A glucose-responsive drug-loaded hydrogel and a preparation method and application thereof

Abstract

The application discloses a drug-loaded hydrogel with glucose responsiveness and a preparation method and application thereof, and relates to the technical fields of chemistry and biological medicine. The preparation method comprises the following steps: reacting epsilon-polylysine and 2-formylphenylboronic acid to obtain a polymer PL-PBA of phenylboronic acid; reacting a CaCl2 solution and yam polysaccharide, then adding a carbonate solution, and continuously reacting to obtain yam polysaccharide-CaCO3 microspheres; dispersing the yam polysaccharide-CaCO3 microspheres in a polyvinyl alcohol solution to obtain a DOP-CaCO3 / PVA mixed solution, adding a hypoglycemic drug solution into the DOP-CaCO3 / PVA mixed solution, uniformly mixing, then adding the PL-PBA, and reacting to obtain the drug-loaded hydrogel. The drug-loaded hydrogel developed in the application has glucose responsiveness, can effectively inhibit the inflammatory reaction of a diabetic wound, promotes wound healing, and has good biocompatibility.

Description

Technical Field

[0001] This invention relates to the fields of chemistry and biomedicine, and in particular to a glucose-responsive drug-loaded hydrogel, its preparation method, and its applications. Background Technology

[0002] Chronic diabetic wounds are a serious complication of diabetes, often leading to high treatment costs and amputation rates. Many studies have attempted to explore the reasons why chronic diabetic wounds fail to heal due to differences in the wound's physiological environment. Normal wound healing involves hemostasis, anti-inflammation, proliferation, and remodeling; however, high glucose concentrations in the wound microenvironment hinder chronic diabetic wounds from entering the tissue remodeling stage. Prolonged hyperglycemia causes the accumulation of advanced glycation end products (AGEs) in skin tissue, leading to a series of histological and cellular changes, such as cellular dysfunction and reduced secretion of various growth factors, particularly inflammatory cell dysfunction, which exacerbates and prolongs the inflammatory phase of chronic diabetic wounds. Furthermore, another factor affecting diabetic wound healing is the irregular shape of the wound after the removal of necrotic tissue; due to its irregular shape, traditional clinical dressings are highly unsuitable, leading to a higher risk of infection. Currently, subcutaneous insulin injection remains the primary treatment for diabetes; however, the poor lipid solubility, easy inactivation, and high molecular weight of insulin reduce its bioavailability, contributing to chronic diabetic complications. Simultaneously, this administration method can cause pain and infection at the injection site, easily leading to psychological stress in diabetic patients. Hydrogels offer unique advantages in insulin delivery systems, enabling targeted and controllable drug delivery, reducing side effects, and mimicking the extracellular matrix as an excellent wound dressing. PBA-based glucose-responsive hydrogels have been extensively studied; however, due to the high pKa value of phenylboronic acid, it cannot competitively bind to glucose molecules under physiological conditions. Furthermore, in vivo, the synthesized drug carriers are easily recognized by macrophages, leading to rejection and preventing their participation in the body's circulatory processes.

[0003] Therefore, it is necessary to develop a dressing that can fully adapt to the shape of the wound and regulate the glucose level of the wound in order to accelerate the healing of wounds caused by chronic diabetes. Summary of the Invention

[0004] The purpose of this invention is to provide a glucose-responsive drug-loaded hydrogel, its preparation method, and its application, in order to solve the problems existing in the prior art. This drug-loaded hydrogel is glucose-responsive, can effectively inhibit the inflammatory response of diabetic wounds, promote wound healing, and has good biocompatibility.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing a glucose-responsive drug-loaded hydrogel, comprising the following steps: (1) ε-polylysine and 2-formylphenylboronic acid react to obtain the polymer PL-PBA of phenylboronic acid; (2) After the CaCl2 solution reacts with the yam polysaccharide, a carbonate solution is added and the reaction continues to obtain yam polysaccharide-CaCO3 microspheres; (3) The yam polysaccharide-CaCO3 microspheres are dispersed in a polyvinyl alcohol solution to obtain a DOP-CaCO3 / PVA mixture. Then, a hypoglycemic drug is added to the DOP-CaCO3 / PVA mixture, and after mixing evenly, the PL-PBA is added. After the reaction, the drug-loaded hydrogel is obtained.

