Hydrogel microsphere system for sequential delivery of drugs as well as preparation method and application of hydrogel microsphere system

Abstract

The invention discloses a hydrogel microsphere system for sequential delivery of drugs as well as a preparation method and application thereof, and particularly relates to the technical field of biomedical materials. The microsphere system comprises inflammation responsive hydrogel microspheres obtained by grafting molecules containing inflammation responsive groups with hyaluronic acid as a skeleton, common microspheres obtained by grafting non-responsive molecules with hyaluronic acid as a skeleton, an anti-inflammatory drug and a healing promoting drug. Wherein the molecular weight of the hyaluronic acid is 74-800kDa, the inflammation responsive hydrogel microspheres are loaded with anti-inflammatory drugs, and the common microspheres are loaded with healing promoting drugs. According to the hydrogel microsphere system, the drug release rate and degradation time of the microspheres are adjusted by limiting the molecular weight of hyaluronic acid, and a molecular basis is provided for subsequent differentiated drug release.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a hydrogel microsphere system for sequential drug delivery, a preparation method thereof, and applications thereof. Background Art

[0002] As the largest organ in the human body, the skin plays a crucial role in protecting internal tissues and defending against pathogen invasion. When the skin is damaged, a healing process begins, consisting of hemostasis, inflammation, proliferation, and remodeling. These four phases occur sequentially and partially overlap, relying on the synergistic action of multiple cells and factors. The hemostasis phase centers on platelet activation and fibrin polymerization to form a clot, effectively sealing blood vessels and laying the foundation for subsequent repair. The inflammation phase recruits immune cells (such as macrophages) through cytokines, releasing proinflammatory cytokines and reactive oxygen species to eliminate pathogens and tissue debris, followed by anti-inflammatory factors that direct repair. During the proliferation and remodeling phases, fibroblasts and keratinocytes migrate and proliferate to seal the wound surface, while secreting collagen to reshape the extracellular matrix. Ultimately, capillary remodeling restores the skin to homeostasis.

[0003] If the dynamic balance of the healing phase is disrupted (e.g., persistent hyperinflammation during the inflammatory phase), chronic wounds can easily develop. Endogenous diseases or bacterial infections often lead to excessive secretion of proinflammatory cytokines and abnormal activation of immune cells, which in turn inhibit the proliferation and migration of tissue cells and trigger matrix metalloproteinase-mediated collagen degradation, hindering the formation of new granulation tissue. Therefore, regulating the inflammatory response is key to promoting healing. It is also necessary to specifically promote angiogenesis and collagen deposition and inhibit scar formation during the proliferation and remodeling phases. Implementing a sequential treatment strategy tailored to different healing stages has become a core challenge in wound treatment.

[0004] Drug therapy is an important means of wound intervention, but traditional drug delivery methods pose risks of drug toxicity, short half-life, and the need for frequent dressing changes. To address this, novel drug carrier dressings (such as sponges, hydrogels, and hydrogel microspheres) have emerged. Hydrogel microspheres are miniature hydrogel particles prepared by mechanical crushing, emulsification, or other methods. With their controllable particle size and surface morphology, they possess great application potential for fluid absorption, wound filling, and sustained drug release. To better target inflammatory environments by releasing anti-inflammatory drugs, researchers have introduced inflammatory-responsive groups into the dressing, enabling the dressing structure to responsively release anti-inflammatory drugs in response to destruction in the inflammatory environment, thereby providing more targeted treatment for the skin wound environment.

[0005] Hyaluronic acid is the main component of the extracellular matrix. It has a wide range of sources, excellent biocompatibility and water retention, and is often used to prepare medical dressings. According to the 2022 version of the "Expert Consensus on the Rational Selection and Application of Hyaluronic Acids of Different Relative Molecular Weights and Structures in Dermatology", the molecular weight of hyaluronic acid can be divided into three levels: high (number of repeating units greater than 5000), medium (number of repeating units between 1000-5000), and low (number of repeating units less than 1000). Hyaluronic acid of different molecular weights has different biological functions and physical properties; such as the Chinese patent CN116808286A, which uses a repeating unit number greater than 5000. Too high a molecular weight will cause the hydrogel microspheres to be too hard, resulting in a short residence time in the wound, requiring further cross-linking, and cross-linking also requires Controlling the degree of cross-linking, the preparation is complicated, and the patent is only loaded with anti-inflammatory drugs, so that the hydrogel is better in anti-inflammatory hemostasis, but the healing effect is not good; Chinese patent CN108912245A, the molecular weight of the hyaluronic acid used is 35-45kDa, but too low molecular weight degradation rate is too fast, causing drug release too fast, thus the patent needs to carry out fluorination trifluoroacetylation to hyaluronic acid, complicated operation, and the hyaluronic acid polyanion characteristic of the patent can form conjugates with cationic drugs, and can also form conjugates with the non-polar drugs wrapped by positive liposomes, which are better for the anti-inflammatory hemostatic effect of wounds, but limited to the healing of subsequent wounds. Therefore, the impact of the molecular weight of balancing hyaluronic acid on hydrogel microspheres is a difficult problem that needs to be solved urgently.

[0006] Current clinical dressings still suffer from a single drug delivery method and an inability to adapt to the dynamic changes of the wound surface. Combining responsive delivery systems (releasing drugs in response to environmental changes) with sustained-release technologies (extending the duration of drug action) has the potential to achieve differentiated, precise drug delivery at different healing stages. Therefore, the development of new functional dressings must ensure maximum biosafety while enabling targeted release of different drugs at different stages of skin wound healing. Summary of the Invention

[0007] To this end, the present invention provides a hydrogel microsphere system for time-sequential drug delivery and a preparation method and application thereof to solve the problems in the prior art.

[0008] In the hydrogel microsphere system of the present invention, the inflammation-responsive hydrogel microsphere system is first destroyed in the skin wound site to release the anti-inflammatory drug, and then the ordinary hydrogel microspheres naturally release the pro-healing drug. The inflammation-responsive microspheres in the hydrogel microsphere system can form a hydrogel after destruction, which further wraps the ordinary microspheres to protect the wound site. The hydrogel microsphere system has a sustained release time of 2-4 days for the anti-inflammatory drug and a sustained release time of 6-10 days for the pro-healing drug.

[0009] In order to achieve the above object, the present invention provides the following technical solutions: According to the first aspect of the present invention, a hydrogel microsphere system for timed drug delivery is provided, wherein the microspheres include inflammation-responsive hydrogel microspheres obtained by grafting molecules containing inflammation-responsive groups onto hyaluronic acid as a skeleton, ordinary microspheres obtained by grafting non-responsive molecules onto hyaluronic acid as a skeleton, anti-inflammatory drugs and pro-healing drugs; wherein the molecular weight of hyaluronic acid is 74-800kDa, the inflammation-responsive hydrogel microspheres are loaded with anti-inflammatory drugs, and the ordinary microspheres are loaded with pro-healing drugs.

