Traditional Chinese medicine hydrogel microneedle for diabetic wounds as well as preparation method and application of traditional Chinese medicine hydrogel microneedle

Abstract

The invention belongs to the technical field of hydrogel microneedles, and discloses a traditional Chinese medicine hydrogel microneedle for diabetic wounds, the traditional Chinese medicine hydrogel microneedle comprises a needle tip and a substrate, and the needle tip is mainly prepared from methacrylated glucan, ligustrazine-loaded polyphenylene sulfide-hyaluronic acid nano-micelle, safflower polysaccharide and a light curing agent. According to the preparation method, methylacryloylated glucan is selected as a drug carrier in the microneedle, traditional Chinese medicine ligustrazine and safflower polysaccharide are selected as active substances, the anti-inflammatory, anti-oxidation and angiogenesis promoting effects of ligustrazine are combined with the immunoregulation and tissue repair functions of safflower polysaccharide, and through a continuous release mechanism of the microneedle, the anti-inflammatory, anti-oxidation and angiogenesis promoting effects of ligustrazine and the anti-inflammatory, anti-oxidation and angiogenesis promoting effects of the safflower polysaccharide are achieved. The composition can be used for promoting healing of diabetic wounds. The invention further discloses a preparation method and application of the traditional Chinese medicine hydrogel microneedle for the diabetic wound.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogel microneedles, and in particular relates to traditional Chinese medicine hydrogel microneedles for diabetic wounds, and a preparation method and application thereof. Background Art

[0002] Diabetic wounds are a common complication in diabetic patients, often occurring on the feet. They are characterized by prolonged healing, high disability rates, and high mortality rates. Studies have shown that approximately 19%-34% of diabetic patients develop diabetic wounds. Wound repair is a multi-layered physiological process, primarily divided into four overlapping transitional stages: hemostasis, inflammation, proliferation, and remodeling. Diabetic wounds are often accompanied by chronic inflammatory responses, oxidative stress, circulatory disorders, and nerve damage, which affect all four steps of skin injury repair and lead to delayed wound repair.

[0003] Wound dressings, as an important tool for treating diabetic wounds, have been widely used clinically. Their primary function is to promote wound healing, reduce infection, alleviate inflammatory responses, and accelerate wound healing by providing a suitable local environment. They also adsorb exudate and maintain a moist environment, accelerating wound healing. Based on their functions and properties, wound dressings can be categorized into various types, with traditional and modern dressings being the most common. Traditional dressings, such as gauze, cotton pads, and plaster, while providing some protection, have relatively limited wound healing effects and are prone to infection and dryness. Modern dressings, including moist dressings, antimicrobial dressings, and biological dressings, hold significant clinical significance in the treatment of diabetic wounds. However, these dressings suffer from the inability to simultaneously address the multiple issues associated with diabetic wounds, limited surface drug delivery, short dressing change cycles, and a lack of comfort. Therefore, an effective material that can address these issues is urgently needed. Summary of the Invention

[0004] The first object of the present invention is to provide a traditional Chinese medicine hydrogel microneedle for diabetic wounds to solve at least one of the above technical problems.

[0005] The second object of the present invention is to provide a method for preparing traditional Chinese medicine hydrogel microneedles for diabetic wounds to solve at least one of the above technical problems.

[0006] The third object of the present invention is to provide an application of traditional Chinese medicine hydrogel microneedles for diabetic wounds to solve at least one of the above technical problems.

[0007] According to the first aspect of the present invention, a traditional Chinese medicine hydrogel microneedle for diabetic wounds is provided, comprising a needle tip and a substrate. The raw materials for preparing the needle tip are composed of methacryloylated dextran (DexMA), polyphenylene sulfide-hyaluronic acid nanomicelles loaded with ligustrazine (TMP@PPS-HA), safflower polysaccharide and a photocuring agent.

[0008] The traditional Chinese medicine hydrogel microneedles for diabetic wounds of the present invention are prepared by selecting methacryloylated dextran as the drug carrier in the microneedles, and selecting ligustrazine and safflower polysaccharide from traditional Chinese medicine as active substances. The anti-inflammatory, antioxidant and angiogenesis-promoting effects of ligustrazine are combined with the immunoregulatory and tissue repair functions of safflower polysaccharide. Through the sustained release mechanism of the microneedles, the microneedles can be used to promote the healing of diabetic wounds.

[0009] In some embodiments, the content of ligustrazine (TMP) in the ligustrazine-loaded polyphenylene sulfide-hyaluronic acid nanomicelles is 5-15 mg / mL.

[0010] In some embodiments, the mass ratio of methacryloylated dextran to safflower polysaccharide is (200-400):1, and the ratio of the amount of polyphenylene sulfide-hyaluronic acid nanomicelles loaded with ligustrazine to the mass of safflower polysaccharide is (1-15) mL:2 mg.

[0011] In some embodiments, the photocuring agent is selected from at least one of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and 2-hydroxy-2-methyl-1-phenyl-1-propanone.

[0012] In some embodiments, the mass of the photocuring agent is 1%-3% of the total mass of methacryloylated dextran, polyphenylene sulfide-hyaluronic acid nanomicelles loaded with ligustrazine (TMP@PPS-HA), safflower polysaccharide and the photocuring agent.

[0013] In some embodiments, methacryloylated dextran is prepared by the following steps:

[0014] (1) dissolving dextran in an organic solvent to obtain a dextran solution, adding 4-dimethylaminopyridine (DMAP) to the dextran solution and stirring until the solution becomes clear and light green, to obtain a mixed solution A;

[0015] (2) Add glycidyl methacrylate (GMA) to the mixed solution A, stir in the dark for 24-72 hours, and then freeze-dry for 48-72 hours to obtain the product.

[0016] In some embodiments, the mass ratio of dextran to 4-dimethylaminopyridine is (5-7):1.

[0017] In some embodiments, polyphenylene sulfide-hyaluronic acid nanomicelles loaded with ligustrazine are prepared by the following steps:

[0018] S1. Preparation of polyphenylene sulfide (PPS)

[0019] (1) dissolving 3-mercaptopropionic acid (3-MPA) in an organic solvent under ice bath conditions, and adding 1,8-diazabicyclo[5.4.0]undec-7-ene to the organic solvent to obtain a reaction mixture;

[0020] (2) adding propylene sulfide dropwise to the reaction mixture under a nitrogen atmosphere and stirring at 55-70° C. overnight, then adding H 2 O to quench the reaction, precipitating the reaction mixture in cold methanol for purification, and evaporating the solvent under reduced pressure to obtain polyphenylene sulfide;

[0021] S2. Preparation of amino-modified polyphenylene sulfide (PPS-NH2)

[0022] (1) dispersing N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) in an organic solvent containing polyphenylene sulfide to obtain a mixture;

[0023] (2) adding ethylenediamine dropwise to the mixture and stirring overnight to obtain a reaction solution, diluting the reaction solution with an organic solvent, washing and drying with a solid desiccant, filtering, and concentrating the filtrate under reduced pressure to obtain amino-modified polyphenylene sulfide;

[0024] S3. Preparation of hyaluronic acid-polypropylene sulfide (PPS-HA)

[0025] (1) dialyzing hyaluronic acid sodium salt in a hydrochloric acid solution and freeze-drying to obtain acidic hyaluronic acid;

[0026] (2) dissolving acidic hyaluronic acid in water, and then dispersing N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in the aqueous solution of acidic hyaluronic acid to obtain a reaction system;

[0027] (3) dissolving the amino-modified polyphenylene sulfide in an organic solvent to obtain an organic solvent containing the amino-modified polyphenylene sulfide;

[0028] (4) adding an organic solvent containing amino-modified polyphenylene sulfide dropwise to the reaction system, stirring and reacting for 24-48 hours under a nitrogen atmosphere to obtain a mixed solution B, dialyzing the mixed solution B and then freeze-drying it to obtain hyaluronic acid-polypropylene sulfide;

[0029] S4. Preparation of polyphenylene sulfide-hyaluronic acid nanomicelles loaded with ligustrazine

[0030] (1) dissolving hyaluronic acid-polypropylene sulfide in PBS to obtain an aqueous phase, and dissolving ligustrazine in acetone-ethanol solution to obtain an organic phase;

[0031] (2) The organic phase is added to the aqueous phase, and the mixture is subjected to rotary evaporation at 30-50° C. for 3-5 minutes, ultrasonicated, filtered with a microporous filter membrane, and the filtrate is collected to obtain polyphenylene sulfide-hyaluronic acid nanomicelles loaded with ligustrazine.