[0006] Further, in step (1), the molar ratio of the ε-polylysine and the 2-formylphenylboronic acid is greater than or equal to 2:1.

[0007] Further, in step (1), the pH value of the reaction is 4.5-5.5, the temperature is 80℃, and the time is 24-72h.

[0008] Further, in step (2), the Ca in the CaCl2 solution 2+ CO3 in the carbonate solution 2- The molar mass ratio of the yam polysaccharide to the polysaccharide is (3-8)×10 5 :(3-8)×10 5 :1.

[0009] Further, in step (3), the mass ratio of polyvinyl alcohol in the polyvinyl alcohol solution to that of the yam polysaccharide-CaCO3 microspheres is 20:(1-4).

[0010] Furthermore, in step (3), the blood glucose-lowering drug includes insulin.

[0011] The present invention also provides a drug-loaded hydrogel prepared according to the above preparation method.

[0012] The present invention also provides the application of the above-described drug-loaded hydrogel in the preparation of a drug that promotes the healing of diabetic wounds.

[0013] The present invention also provides a medicament for promoting the healing of diabetic wounds, comprising the above-described drug-loaded hydrogel.

[0014] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0015] The present invention discloses the following technical effects: Glucose-responsive systems can be applied to human blood glucose detection and the controlled release of diabetes medications. With technological advancements, the precise control of these systems is receiving increasing attention. To achieve accurate control, the system must first respond to glucose at the human physiological pH of 7.4; secondly, its response at this pH must be sufficiently sensitive to adapt to human blood glucose levels. Addressing the issues of phenylboronic acid's pKa (approximately 8-9), which is far higher than the human physiological pH, the low glucose sensitivity of phenylboronic acid systems, and low biocompatibility, this invention uses yam polysaccharide, a natural medicinal herb, as a base material. By utilizing the cross-linking effect of phenylboronic acid forming cyclic esters with sugar units, the apparent pKa value of phenylboronic acid is reduced. This allows for the design and synthesis of a gel-loaded drug system with extremely high glucose sensitivity, biodegradability, and good biocompatibility at the human physiological pH of 7.4. Furthermore, yam polysaccharide is one of the effective active ingredients in the natural medicinal herb yam, exhibiting excellent hypoglycemic activity.