[0010] Furthermore, the inflammation-responsive group includes one or more of a diselenide bond, a disulfide bond, a phenylboronic acid ester bond, a thioether bond, an oxalic acid polyester, an acylpyrrolidine-2-carboxamide group, a ferrocene-cyclodextrin self-assembly structure group, a ketal thiol, concanavalin, a metalloproteinase-responsive peptide chain, a Schiff base bond, and a pH-responsive peptide chain.

[0011] Furthermore, the non-responsive molecule includes one or more of hexamethylenediamine, ethylenediamine, and polyethylene glycol.

[0012] Furthermore, the anti-inflammatory drug includes one or more of gallic acid, epigallocatechin gallate, triamcinolone acetonide, berberine, curcumin, tea polyphenols, rhein, glycyrrhizic acid, quercetin, myricetin, dihydromyricetin, tannic acid, polydopamine, shikonin, celecoxib, and amoxicillin.

[0013] Furthermore, the healing-promoting drug includes one or more of astragaloside IV, asiaticoside, madecassoside, lidocaine, vascular endothelial growth factor, fibroblast exosomes, adipose stem cell exosomes, minoxidil, and vertepofol.

[0014] Furthermore, the diameter of the hydrogel microspheres is 20-500 microns. The inflammation-responsive hydrogel microspheres release 65-80% of the anti-inflammatory drug within 24 hours in the inflammatory environment of the wound site. The conventional hydrogel microspheres release 25-35% of the healing-promoting drug within 48 hours in the wound site.

[0015] Furthermore, the hydrogel for sequential drug delivery has a maximum deformation of 300%-600%, a modulus of 200-1000 Pascals, and good self-healing properties.

[0016] According to a second aspect of the present invention, a method for preparing a hydrogel microsphere system for time-sequential drug delivery is provided, the method comprising: Step 1: Preparation of inflammation-responsive hydrogel microspheres and ordinary microspheres Hyaluronic acid and inflammatory responsive molecules are mixed and dissolved in proportion to prepare an inflammation-responsive hydrogel aqueous phase W1; hyaluronic acid and non-responsive molecules are mixed and dissolved in proportion to prepare a normal hydrogel aqueous phase W2; Mix mineral oil and Span 80 in proportion to obtain oil phase O; The inflammation-responsive hydrogel aqueous phase W1 and the ordinary hydrogel aqueous phase W2 are mixed and stirred with the oil phase O in proportion to obtain the inflammation-responsive hydrogel emulsion C1 and the ordinary hydrogel emulsion C2; The inflammation-responsive hydrogel emulsion and the ordinary hydrogel emulsion were washed with isopropyl alcohol and centrifuged to obtain unloaded inflammation-responsive hydrogel microspheres B1 and unloaded ordinary hydrogel microspheres B2; Step 2: Drug loading The unloaded inflammation-responsive hydrogel microspheres are soaked in an anti-inflammatory drug solution to obtain a drug-loaded inflammation-responsive hydrogel microsphere mixture; the unloaded ordinary hydrogel microspheres are soaked in a healing-promoting drug solution to obtain a drug-loaded ordinary hydrogel microsphere mixture; the drug-loaded inflammation-responsive hydrogel microsphere mixture and the drug-loaded ordinary hydrogel microsphere mixture are freeze-dried and then sieved to obtain drug-loaded inflammation-responsive hydrogel microspheres a and drug-loaded ordinary hydrogel microspheres b, respectively; The drug-loaded inflammation-responsive hydrogel microspheres a and the drug-loaded ordinary hydrogel microspheres b are mixed in proportion to obtain a hydrogel microsphere system for sequential drug delivery.

[0017] Furthermore, in step 1, the concentration of hyaluronic acid is 1-10 wt%, the molar ratio of hyaluronic acid to inflammation-responsive molecules is 1:0.5-1:2; and the molar ratio of hyaluronic acid to non-responsive molecules is 1:0.5-1:2.

[0018] In order to make the reaction faster, one or more molecules including 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and N,N'-carbonyldiimidazole can be added as activators. The specific addition ratio is determined by the reaction system.

[0019] As an example, preferably, mineral oil and Span 80 are mixed in a volume ratio of 1:50-1:150; W1, W2 and oil phase O are mixed in a volume ratio of 1:5-1:20; the centrifugation conditions are: a rotation speed of 1000-3000 rpm, and a centrifugation time of 3-10 min; the microspheres B1 are soaked in an anti-inflammatory drug solution with a mass ratio of 1:20-1:100, wherein the concentration of the anti-inflammatory drug solution is 0.5-10 wt%; the microspheres B2 are soaked in a pro-healing drug solution with a mass ratio of 1:15-1:80, wherein the concentration of the pro-healing drug solution is 0.05-5 wt%; the freeze dryer temperature is -40°C to -60°C; the inflammation-responsive hydrogel microspheres and ordinary hydrogel microspheres are mixed in a mass ratio of 1:0.5-1:2.

[0020] According to the third aspect of the present invention, a hydrogel microsphere system for time-sequential drug delivery is provided for use in preparing a drug for treating skin wounds.

[0021] The skin wound is any one of pressure ulcer, venous ulcer, bedsore wound, burn wound, surgical wound and diabetic foot.

[0022] Principle: After the hydrogel microsphere system of the present invention is applied to the skin surface, the inflammation-responsive microspheres are destroyed to form a hydrogel, which further wraps the ordinary microspheres to protect the wound site; the anti-inflammatory drug in the inflammation-responsive microspheres loaded with anti-inflammatory drugs is continuously released for 2-4 days, and the healing-promoting drug is continuously released for 6-10 days.

[0023] The hydrogel microsphere system of the present invention has a complete degradation time of 10-14 days, and can be administered once, with long-term drug retention. The hydrogel microsphere system of the present invention can be directly placed on the skin wound site in a freeze-dried state, and can continuously absorb inflammatory exudate to a certain extent. At the same time, the inflammation-responsive microspheres in the hydrogel system release anti-inflammatory drugs to treat the early inflammation of the wound site, allowing the wound to smoothly pass through the inflammatory period. The ordinary microspheres in the hydrogel system release pro-healing drugs with a delayed release, allowing the wound to heal smoothly during the proliferation and remodeling periods.