[0032] In some embodiments, in step S1, the molar ratio of 3-mercaptopropionic acid, 1,8-diazabicyclo[5.4.0]undec-7-ene and propylene sulfide is (1-1.5):(3-3.5):(20-25).

[0033] In some embodiments, in step S2, the molar ratio of polyphenylene sulfide, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and ethylenediamine is 1:(1.5-2.5):(2-3):(15-25).

[0034] In some embodiments, in step S3, the concentration of the hydrochloric acid solution is 0.01-0.02 mol / L.

[0035] In some embodiments, in step S3, the mass ratio of acidic hyaluronic acid, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and amino-modified polyphenylene sulfide is (90-110):(5-10):(10-20):(30-50).

[0036] In some embodiments, in step S4, in the acetone-ethanol solution, the volume ratio of acetone to ethanol is (2.6-4.5):(1.5-2.4); preferably, the volume ratio of acetone to ethanol is 3:2.

[0037] In some embodiments, in step S4, the rotation speed used for rotary evaporation is 40-50 rpm; preferably, the rotation speed is 45 rpm.

[0038] In some embodiments, the solid desiccant is selected from at least one of anhydrous magnesium sulfate and anhydrous calcium chloride.

[0039] In some embodiments, the organic solvent is selected from at least one of dimethyl sulfoxide (DMSO), tetrahydrofuran, anhydrous tetrahydrofuran, and dichloromethane.

[0040] According to a second aspect of the present invention, a method for preparing a Chinese medicine hydrogel microneedle for treating diabetic wounds is provided, comprising the following steps:

[0041] Dissolving safflower polysaccharide and methacryloylated dextran in water to obtain a mixed solution C, and sequentially adding ligustrazine-loaded polyphenylene sulfide-hyaluronic acid nanomicelles and a light curing agent to the mixed solution C to obtain a gel precursor solution;

[0042] After dripping the gel precursor solution into the microneedle mold, the mold is transferred to a vacuum degassing box, and the vacuum degree of the vacuum degassing box is adjusted to -90 to -110 kPa for vacuum degassing. The microneedle mold is cured under 360-370 nm ultraviolet light to obtain a needle tip, and then hyaluronic acid solution is added to the microneedle mold, dried at 36-40 ° C for 48-96 hours to obtain a base, and demolded.

[0043] The present invention first prepares the needle tip of the hydrogel microneedle by combining safflower polysaccharide, methacryloylated dextran, polyphenylene sulfide-hyaluronic acid nano-micelles loaded with ligustrazine and a light curing agent, and then adds hyaluronic acid to obtain the microneedle base. The prepared hydrogel microneedle has good mechanical properties and can penetrate the stratum corneum to reach the epidermis and upper dermis to achieve transdermal delivery of drugs.

[0044] In some embodiments, the mass volume percentage concentration of the hyaluronic acid solution is 1%-3%.

[0045] According to a third aspect of the present invention, there is provided use of traditional Chinese medicine hydrogel microneedles for diabetic wounds in the preparation of a medicament for promoting healing of diabetic wounds.

[0046] The beneficial effects of the present invention are:

[0047] (1) The Chinese medicine hydrogel microneedles for diabetic wounds of the present invention are prepared with methacryloylated dextran, polyphenylene sulfide-hyaluronic acid nano-micelles loaded with ligustrazine, brown sugar polysaccharide and light curing agent as raw materials to obtain needle tips, which can effectively deliver drugs to the deep layer of the wound. Through the sustained release mechanism of the microneedles, multiple problems such as chronic inflammation, oxidative stress, blood circulation disorders and delayed tissue repair in diabetic wounds can be solved at the same time, and long-term stable release of drugs can be achieved, thereby extending the drug action time and reducing the need for frequent dressing changes, thereby improving the treatment effect and patient treatment compliance;

[0048] (2) The present invention uses polyphenylene sulfide-hyaluronic acid nanomicelles loaded with ligustrazine as the raw material for preparation. The constructed polyphenylene sulfide-hyaluronic acid nanomicelles loaded with ligustrazine have the dual functions of reactive oxygen species response release and reactive oxygen species scavenging, which can effectively improve the common chronic inflammation, oxidative stress and other problems in the healing process of diabetic wounds, thereby facilitating drug release and absorption;

[0049] (3) The present invention selects the traditional Chinese medicine ligustrazine and safflower polysaccharide as active drugs, combines the anti-inflammatory, antioxidant and angiogenesis-promoting effects of ligustrazine with the immunomodulatory and tissue repair functions of safflower polysaccharide, and has a synergistic therapeutic effect of promoting the healing of diabetic wounds, and can be used to prepare drugs that promote the healing of diabetic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1(A) is the H NMR spectrum of the dextran and methacryloylated dextran in Example 1 of the present invention, Figure 1 (B) is the H NMR spectrum of polyphenylene sulfide and hyaluronic acid-polypropylene sulfide in Example 1 of the present invention;

[0051] Figure 2 (A) is a transmission electron microscope image of PPS-HA in Example 1 of the present invention, Figure 2 (B) Transmission electron microscopy image of TMP@PPS-HA;

[0052] Figure 3 This is a white light image of the traditional Chinese medicine hydrogel microneedle used for diabetic wounds in Example 1 of the present invention;

[0053] Figure 4 Inverted microscope images of the Chinese medicine hydrogel microneedles used for diabetic wounds in Example 1 of the present invention taken at different angles;

[0054] Figure 5 3D structural images of the traditional Chinese medicine hydrogel microneedles used for diabetic wounds at different angles in Example 1 of the present invention;

[0055] Figure 6 The mechanical strength test results of the hydrogel microneedles of Example 1 and Comparative Examples 1-3 of the present invention are shown;

[0056] Figure 7 This is an image of the Chinese medicine hydrogel microneedles used for diabetic wounds in Example 1 of the present invention being inserted into pig skin;

[0057] Figure 8 Fluorescence images of different depths of the FITC-labeled traditional Chinese medicine hydrogel microneedle for diabetic wounds after being inserted into pig skin slices;

[0058] Figure 9 (A) is the number of Escherichia coli and Staphylococcus aureus colonies after co-culture on different hydrogels. Figure 9 (B) Plate count images of Escherichia coli and Staphylococcus aureus in different hydrogels;

[0059] Figure 10 (A) Fluorescence images of HT22 cells in each group; Figure 10 (B) Quantitative analysis of ROS fluorescence intensity in HT22 cells;

[0060] Figure 11 (A) Transwell cell migration of HUVEC cells after treatment in each group; Figure 11 (B) Quantitative analysis of the number of migrating cells in the Transwell migration assay;

[0061] Figure 12The wound images of diabetic rats during the hydrogel microneedle drug delivery process in each group;

[0062] Figure 13 This is the wound healing rate curve of each group of hydrogel microneedle treatment of diabetic wounds;

[0063] Figure 14 (A) is the IL-1β expression level in the wound tissue of each group 3 and 7 days after surgery, Figure 14 (B) The expression levels of TNF-α in wound tissues of each group at 3 and 7 days after surgery. DETAILED DESCRIPTION

[0064] The present invention will be further described in detail below with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto. The raw materials and reagents involved in the following examples can all be obtained from commercial channels.

[0065] Example 1

[0066] This embodiment provides a method for preparing a traditional Chinese medicine hydrogel microneedle (DexMA / SPS / TMP@PPS-HA hydrogel microneedle) for diabetic wounds, comprising the following steps:

[0067] 1. Preparation of methacryloylated dextran (DexMA)

[0068] 5.0 g of dextran was weighed and added to a beaker containing 50 mL of DMSO. The beaker was sealed with plastic wrap to prevent solvent evaporation and stirred on a magnetic stirrer for 1 hour until a clear, light yellow, homogeneous solution was formed. Then, 1 g of DMAP was added to the solution and stirred for 0.5 hour until the solution became clear, light green, and homogeneous. Then, 1.5 mL of GMA was added to the solution and stirred for 48 hours in the dark. The reaction solution was then transferred to a 14,000 Da dialysis bag and dialyzed in deionized water for 3 days to remove by-products. The dialyzed reaction solution was freeze-dried for 48 hours to obtain DexMA.