[0016] This invention first synthesizes yam polysaccharide-CaCO3 (DOP-CaCO3) microspheres using yam polysaccharide (DOP), and then incorporates these microspheres into a bio-based polylysine-polyvinyl alcohol (PL-PVA) hydrogel, loading insulin into the hydrogel network. Phenylboronic acid-functionalized polylysine (PL) can crosslink with PVA within seconds to form a stable hydrogel, and the microsphere doping significantly improves the mechanical properties of the hydrogel. The formation of dynamic borate ester bonds endows the hydrogel with glucose-responsive properties, allowing for the slow release of insulin under high glucose concentrations. Furthermore, while regulating insulin release according to in vivo glucose concentration, the drug-loaded hydrogel can also release DOP with hypoglycemic activity and CaCO3 with anti-inflammatory effects. 2+ This invention utilizes in vitro cell experiments and erythrocyte hemolysis experiments to demonstrate that the DOP-CaCO3-doped hydrogel possesses good biocompatibility and promotes the proliferation of rat fibroblasts (NIH / 3T3). Furthermore, DOP can synergistically work with released insulin to promote glucose consumption by fibroblasts, alleviate oxidative stress induced by high glucose levels, and reduce the expression of intracellular inflammatory factors (TNF-α and IL-6). Application of this hydrogel to wound dressings in diabetic rats shows that the hydrogel synthesized in this invention can serve as a safe and effective wound dressing that inhibits inflammatory responses in diabetic rat wounds and promotes wound healing. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 FT-IR (A) spectra of 2-FPBA, ε-PL and PL-PBA, and the structural formula and 1H NMR spectrum of PL-PBA (B); in B, (a) is ε-PL and (b) is PL-PBA; Figure 2 XRD spectra of CaCO3 and DOP-CaCO3 microspheres; Figure 3 SEM images of CaCO3 (a), DOP-CaCO3 (b) microspheres and PL-PVA / DOP-CaCO3 hydrogels with different concentrations of PL-PBA (c); Figure 4 Rheological analysis of PL-PVA hydrogels prepared with different concentrations of PL-PBA: time-scan (A) mode and frequency-scan (B) mode; rheological analysis of PL-PVA / DOP-CaCO3 hydrogels doped with different concentrations of DOP-CaCO3 microspheres in frequency-scan mode (C); self-healing and tensile properties of PL-PVA / DOP-CaCO3 hydrogels (D); swelling ratio of PL-PVA and PL-PVA / DOP-CaCO3 hydrogels (E). Figure 5 Degradation of PL-PVA / DOP-CaCO3 hydrogel at different glucose concentrations (A) Cumulative release of insulin in the hydrogel (B); Figure 6 Cell viability and hemolysis rate of different hydrogels and DOP-CaCO3 after treatment at 24 h (A) and 48 h (B) were compared (C). Hydrogel Ⅰ was PL-PVA / DOP-CaCO3 hydrogel, Hydrogel Ⅱ was PL-PVA hydrogel, INS was insulin, and PBS was pH 7.4 phosphate buffer. Figure 7 The effects of different glucose concentrations on the survival rate of 3T3 cells (A), glucose consumption of 3T3 cells induced by high glucose (B), and cell migration of 3T3 cells cultured for 12 h and 24 h in different sample groups (C); * P < 0.05, ** P < 0.01, compared with normal group; # P < 0.05, ## P < 0.01, compared with control group; Figure 8 The expression of TNF-α (A) and IL-6 in RAW264.7 cells after different hydrogel treatments (B); Figure 9Photographs of rat wounds (A) and analysis of wound healing traces (B) for rats treated in different experimental groups; Figure 10 Wound healing rates of rats treated in different experimental groups at 3, 7, 14, and 21 days; Figure 11 H&E staining image of regenerated skin tissue; yellow dashed lines indicate the epithelial-dermal boundary, green arrows indicate capillaries, and red arrows indicate hair follicles; scale bar: 100 μm. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] The yam polysaccharide (DOP) used in the following examples is Huai yam polysaccharide with a purity greater than 40%, and the extraction method is referenced in "Food Chemistry, 227, 64-72".

[0025] Example 1

[0026] A method for preparing glucose-responsive yam polysaccharide-CaCO3 microsphere-doped hydrogels that can be used to accelerate the healing of diabetic wounds, comprising the following steps: (1) Accurately weigh 600 mg (4.68 mmol) of ε-polylysine (ε-PL) and 351 mg (2.34 mmol) of 2-formylphenylboronic acid (2-FPBA) and completely dissolve them in 100 mL of acetic acid / sodium acetate buffer at pH 4.5. React at 80 °C for 72 h. After the reaction is complete, transfer the reaction solution to a dialysis belt (MW1000) and dialyze for 7 days to remove small molecule impurities. Finally, freeze-dry the dialyzed solution to obtain a sponge-like solid, which is PL-PBA.

[0027] The molar ratio of ε-PL to 2-FPBA needs to be greater than or equal to 2:1; otherwise, the synthesized PL-PBA will have poor water solubility and cannot be used for the next synthesis step.

[0028] The infrared spectrum and proton nuclear magnetic resonance spectrum of PL-PBA are as follows: Figure 1 As shown.