[0024] The hydrogel microspheres of the present invention can be directly added to the skin wound site in a freeze-dried form for treatment. The hydrogel absorbs the inflammatory exudate at the wound site, undergoes the above-mentioned changes, and releases different drugs in stages, thereby achieving full-process wound healing treatment.

[0025] The hydrogel microsphere system of the present invention is obtained by soaking the obtained preliminary microspheres in a drug solution and then freeze-drying them, which increases the drug loading capacity of the microspheres. At the same time, compared with microspheres soaked in liquid form, it can better prolong the residence time of the drug in the microspheres, which helps the microspheres release the drug after absorbing exudate at the wound and transforming into a macroscopic hydrogel state.

[0026] The present invention has the following advantages: 1) The hydrogel microsphere system of the present invention regulates the drug release rate and degradation time of the microspheres by limiting the molecular weight of hyaluronic acid, providing a molecular basis for subsequent differentiated drug release.

[0027] 2) The hydrogel microsphere system of the present invention is in a freeze-dried form and can be directly covered on the wound site to absorb the exudate from the inflammatory site and reduce the retention of the inflammatory exudate in the inflammatory site.

[0028] 3) The inflammation-responsive microspheres in the hydrogel microsphere system of the present invention can swell after inflammation-responsive destruction to further absorb inflammatory exudate, and can also cross-link to form a hydrogel after swelling to protect the inflammation site.

[0029] 4) The skeleton of the hydrogel microsphere system of the present invention is a natural macromolecule, which has good biocompatibility and water retention, and enhances the absorption of exudate by the microspheres.

[0030] 5) The inflammation-responsive microspheres in the hydrogel microsphere system of the present invention can quickly release anti-inflammatory drugs in a strong inflammatory environment, ensuring a smooth transition from the traumatic inflammation period.

[0031] 6) The ordinary microspheres in the hydrogel microsphere system of the present invention can slowly release the healing-promoting drugs, and as the microspheres degrade, the relevant drugs are released in a targeted manner during the proliferation and remodeling phases of wound healing.

[0032] 7) The medical dressing of the present invention can meet the needs of all stages of wound healing with a single administration, thus avoiding the pain and discomfort of patients caused by repeated administration.

[0033] 8) The product formed by the medical dressing of the present invention after absorbing the inflammatory exudate and causing destruction is a hydrogel, which has good self-healing properties, can effectively cover the wound inflammatory environment and meet the requirements of use in a dynamic physiological environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0035] The structures, proportions, sizes, etc. illustrated in this specification are intended only to complement the contents disclosed herein and to facilitate understanding and reading by persons familiar with the art. They are not intended to limit the conditions under which the present invention may be implemented and therefore have no substantive technical significance. Any structural modifications, changes in proportions, or adjustments in sizes, without affecting the efficacy and objectives of the present invention, shall still fall within the scope of the technical contents disclosed herein.

[0036] Figure 1 A schematic diagram of a system for preparing hydrogel microspheres provided in Example 1 of the present invention; Figure 2 Schematic diagram of the hydrogel microsphere system used in Experimental Example 2 of the present invention to repair mouse wounds; Figure 3 This is a macroscopic image of the hydrogel microsphere system provided in Experimental Example 3 of the present invention, which simulates the absorption of oxidative inflammatory exudate in vitro and gradually cross-links to form hydrogel-encapsulated ordinary microspheres; Figure 4This is a scanning electron micrograph of the hydrogel formed by the hydrogel microsphere system provided in Experimental Example 4 of the present invention, which simulates the absorption of oxidative inflammatory exudate and cross-linking in vitro; Figure 5 This is the experimental result obtained by performing a "high strain-low strain" alternating rheological test on the hydrogel microsphere system provided in Experimental Example 5 of the present invention by simulating the absorption of oxidative inflammatory exudate in vitro and cross-linking the formed hydrogel using a rotational rheometer. DETAILED DESCRIPTION

[0037] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0038] Example 1 In this example, a hydrogel microsphere system was prepared, including inflammation-responsive hydrogel microspheres a with diselenide bonds as inflammation-responsive groups and ordinary microspheres b with hexamethylenediamine as cross-linking molecules. The hyaluronic acid in microsphere a had a molecular weight of 200 kDa, delivered berberine, and had a diameter of 120 μm. The hyaluronic acid in microsphere b had a molecular weight of 250 kDa, delivered asiaticoside, and had a diameter of 180 μm.

[0039] The specific steps are as follows: Figure 1 As shown: S1. Prepare a 1.5 wt% hyaluronic acid solution, then mix and dissolve hyaluronic acid (CAS No. 9004-61-9): selenocystamine (CAS No. 2697-61-2): 1-ethyl-(3-dimethylaminopropyl)carbodiimide (CAS No. 1892-57-5) in a molar ratio of 1:0.6:1 to prepare an aqueous phase W1 for preparing microspheres a; S2. A 3 wt% hyaluronic acid solution was prepared, and then hyaluronic acid: hexamethylenediamine (CAS No.: 124-09-4): 1-ethyl-(3-dimethylaminopropyl)carbodiimide (CAS No.: 1892-57-5) were mixed and dissolved in a molar ratio of 1:0.6:1 to prepare an aqueous phase W2 for preparing microspheres b. S3. Mix mineral oil (CAS No.: 8042-47-5) and the amphiphilic molecule Span 80 (CAS No.: 1338-43-8) in a volume ratio of 1:80 to obtain an oil phase O; S4. The aqueous phase W1, W2 and the oil phase O were mixed and stirred in a volume ratio of 20:80 to obtain mixed solutions C1 and C2, with a stirring speed of 300 rpm and a reaction time of 12 h; S5. After the reaction is complete, wash the mixed solutions C1 and C2 with isopropyl alcohol (CAS No. 67-63-0), transfer them to a 50 mL centrifuge tube, and centrifuge at 1500 rpm for 3 min. Discard the liquid to obtain microspheres B1 and B2, respectively. S6. Soak the microspheres B1 obtained by centrifugation in a 1:50 solution of berberine (CAS No. 2086-83-1) at a concentration of 2 wt% to obtain a microsphere B1 suspension. Soak the microspheres B2 in a 1:25 solution of asiaticoside (CAS No. 16830-15-2) at a concentration of 0.5 wt% to obtain a microsphere B2 suspension. The microsphere B1 and microsphere B2 suspensions were then transferred to a freeze dryer and completely freeze-dried at -55°C to obtain inflammation-responsive hydrogel microspheres a and conventional microspheres b. S7. The freeze-dried microspheres were screened with a sieve having an upper limit of 200 μm and a lower limit of 100 μm. S8. Mix hydrogel microspheres a and ordinary microspheres b in a mass ratio of 1:1 to obtain a hydrogel microsphere system for timed drug delivery.

