Antioxidant performance test of Que@HZ hydrogel The DPPH free radical scavenging ability of the materials was determined using 1,1-diphenyl-2-picrylhydrazine (DPPH). A 0.04 mg / mL DPPH working solution was prepared with anhydrous ethanol. The extract of the experimental group was mixed with an equal volume of DPPH ethanol solution, and incubated in the dark for 30 min. The absorbance at 517 nm was then measured using a UV-Vis spectrophotometer.

[0036] The ABTS radical scavenging ability of the materials was determined using 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS). A 7 mM ABTS solution was mixed with a 2.45 mM potassium persulfate (KPS) solution at a 1:1 volume ratio and incubated overnight at 4°C in the dark. The mixture was then diluted 10 times and mixed with an equal volume of the experimental group solution. After incubation in the dark for 30 min, the absorbance at 517 nm was measured using a UV-Vis spectrophotometer.

[0037] The scavenging ability of the material for ·OH was determined using a TMB probe. The experimental extract was incubated with 0.1 MH₂O₂ and 0.1 MFeCl₂ at 37 °C for 30 minutes. 100 μL of the supernatant was mixed with 900 μL of 0.1 mM TMB solution (TMB dissolved in DMSO and diluted with acetic acid buffer at pH 3.6) for 10 minutes. The absorbance at 650 nm was measured using a UV-Vis spectrophotometer.

[0038] Example 8: Anti-inflammatory performance test of Que@HZ hydrogel RAW264.7 cells (6 × 10⁻⁶) 5 (10 cells / well) were seeded in a 6-well plate. After overnight incubation, 1 μg / mL of the solution was added. -1 Cells were stimulated with LPS for 24 hours. Cell wells containing only culture medium served as a negative control group, as did cell wells containing LPS culture medium. 50 μL of supernatant was mixed with 50 μL of LGriess reagent I and 50 μL of LGriess reagent II at room temperature. After incubation for 30 minutes, the OD540 value was measured using a microplate reader.

[0039] The secretion of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) was analyzed using an ELISA kit. Cell groups and treatment methods were as described above, and the levels of IL-6 and TNF-α in the supernatant were detected according to recommended methods.

[0040] Example 9: Evaluation of the healing effect of Que@HZ hydrogel on chronically infected wounds in diabetic rats. Male SD rats (6-8 weeks old) were fed a high-glucose, high-fat diet for 4 weeks. Type II diabetic rats were induced by intraperitoneal injection of STZ (35 mg / kg) for 3 consecutive days. Fasting blood glucose was measured three days after the last STZ injection. A blood glucose level ≥ 14 mM / kg was considered a successful model. A standard biopsy puncture site was created on the rat's back using an 8 mm diameter biopsy puncturist. 100 μL of Staphylococcus aureus (1 × 10⁻⁶) was injected into the wound. 8(CFU / mL) After 24 hours of infection, the wounds were treated with PBS, commercial HA gel (C-HA), HZ, and Que@HZ hydrogel, respectively. The PBS-treated group was designated as the Control group, and the wounds were fixed with Tegdem™ medical film. Changes in wound area shrinkage were observed during the animal experiments.

[0041] Experimental results: 1. Characterization of HA-Nor Depend on Figure 1 pass 1 HNMR and FT-IR showed that norbornene-modified hyaluronic acid was obtained by esterification of norbornene anhydride onto the HA backbone, yielding HA-Nor. 1 The four new sharp peaks appearing at 6.02–6.33 ppm on the HNMR spectrum are the vinyl proton peaks of the norbornene group, and the two small peaks appearing at 1.57–1.27 ppm are the bridging and cyclic proton peaks of the norbornene group. Figure 1 a), FT-IR spectrum ( Figure 1 b) This also confirms the successful preparation of HA-Nor materials.

[0042] 2. Characterization of QueNPs Depend on Figure 2 SEM observation revealed that QueNPs were regularly spherical with a uniform particle size distribution. Figure 2 a) The particle size of QueNPs is mainly concentrated in the range of 190-255 nm. Figure 2 (b) According to the formula, the drug loading of QueNPs is 9.70±1.08%; the encapsulation efficiency is 93.19±2.91%.