[0029] For ε-PL and PL-PBA, 2934 cm -1 The absorption peak at 1678 cm⁻¹ is the stretching vibration peak of CH on the ε-PL backbone. -1 The absorption peak at 1568 cm⁻¹ is caused by the tensile vibration of C=O. -1 The peak value at 1264 cm is attributed to the tensile vibration of C=N, while the peak value at 1264 cm is attributed to the tensile vibration of C=N. -1 The peak at 832 cm⁻¹ is a characteristic absorption peak for the BOH bond in PBA. Because of the presence of a benzene ring in PBA, this peak is observed at 832 cm⁻¹. -1 726 cm -1 A vibrational peak indicating out-of-plane deformation of the benzene ring was observed at this point. This indicates that PBA was successfully grafted onto ε-PL.

[0030] For ε-PL and PL-PBA in D2O 1The 1H NMR spectrum is shown in 1B. The proton peak at 3.93 ppm (d, 2H) corresponds to proton signals from C1 and C9, the proton peak at 1.86 ppm (d, 4H) to H signals from C2 and C10, the proton peak at 1.40 ppm (d, 4H) to H signals from C3 and C11, the proton peak at 1.59 ppm (d, 4H) to H signals from C4 and C12, and the proton peak at 3.24 ppm (d, 4H) to H signals from C5 and C13. Compared with ε-PL, a very obvious characteristic peak appears at 7.52 ppm (t, 4H), which corresponds to the proton peak of H in the benzene ring skeleton. A new proton peak also appears near 6.02 ppm (d, 1H), which corresponds to the proton signal from the carbon atom in the CN double bond.

[0031] The above results further demonstrate the successful synthesis of PL-PBA.

[0032] (2) Weigh out Na₂CO₃ and CaCl₂ separately, and dissolve them in 10 mL of deionized water to obtain 0.33 M Na₂CO₃ solution and 0.33 M CaCl₂ solution. First, mix the CaCl₂ solution with 10 mg DOP (n Ca :n DOP ≈3×10 5 :1) Stir at room temperature for 1 min, then rapidly inject Na2CO3 solution into the reaction solution under magnetic stirring, and continue stirring at 800 rpm for 10 min. Finally, centrifuge the reaction solution and wash the precipitate with deionized water, then vacuum dry to obtain DOP-CaCO3 microspheres.

[0033] Figure 2 The XRD spectra of CaCO3 and DOP-CaCO3 microspheres are shown. As can be seen from the figure, CaCO3 is calcite type, and DOP-CaCO3 microspheres are a mixed crystal type of calcite and aragonite. The diffraction angle 2θ=29.40° mainly corresponds to the (104) crystal plane of calcite, while DOP-CaCO3 microspheres show the lattice diffraction peaks of aragonite (110), (112), and (114).

[0034] The microstructure of DOP-CaCO3 microspheres was observed using SEM. For example... Figure 3 As shown in Figures a and b, CaCO3 without DOP exhibits a cubic structure, while the CaCO3 with DOP exhibits a uniform spherical shape, indicating that DOP-CaCO3 microspheres have been successfully synthesized.

[0035] (3) Weigh 4 g of PVA (polyvinyl alcohol, Mw~27000) and add 36 mL of deionized water to a round-bottom flask. Stir at 60 °C for 2 h, then heat to 95 °C and continue reflux for 15 min to obtain a 10% (W / V) PVA solution. Cool the solution and then add 1 mg of DOP-CaCO3 microspheres obtained in step (2) to 200 µL of the above 10% PVA solution. Once the microspheres are evenly dispersed, immediately add 200 µL of 2% (W / V) PL-PBA solution with pH adjusted to 7 using NaOH solution (pH 7-8 can achieve the same effect). Stir rapidly, and a hydrogel (PL-PVA / DOP-CaCO3) will be formed within seconds.

[0036] The microstructure and porosity of PL-PVA / DOP-CaCO3 hydrogels with different concentrations of PL-PBA polymer were observed using scanning electron microscopy (SEM), such as... Figure 3 As shown in Figure c, due to the cross-linking effect of dynamic borate ester bonds, a continuous porous microstructure can be observed in all PL-PVA / DOP-CaCO3 hydrogels. Furthermore, with increasing PL-PBA polymer concentration, the porous structure becomes denser, and the pore size decreases.