[0040] Example 2 In this example, a hydrogel microsphere system was prepared, including inflammation-responsive hydrogel microspheres a with phenylboronic acid bonds as inflammation-responsive groups and ordinary microspheres b with ethylenediamine as cross-linking molecules. The hyaluronic acid in microsphere a had a molecular weight of 350 kDa, delivered myricetin, and had a diameter of 80 μm. The hyaluronic acid in microsphere b had a molecular weight of 400 kDa, delivered madecassoside, and had a diameter of 120 μm.

[0041] The specific steps are as follows: S1. Prepare a 1 wt% hyaluronic acid solution, then mix and dissolve oxidized hyaluronic acid and myricetin-cross-linked aminophenylboronic acid (CAS No. 80460-73-7) in a molar ratio of 1:1 to prepare an aqueous phase W1 for preparing microspheres a; S2. Prepare a 1 wt% hyaluronic acid solution, and then mix and dissolve hyaluronic acid: ethylenediamine: 1-ethyl-(3-dimethylaminopropyl)carbodiimide in a molar ratio of 1:0.5:1 to prepare an aqueous phase W2 for preparing microspheres b; S3. The mineral oil and the amphiphilic molecule Span 80 were mixed in a volume ratio of 1:75 to obtain an oil phase O; S4. The aqueous phases W1, W2, and the oil phase O were mixed and stirred at a volume ratio of 10:90 to obtain mixed solutions C1 and C2, respectively. The stirring speed was 400 rpm and the reaction time was 24 h. S5. After the reaction is complete, the mixed solutions C1 and C2 are washed with isopropanol and then transferred to a 50 mL centrifuge tube and centrifuged at 1000 rpm for 3 min. The liquid is discarded to obtain microspheres B1 and B2, respectively. S6. Microspheres B1 obtained by centrifugation were further soaked in a 1:45 solution of myricetin (CAS No. 529-44-2) at a concentration of 0.5 wt% to obtain a microsphere B1 suspension. Microspheres B2 were then soaked in a 1:20 solution of madecassoside (CAS No. 34540-22-2) at a concentration of 1 wt% to obtain a microsphere B2 suspension. The microsphere B1 and microsphere B2 suspensions were then transferred to a freeze dryer and completely freeze-dried at -55°C to obtain inflammation-responsive hydrogel microspheres a and conventional microspheres b. S7. The freeze-dried microspheres were screened with a mesh having an upper limit of 150 μm and a lower limit of 50 μm; S8. Mix hydrogel microspheres a and ordinary microspheres b in a mass ratio of 1:1 to obtain a hydrogel microsphere system for timed drug delivery.

[0042] Example 3 In this example, a hydrogel microsphere system was prepared, including inflammation-responsive hydrogel microspheres a with Schiff base bonds as inflammation-responsive groups and conventional microspheres b with ethylenediamine as cross-linking molecules. The hyaluronic acid in microsphere a had a molecular weight of 150 kDa, delivered dihydromyricetin (CAS No. 27200-12-0), and had a diameter of 150 μm. The hyaluronic acid in microsphere b had a molecular weight of 300 kDa, delivered minoxidil (CAS No. 38304-91-5), and had a diameter of 180 μm.

[0043] The specific steps are as follows: S1. Prepare a 0.5 wt% oxidized hyaluronic acid solution, then mix and dissolve oxidized hyaluronic acid: dihydromyricetin-cross-linked aminophenylboronic acid (CAS No.: 3945-69-5) in a molar ratio of 1:1 to prepare an aqueous phase W1 for preparing microspheres a; S2. Prepare a 1.5 wt% hyaluronic acid solution, then mix and dissolve hyaluronic acid: ethylenediamine: 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (CAS No.: 80460-73-7) in a molar ratio of 1:0.5:1 to prepare an aqueous phase W2 for preparing microspheres b; S3. The mineral oil and the amphiphilic molecule Span 80 were mixed in a volume ratio of 1:70 to obtain an oil phase O; S4. The aqueous phases W1, W2, and the oil phase O were mixed and stirred at a volume ratio of 10:90 to obtain mixed solutions C1 and C2, respectively. The stirring speed was 300 rpm and the reaction time was 12 h. S5. After the reaction is complete, the mixed solutions C1 and C2 are washed with isopropanol, then transferred to a 50 mL centrifuge tube and centrifuged at 1200 rpm for 5 min. The liquid is discarded to obtain microspheres B1 and B2, respectively. S6. The microspheres B1 obtained by centrifugation were further soaked in a dihydromyricetin solution at a mass ratio of 1:30, wherein the dihydromyricetin solution had a concentration of 0.5 wt% to obtain a microsphere B1 suspension. The microspheres B2 were then soaked in a minoxidil solution at a mass ratio of 1:25, wherein the minoxidil solution had a concentration of 1.5 wt%, to obtain a microsphere B2 suspension. The microsphere B1 suspension and the microsphere B2 suspension were then transferred to a freeze dryer for complete freeze drying to obtain inflammation-responsive hydrogel microspheres a and ordinary microspheres b. The freeze dryer temperature was -55°C. S7. The freeze-dried microspheres were screened with a sieve having an upper limit of 200 μm and a lower limit of 100 μm. S8. Mix hydrogel microspheres a and ordinary microspheres b in a mass ratio of 1:1 to obtain a hydrogel microsphere system for timed drug delivery.

[0044] Example 4 In this example, a hydrogel microsphere system was prepared, including inflammation-responsive hydrogel microspheres a containing matrix metalloproteinase-responsive peptide chains as inflammation-responsive groups and conventional microspheres b containing hexamethylenediamine as cross-linking molecules. The hyaluronic acid in microspheres a had a molecular weight of 200 kDa and delivered curcumin (CAS No. 458-37-7), with a diameter of 120 μm. The hyaluronic acid in microspheres b had a molecular weight of 300 kDa and delivered astragaloside IV (CAS No. 83207-58-3), with a diameter of 180 μm.

