A hydrogel microsphere system for time-sequential drug delivery, preparation method, and application thereof

Abstract

This invention discloses a time-sequential drug delivery hydrogel microsphere system, its preparation method, and its applications, specifically relating to the field of biomedical materials technology. The microsphere system includes inflammatory-responsive hydrogel microspheres obtained by grafting molecules containing inflammatory-responsive groups onto a hyaluronic acid backbone, ordinary microspheres obtained by grafting non-responsive molecules onto a hyaluronic acid backbone, an anti-inflammatory drug, and a healing-promoting drug; wherein the molecular weight of hyaluronic acid is 74-800 kDa, the inflammatory-responsive hydrogel microspheres are loaded with anti-inflammatory drugs, and the ordinary microspheres are loaded with healing-promoting drugs. The hydrogel microsphere system of this 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.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a time-delivery hydrogel microsphere system for drug delivery, its preparation method, and its applications. Background Technology

[0002] As the largest organ in the human body, the skin plays a vital role in protecting internal tissues and resisting the invasion of pathogens. When the skin is damaged, it initiates a healing process consisting of four phases: hemostasis, inflammation, proliferation, and remodeling. These four phases occur sequentially and partially overlap, relying on the synergistic effects of various cells and factors. The hemostasis phase is centered on platelet activation and fibrin polymerization to form a clot, effectively sealing blood vessels and laying the foundation for subsequent repair. In the inflammation phase, cytokines recruit immune cells (such as macrophages), whose released pro-inflammatory factors and reactive oxygen species clear pathogens and tissue debris, followed by anti-inflammatory factors leading the repair process. In the proliferation and remodeling phases, fibroblasts and keratinocytes migrate and proliferate to seal the wound, while secreting collagen to remodel the extracellular matrix, ultimately restoring the skin to homeostasis through capillary remodeling.

[0003] If the dynamic balance of the healing process is disrupted (e.g., persistent hyperactivity during the inflammatory phase), it can easily develop into a chronic wound. Endogenous diseases or bacterial infections often lead to excessive secretion of pro-inflammatory factors and abnormal activation of immune cells, which in turn inhibits tissue cell proliferation and migration, and triggers matrix metalloproteinase-mediated collagen degradation, hindering the formation of new granulation tissue. Therefore, regulating the inflammatory response is key to promoting healing, while simultaneously promoting angiogenesis and collagen deposition and inhibiting scar formation during the proliferative and remodeling phases. Achieving a sequential treatment strategy targeting different healing stages has become a core challenge in trauma treatment.

[0004] Drug therapy is an important means of wound intervention, but traditional drug delivery methods have problems such as drug toxicity risks, short half-lives, and the need for frequent dressing changes. Therefore, novel drug carrier dressings (such as sponges, hydrogels, and hydrogel microspheres) have emerged. Among them, hydrogel microspheres are miniature hydrogel particles prepared by mechanical crushing, emulsification, or other methods. With their controllable particle size and surface morphology, they possess the combined functions of exudate absorption, wound filling, and sustained drug release, showing great application potential. Meanwhile, to better target the inflammatory environment by releasing anti-inflammatory drugs, researchers have introduced inflammatory-responsive groups into dressings. This allows the dressing structure to react responsively to the inflammatory environment, releasing anti-inflammatory drugs and thus more specifically treating the skin wound environment.

[0005] Hyaluronic acid is a major component of the extracellular matrix, with wide availability, excellent biocompatibility, and water retention properties, and is often used to prepare medical dressings. According to the 2022 edition of the "Expert Consensus on the Rational Selection and Application of Hyaluronic Acids with Different Relative Molecular Weights and Structures in Dermatology," the molecular weight of hyaluronic acid can be divided into three levels: high (repeat unit number greater than 5000), medium (repeat unit number between 1000-5000), and low (repeat unit number less than 1000). Hyaluronic acid with different molecular weights has different biological functions and physical properties. For example, Chinese patent CN116808286A uses hyaluronic acid with a repeat unit number greater than 5000. Excessively high molecular weight can lead to overly rigid hydrogel microspheres, resulting in a short residence time at the wound site, requiring further cross-linking. This cross-linking process also requires… Controlling the degree of crosslinking is complex in preparation, and this patent only loads anti-inflammatory drugs, making the hydrogel more effective in anti-inflammatory and hemostatic effects, but its healing effect is poor. Chinese patent CN108912245A uses hyaluronic acid with a molecular weight of 35-45 kDa, but the low molecular weight leads to rapid degradation and excessive drug release. Therefore, this patent requires fluorination and trifluoroacetylation of the hyaluronic acid, which is complex. Furthermore, the multi-anionic nature of the hyaluronic acid in this patent allows it to form conjugates with cationic drugs and non-polar drugs encapsulated in positive liposomes, resulting in better anti-inflammatory and hemostatic effects on wounds, but limited subsequent wound healing. Therefore, balancing the influence of hyaluronic acid molecular weight on hydrogel microspheres is a pressing problem that needs to be solved.