[0037] The mechanical properties of the hydrogel were further evaluated using a rheometer, and the results are as follows: Figure 4 As shown. In time scan mode ( Figure 4 In Figure A), the elastic modulus (G') of all hydrogels was greater than the viscous modulus (G''), and the G' and G'' values ​​of each hydrogel remained constant throughout the test time, indicating that the hydrogels were successfully synthesized and possess good mechanical stability. The mechanical strength of the hydrogels is reflected by the G' value. The mechanical strength of the hydrogels was further determined by oscillation frequency scanning, such as... Figure 4 As shown in Figure B, the higher the concentration of PL-PBA, the higher the mechanical strength of the PL-PVA hydrogel. With the increase of PL-PBA concentration, more PBA groups can chemically crosslink with PVA, thus making the PL-PVA hydrogel exhibit stronger mechanical strength. Secondly, hydrogels with different DOP-CaCO3 microsphere contents were prepared to investigate the effect of microsphere doping on the mechanical properties of the hydrogel. Figure 4 As shown in Figure C, doping with DOP-CaCO3 microspheres can significantly increase the elastic modulus (G') of the hydrogel. The elastic modulus (G') of the hydrogel is the highest and remains stable when the microspheres are doped with 0.25% (W / V). Figure 4The D-axis demonstrates the self-healing and tensile properties of the hydrogel. Unlike some hydrogels formed by rigid covalent networks, the PL-PVA / DOP-CaCO3 hydrogel exhibits remodeling and self-healing properties due to the formation-breakage cycle of borate ester bonds. When stretched, the hydrogel deforms rather than peels off from the skin surface, indicating that the hydrogel, as a dynamic network, dissipates energy through deformation, leading to a reduction in forces acting on the adhesive interface and thus enhancing the adhesive strength of the hydrogel. The tensile properties of the hydrogel show that it does not detach with joint movement at active joints, suggesting that the PL-PVA / DOP-CaCO3 hydrogel can be used as a good dressing for wounds. Figure 4 The image shows the effect of DOP-CaCO3 microsphere doping on the swelling properties of PL-PVA hydrogel. The PL-PVA hydrogel reached swelling equilibrium (≈448%) within 90 minutes, after which degradation began. The PL-PVA / DOP-CaCO3 hydrogel reached swelling equilibrium within 75 minutes, with a significantly increased swelling rate (≈502%), and degradation began after 180 minutes. This indicates that DOP-CaCO3 microsphere doping significantly improves the swelling properties of the hydrogel. The PL-PVA / DOP-CaCO3 hydrogel can rapidly absorb wound exudate and blood while maintaining a stable shape and volume, thus creating a moist healing environment and accelerating wound healing.

[0038] (4) Loading of insulin onto the hydrogel: Insulin was dissolved in 0.1 M HCl and brought to a final volume with PBS to prepare an insulin solution of 2 mg / mL. The preparation process of the drug-loaded hydrogel was the same as in (3). Before adding the PL-PBA solution, 100 µL of insulin solution was added dropwise to 200 µL of DOP-CaCO3 / PVA mixture. After mixing evenly, 200 µL of PL-PBA solution was quickly added and stirred evenly to form a drug-loaded hydrogel (INS@PL-PVA / DOP-CaCO3).

[0039] In vitro release of the hydrogel: The drug-loaded hydrogel was placed in 2.5 mL of PBS solution with different concentrations of glucose (0 mg / mL, 1.0 mg / mL, and 4.0 mg / mL), shaken at 37 °C, and 250 µL of the medium was drawn at regular intervals to detect the amount of insulin released. The same volume of fresh solution was added simultaneously to maintain a constant volume of the release medium. The amount of insulin released was detected using an ELISA kit.