[0045] The specific steps are as follows: S1. Prepare a 1.5 wt% hyaluronic acid solution, then mix and dissolve hyaluronic acid: matrix metalloproteinase-responsive peptide chain (CAS No.: 9000-70-8): 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride in a molar ratio of 1:1:1 to prepare an aqueous phase W1 for preparing microspheres a; S2. Prepare a 3 wt% hyaluronic acid solution, and then mix and dissolve the solution in a molar ratio of hyaluronic acid: hexamethylenediamine: activator = 1:0.5:1 to prepare an aqueous phase W2 for preparing microspheres b; S3. The mineral oil and the amphiphilic molecule Span 80 were mixed in a volume ratio of 1:80 to obtain an oil phase O; S4. The aqueous phases W1 and W2 were mixed with the oil phase O in a volume ratio of 15:85 to obtain mixed solutions C1 and C2, respectively. The stirring speed was 250 rpm and the reaction time was 12 h. S5. After the reaction is complete, the mixed solutions C1 and C2 are washed with isopropanol, then transferred to a 50 mL centrifuge tube and centrifuged at 1500 rpm for 3 min. The liquid is discarded to obtain microspheres B1 and B2, respectively. S6. Microspheres B1 obtained by centrifugation were further soaked in a curcumin solution at a mass ratio of 1:50, wherein the dihydromyricetin solution concentration was 0.5 wt%, to obtain a microsphere B1 suspension. Microspheres B2 were then soaked in an astragaloside IV solution at a mass ratio of 1:30, wherein the astragaloside IV concentration was 1 wt%, to obtain a microsphere B2 suspension. The microsphere B1 suspension and the microsphere B2 suspension were then transferred to a freeze dryer and completely freeze-dried at -55°C to obtain inflammation-responsive hydrogel microspheres a and conventional microspheres b. S7. The freeze-dried microspheres were screened with a sieve having an upper limit of 200 μm and a lower limit of 100 μm. S8. Mix hydrogel microspheres a and ordinary microspheres b in a mass ratio of 1:1 to obtain a hydrogel microsphere system for timed drug delivery.

[0046] Example 5 In this example, a hydrogel microsphere system was prepared, including inflammation-responsive hydrogel microspheres a with disulfide bonds as inflammation-responsive groups and conventional microspheres b with hexamethylenediamine as cross-linking molecules. The hyaluronic acid in microsphere a had a molecular weight of 150 kDa, delivered the drug shikonin (CAS No. 517-89-5), and had a diameter of 80 μm. The hyaluronic acid in microsphere b had a molecular weight of 200 kDa, delivered the drug lidocaine (CAS No. 137-58-6), and had a diameter of 85 μm.

[0047] The specific steps are as follows: S1. Prepare a 1.5 wt% hyaluronic acid solution, and then mix and dissolve hyaluronic acid: cystamine (CAS No.: 51-85-4): 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride in a molar ratio of 1:0.6:1 to prepare an aqueous phase W1 for preparing microspheres a; S2. A 2 wt% hyaluronic acid solution was prepared, and then hyaluronic acid: hexamethylenediamine: 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride was mixed and dissolved in a molar ratio of 1:0.6:1 to prepare an aqueous phase W2 for preparing microspheres b; S3. The mineral oil and the amphiphilic molecule Span 80 were mixed in a volume ratio of 1:80 to obtain an oil phase O; S4. The aqueous phases W1 and W2 and the oil phase O were mixed and stirred at a volume ratio of 20:80 to obtain mixed solutions C1 and C2, respectively. The stirring speed was 250 rpm and the reaction time was 12 h. S5. After the reaction is complete, the mixed solutions C1 and C2 are washed with isopropanol and then transferred to a 50 mL centrifuge tube and centrifuged at 1000 rpm for 3 min. The liquid is discarded to obtain microspheres B1 and B2, respectively. S6. Soak the microspheres B1 obtained by centrifugation in a 1:50 shikonin solution with a concentration of 1.5 wt% to obtain a microsphere B1 suspension. Soak the microspheres B2 in a 1:20 lidocaine solution with a concentration of 2 wt% to obtain a microsphere B2 suspension. The microsphere B1 and B2 suspensions were then transferred to a freeze dryer and completely freeze-dried at -55°C to obtain inflammation-responsive hydrogel microspheres a and ordinary microspheres b. S7. The freeze-dried microspheres were screened with a mesh having an upper limit of 100 μm and a lower limit of 50 μm; S8. Mix hydrogel microspheres a and ordinary microspheres b in a mass ratio of 1:1 to obtain a hydrogel microsphere system for timed drug delivery.

[0048] Example 6 In this example, a hydrogel microsphere system was prepared, including inflammation-responsive hydrogel microspheres a with a ferrocene-cyclodextrin self-assembled structure as the inflammation-responsive group and ordinary microspheres b with ethylenediamine as the cross-linking molecule. The hyaluronic acid in microsphere a had a molecular weight of 250 kDa, and the drug delivered was triamcinolone acetonide (CAS No. 124-94-7), with a diameter of 110 μm. The hyaluronic acid in microsphere b had a molecular weight of 450 kDa, and the drug delivered was fibroblast exosomes, with a diameter of 150 μm.

[0049] The specific steps are as follows: S1. Prepare a 1 wt% oxidized hyaluronic acid solution, then mix and dissolve oxidized hyaluronic acid: ferrocene-cyclodextrin self-assembled molecules (CAS No.: 12176-38-4; CAS No.: 12619-70-4): N,N'-carbonyldiimidazole (CAS No.: 530-62-1) in a molar ratio of 1:1:1 to prepare an aqueous phase W1 for preparing microspheres a; S2. A 1.5 wt% hyaluronic acid solution was prepared, and then hyaluronic acid: hexamethylenediamine: (4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride was mixed and dissolved in a molar ratio of 1:0.5:1 to prepare an aqueous phase W2 for preparing microspheres b; S3. The mineral oil and the amphiphilic molecule Span 80 were mixed in a volume ratio of 1:80 to obtain an oil phase O; S4. The aqueous phases W1 and W2 were mixed with the oil phase O in a volume ratio of 10:90 to obtain mixed solutions C1 and C2, respectively. The stirring speed was 350 rpm and the reaction time was 12 h. S5. After the reaction is complete, the mixed solutions C1 and C2 are washed with isopropanol, then transferred to a 50 mL centrifuge tube and centrifuged at 1200 rpm for 5 min. The liquid is discarded to obtain microspheres B1 and B2, respectively. S6. The microspheres B1 obtained by centrifugation were further soaked in a triamcinolone acetonide solution at a mass ratio of 1:30, wherein the triamcinolone acetonide solution concentration was 1 wt% to obtain a microsphere B1 suspension. The microspheres B2 were soaked in a fibroblast exosome solution at a mass ratio of 1:30, wherein the fibroblast exosome solution concentration was 0.1 wt% to obtain a microsphere B1 suspension. The microsphere B1 suspension and the microsphere B2 suspension were then transferred to a freeze dryer for complete freeze drying to obtain inflammation-responsive hydrogel microspheres a and ordinary microspheres b. The freeze dryer temperature was -55°C. S7. The freeze-dried microspheres were screened with a sieve having an upper limit of 200 μm and a lower limit of 100 μm. S8. Mix hydrogel microspheres a and ordinary microspheres b in a mass ratio of 1:1 to obtain a hydrogel microsphere system for timed drug delivery.