[0043] 3. Characterization of Que@HZ hydrogel A schematic diagram of the preparation of Que@HZ hydrogel is shown below. Figure 3 First, QueNPs were physically loaded, and then HS-PEG-SH and DTT were used as crosslinking agents. After UV curing, the mixture was immersed in ZnCl2 solution to prepare a double-crosslinked network hydrogel. This crosslinked network includes both chemical and physical crosslinking mechanisms, including thiol-ene click reactions and metal coordination interactions. After UV irradiation, the Que@HZ hydrogel changed from a liquid state to a gel state. Figure 3 b), by Figure 3 SEM observation revealed that all Que@HZ hydrogels exhibited similar pore structures. The addition of QueNPs nanoparticles made the surface of the hydrogel pores rougher, without significantly altering the three-dimensional network structure of the hydrogel. Figure 3c). This three-dimensional network structure is an ECM-like structure, and hyaluronic acid exhibits components similar to ECM, thus mimicking the microenvironment for cell growth and function, thereby promoting cell attachment, proliferation, and migration. Good mechanical and elastic properties allow the hydrogel to maintain its structural integrity and function under external forces. The mechanical properties of hydrogel dressings are crucial for protecting wounds from external forces. With the introduction of QueNPs nanoparticles, the compressibility and storage modulus of Que@HZ are further improved. Figure 3 d and Figure 3 e). The reason may be the nano-reinforcement effect. To optimize the crosslinking network of the hydrogel, this invention investigated Zn. 2+ The effect of Zn concentration on hydrogels was investigated by measuring the storage modulus (G') of four hydrogels: HZ-0, HZ-0.01, HZ-0.05, and HZ-0.1. The results were 3258±891 Pa, 5480±2543 Pa, 8489±737 Pa, and 9112±3191 Pa, respectively. The results indicate that Zn... 2+ The incorporation of Zn leads to an increase in the storage modulus of the hydrogel, which is due to the presence of Zn in the hydrogel. 2+ More metal coordination bonds are formed with HA, increasing the degree of cross-linking. After 0.05 mM, with the increase of Zn... 2+ With the increase of [amount], the increase in the storage modulus of the hydrogel tended to be gradual and showed no significant improvement. Considering the potential toxicity risks posed by metal ions, 0.05 mM was ultimately chosen as the final concentration of the HZ hydrogel to balance mechanical properties and biocompatibility. The swelling values ​​of the HZ and Que@HZ hydrogels were 2023.09 ± 70.44% and 1855.94 ± 36.48%, respectively. Figure 3 (f) This indicates that both HZ hydrogel and Que@HZ hydrogel have good swelling capacity, which is beneficial for absorbing exudate from the wound surface. The water retention rates of HZ and Que@HZ hydrogels are greater than 95%, and there is no significant difference in water content between the two types of hydrogels. Therefore, hydrogels can, through their inherent wetting properties, reduce the increase in skin temperature caused by infection in refractory wounds, thereby accelerating the wound healing process.

[0044] Wound infection can cause pH changes. Studies have reported that the pH of chronic wounds is approximately 7.3-10. Therefore, we investigated the in vitro release of quercetin from the Que@HZ hydrogel at different pH values ​​(pH 5, pH 7, and pH 9), such as... Figure 3 Quercetin exhibited slow and continuous release over 12 days in PBS buffers at different pH values. The release rate was fastest at pH 9, with a cumulative release rate of 71.36 ± 1.06%, while the release rate was slowest at pH 5, with a cumulative release rate of only 58.12 ± 0.95%.

[0045] 4. Biocompatibility evaluation of Que@HZ hydrogel Ideal wound dressings should possess good biocompatibility. First, we evaluated the blood compatibility of the hydrogel using a hemolysis test. The hemolysis rate of Que@HZ hydrogel was 0.80 ± 0.14% (…). Figure 4 a) meets the relevant national standards (<5%). Secondly, we used the CCK-8 assay to evaluate the cell compatibility of the hydrogel with L929 cells. Within 7 days, the cell viability values ​​of the experimental groups were all greater than 90% ( Figure 4 (b) The above results all indicate that Que@HZ has good biocompatibility.