[0040] The degree of degradation of PL-PVA / DOP-CaCO3 hydrogel at different glucose concentrations is as follows: Figure 5As shown in Figure A, the degradation rate of the hydrogel increases with increasing glucose concentration. At a glucose concentration of 4 mg / mL, 87% of the hydrogel degraded within 24 hours, while in PBS, only 69% of the hydrogel degraded within 24 hours. This is because glucose competitively binds to boronic acid molecules, leading to the cleavage of dynamic boronic acid ester bonds in the hydrogel, resulting in hydrogel collapse and degradation. The in vitro degradation results fully demonstrate that the PL-PVA / DOP-CaCO3 hydrogel has good glucose responsiveness and can be used as a biodegradable and glucose-sensitive wound healing dressing for diabetes. Insulin release curves in different glucose concentrations are shown in Figure A. Figure 5 As shown in Figure B, in glucose-free PBS medium, the cumulative insulin release over 48 hours was 53.77 ± 2.43%, possibly due to swelling of the hydrogel in PBS, which increased the pore size and led to insulin leakage. When the glucose concentration increased from 0 to 4 mg / mL, the cumulative insulin release over 48 hours increased from 53.77 ± 2.43% to 91.91 ± 5.27%, continuing a slow and sustained release over three days. Furthermore, the cumulative insulin release gradually increased with increasing glucose concentration, indicating that under high glucose concentrations, glucose competitively binds to boronic acid bonds, disrupting the 3D network structure of the hydrogel. The gradual disintegration of the hydrogel allows for sustained insulin release in response to high blood glucose levels.

[0041] The cytotoxicity of hydrogels can be used to assess their biocompatibility. The MTT assay was used to detect the effects of PL-PVA / DOP-CaCO3 hydrogel (Hydrogel I), PL-PVA hydrogel (Hydrogel II), and DOP-CaCO3 microspheres on the cell viability of rat fibroblasts (3T3). The results are as follows: Figure 6 As shown in the figure. Compared with the blank group, the cell viability of extracts from different concentrations of hydrogel was around 100% within 48 h, showing a high cell viability rate. Furthermore, the cell viability of the high-concentration hydrogel I group was higher than 100%, indicating that high concentrations of hydrogel I may promote fibroblast proliferation. For the DOP-CaCO3 group, the cell viability of DOP-CaCO3 microspheres at different concentrations was higher than 95.0% within 48 h. The blood compatibility of the hydrogel is as follows... Figure 6 As shown in Figure C, compared with the positive control group, the hemolysis rate of each hydrogel group was much lower than that of the positive control group (<2%). Therefore, the hemolysis rate of each hydrogel group can be ignored. This shows that the hydrogel has good biocompatibility and can be used as a safe dressing material for in vivo wounds.

[0042] The effects of different glucose concentrations on cells were investigated, and the results are as follows: Figure 7As shown, cell viability began to decrease (93.72%) when glucose concentration exceeded 6 mg / mL; therefore, a glucose concentration of 5 mg / mL was selected as the model for high glucose injury. Compared with the high glucose model group, 3T3 cells co-cultured with INS@Hydrogel I hydrogel for 24 h showed a 24.56% increase in glucose consumption under 5 mg / mL glucose conditions, while the glucose consumption of 3T3 cells with high insulin concentration (0.32 mg / mL) increased by only 13.42%. This may be because high glucose concentrations can cause hydrogel lysis and slow release of insulin, and the insulin encapsulated in the hydrogel is more active, thus promoting glucose consumption in cells. Cell scratch healing results showed that, compared with the control group, cells treated with hydrogel gradually healed within 24 h. This may be due to the properties of the hydrogel mimicking the extracellular matrix (ECM), thereby promoting cell adhesion. Fibroblasts in the Hydrogel I and INS@Hydrogel I groups showed increased migration ability and significant scratch healing within 24 h.

[0043] Example 1 of effect verification In vitro anti-inflammatory effects of hydrogels: First, RAW 264.7 macrophages were cultured at 2 × 10⁶ cells per well. 5 RAW 264.7 macrophages were seeded at a density of 1000 cells / well in 6-well plates. After cell adhesion, DMEM medium containing 1 µg / mL LPS was added, and the cells were cultured for another 24 h. Then, RAW 264.7 macrophages were incubated in DMEM medium containing 1 µg / mL LPS and 0.1 g / mL hydrogel for another 24 h. Finally, the levels of TNF-α and IL-6 in each group of cells were detected using an ELISA kit.