[0069] 2. Preparation of Tetramethylpyrazine-loaded Polyphenylene Sulfide-Hyaluronic Acid Nanomicelles (TMP-PPS@HA)

[0070] 2.1 Preparation of polyphenylene sulfide (PPS)

[0071] Under ice bath conditions, 100 μL (1.15 mmol) of 3-MPA was added to 30 mL of anhydrous tetrahydrofuran and mixed by magnetic stirring; 524 μL (3.45 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was then added, and the reaction mixture was stirred under a nitrogen atmosphere for 30 min; 1.9 mL (21.15 mmol) of propylene sulfide was then added dropwise, and the reaction mixture was stirred at 60°C overnight; 5 mL of H2O was then added to the reaction mixture to quench the reaction, and the product was purified by precipitation in cold methanol, and the solvent was evaporated under reduced pressure to obtain PPS as a yellow oil.

[0072] 2.2 Preparation of amino-modified polyphenylene sulfide (PPS-NH2)

[0073] 158.6 mg (100 μmol) of dried PPS was added to 20 mL of dichloromethane and dissolved by magnetic stirring; then 23 mg (200 μmol) of NHS and 48 mg (250 μmol) of EDCI were added to PPS and stirred at room temperature for 30 min until the solid dissolved; then 133 μL (2 mmol) of ethylenediamine was added dropwise to the mixture and continued to stir overnight at room temperature; then 20 mL of dichloromethane was added to the mixture to dilute the reaction solution, washed with H2O and saturated NaCl in sequence, dried over MgSO4 and filtered, and the solution was concentrated under reduced pressure to obtain PPS-NH2, which was dried for later use.

[0074] 2.3 Preparation of hyaluronic acid-polypropylene sulfide (PPS-HA)

[0075] The sodium salt of hyaluronic acid was dialyzed against a 0.01 mol / L HCl solution overnight and then freeze-dried to obtain acidic hyaluronic acid; 10 mL of H2O and 100 mg of acidic hyaluronic acid were then added to a 50 mL beaker and magnetically stirred for dissolution; 7 mg (60 μmol) of NHS and 14.5 mg (75 μmol) of EDCI were then added to the beaker, and the mixture was stirred at room temperature for 30 min until the solid was completely dissolved to obtain a reaction system; 40 mg of PPS-NH2 was then dissolved in 1 mL of tetrahydrofuran and added dropwise to the reaction system; the reaction was then stirred at room temperature under nitrogen protection for 24 h; the mixture was then dialyzed against water / methanol at a ratio of 1:1 three times for one day and then against distilled water three times for one day; the solvent was then removed by freeze-drying to obtain a PPS-HA conjugate.

[0076] 2.4 Preparation of Tetramethylpyrazine-loaded Polyphenylene Sulfide-Hyaluronic Acid Nanomicelles (TMP@PPS-HA)

[0077] 0.005 g PPS-HA was dissolved in 4 mL PBS to obtain an aqueous phase; 0.01 g TMP was dissolved in 1 mL acetone-ethanol solution (the volume ratio of acetone to ethanol was 3:2) to obtain an organic phase; the aqueous phase was then transferred to a rotary evaporator, the organic phase was added to the stirred aqueous phase, rotary evaporated for 3-5 minutes (40°C, 45 rpm), ultrasonicated for 10 minutes, and filtered through a 0.45 μm microporous filter membrane to obtain TMP@PPS-HA with a TMP content of 5 mg / mL.

[0078] 3. Preparation of DexMA / SPS / TMP@PPS-HA hydrogel microneedles

[0079] 2 mg of safflower polysaccharide (SPS) and 400 mg of DexMA were dissolved in 1 mL of deionized water to form a DexMA / SPS solution; then 1 mL of TMP@PPS-HA nanomicelles was added to the DexMA / SPS solution to obtain a prepolymer solution, and then 2% LAP was added to the prepolymer solution to obtain a gel precursor solution; the gel precursor solution was mixed evenly and then dripped into a polydimethylsiloxane microneedle mold, and the mold was transferred to an acrylic vacuum degassing box; then the vacuum degree in the acrylic vacuum degassing box was adjusted to -100 kPa to fill the mold with the gel precursor solution, and vacuum degassing was repeated several times to expel the air in the mold and the solution; the gel precursor solution was cured by ultraviolet light (365 nm) to obtain a hydrogel needle tip, and then a HA solution with a mass volume percentage concentration of 2% was added to the mold, and the base was obtained by drying at 37°C for 72 h. After demolding, the hydrogel microneedles were obtained, which were recorded as DexMA / SPS / TMP@PPS-HA hydrogel microneedles or D / SPS / T@PH.

[0080] Dextran and methacryloyl dextran were subjected to nuclear magnetic resonance hydrogen spectrum detection respectively. The results are as follows Figure 1 (A) As shown. Figure 1 (A) It can be seen that compared with the H NMR spectrum of dextran, the H NMR spectrum of the modified methacryloyl dextran has obvious chemical shifts of the active hydrogen of the methacryloyl group at 5.6 ppm and 6.1 ppm, indicating the successful synthesis of methacryloyl dextran.

[0081] Polyphenylene sulfide and hyaluronic acid-polypropylene sulfide were tested by nuclear magnetic resonance hydrogen spectrum respectively. The results are as follows Figure 1 (B) is shown. Figure 1(B) It can be seen that the nuclear magnetic resonance hydrogen spectrum of the modified hyaluronic acid-polypropylene sulfide shows methyl peaks (-CH3) at 1.2ppm and 2.5ppm belonging to polyphenylene sulfide, and a methyl peak (-CH3) belonging to hyaluronic acid appears at 1.9ppm. This is because the carboxyl group of hyaluronic acid and the amino group of PPS-NH2 form an amide bond under the catalysis of NHS and EDCI, thereby combining hyaluronic acid with PPS-NH2, indicating the successful synthesis of hyaluronic acid-polypropylene sulfide.

[0082] PPS-HA and TMP@PPS-HA were characterized by transmission electron microscopy. Figure 2 (A) and Figure 2 As shown in (B), Figure 2 (A) is a transmission electron microscope image of PPS-HA. Figure 2 (B) is the transmission electron microscopy image of TMP@PPS-HA. Figure 2 It can be seen that PPS-HA is a nano-spherical micelle. After PPS-HA is loaded with TMP, the TMP@PPS-HA obtained is still a nano-spherical micelle, but the size of TMP@PPS-HA is significantly larger than that of PPS-HA.

[0083] Comparative Example 1

[0084] The preparation method of the hydrogel microneedles provided in this comparative example is the same as that in Example 1, except that the hydrogel microneedles are not loaded with ligustrazine and safflower polysaccharide active ingredients, and comprises the following steps:

[0085] 1. Preparation of DexMA

[0086] 5.0 g of dextran was weighed and added to a beaker containing 50 mL of DMSO. The beaker was sealed with plastic wrap to prevent solvent evaporation and stirred on a magnetic stirrer for 1 hour until a clear, light yellow, homogeneous solution was formed. Then, 1 g of DMAP was added to the solution and stirred for 0.5 hour until the solution became clear, light green, and homogeneous. Then, 1.5 mL of GMA was added to the solution and stirred for 48 hours in the dark. The reaction solution was then transferred to a 14,000 Da dialysis bag and dialyzed in deionized water for 3 days to remove by-products. The dialyzed reaction solution was freeze-dried for 48 hours to obtain DexMA.