[0046] 5. Antibacterial performance test of Que@HZ hydrogel Recurrent infections by Staphylococcus aureus and Escherichia coli are a significant contributing factor to chronic, non-healing wounds. Therefore, we evaluated the antibacterial efficacy of Que@HZ hydrogel against these two bacteria. First, we assessed the hydrogel's bactericidal ability using plate colony counting, comparing QueNPs and Zn... 2+ The number of Staphylococcus aureus colonies was significantly reduced after the hydrogel treatment. Figure 5 a), especially through Zn 2+ The treated hydrogel showed a 100% inhibition rate against both Staphylococcus aureus and Escherichia coli. Figure 5 b).

[0047] 6. Antioxidant performance test of Que@HZ hydrogel Reactive oxygen species (ROS) refer to a general term for oxygen-containing molecules or free radicals with strong oxidizing capabilities, such as hydrogen peroxide (H2O2) and superoxide anion (O2). - • and hydroxyl radicals (·OH). Excessive ROS production in chronic wounds leads to oxidative stress, resulting in a persistent inflammatory response and hindering tissue regeneration and repair. Therefore, we investigated the effects of Que@HZ hydrogel on DPPH· radicals and ABTS. + Scavenging ability of · free radicals and ·OH free radicals.

[0048] First, with DPPH· and ABTS + The free radical scavenging ability of Que@HZ hydrogel was evaluated using reactive nitrogen (RNS) as an indicator. DPPH· had a characteristic absorption peak at 517 nm, while the absorption peaks of QueNPs and Que@HZ hydrogel at 517 nm were significantly reduced. The scavenging rate of DPPH by Que@HZ hydrogel reached 90.91±0.77%, while the scavenging rate of HZ hydrogel was less than 50%. Figure 6 a), ABTS +The characteristic absorption peak of · is located at 734 nm. After treatment with QueNPs and Que@HZ hydrogel, ABTS + The absorption peak of · decreased significantly and faded, indicating that its ABTS + • The clearance rate exceeds 95% ( Figure 6 b). These results demonstrate that the Que@HZ hydrogel possesses excellent and rapid RNS scavenging ability.

[0049] •OH radicals are among the most toxic and harmful reactive oxygen species (ROS) to biological systems. They damage carbohydrates, amino acids, proteins, nucleic acids, and lipids, causing oxidative damage. The hydroxyl radical scavenging ability of hydrogels was evaluated using a TMB probe. The peak intensities of QueNPs and HZ hydrogels were significantly reduced, and the Que@HZ hydrogel group showed almost no characteristic absorption peak at 650 nm. Figure 6 c). The Que@HZ hydrogel exhibits a ·OH scavenging rate of 94.52±1.25%, while the ·OH scavenging rates of both Gelatin NPs and HZ are less than 50%. This is attributed to the strong antioxidant properties of the Que NPs loaded on the hydrogel.

[0050] 7. Anti-inflammatory properties test of Que@HZ hydrogel Macrophages can regulate the inflammatory process and can be divided into two phenotypes: pro-inflammatory M1 and anti-inflammatory M2. M1 can release various pro-inflammatory factors, such as TNF-α and IL-6, while M2 can release anti-inflammatory factors such as IL-10. Therefore, we measured IL-6 and IL-10 using an ELISA kit. As shown in the figure, IL-6 was significantly increased in the positive control group after LPS treatment. Compared with the positive control group, IL-6 was slightly decreased in Gelatin NPs and HZ, while IL-6 was significantly decreased after Que NPs and Que@HZ treatment, especially in the Que@HZ group from 379.63±40.42 pg / ml. -1 Decreased to 114.00±11.42 pg / mL -1 Similarly, compared to the LPS group (43.13±23.14 pg / mL) -1 IL-10 was significantly elevated in the QueNPs and Que@HZ groups (189.60±53.75 pg / mL). -1 and 230.00±59.17 pg / mL -1These results indicate that the Que released by the Que@HZ hydrogel exerts its anti-inflammatory effect by reducing pro-inflammatory factors and increasing the release of anti-inflammatory factors. In the mid-to-late stages of inflammation, with the persistence of inflammatory signals, inducible nitric oxide synthase is induced to express, and macrophages produce large amounts of NO. Measuring NO levels reflects the mid-to-late stages of inflammation. Under LPS stimulation, after treatment with QueNPs and Que@HZ, NO expression decreased from the original 8.43±0.64 μM to 4.58±0.31 μM and 3.59±0.42 μM, respectively, representing reductions of 45.67% and 57.41%, respectively. This demonstrates that the Que released from the Que@HZ gel can exert an anti-inflammatory effect.