[0044] Persistent hyperglycemia is the most prominent characteristic of diabetes, and long-term persistent hyperglycemia makes wounds in chronic diabetes more susceptible to chronic inflammation. Therefore, the expression of pro-inflammatory cytokines TNF-α and IL-6 was examined to demonstrate the anti-inflammatory capabilities of the hydrogel. Figure 8 As shown, LPS induction significantly increased the secretion of TNF-α and IL-6 in RAW 264.7 cells, while hydrogel treatment downregulated the secretion of both TNF-α and IL-6. Hydrogel I exhibited a superior inhibitory effect on TNF-α secretion compared to Hydrogel II, likely due to the enhanced anti-inflammatory properties of the gel from the doping of DOP-CaCO3 microspheres. Compared to the LPS group, the INS@Hydrogel I group showed a 48.35% and 46.41% reduction in TNF-α and IL-6 secretion, respectively, demonstrating a better anti-inflammatory effect than free insulin. This indicates that the hydrogel possesses superior anti-inflammatory activity due to the effective release of insulin.

[0045] Effect of hydrogel on wound healing in diabetic rats: A full-thickness skin defect model in diabetic rats was used to evaluate the effect of hydrogel on wound healing in vivo. Male SD rats, weighing 200-220 g, were first fed a high-sugar, high-fat diet for 4 weeks, and then injected intraperitoneally with streptozotocin solution (STZ, 30 mg / kg BW, dissolved in 0.01 M sodium citrate buffer, pH 4.4). After STZ induction for 72 hours, fasting blood glucose levels were measured at the tail. Rats with fasting blood glucose (FBG) ≥11.1 mmol / L were selected as T2DM model rats. After 7 days of stable blood glucose, a full-thickness skin defect model was established, and blood glucose levels in SD rats were recorded until the wound was completely healed. Diabetic rats were randomly divided into five groups (n≥8). After anesthetizing and shaving the rats, a complete skin defect lesion (7 mm in diameter) was formed on the back of each rat. After rinsing with physiological saline, rats were treated in different ways: (1) wounds were bandaged with gauze (gauze group, Control group); (2) wounds were treated with commercial dressings (levofloxacin gel dressing group, Positive control group); (3) wounds were treated with 0.4 mL PL-PVA / DOP-CaCO3 hydrogel (Hydrogel I group); (4) wounds were treated with 0.4 mL PL-PVA hydrogel (Hydrogel II group); (5) wounds were treated with 0.4 mL INS@PL-PVA / DOP-CaCO3 hydrogel (INS@Hydrogel I group). A normal rat wound healing group (Normal group) was also set up. All hydrogels completely covered the wounds with skin defects. During the healing process, the hydrogels were changed every 48 h. The wound area was recorded using a camera, and blood glucose levels were continuously monitored during the process. On days 0, 7, 14, and 21 after drug administration, the wounds of rats in each group were photographed and recorded, the wound area of ​​each group was measured, and the wound healing rate was calculated. On days 7, 14, and 21 after drug administration, the animals were euthanized, and intact dermal tissue and surrounding tissue were harvested from the wound, approximately 1 cm in diameter. The tissue was fixed in 4% paraformaldehyde and left overnight. Wound sections from the rats were stained with H&E and observed and photographed.