[0087] 2. Preparation of DexMA hydrogel microneedles

[0088] 200 mg of DexMA was weighed and placed in a centrifuge tube. 0.9 mL of deionized water was added and vortexed to completely dissolve the DexMA. 2% LAP was then added and mixed thoroughly using a vortexer to obtain a 20% DexMA gel precursor solution. The gel precursor solution was dripped into a polydimethylsiloxane microneedle mold, and the mold was transferred to an acrylic vacuum degassing box. The vacuum degree in the acrylic vacuum degassing box was then adjusted to -100 kPa to fill the mold with the gel precursor solution. Vacuum degassing was repeated several times to expel air from the mold and the solution. The gel precursor solution was cured by UV light (365 nm) for about 60 seconds to obtain a 20% DexMA hydrogel needle tip. 2% HA solution was then added to the mold and dried at 37°C for 72 h to obtain a substrate. After demolding, the hydrogel microneedles were obtained, which were recorded as DexMA hydrogel microneedles.

[0089] Comparative Example 2

[0090] The preparation method of the hydrogel microneedles provided in this comparative example is the same as that in Example 1, except that the microneedles in the hydrogel are not loaded with ligustrazine. Specifically, the method includes the following steps:

[0091] 1. Preparation of DexMA

[0092] 5.0 g of dextran was weighed and added to a beaker containing 50 mL of DMSO. The beaker was sealed with plastic wrap to prevent solvent evaporation and stirred on a magnetic stirrer for 1 hour until a clear, light yellow, homogeneous solution was formed. Then, 1 g of DMAP was added to the solution and stirred for 0.5 hour until the solution became clear, light green, and homogeneous. Then, 1.5 mL of GMA was added to the solution and stirred for 48 hours in the dark. The reaction solution was then transferred to a 14,000 Da dialysis bag and dialyzed in deionized water for 3 days to remove by-products. The dialyzed reaction solution was freeze-dried for 48 hours to obtain DexMA.

[0093] 2. Preparation of DexMA / SPS hydrogel microneedles

[0094] 2 mg of safflower polysaccharide (SPS) and 400 mg of DexMA were dissolved in 1 mL of deionized water to form a DexMA / SPS solution; then 1 mL of TMP@PPS-HA nanomicelles was added to the DexMA / SPS solution to obtain a prepolymer solution, and then 2% LAP was added to the prepolymer solution to obtain a gel precursor solution; the gel precursor solution was mixed evenly and then dripped into a polydimethylsiloxane microneedle mold, and the mold was transferred to an acrylic vacuum degassing box; then the vacuum degree in the acrylic vacuum degassing box was adjusted to -100 kPa to fill the mold with the gel precursor solution, and vacuum degassing was repeated several times to expel the air in the mold and the solution; the gel precursor solution was cured by ultraviolet light (365 nm) to obtain a hydrogel needle tip, and then a HA solution with a mass volume percentage concentration of 2% was added to the mold, and the base was obtained by drying at 37°C for 72 h. After demolding, the hydrogel microneedle was obtained, which was recorded as DexMA / SPS hydrogel microneedle or D / SPS.

[0095] Comparative Example 3

[0096] The preparation method of the hydrogel microneedles provided in this comparative example is the same as that in Example 1, except that the microneedles in the hydrogel are not loaded with safflower polysaccharide, and comprises the following steps:

[0097] 1. Preparation of DexMA

[0098] 5.0 g of dextran was weighed and added to a beaker containing 50 mL of DMSO. The beaker was sealed with plastic wrap to prevent solvent evaporation and stirred on a magnetic stirrer for 1 hour until a clear, light yellow, homogeneous solution was formed. Then, 1 g of DMAP was added to the solution and stirred for 0.5 hour until the solution became clear, light green, and homogeneous. Then, 1.5 mL of GMA was added to the solution and stirred for 48 hours in the dark. The reaction solution was then transferred to a 14,000 Da dialysis bag and dialyzed in deionized water for 3 days to remove by-products. The dialyzed reaction solution was freeze-dried for 48 hours to obtain DexMA.

[0099] 2. Preparation of TMP-PPS@HA

[0100] 2.1 Preparation of PPS

[0101] Under ice bath conditions, 100 μL (1.15 mmol) of 3-MPA was added to 30 mL of anhydrous tetrahydrofuran and mixed by magnetic stirring; 524 μL (3.45 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was then added, and the reaction mixture was stirred under a nitrogen atmosphere for 30 min; 1.9 mL (21.15 mmol) of propylene sulfide was then added dropwise, and the reaction mixture was stirred at 60°C overnight; 5 mL of H2O was then added to the reaction mixture to quench the reaction, and the product was purified by precipitation in cold methanol, and the solvent was evaporated under reduced pressure to obtain PPS as a yellow oil.

[0102] Preparation of 2.2PPS-NH2

[0103] 158.6 mg (100 μmol) of dried PPS was added to 20 mL of dichloromethane and dissolved by magnetic stirring; then 23 mg (200 μmol) of NHS and 48 mg (250 μmol) of EDCI were added to PPS and stirred at room temperature for 30 min until the solid dissolved; then 133 μL (2 mmol) of ethylenediamine was added dropwise to the mixture and continued to stir overnight at room temperature; then 20 mL of dichloromethane was added to the mixture to dilute the reaction solution, washed with H2O and saturated NaCl in sequence, dried over MgSO4 and filtered, and the solution was concentrated under reduced pressure to obtain PPS-NH2, which was dried for later use.

[0104] Preparation of 2.3PPS-HA

[0105] The sodium salt of hyaluronic acid was dialyzed against a 0.01 mol / L HCl solution overnight and then freeze-dried to obtain acidic hyaluronic acid; 10 mL of H2O and 100 mg of acidic hyaluronic acid were then added to a 50 mL beaker and magnetically stirred for dissolution; 7 mg (60 μmol) of NHS and 14.5 mg (75 μmol) of EDCI were then added to the beaker, and the mixture was stirred at room temperature for 30 min until the solid was completely dissolved to obtain a reaction system; 40 mg of PPS-NH2 was then dissolved in 1 mL of tetrahydrofuran and added dropwise to the reaction system; the reaction was then stirred at room temperature under nitrogen protection for 24 h; the mixture was then dialyzed against water / methanol at a ratio of 1:1 three times for one day and then against distilled water three times for one day; the solvent was then removed by freeze-drying to obtain a PPS-HA conjugate.

[0106] 2.4 Preparation of TMP@PPS-HA

[0107] 0.005 g PPS-HA was dissolved in 4 mL PBS to obtain an aqueous phase; 0.01 g TMP was dissolved in 1 mL acetone-ethanol solution (the volume ratio of acetone to ethanol was 3:2) to obtain an organic phase; the aqueous phase was then transferred to a rotary evaporator, the organic phase was added to the stirred aqueous phase, rotary evaporated for 3-5 minutes (40°C, 45 rpm), ultrasonicated for 10 minutes, and filtered through a 0.45 μm microporous filter membrane to obtain TMP@PPS-HA with a TMP content of 5 mg / mL.

[0108] 3. Preparation of DexMA / TMP@PPS-HA hydrogel microneedles

[0109] 200 mg of DexMA was dissolved in 1 mL of deionized water to form a DexMA solution; then 1 mL of TMP@PPS-HA nanomicelles was added to the DexMA solution to obtain a prepolymer solution, and then 2% LAP was added to the prepolymer solution to obtain a gel precursor solution; the gel precursor solution was mixed evenly and then dripped into a polydimethylsiloxane microneedle mold, and the mold was transferred to an acrylic vacuum degassing box; then the vacuum degree in the acrylic vacuum degassing box was adjusted to -100 kPa to fill the mold with the gel precursor solution, and vacuum degassing was repeated several times to expel the air in the mold and the solution; the gel precursor solution was cured by ultraviolet light (365 nm) to obtain a hydrogel needle tip, and then a HA solution with a mass volume percentage concentration of 2% was added to the mold, and the base was obtained by drying at 37°C for 72 h. After demolding, the hydrogel microneedles were obtained, which were recorded as DexMA / TMP@PPS-HA hydrogel microneedles or D / T@PH.