[0050] Comparative Example 1 This comparative example prepares a hydrogel microsphere system including inflammation-responsive hydrogel microspheres a with diselenide bonds as inflammation-responsive groups and ordinary microspheres b with hexamethylenediamine as cross-linking molecules, wherein the molecular weight of the hyaluronic acid in microsphere a is 20 kDa, the drug delivered is berberine, and the diameter is 110 microns; the molecular weight of the hyaluronic acid in microsphere b is 50 kDa, the drug delivered is asiaticoside, and the diameter is 160 microns.

[0051] The specific steps are as follows: Steps S1-S8 are the same as in Example 1.

[0052] Comparative Example 2 This comparative example prepares a hydrogel microsphere system including inflammation-responsive hydrogel microspheres a with diselenide bonds as inflammation-responsive groups and ordinary microspheres b with hexamethylenediamine as cross-linking molecules, wherein the molecular weight of the hyaluronic acid in microsphere a is 1000 kDa, the drug delivered is berberine, and the diameter is 140 microns; the molecular weight of the hyaluronic acid in microsphere b is 900 kDa, the drug delivered is asiaticoside, and the diameter is 160 microns.

[0053] The specific steps are as follows: Steps S1-S8 are the same as in Example 1.

[0054] Comparative Example 3 This comparative example is the hydrogel microsphere a prepared in Example 1.

[0055] Comparative Example 4 This comparative example is the ordinary microsphere b prepared in Example 1.

[0056] Comparative Example 5 In this example, a hydrogel microsphere system is prepared, including an inflammation-responsive hydrogel patch a with a diselenide bond as the inflammation-responsive group and ordinary microspheres b with hexamethylenediamine as the cross-linking molecule, wherein the hyaluronic acid of microsphere a has a molecular weight of 200 kDa, the drug delivered is berberine, and the diameter is 120 μm; the hyaluronic acid of microsphere b has a molecular weight of 250 kDa, the drug delivered is asiaticoside, and the diameter is 180 μm.

[0057] The preparation steps of microsphere b are the same as those in Example 1. The specific preparation steps of hydrogel patch a are as follows: S1. Prepare a 1.5 wt% hyaluronic acid solution, then mix and dissolve hyaluronic acid: selenocystamine: 1-ethyl-(3-dimethylaminopropyl)carbodiimide (CAS No.: 1892-57-5): berberine in a molar ratio of 1:0.6:1:0.1 to prepare an aqueous phase W1 for preparing patch a; S2. Add microspheres b to the aqueous phase W1 in a 1:1 ratio of hyaluronic acid mass and mix well to obtain W2; S3. Transfer the aqueous phase W2 to the mold and react for 12 hours to obtain the hydrogel microsphere system.

[0058] Comparative Example 6 In this comparative example, a hydrogel microsphere system comprising inflammation-responsive hydrogel microspheres a with diselenide bonds as inflammation-responsive groups and pure asiaticoside drug powder was prepared, wherein the index parameters of microspheres a were the same as those in Example 1.

[0059] The specific steps are as follows: the preparation steps of inflammation-responsive microspheres a are the same as S1-S7 of Example 1; S8. Mix the hydrogel microspheres a and the asiaticoside powder of the same pharmaceutical quality as in Example 1 according to the relevant proportions in Example 1.

[0060] Comparative Example 7 In this comparative example, a hydrogel microsphere system including ordinary microspheres b using hexamethylenediamine as a cross-linking molecule and pure berberine drug powder was prepared, wherein the index parameters of the microspheres b were the same as those in Example 1.

[0061] The specific steps are as follows: the preparation steps of ordinary microspheres b are the same as S1-S7 of Example 1; S8. Mix the hydrogel microspheres b and the berberine drug powder of the same drug mass as that of Example 1 according to the relevant proportions in Example 1.

[0062] Experimental Example 1 The hydrogel microsphere system obtained in Example 1 simulates the process of absorbing oxidative inflammatory exudate and gradually cross-linking to form hydrogel-encapsulated ordinary microspheres in vitro: For the hydrogel system obtained in Example 1, 1 mM hydrogen peroxide solution was used to simulate the reactive oxygen species at the inflammation site, such as Figure 3 As shown, it can be seen that the hydrogel microsphere system rapidly undergoes differentiated deformation in the oxidative exudate environment. The inflammatory-responsive microspheres absorb the exudate and transform into a macroscopic hydrogel form, while the ordinary microspheres exist stably, forming a jelly structure similar to fruit grains. The surface hydrogel microsphere system shows differentiated responsiveness in the inflammatory exudate environment, which is helpful for the subsequent staged treatment of the wound.

[0063] Experimental Example 2 Scanning electron microscopy of the hydrogel microsphere system of Example 1, which simulates the absorption of oxidative inflammatory exudate and cross-linking of the hydrogel. Specifically, the hydrogel obtained in Example 1 after reactive oxygen species were freeze-dried, quenched with liquid nitrogen and broken, and the surrounding structure of the hydrogel was observed using a scanning electron microscope. Figure 4 It can be seen that a structure of hydrogel-wrapped microspheres is formed. The hydrogel is loose and porous, while the microspheres are relatively dense in morphology, indicating that no obvious changes have occurred.

[0064] Experimental Example 3 The hydrogel microsphere system of Example 1 was subjected to a "high strain-low strain" alternating rheological test using a rotational rheometer to simulate the absorption of oxidative inflammatory exudate and the cross-linked hydrogel to observe the self-healing properties of the hydrogel. Figure 5 As shown in the figure, when high strain occurs, the hydrogel is destroyed, the elastic modulus is lower than the viscous modulus, and the whole is in a sol state. When the strain is restored to low, the hydrogel quickly returns to a gel state, and the elastic modulus is greater than the viscous modulus. These results show that the hydrogel has excellent self-healing properties and can recover to a gel state after damage, which is beneficial for the use of hydrogels in dynamic wound environments.

[0065] Experimental Example 4 Biocompatibility test: The hydrogel microsphere system from Example 1 was co-cultured with mouse fibroblast L929 cells in a transwell culture chamber for 24 hours. The cytotoxicity of the hydrogel microsphere system was then measured using thiazolyl blue. The results showed that the cell survival rate after co-culture with the hydrogel microsphere system from Example 1 was over 95%, meeting biosafety standards.