[0046] Photos of wound healing Figure 9 As shown, the healing rate is as follows Figure 10As shown, with the extension of time, the wound size of all treatment groups showed a decreasing trend, while the wound recovery in the control group was relatively slow. After 3 days of treatment, the wound closure rate of the INS@Hydrogel I hydrogel group and the levofloxacin gel dressing group was significantly higher than that of the control group, reaching 36.01% and 37.89% respectively, while the other diabetic groups showed little change. After 7 days, the wound closure rate of the Hydrogel I and INS@Hydrogel I hydrogel groups was between 65% and 75%, both significantly higher than that of the control group (21.06%), and the scabs fell off naturally. This may be because INS@Hydrogel I releases insulin under the high glucose concentration conditions around diabetic wounds, while the hydrogel exerts its extracellular matrix properties, timely stopping bleeding and relieving inflammation, promoting cell adhesion and proliferation, thereby promoting wound healing in vivo. After 14 days of treatment, all wounds were not fully healed. The wound healing rate in the levofloxacin gel dressing group was 96.38%, while the wound healing rates in the Hydrogel I and INS@Hydrogel I groups were 96.18% and 99.35%, respectively, significantly higher than the control group. The wounds were largely healed and showed signs of crusting. After 21 days of treatment, all wounds had completely healed. These results indicate that the insulin-loaded hydrogel dressing is superior to commercial dressings and accelerates wound healing.

[0047] To assess the healing effect of the hydrogel dressing from a histological perspective, regenerated skin tissue was collected on days 7, 14, and 21 for H&E staining. Figure 11 As shown in the diagram. On day 7 of administration, a large amount of inflammatory cell-infiltrated granulation tissue was observed in the positive control group, while fewer inflammatory cells were found in the wound tissue of the Hydrogel I group and the INS@Hydrogel I group. This may be because the higher glucose concentration around the chronic diabetic wound causes the hydrogel to cleave, releasing insulin and polysaccharides, thus exerting an anti-inflammatory effect. On day 14, compared with the untreated control group, the wounds treated in the Hydrogel I group and the INS@Hydrogel I group showed hair follicle tissue and gradual epidermal thickening, similar to the normal wounds, indicating good growth. On day 21, complete epithelium had formed in all groups, and the formation of skin appendages was clearly visible in all three hydrogel groups (red arrows), indicating that the healed tissue was highly similar to healthy skin. In conclusion, the INS@Hydrogel I gel treatment for diabetic wounds was significantly better than other groups in terms of wound repair. This demonstrates that insulin-loaded hydrogels can promote the proliferation, migration, and secretion of keratinocytes, endothelial cells, and fibroblasts, accelerating wound healing.

[0048] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a glucose-responsive drug-loaded hydrogel, characterized in that, Includes the following steps: (1) ε-polylysine and 2-formylphenylboronic acid are reacted at pH 4.5-5.5 and 80℃ for 24-72 h to obtain the polymer PL-PBA of phenylboronic acid; the molar ratio of ε-polylysine and 2-formylphenylboronic acid is greater than or equal to 2:1; (2) After the CaCl2 solution reacts with the yam polysaccharide, a carbonate solution is added to obtain yam polysaccharide-CaCO3 microspheres; the Ca in the CaCl2 solution... 2+ CO3 in the carbonate solution 2- The molar ratio of the yam polysaccharide to the polysaccharide is (3-8)×10. 5 :(3-8)×10 5 :1; (3) The yam polysaccharide-CaCO3 microspheres are dispersed in a polyvinyl alcohol solution to obtain a DOP-CaCO3 / PVA mixture. Then, a hypoglycemic drug is added and mixed evenly. PL-PBA is then added, and a drug-loaded hydrogel is formed through a dynamic borate ester bond crosslinking reaction. The mass ratio of polyvinyl alcohol in the polyvinyl alcohol solution to the yam polysaccharide-CaCO3 microspheres is 20:(1-4). The hypoglycemic drug is insulin.

2. A drug-loaded hydrogel prepared by the preparation method according to claim 1, characterized in that, It has a porous microstructure containing yam polysaccharide-CaCO3 microspheres, insulin, and a PL-PBA / PVA cross-linked network.

3. The use of the drug-loaded hydrogel as described in claim 2 in the preparation of a medicament for promoting the healing of diabetic wounds.

4. A drug for promoting wound healing in diabetic patients, characterized in that, Includes the drug-loaded hydrogel of claim 2 and pharmaceutically acceptable excipients.

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