[0110] 1. Morphology test

[0111] The morphology of the Chinese medicine hydrogel microneedles for diabetic wounds prepared in Example 1 was characterized. Figure 3 、 Figure 4 and Figure 5 shown. Figure 3 White light image of Chinese medicine hydrogel microneedle used for diabetic wounds, Figure 3 It can be observed that each microneedle patch has a 10×10 needle array. Figure 4 Inverted microscope images of Chinese medicine hydrogel microneedles used on diabetic wounds from different angles. Figure 4 The prepared microneedles exhibited well-defined conical tips, with the tips evenly distributed throughout the array. Each microneedle was pyramidal, 800 μm high, with a base diameter of approximately 200 μm and a spacing of 500 μm. The arrangement formed a regular array with a smooth surface. Figure 5 3D structural images of Chinese medicine hydrogel microneedles used for diabetic wounds from different angles. Figure 5 It can be observed that the microneedles have regular morphology and are evenly arranged on the backing. The needle tips are sharp and have the ability to effectively penetrate the stratum corneum of the skin.

[0112] 2. Mechanical strength test

[0113] The Chinese medicine hydrogel microneedles for diabetic wounds prepared in Example 1 and the hydrogel microneedles prepared in Comparative Examples 1-3 were placed upside down on the sample stage of a universal testing machine, with the needle tip facing upward, and compressed downward at a constant rate of 0.05 mm / s. The mechanical strength test results are shown in FIG. Figure 6 shown.

[0114] from Figure 6 It can be seen that the mechanical properties of the traditional Chinese medicine hydrogel microneedles for diabetic wounds prepared by simultaneously introducing ligustrazine and brown sugar polysaccharide in Example 1 are only lower than those of the hydrogel microneedles in Comparative Example 1, indicating that the traditional Chinese medicine hydrogel microneedles for diabetic wounds have strong mechanical strength, indicating that they have sufficient mechanical properties to penetrate the skin.

[0115] 3. In vitro microneedle simulated puncture and skin penetration depth test

[0116] 1. Experimental Preparation

[0117] Trim the pig skin tissue into 1.5×1.5 cm 2 The frozen pig skin was moistened in 37°C water and air-dried for 30 min before use.

[0118] 2. In vitro microneedle simulated puncture

[0119] The Chinese medicine hydrogel microneedle patch for diabetic wound prepared in Example 1 was inserted into the pig skin. Figure 7 As shown. Figure 7 An array of microneedle holes can be observed on the pig skin. The pig skin surface is punctured with holes of uniform size and the same number as the microneedle tips, indicating that the hydrogel microneedles prepared by the present invention can penetrate the skin.

[0120] 3. Skin penetration depth test

[0121] Fluorescein isothiocyanate (FITC)-labeled traditional Chinese medicine hydrogel microneedles for diabetic wounds were applied to pig skin to evaluate the transdermal release ability of the microneedles.

[0122] After FITC-labeled traditional Chinese medicine hydrogel microneedles for diabetic wounds were inserted into pig skin, confocal microscopy was used to scan the coronal surface of the pig skin slices (from the stratum corneum to the dermis) layer by layer at 20 μm intervals to observe the skin penetration of the microneedles. The results are as follows: Figure 8 As shown. Figure 8It can be seen that the insertion depth of the traditional Chinese medicine hydrogel microneedles used for diabetic wounds in the pig skin is 220μm, indicating that the traditional Chinese medicine hydrogel microneedles used for diabetic wounds can penetrate the stratum corneum to reach the epidermis and upper dermis to achieve transdermal delivery of drugs.

[0123] IV. In vitro antibacterial test

[0124] 1. Bacteria culture

[0125] The revived Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were cultured to the logarithmic growth phase, the culture medium was removed by centrifugation, and the bacteria were resuspended in PBS to adjust the bacterial concentration to 1×10 8 CFU / mL, and Escherichia coli and Staphylococcus aureus bacterial liquids were obtained.

[0126] 2. Experimental methods

[0127] 200 μL of the gel precursor solutions prepared in Comparative Examples 1-3 and Example 1 were respectively added to a 24-well plate, and the operation was repeated twice. The hydrogels of Example 1 and Comparative Examples 1-3 were cured by ultraviolet light (365 nm) to obtain hydrogels, respectively. Each hydrogel had 2 wells. Two wells were selected as blank groups, which were not treated with any drug and were sterilized by ultraviolet irradiation overnight.

[0128] 100 μL of 1×10 8 CFU / mL of Escherichia coli solution, and then add 100 μL of 1×10 8 CFU / mL of Staphylococcus aureus liquid was added to each well surface and 1 mL of sterile saline was added. The plate was then placed in a 37°C constant temperature shaker for 24 hours. After the incubation, the liquid was diluted with sterile saline for gradient dilution. 6 The dilution was spread on LB agar plates and incubated upside down in a 37°C incubator for 24 hours. Finally, the number of colonies was counted and photos were taken to record the growth of the colonies on the agar plates. Figure 9 shown. Figure 9 (A) is the number of Escherichia coli and Staphylococcus aureus colonies after co-culture on different hydrogels. Figure 9 (B) Plate count images of Escherichia coli and Staphylococcus aureus in different hydrogels.

[0129] In vitro antibacterial experiments were conducted to evaluate the in vitro antibacterial effects of hydrogel microneedles on Escherichia coli and Staphylococcus aureus. Figure 9 (A) It can be observed that the hydrogels prepared in Comparative Example 3 and Example 1 have significant antibacterial effects on Escherichia coli and Staphylococcus aureus. Figure 9(B) It can be seen that compared with the blank group, the number of Escherichia coli and Staphylococcus aureus in the hydrogel of Example 1, Comparative Example 2 and Comparative Example 3 was significantly reduced, indicating that the addition of red sugar polysaccharide or polyphenylene sulfide-hyaluronic acid nanomicelles loaded with ligustrazine to the hydrogel can reduce the number of colonies. No growth of Escherichia coli and Staphylococcus aureus colonies was observed on the agar plate corresponding to the bacterial liquid cultured with the hydrogel of Example 1, indicating that the hydrogel obtained by adding red sugar polysaccharide and polyphenylene sulfide-hyaluronic acid nanomicelles loaded with ligustrazine has the best antibacterial effect on Escherichia coli and Staphylococcus aureus.

[0130] 5. Effect of hydrogel on ROS expression level in HT22 cells

[0131] In this experimental example, the DCFH-DA probe was used to detect the ROS level in HT22 cells to evaluate the ROS scavenging ability of the hydrogel microneedles prepared in Example 1 and Comparative Examples 1-3 at the cellular level.

[0132] The gel precursor solutions in Example 1 and Comparative Examples 1-3 were cured under 365 nm ultraviolet light to obtain the hydrogels in Example 1 and Comparative Examples 1-3.

[0133] HT22 cells were plated at 5 × 10 3 The cells were seeded in a 48-well plate at a density of 100 μg / mL and cultured for 24 hours to allow them to fully adhere to the wall. HT22 cells were divided into Control group, PMA group, DexMA group, D / SPS group, D / T@PH group, and D / SPS / T@PH group. Among them, the Control group was a negative control group, and HT22 cells were treated with PBS; the PMA group was a positive control group, and HT22 cells were treated with phorbol myristate acetate (PMA); the DexMA group treated HT22 cells with a culture medium containing the hydrogel extract of Comparative Example 1 for 24 hours; the D / SPS group treated HT22 cells with a culture medium containing the hydrogel extract of Comparative Example 2 for 24 hours; the D / T@PH group treated HT22 cells with a culture medium containing the hydrogel extract of Comparative Example 3 for 24 hours; and the D / SPS / T@PH group treated HT22 cells with a culture medium containing the hydrogel extract of Example 1 for 24 hours.

[0134] After each treatment, the HT22 cells were washed 2-3 times with PBS to remove the residual culture medium, and then stained with DCFH-DA for 20 minutes. After the staining was completed, the HT22 cells in each group were washed again with PBS to remove the unbound probe. Finally, the ROS scavenging ability of each group was imaged using a fluorescence microscope. The results are shown in Figure 2. Figure 10 (A) and the mean fluorescence intensity was quantified by Image J software. The quantitative analysis results are shown in Figure 10 (B) and Table 1.