[0006] Current clinical dressings still suffer from limitations such as a single delivery method and an inability to adapt to dynamic changes in the wound surface. Combining responsive delivery systems (releasing drugs according to environmental changes) with sustained-release technology (prolonging the duration of drug action) holds promise for achieving precise, differentiated drug delivery at different stages of healing. Therefore, the development of novel functional dressings needs to meet biosafety requirements as much as possible while being able to release different drugs specifically at different stages of skin wound healing. Summary of the Invention

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

[0008] In this invention, the hydrogel microsphere system first breaks down at the site of skin trauma, releasing an anti-inflammatory drug, followed by a natural sustained release of a healing-promoting drug from ordinary hydrogel microspheres. After the inflammatory-responsive microspheres in the hydrogel microsphere system are destroyed, they form a hydrogel that further encapsulates the ordinary microspheres, protecting the wound site. The sustained release time of the anti-inflammatory drug in the hydrogel microsphere system is 2-4 days, and the sustained release time of the healing-promoting drug is 6-10 days.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] According to a first aspect of the present invention, a time-delivery hydrogel microsphere system for drug delivery is provided, wherein the microspheres include inflammatory-responsive hydrogel microspheres obtained by grafting molecules containing inflammatory-responsive groups onto a hyaluronic acid backbone, ordinary microspheres obtained by grafting non-responsive molecules onto a hyaluronic acid backbone, an anti-inflammatory drug, and a healing-promoting drug; wherein the molecular weight of the hyaluronic acid is 74-800 kDa, the inflammatory-responsive hydrogel microspheres are loaded with the anti-inflammatory drug, and the ordinary microspheres are loaded with the healing-promoting drug.

[0011] Furthermore, the inflammatory-responsive group includes one or more of the following: diselenylene bond, disulfide bond, phenylboronic acid ester bond, thioether bond, oxalate polyester, acylpyrrolidine-2-carboxamide group, ferrocene-cyclodextrin self-assembled structural group, ketthiol, concanavalin A, metalloproteinase-responsive peptide chain, Schiff base bond, and pH-responsive peptide chain.

[0012] Furthermore, the non-responsive molecule includes one or more of hexamethylenediamine, ethylenediamine, and polyethylene glycol.

[0013] Furthermore, the anti-inflammatory drugs include one or more of gallic acid, epigallocatechin gallate, triamcinolone, berberine, curcumin, tea polyphenols, rhein, glycyrrhizic acid, quercetin, myricetin, dihydromyricetin, tannic acid, polydopamine, shikonin, celecoxib, and amoxicillin.

[0014] Furthermore, the healing-promoting drugs include one or more of astragaloside A, asiaticoside, hydroxyasiaticoside, lidocaine, vascular endothelial growth factor, fibroblast exosomes, adipose stem cell exosomes, minoxidil, and verteporphyrin.

[0015] Furthermore, the diameter of the hydrogel microspheres is 20-500 micrometers. The inflammatory-responsive hydrogel microspheres exhibit a 65-80% release rate of anti-inflammatory drugs within 24 hours in the inflammatory environment of the wound site. The ordinary hydrogel microspheres exhibit a 25-35% release rate of healing-promoting drugs within 48 hours at the wound site.

[0016] Furthermore, the hydrogel for time-delivering drugs has a maximum deformation of 300%-600% and a modulus of 200-1000 Pascals, exhibiting good self-healing properties.

[0017] According to a second aspect of the present invention, a method for preparing a time-delivery drug hydrogel microsphere system is provided, the method comprising:

[0018] Step 1: Preparation of inflammatory-responsive hydrogel microspheres and ordinary microspheres

[0019] Hyaluronic acid and inflammatory-responsive molecules were mixed and dissolved in a certain proportion to prepare an inflammatory-responsive hydrogel aqueous phase W1; hyaluronic acid and non-responsive molecules were mixed and dissolved in a certain proportion to prepare a normal hydrogel aqueous phase W2.

[0020] Mineral oil and Span 80 were mixed evenly in a certain proportion to obtain oil phase O;

[0021] The aqueous phases W1 and W2 of the inflammatory-responsive hydrogel and the oil phase O were mixed and stirred in a certain proportion to obtain the inflammatory-responsive hydrogel emulsion C1 and the ordinary hydrogel emulsion C2.

[0022] The inflammatory-responsive hydrogel emulsion and the ordinary hydrogel emulsion were washed with isopropanol and centrifuged to obtain drug-free inflammatory-responsive hydrogel microspheres B1 and drug-free ordinary hydrogel microspheres B2.