[0066] Experimental Example 5 The hydrogel microsphere system from Example 1 was immersed in a PBS solution and a fluid simulating an inflammatory wound environment for 24 hours. The mass before and after immersion was measured to determine the hydrogel microsphere system's fluid absorption rate. The results showed that the hydrogel microsphere system had a fluid absorption rate of 152% in the PBS solution and 541% in the fluid simulating an inflammatory wound environment. These results demonstrate that the hydrogel microsphere system is more effective in absorbing exudate and converting it into a hydrogel in an inflammatory environment, which may aid in the treatment of wounds with highly inflammatory conditions.

[0067] Experimental Example 6 Different drugs require different release rates. The inflammatory phase of a wound primarily lasts for 4-6 days, so anti-inflammatory drugs need to be released preferentially in the inflammatory environment. Healing-promoting drugs, on the other hand, primarily target the proliferation and remodeling phases of the wound, so their initial release needs to be as slow as possible or their fastest release needs to be shifted to a later time. This differentiated release method ensures that a single dose can meet the full healing process.

[0068] The microspheres of Examples 1-6 and Comparative Examples 1-7 were placed in a solution simulating a wound environment. The microspheres were weighed at different times and their degradation time was measured. The drug release was measured using a microplate reader after the microspheres were placed in a solution simulating a wound environment. The results are shown in Table 1.

[0069] Table 1

[0070] As shown in Table 1, Comparative Examples 1 and 2 demonstrate the effects of low (less than 74 kDa) and high (greater than 800 kDa) hyaluronic acid molecular weights on hydrogel degradation time and drug release, respectively. As shown in Comparative Example 1, when the molecular weight is too low, the microspheres degrade within two days, failing to effectively treat the inflammatory phase. Furthermore, structural damage results in excessively rapid drug release, with the healing-promoting drug completely released by 48 hours, rendering it ineffective during the proliferation and healing phases. Conversely, when the molecular weight is too high, the anti-inflammatory drug is not effectively released. In Comparative Example 2, only 31% of the anti-inflammatory drug is released within 48 hours, failing to effectively treat inflammation.

[0071] The hyaluronic acid backbone of the hydrogel microspheres constructed in Comparative Examples 3-5 and Examples 1-6 of the present invention has an appropriate molecular weight (74-800 kDa), preventing rapid microsphere degradation or slow drug release. Furthermore, the inflammation-responsive microspheres in the hydrogel microsphere system responsively release drugs in a simulated wound environment, reaching 40%-52% within 12 hours, effectively curbing hyperinflammation in the wound. The drug release rate then slows, but remains significantly faster than that of Comparative Example 2. The healing-promoting drug is also slowly released, reaching 28%-30% within 48 hours. The release rate accelerates during the proliferation phase, increasing by over 40% within 96 hours, effectively delaying release and achieving targeted treatment. More importantly, the degradation time is 10-14 days, meeting the requirements of a single-dose treatment. It should be noted that the inflammation-responsive microspheres from Comparative Examples 3 and 4 degrade faster than conventional microspheres, meeting the design requirements of the patented material. However, single microspheres cannot meet the requirements for targeted drug release and the synergistic effect of multiple drugs. For example, Comparative Examples 3 and 4 can only load a single drug and cannot achieve the combined effects of multiple drugs. Comparative Examples 6 and 7 use non-microspheroidized berberine or asiaticoside. Although these also have anti-inflammatory and healing effects, the non-microspheroidization process results in excessively rapid drug release, failing to achieve the desired release and healing times.

[0072] Traditional wound treatment dressings (gauze, sponges, etc.) can quickly lose effectiveness due to rapid degradation or drug release, preventing subsequent treatment and even causing physiological toxicity from burst drug release. However, excessively slow degradation or drug release (e.g., Comparative Examples 3-7) can lead to ineffective drug efficacy or foreign body reactions, delaying or hindering wound healing. Therefore, it is necessary to balance drug degradation rate and drug release within an appropriate range. The present invention effectively achieves this requirement by regulating the molecular weight of hyaluronic acid. When the molecular weight of hyaluronic acid is maintained within an appropriate range, its physical entanglement effectively regulates the drug release rate while also allowing for stable degradation under the degradation of hyaluronidase in the organism.

[0073] Experimental Example 7 To investigate the effects of different drug release characteristics at different wound healing stages in Examples 1-6 and Comparative Examples 1-7, a diabetic full-thickness dorsal wound model was constructed in C57BL / 6J mice. Diabetic wounds experience severe inflammation due to a persistent high-sugar environment, further damaging cells and blood vessels and hindering wound healing. Therefore, it is necessary to release anti-inflammatory drugs during the inflammatory phase to alleviate inflammation, followed by the release of pro-healing drugs during the proliferation and remodeling phases of the wound to ensure comprehensive wound healing.

[0074] The materials from Comparative Examples 1-7 and Examples 1-6 were placed on the wound site and wrapped with 3M film to prevent the materials from detaching from the tissue. Tissues were collected on day 6, homogenized, and flow cytometry was performed to measure the ratio of CD86 positivity (M1 macrophages) to CD206 positivity (M2 macrophages). Wound healing rates were photographed and calculated on day 10. The results are shown in Table 2: Table 2

[0075] CD86 is a marker for M1 macrophages and a pro-inflammatory indicator; CD206 is a marker for M2 macrophages and an anti-inflammatory indicator. A high CD86 / CD206 ratio indicates that M1 macrophages predominate in the wound site, creating a strong inflammatory atmosphere that is detrimental to wound healing. Conversely, a low ratio indicates that the wound is in remission, nearing the end of the inflammatory phase and transitioning to the proliferation and remodeling phases, which facilitate wound healing.

[0076] From Comparative Example 1, it can be seen that when the hydrogel microspheres degrade too quickly, the drug cannot continue to work. The CD86 / CD206 ratio is 129%, which is higher than 100%. The inflammation is suppressed, but the overall environment is still pro-inflammatory. The wound healing rate on the 10th day is only 55%. From Comparative Example 2, it can be seen that when the hydrogel degrades too slowly, although the inflammation can be effectively relieved on the 7th day, the CD86 / CD206 ratio is 44%, but because the healing drug is not released in time, the wound tissue cannot heal quickly during the proliferation and remodeling phases. The final wound healing rate on the 10th day is only 66%. From Comparative Examples 3 and 4, it can be seen that simply controlling inflammation or promoting the repair of tissues related to the proliferation and remodeling phases cannot effectively promote wound healing. The healing rates of both are less than 50%, and the wound healing process is significantly slowed down. From Comparative Example 5, it can be seen that the hydrogel patch technology can also effectively differentiate the delivery of drugs to promote wound healing, but due to ignoring the absorption of wound exudate during the inflammatory phase, the effect of controlling inflammation is poor, and the final healing rate is only 71%. Comparative Examples 6 / 7 used non-microspheroidization treatment, which failed to release the anti-inflammatory drug (Comparative Example 6) and the healing-promoting drug (Comparative Example 7) at the expected drug release rate, resulting in the final healing rates of mice being only 47% and 42%.