[0135] Table 1 Comparison of ROS levels in HT22 cells in each group

[0136] Group DCFH-DA mean fluorescence intensity PMA group 644.49±43.04 Control group 222.56±56.15 DexMA group 128.18±9.65 D / SPS group 56.02±16.60 D / T@PH group 106.42±8.98 D / SPS / T@PH group 26.81±19.95

[0137] from Figure 10 (A) It can be seen that the fluorescence intensity of DCFH-DA in the PMA group was the highest. Compared with the PMA group, the fluorescence intensity of DCFH-DA in the D / SPS group and the D / SPS / T@PH group was significantly reduced. Figure 10 As shown in (B) and Table 1, compared with the control group, the ROS level of HT22 cells in the D / T@PH group was reduced (P < 0.05), the ROS level of HT22 cells in the D / SPS group was significantly reduced (P < 0.01), and the ROS level of HT22 cells in the D / SPS / T@PH group was significantly reduced (P < 0.001), with statistically significant differences. However, the ROS level of HT22 cells in the DexMA group was not statistically different from that in the control group. Compared with the PMA group, the ROS levels of HT22 cells in the control group, DexMA group, D / SPS group, D / T@PH group, and D / SPS / T@PH group were significantly reduced, with statistically significant differences.

[0138] VI. Transwell cell migration assay

[0139] The gel precursor solutions in Example 1 and Comparative Examples 1-3 were cured under 365 nm ultraviolet light to obtain the hydrogels in Example 1 and Comparative Examples 1-3.

[0140] HUVEC cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) in an environment of 5% CO2 and a temperature of 37°C. 4 HUVECs were seeded into the upper chamber of the Transwell plate. The HUVEC cells were divided into Control group, DexMA group, D / SPS group, D / T@PH group, and D / SPS / T@PH group. Among them, PBS solution was added to the Control group; the DexMA group added the hydrogel of Comparative Example 1; the D / SPS group added the hydrogel of Comparative Example 2; the D / T@PH group added the hydrogel of Comparative Example 3; and the D / SPS / T@PH group added the hydrogel of Example 1. After the lower chamber was treated according to the above grouping, the cells were cultured for 12 hours. Subsequently, the migrating cells that had passed through the basolateral membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Cell invasion was observed and recorded using an optical microscope, and the results are shown in the figure. Figure 11 shown. Figure 11 (A) Transwell cell migration of HUVEC cells after treatment in each group; Figure 11(B) Quantitative analysis of the number of migrating cells in the Transwell migration assay.

[0141] As can be seen from Figure 11(A), after 4 hours of culture, the D / SPS, D / T@PH, and D / SPS / T@PH groups all had a significant effect on promoting angiogenesis. The tubular structure could be clearly observed under the bright field of the microscope. At the same time, the number of vascular branches, the number of obvious nodes, and the length of the blood vessels under the field of view were counted to illustrate the effects of different hydrogel groups on the formation of new blood vessels. It was found that after 4 hours of culture, the number of vascular branches in the D / SPS / T@PH group was (200.70±11.24), the number of obvious nodes was (360.66±39.59), and the length of the blood vessels was (32745.83±2560.06) μm, which were significantly higher than those of the other groups.

[0142] VII. Experiment on promoting full-thickness skin wound healing in diabetic rats with hydrogel microneedles

[0143] 1. Main reagents

[0144] The main reagents used in the experiment are shown in Table 2.

[0145] Table 2 Main reagents

[0146] Reagent name Manufacturer Anhydrous ethanol Sinopharm Chemical Reagent Co., Ltd. Xylene Sinopharm Chemical Reagent Co., Ltd. Hematoxylin-eosin stain Wuhan Savier Biotechnology Co., Ltd. hydrochloric acid Sinopharm Chemical Reagent Co., Ltd. ammonia Sinopharm Chemical Reagent Co., Ltd. Neutral gum Sinopharm Chemical Reagent Co., Ltd. HRP-labeled goat anti-rabbit Wuhan Sanying Biotechnology Co., Ltd. HRP-labeled goat anti-mouse Wuhan Sanying Biotechnology Co., Ltd. EDTA (PH8.0) antigen retrieval solution Wuhan Savier Biotechnology Co., Ltd. EDTA (PH=9.0) antigen retrieval solution Wuhan Savier Biotechnology Co., Ltd. Citric acid (PH6.0) antigen retrieval solution Wuhan Savier Biotechnology Co., Ltd. PBS buffer Wuhan Savier Biotechnology Co., Ltd. Bovine serum albumin (BSA) Beijing Solebow Technology Co., Ltd. HRP-labeled goat anti-rabbit IgG Wuhan Savier Biotechnology Co., Ltd. HRP-labeled goat anti-mouse IgG Wuhan Savier Biotechnology Co., Ltd. Anti-fluorescence fading mounting medium Wuhan Savier Biotechnology Co., Ltd. Streptozotocin (STZ) Shanghai Aladdin Reagent Co., Ltd.

[0147] 2. Experimental instruments

[0148] The instruments and equipment used in the experiment are shown in Table 3.

[0149] Table 3 Experimental instruments

[0150] name model Manufacturer Dehydrator JJ-12J Wuhan Junjie Electronics Co., Ltd. Embedding Machine JB-P5 Wuhan Junjie Electronics Co., Ltd. Pathology slicer RM2016 Shanghai Leica Instruments Co., Ltd. Frozen Table JB-L5 Wuhan Junjie Electronics Co., Ltd. Tissue spreading machine KD-P Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd. oven DHG-9140A Shanghai Huitai Instrument Manufacturing Co., Ltd. Slides and coverslips 10212432C Jiangsu Shitai Experimental Equipment Co., Ltd. High-speed refrigerated centrifuge TGL-16 Hunan Xiangyi Laboratory Instrument Development Co., Ltd. -80℃ Refrigerator DW-HL218 Zhongke Meiling Cryogenic Technology Co., Ltd. Decolorization shaker (pendulum type) TSY-B Wuhan Savier Biotechnology Co., Ltd. Decolorization shaker (horizontal) TSY-A Wuhan Savier Biotechnology Co., Ltd. Constant temperature water bath B-260 Shanghai Yarong Biochemical Instrument Factory tissue grinder Tissuelyser-24 Shanghai Jingxin Industrial Development Co., Ltd. Ice Maker JKG-20 Changshu Linke Electric Co., Ltd. Inverted fluorescence microscope Nikon Eclipse Ti-SR Nikon Corporation of Japan

[0151] 3. Experimental Animals

[0152] Male Sprague-Dawley rats weighing 210–240 g were used in this experiment and were purchased from Guangzhou Ruige Biotechnology Co., Ltd. (license number: SCKX(Yue)2023-0059, quality certificate: NO. 44827200012122). All animal experimental protocols were reviewed and approved by the Ruige Biological Laboratory Animal Ethics Committee (approval number: 20240607-002).

[0153] 4. Construction of diabetic rat model

[0154] Twenty-five male Sprague-Dawley rats were fasted for 12 hours before the experiment and given a fixed amount of water. On the day of the experiment, the rats were weighed, and blood and urine were collected from the tail vein. Baseline blood glucose and urine glucose levels were measured using a glucometer and test strips, respectively. Solution A was prepared by adding 2.1g of citric acid to 100mL of double-distilled water; solution B was prepared by adding 2.94g of sodium citrate to 100mL of double-distilled water. Solution A and solution B were mixed in a 1:1 ratio and the pH was adjusted to 4.2-4.5 to prepare a sodium citrate buffer solution. Streptozotocin (STZ) was dissolved in the sodium citrate buffer solution to prepare a 1% STZ solution by mass volume. A single intraperitoneal injection of 60mg / kg of STZ solution was administered. If the rat's blood glucose level remained >16.65mmol / L for two weeks, the diabetic rat model was considered successfully established and could be used for subsequent experiments.

[0155] 5. Construction of diabetic rat wound model and drug administration

[0156] Twenty-five male SD rats with successfully established diabetic models were anesthetized with an intraperitoneal injection of 0.5 mL of 1% sodium pentobarbital solution and an intramuscular injection of 0.02 mL of Sumenxin. The hair on the back of the rats was then shaved using a shaver, covering an area of ​​5 cm x 7 cm. Further hair removal was performed with a depilatory cream and the rats were cleaned. The exposed skin on the back of the rats was then disinfected with iodine. After disinfection, the skin was lifted along the midline of the back, and four circular, full-thickness wounds, each 1 cm in diameter, were created symmetrically on the back using a skin scraper.