[0051] 8. Evaluation of the healing effect of Que@HZ hydrogel on chronically infected wounds in diabetic rats To investigate whether Que@HZ hydrogel could promote wound healing in patients with chronic diabetes, we created an 8mm circular full-thickness excision wound on the back of SD rats and then infected the wound with Staphylococcus aureus. The wounds were treated with PBS (control group), blue gel (C-HZ, a commercially available hyaluronic acid gel), HZ, and Que@HZ gel, respectively. Digital images of the wounds showed that Que@HZ gel exhibited a good effect in promoting wound closure. Figure 8 a) On day 3, the wound healing rate of Que@HZ gel reached 43.24±2.50%, and on day 7, the wound healing rate reached 86.13±3.66% (a). Figure 8 (b) By day 14, the wound had largely healed. In contrast, the wound healing rate in the C-HZ group was only 30.41±2.58% on day 3 and 66.26±2.90% on day 7. Furthermore, compared to HZ gel, Que@HZ gel showed a significantly better healing-promoting effect, meaning that Que can play an additional role in promoting wound repair.

[0052] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of protection of this patent.

[0053] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. Application of hydrogel formulations loaded with quercetin nanoparticles in the preparation of therapeutic materials for chronic, difficult-to-heal wounds.

2. The application according to claim 1, characterized in that, The hydrogel formulation loaded with quercetin nanoparticles consists of quercetin nanoparticles (QueNPs) loaded on cross-linked Zn. 2+ Que@HZ hydrogel was obtained by modifying hyaluronic acid.

3. The application according to claim 2, characterized in that, The modified hyaluronic acid mentioned is norbornene-modified hyaluronic acid (HA-Nor).

4. The application according to claim 2, characterized in that, The cross-linked Zn 2+ The concentration is 0.05M.

5. The application according to claim 2, characterized in that, The concentration of quercetin nanoparticles (QueNPs) in the Que@HZ hydrogel is 2%wt.

6. The application according to claim 2, characterized in that, The quercetin nanoparticles (QueNPs) are prepared through the following steps: quercetin (Que) is dissolved in anhydrous ethanol and sonicated; gelatin is dissolved in deionized water and heated with stirring in a constant temperature water bath; and the anhydrous ethanol solution of quercetin is slowly and uniformly added dropwise. After the reaction was complete, the pH of the system was controlled at 7, and the mixture was stirred until completely homogeneous and cooled to room temperature. Formaldehyde was added under ice bath conditions, and stirring was continued. The precipitate was collected by centrifugation, washed with deionized water and ethanol respectively, and then freeze-dried to obtain QueNPs precipitate.

7. The application according to claim 6, characterized in that, The concentration of quercetin is 4 mg / mL.

8. The application according to claim 6, characterized in that, The anhydrous quercetin ethanol solution was added slowly and uniformly at a rate of 3 mL / h.

9. The application according to claim 6, characterized in that, The hydrogel formulation loaded with quercetin nanoparticles was prepared by the following steps: HA-Nor was dissolved in phosphate buffer containing photoinitiator PI2959 at room temperature; then, dithiol-functionalized polyethylene glycol HS-PEG-SH was added to the HA-Nor solution until completely dissolved; QueNPs were dispersed in the above solution at a concentration of 2%; the mixture was stirred at 1500 rpm to obtain a homogeneous solution system, which was then exposed to 15 mW·cm⁻¹. -2 Under ultraviolet light, the obtained hydrogel was immersed in a 0.05M ZnCl2 solution to obtain the hydrogel formulation Que@HZ loaded with quercetin nanoparticles.

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