[0077] From Examples 1-6, it can be seen that when the differentiated delivery rate of microsphere drugs is appropriate, on the one hand, inflammation can be effectively controlled, and the CD86 / CD206 ratio is 29%-38%, which is at a very low level, ensuring a smooth transition from the inflammatory phase. On the other hand, the healing-promoting drugs can act precisely during the proliferation and remodeling phases, and the wound healing rate can be effectively accelerated. On the 10th day, the wound healing rate is 83%-88%, which means that the wound is about to close. The specific healing process diagram is as follows Figure 2 shown.

[0078] These results demonstrate that diabetic wound healing is a symbiotic process across various phases. Simply controlling or targeting tissue remodeling during the proliferation and remodeling phases is ineffective in promoting wound healing. Slow drug release can also occur when drug release is too rapid or too slow to effectively exert its effect. The timed drug delivery hydrogel microsphere system designed in this invention effectively addresses the diverse therapeutic needs of different wound healing phases, delivering targeted drug release to promote wound healing.

[0079] The hydrogel system of Example 1 of the present invention can absorb inflammatory exudate in the wound area in the early stage and responsively release berberine drugs, eliminate reactive oxygen species and inhibit inflammation in the wound; at the same time, ordinary microspheres slowly release asiaticoside, promote cell migration, angiogenesis and promote wound healing, thereby improving the quality of healing.

[0080] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A hydrogel microsphere system for time-sequential drug delivery, characterized in that: The microsphere system includes inflammation-responsive hydrogel microspheres obtained by grafting molecules containing inflammation-responsive groups onto hyaluronic acid as a skeleton, ordinary microspheres obtained by grafting non-responsive molecules onto hyaluronic acid as a skeleton, anti-inflammatory drugs and pro-healing drugs; wherein, the molecular weight of hyaluronic acid is 74-800kDa, the inflammation-responsive hydrogel microspheres are loaded with anti-inflammatory drugs, and the ordinary microspheres are loaded with pro-healing drugs.

2. The hydrogel microsphere system for timed drug delivery according to claim 1, characterized in that: The inflammation-responsive groups include one or more of a diselenide bond, a disulfide bond, a phenylboronic acid ester bond, a thioether bond, an oxalic acid polyester, an acylpyrrolidine-2-carboxamide group, a ferrocene-cyclodextrin self-assembly structure group, a ketal thiol, concanavalin, a metalloproteinase-responsive peptide chain, a Schiff base bond, and a pH-responsive peptide chain.

3. The hydrogel microsphere system for timed drug delivery according to claim 1, characterized in that: The non-responsive molecules include one or more of hexamethylenediamine, ethylenediamine, and polyethylene glycol.

4. The hydrogel microsphere system for timed drug delivery according to claim 1, characterized in that: The anti-inflammatory drugs include one or more of gallic acid, epigallocatechin gallate, triamcinolone acetonide, berberine, curcumin, tea polyphenols, rhein, glycyrrhizic acid, quercetin, myricetin, dihydromyricetin, tannic acid, polydopamine, shikonin, celecoxib, and amoxicillin.

5. The hydrogel microsphere system for time-sequential drug delivery according to claim 1, characterized in that: The healing-promoting drugs include one or more of astragaloside IV, asiaticoside, madecassoside, lidocaine, vascular endothelial growth factor, fibroblast exosomes, adipose stem cell exosomes, minoxidil, and vertepofol.

6. The hydrogel microsphere system for timed drug delivery according to claim 1, characterized in that: The diameter of the hydrogel microspheres is 20-500 microns.

7. The hydrogel microsphere system for time-sequential drug delivery according to claim 1, characterized in that: The maximum deformation of the inflammation-responsive hydrogel is 300%-600%, and the modulus is 200-1000 Pascals.

8. A method for preparing a hydrogel microsphere system for time-sequential drug delivery, characterized in that: The method comprises: Step 1: Preparation of inflammation-responsive hydrogel microspheres and ordinary microspheres Hyaluronic acid and inflammatory responsive molecules are mixed and dissolved in proportion to prepare an inflammation-responsive hydrogel aqueous phase W1; hyaluronic acid and non-responsive molecules are mixed and dissolved in proportion to prepare a normal hydrogel aqueous phase W2; Mix mineral oil and Span 80 in proportion to obtain oil phase O; The inflammation-responsive hydrogel aqueous phase W1 and the ordinary hydrogel aqueous phase W2 are mixed and stirred with the oil phase O in proportion to obtain the inflammation-responsive hydrogel emulsion C1 and the ordinary hydrogel emulsion C2; The inflammation-responsive hydrogel emulsion and the ordinary hydrogel emulsion were washed with isopropyl alcohol and centrifuged to obtain unloaded inflammation-responsive hydrogel microspheres B1 and unloaded ordinary hydrogel microspheres B2; Step 2: Drug loading The unloaded inflammation-responsive hydrogel microspheres are soaked in an anti-inflammatory drug solution to obtain a drug-loaded inflammation-responsive hydrogel microsphere mixture; the unloaded ordinary hydrogel microspheres are soaked in a healing-promoting drug solution to obtain a drug-loaded ordinary hydrogel microsphere mixture; the drug-loaded inflammation-responsive hydrogel microsphere mixture and the drug-loaded ordinary hydrogel microsphere mixture are freeze-dried and then sieved to obtain drug-loaded inflammation-responsive hydrogel microspheres a and drug-loaded ordinary hydrogel microspheres b, respectively; The drug-loaded inflammation-responsive hydrogel microspheres a and the drug-loaded ordinary hydrogel microspheres b are mixed in proportion to obtain an inflammation-responsive hydrogel microsphere system.

9. The method for preparing a hydrogel microsphere system for timed drug delivery according to claim 8, characterized in that: In step 1, the concentration of hyaluronic acid is 1-10 wt %, the molar ratio of hyaluronic acid to inflammation-responsive molecules is 1:0.5-1:2; and the molar ratio of hyaluronic acid to non-responsive molecules is 1:0.5-1:

2.

10. Application of a hydrogel microsphere system for time-sequential drug delivery in the preparation of drugs for treating skin wounds.

Images