[0157] Diabetic rats with a successfully established full-thickness skin wound model were randomly divided into five groups: Control, DexMA, D / SPS, D / T@PH, and D / SPS / T@PH. The Control group served as a blank control group, directly exposed to the external environment without any other treatment. The other groups served as drug-treated groups: the DexMA group was treated with the DexMA hydrogel microneedle patch of Comparative Example 1; the D / SPS group was treated with the DexMA / SPS hydrogel microneedle patch of Comparative Example 2; the D / T@PH group was treated with the DexMA / SPS / TMP@PPS-HA hydrogel microneedle patch of Comparative Example 3; and the D / SPS / T@PH group was treated with the traditional Chinese medicine hydrogel microneedle patch for diabetic wounds of Example 1. After a simple debridement with sterile saline, hydrogel microneedles were applied to the wound surface according to the corresponding group. The wounds of rats in each group were covered and fixed with two layers of sterile dry gauze, and the dressings were changed daily. After all treatments were completed, the rats were returned to their cages for maintenance.

[0158] 6. Effects of hydrogel microneedles on full-thickness wounds in diabetic rats

[0159] The wound area was recorded using a digital camera after treatment on days 0, 3, 7, 10, and 14. Figure 12 As shown. Figure 12 It can be seen that the wounds in all groups gradually shrank over time, however, there were differences in the speed of wound healing between the groups. On the third day, the wound area of ​​the D / SPS / T@PH group was significantly reduced, and its healing speed was significantly accelerated compared with the Control and DexMA groups, showing a better repair effect. By the seventh day, the wound area of ​​the D / SPS / T@PH group was further reduced, the wound edges were smoother, and the tissue contraction was tighter, indicating that its healing process was more orderly and efficient. In contrast, the wound healing progress of the Control and DexMA groups was slower. By the 14th day, the wounds of the D / SPS / T@PH group were almost completely healed, showing the best healing effect, while the wounds of the Control and DexMA groups had not yet completely closed, further highlighting the significant advantage of the D / SPS / T@PH group in promoting diabetic wound healing.

[0160] Analysis using ImageJ Figure 12 The wound areas of the DexMA group, D / SPS group, D / T@PH group, and D / SPS / T@PH group on days 0, 3, 7, and 14 were used to calculate the wound healing rates using the following formula:

[0161]

[0162] S t is the wound area on day t after treatment, and S0 is the initial wound area. Based on the results, the wound healing rate curves of each group were drawn, as shown in Figure 13 As shown. Figure 13It was found that in promoting diabetic wound healing, the wound healing rate of the D / SPS / T@PH group was significantly higher than that of the other groups throughout the healing process. On the third day of administration, the wound healing rate of the control group was only (31.72±3.80)%, the wound healing rate of the D / SPS group was (46.44±5.36)%, and the wound healing rate of the D / T@PH group was (44.96±0.77)%. However, the wound healing rate of the D / SPS / T@PH group reached (51.14±2.09)%, significantly higher than that of the other groups. On the seventh day of administration, the wound healing rate of the control group was (60.41±8.16)%, the wound healing rate of the DexMA group was (61.26±4.27)%, and the wound healing rate of the D / SPS / T@PH group further increased to (74.88±2.89)%. The wound healing rate of the D / SPS / T@PH group was much higher than that of the control and DexMA groups. On day 10 of administration, the wound healing rates in the control group were (83.99±3.52)%, and those in the DexMA group were (86.46±1.09)%. The wound healing rates in the D / SPS / T@PH group were significantly superior to those in both the control and DexMA groups, reaching (91.87±2.04)%, approaching complete healing. These data demonstrate that the D / SPS / T@PH hydrogel microneedles significantly promote wound healing in the early, middle, and late stages of diabetic wounds, accelerating wound closure and improving wound healing quality.

[0163] 7. Effects of hydrogel microneedles on wound skin tissue in diabetic rats

[0164] Skin tissues from the wounds of diabetic rats in each group were collected on the 3rd and 7th days of treatment and subjected to immunohistochemical staining. The processing process was as follows:

[0165] (1) Paraffin-embedded tissue sections

[0166] Sampling: Fresh tissue was fixed in 4% paraformaldehyde for at least 24 hours. The tissue was removed from the fixative and trimmed flat with a scalpel in a fume hood. The trimmed tissue and the corresponding label were placed in a dehydration box.

[0167] Dehydration: Place the dehydration box in a hanging basket and place it in a dehydrator for gradient alcohol dehydration treatment: 75% alcohol for 4 hours; 85% alcohol for 2 hours; 90% alcohol for 2 hours; 95% alcohol for 1 hour; anhydrous ethanol I for 30 minutes; anhydrous ethanol II for 30 minutes; alcohol benzene for 5-10 minutes; xylene I for 5-10 minutes; xylene II for 5-10 minutes; wax I for 1 hour; wax II for 1 hour; wax III for 1 hour.

[0168] Embedding: After dehydration, the tissue is immersed in melted wax and then transferred to an embedding machine for embedding. Melted wax is poured into the embedding frame. When the wax is about to solidify, the tissue is removed from the dehydration box and placed in the embedding frame according to the embedding instructions and labeled. The embedding frame is placed in a -20°C freezer to cool. Once the wax is completely solidified, the wax block is removed and trimmed.

[0169] Sectioning: Place the trimmed wax block on a paraffin microtome and cut into 4 μm thick slices. Place the slices on a 40°C warm water surface on the microtome to fully flatten them. Use a glass slide to pick up the tissue slices and bake them in a 60°C oven until the water evaporates and the wax is completely melted. Remove the slices and store at room temperature until needed.

[0170] (2) Immunohistochemical staining

[0171] Dewax the paraffin sections to water: place the sections in xylene I for 15 minutes, xylene II for 15 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and finally rinse with distilled water.

[0172] Antigen retrieval: Place the tissue sections in a retrieval box filled with EDTA antigen retrieval buffer (pH 9.0) and perform antigen retrieval in a microwave oven. Heat on medium heat for 8 minutes until boiling, then reduce heat and hold for 8 minutes. Then, switch to medium-low heat for 7 minutes. During this process, prevent excessive evaporation of the buffer to prevent drying of the slides. After cooling naturally, place the slides in PBS and wash three times for 5 minutes each on a decolorizing shaker.

[0173] Block endogenous peroxidase: Place the sections in 3% hydrogen peroxide solution (hydrogen peroxide: pure water = 1:9) and incubate at room temperature in the dark for 25 minutes. Place the slides in PBS and wash them three times on a decolorizing shaker, each time for 5 minutes.

[0174] BSA or serum blocking: After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue to prevent the antibody from flowing away. Add 3% BSA or 10% normal rabbit serum in the circle to evenly cover the tissue, and block at room temperature for 30 minutes.

[0175] Add primary antibody: Gently shake off the blocking solution and add the primary antibody prepared in PBS at a specific ratio to the sections. Place the sections flat in a humidified chamber and incubate overnight at 4°C. The primary antibody against IL-1β is HRP-conjugated goat anti-rabbit at a volume ratio of 1:100; the primary antibody against TNF-α is HRP-conjugated goat anti-mouse at a volume ratio of 1:100.

[0176] Add secondary antibody: Wash slides three times in PBS on a decolorizing shaker for 5 minutes each wash. After briefly drying the sections, add a secondary antibody of the same species as the primary antibody to the tissue and incubate at room temperature for 50 minutes. The secondary antibody for IL-1β is HRP-conjugated goat anti-rabbit IgG at a 1:200 volume ratio; the secondary antibody for TNF-α is HRP-conjugated goat anti-mouse IgG at a 1:200 volume ratio.

[0177] DAB staining: Wash slides three times in PBS on a destaining shaker for 5 minutes each time. After the sections are lightly dried, add freshly prepared DAB staining solution to the circle. Monitor the color development time under a microscope. A positive color is brownish-yellow. Rinse the sections with tap water to terminate the color development.

[0178] Counterstaining of cell nuclei: After counterstaining with Harris hematoxylin for 3 minutes, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse again with tap water, return to blue with ammonia water, and finally rinse with running water.

[0179] Dehydration and sealing: Place the slices in 75% alcohol for 6 minutes, 85% alcohol for 6 minutes, anhydrous ethanol I for 6 minutes, anhydrous ethanol II for 6 minutes, and xylene I for 5 minutes to dehydrate and make them transparent. Take out the slices from xylene, dry them slightly, and then seal them with neutral gum.

[0180] After immunohistochemical staining, the sections were observed under an optical microscope, and images were collected and analyzed. Figure 14 (A) and Figure 14 As shown in (B), Figure 14 (A) is the IL-1β expression level in the wound tissue of each group 3 and 7 days after surgery, Figure 14 (B) The expression levels of TNF-α in wound tissues of each group at 3 and 7 days after surgery.

[0181] from Figure 14As can be seen in the IL-1β staining results, the control group displayed a strong brown staining signal on both days 3 and 7, indicating high expression of the inflammatory cytokine IL-1β. IL-1β expression levels in the DexMA and D / SPS groups were slightly lower than in the control group. The IL-1β staining signals in the D / T@PH and D / SPS / T@PH groups were the weakest, indicating a greater decrease in IL-1β expression in these groups compared to the control group. Regarding TNF-α staining, the control group displayed a strong brown staining signal on day 3, indicating high TNF-α expression. The TNF-α signal in the DexMA and D / SPS groups weakened somewhat, while the D / T@PH and D / SPS / T@PH groups had the weakest signal, indicating a significant decrease in TNF-α expression. These data demonstrate that TCM hydrogel microneedles applied to diabetic wounds can reduce the expression of inflammatory cytokines, such as IL-1β and TNF-α, in the skin tissue of diabetic rat wounds.

[0182] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A Chinese medicine hydrogel microneedle for diabetic wounds, comprising a needle tip and a base, characterized in that: The raw materials for preparing the needle tip consist of methacryloyl dextran, polyphenylene sulfide-hyaluronic acid nano-micelles loaded with ligustrazine, safflower polysaccharide and a light curing agent.

2. The Chinese medicine hydrogel microneedle for diabetic wounds according to claim 1, characterized in that: The mass ratio of the methacryloylated dextran to safflower polysaccharide is (200-400):1, and the mass ratio of the polyphenylene sulfide-hyaluronic acid nanomicelles loaded with ligustrazine to safflower polysaccharide is (1-15) mL:2 mg.

3. The Chinese medicine hydrogel microneedle for diabetic wounds according to claim 1 or 2, characterized in that: The content of ligustrazine in the ligustrazine-loaded polyphenylene sulfide-hyaluronic acid nanomicelles is 5-15 mg / mL.

4. The Chinese medicine hydrogel microneedle for diabetic wounds according to claim 1, characterized in that: The photocuring agent is selected from at least one of phenyl-2,4,6-trimethylbenzoyl phosphinate lithium and 2-hydroxy-2-methyl-1-phenyl-1-propanone; the mass of the photocuring agent is 1%-3% of the total mass of methacryloylated dextran, polyphenylene sulfide-hyaluronic acid nano-micelles loaded with ligustrazine, safflower polysaccharide and the photocuring agent.

5. The Chinese medicine hydrogel microneedle for diabetic wounds according to claim 1, characterized in that: The methacryloyl dextran is prepared by the following steps: (1) dissolving dextran in dimethyl sulfoxide to obtain a dextran solution, adding 4-dimethylaminopyridine to the dextran solution and stirring until the solution turns clear and light green, to obtain a mixed solution A; (2) Add glycidyl methacrylate to the mixed solution A, stir in the dark for 24-72 hours, and then freeze-dry for 48-72 hours to obtain the product.

6. The Chinese medicine hydrogel microneedle for diabetic wounds according to claim 5, characterized in that: The mass ratio of dextran to 4-dimethylaminopyridine is (5-7):

1.

7. The Chinese medicine hydrogel microneedle for diabetic wounds according to claim 1, characterized in that: The polyphenylene sulfide-hyaluronic acid nano-micelles loaded with ligustrazine are prepared by the following steps: Preparation of amino-modified polyphenylene sulfide: (1) dissolving polyphenylene sulfide in dichloromethane, and then dispersing N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in the dichloromethane containing polyphenylene sulfide to obtain a mixture; (2) adding ethylenediamine dropwise to the mixture and stirring overnight to obtain a reaction solution, diluting the reaction solution with dichloromethane, washing and drying with a solid desiccant, filtering, and concentrating the filtrate under reduced pressure to obtain amino-modified polyphenylene sulfide; Preparation of hyaluronic acid-polypropylene sulfide: (1) dialyzing hyaluronic acid sodium salt against a 0.01-0.02 mol / L hydrochloric acid solution and freeze-drying to obtain acidic hyaluronic acid; (2) dissolving acidic hyaluronic acid in water to obtain an aqueous solution of acidic hyaluronic acid, and then dispersing N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in the aqueous solution of acidic hyaluronic acid to obtain a reaction system; (3) dissolving the amino-modified polyphenylene sulfide in tetrahydrofuran to obtain tetrahydrofuran containing the amino-modified polyphenylene sulfide; (4) adding tetrahydrofuran containing amino-modified polyphenylene sulfide dropwise to the reaction system, stirring and reacting for 24-48 hours under a nitrogen atmosphere to obtain a mixed solution B, dialyzing the mixed solution B and then freeze-drying it to obtain hyaluronic acid-polypropylene sulfide; Preparation of polyphenylene sulfide-hyaluronic acid nanomicelles loaded with ligustrazine: (1) dissolving hyaluronic acid-polypropylene sulfide in PBS to obtain an aqueous phase, and dissolving ligustrazine in acetone-ethanol solution to obtain an organic phase; (2) adding the organic phase to the aqueous phase, rotary evaporating for 3-5 min at a temperature of 30-50° C. and a rotation speed of 40-50 rpm, ultrasonicating, filtering with a microporous filter membrane and collecting the filtrate to obtain polyphenylene sulfide-hyaluronic acid nanomicelles loaded with ligustrazine.

8. The Chinese medicine hydrogel microneedle for diabetic wounds according to claim 7, characterized in that: When preparing amino-modified polyphenylene sulfide, the molar ratio of polyphenylene sulfide, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and ethylenediamine is 1:(1.5-2.5):(2-3):(15-25); When preparing hyaluronic acid-polypropylene sulfide, the mass ratio of acidic hyaluronic acid, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and amino-modified polyphenylene sulfide is (90-110):(5-10):(10-20):(30-50); When preparing polyphenylene sulfide-hyaluronic acid nano-micelles loaded with ligustrazine, the volume ratio of acetone to ethanol in the acetone-ethanol solution is (2.6-4.5): (1.5-2.4).

9. The method for preparing the traditional Chinese medicine hydrogel microneedle for diabetic wounds according to any one of claims 1 to 8, characterized in that: The following steps are involved: Dissolving safflower polysaccharide and methacryloylated dextran in water to obtain a mixed solution C, and sequentially adding ligustrazine-loaded polyphenylene sulfide-hyaluronic acid nanomicelles and a light curing agent to the mixed solution C to obtain a gel precursor solution; After dripping the gel precursor solution into the microneedle mold, the mold is transferred to a vacuum degassing box, and the vacuum degree of the vacuum degassing box is adjusted to -90 to -110 kPa for vacuum degassing. The microneedle mold is cured under 360-370 nm ultraviolet light to obtain a needle tip. Then, a hyaluronic acid solution is added to the microneedle mold, and the mold is dried at 36-40 ° C for 48-96 hours to obtain a base. The mold is then demolded. The mass volume percentage concentration of the hyaluronic acid solution is 1%-3%.

10. Use of the traditional Chinese medicine hydrogel microneedle for diabetic wounds according to any one of claims 1 to 8 in the preparation of a medicament for promoting healing of diabetic wounds.

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