[0023] Step 2, drug loading

[0024] Unloaded inflammatory-responsive hydrogel microspheres were soaked in an anti-inflammatory drug solution to obtain a mixture of drug-loaded inflammatory-responsive hydrogel microspheres; unloaded ordinary hydrogel microspheres were soaked in a healing-promoting drug solution to obtain a mixture of drug-loaded ordinary hydrogel microspheres; the mixtures of drug-loaded inflammatory-responsive hydrogel microspheres and drug-loaded ordinary hydrogel microspheres were freeze-dried and then screened to obtain drug-loaded inflammatory-responsive hydrogel microspheres a and drug-loaded ordinary hydrogel microspheres b, respectively.

[0025] A time-delivery drug-loaded inflammatory-responsive hydrogel microsphere a and a drug-loaded ordinary hydrogel microsphere b were mixed in a certain proportion to obtain a hydrogel microsphere system.

[0026] Furthermore, in step one, the concentration of hyaluronic acid is 1-10 wt%, the molar ratio of hyaluronic acid to inflammatory 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.

[0027] To accelerate the reaction, one or more molecules selected from 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, with the specific addition ratio determined by the reaction system.

[0028] As an example, preferably, mineral oil and Span 80 are mixed at a volume ratio of 1:50-1:150; W1, W2 and oil phase O are mixed at a volume ratio of 1:5-1:20; the centrifugation conditions are: 1000-3000 rpm, centrifugation time is 3-10 min; microspheres B1 are soaked in an anti-inflammatory drug solution at a mass ratio of 1:20-1:100, wherein the concentration of the anti-inflammatory drug solution is 0.5-10 wt%; microspheres B2 are soaked in a healing-promoting drug solution at a mass ratio of 1:15-1:80, wherein the concentration of the healing-promoting drug solution is 0.05-5 wt%; the freeze dryer temperature is -40℃ to -60℃; and inflammatory-responsive hydrogel microspheres and ordinary hydrogel microspheres are mixed at a mass ratio of 1:0.5-1:2.

[0029] The application of a time-delivery hydrogel microsphere system for treating skin trauma, provided by a third aspect of the present invention.

[0030] The skin trauma is any one of the following: pressure ulcer, venous ulcer, bedsore, burn wound, surgical wound, or diabetic foot.

[0031] Principle: When the hydrogel microsphere system of the present invention is applied to the skin surface, the inflammatory-responsive microspheres are broken down to form a hydrogel, which further encapsulates the ordinary microspheres and protects the wound site; the anti-inflammatory drugs in the inflammatory-responsive microspheres loaded with anti-inflammatory drugs are continuously released for 2-4 days, and the healing-promoting drugs are continuously released for 6-10 days.

[0032] The hydrogel microsphere system of this invention has a complete degradation time of 10-14 days, allowing for single-dose administration and prolonged drug retention. This hydrogel microsphere system can be directly placed on skin wounds in a lyophilized state, enabling continuous absorption of inflammatory exudate to a certain extent. Simultaneously, the inflammatory-responsive microspheres in the hydrogel system release anti-inflammatory drugs to treat early-stage inflammation at the wound site, allowing the wound to successfully pass through the inflammatory phase. The ordinary microspheres in the hydrogel system release healing-promoting drugs with a delay, facilitating successful wound healing during the proliferative and remodeling phases.

[0033] The hydrogel microspheres of the present invention can be directly added to the skin wound site in 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 to achieve a complete wound healing treatment.

[0034] The hydrogel microsphere system of the present invention is obtained by lyophilizing the initially obtained microspheres in a drug solution, thereby increasing 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 absorb exudate at the wound site, transform into a macroscopic hydrogel state, and release the drug.

[0035] The present invention has the following advantages:

[0036] 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.

[0037] 2) The hydrogel microsphere system of the present invention is in the form of freeze-dried gel, which can be directly applied to the wound site to absorb the exudate from the inflamed area and reduce the retention of inflammatory exudate in the inflamed area.

[0038] 3) The inflammatory-responsive microspheres in the hydrogel microsphere system of the present invention can expand to further absorb inflammatory exudate after inflammatory response is disrupted. At the same time, after expansion, they can cross-link to form hydrogels to protect the inflammatory site.

[0039] 4) The framework 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 microsphere.

[0040] 5) The inflammatory-responsive microspheres in the hydrogel microsphere system of the present invention can rapidly release anti-inflammatory drugs in a strong inflammatory environment, ensuring a smooth transition during the traumatic inflammatory phase.

[0041] 6) The ordinary microspheres in the hydrogel microsphere system of the present invention can release healing-promoting drugs in a sustained manner, and release relevant drugs in a targeted manner during the proliferation and remodeling phases of wound healing as the microspheres degrade.

[0042] 7) The medical dressing of the present invention can meet the needs of the entire wound healing process with a single administration, avoiding the pain and discomfort caused by repeated administration.

[0043] 8) The product formed after the medical dressing of the present invention absorbs inflammatory exudate and is destroyed is a hydrogel, which has good self-healing properties, can effectively cover the wound inflammatory environment and meet the requirements for use in dynamic physiological environments. Attached Figure Description

[0044] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0045] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0046] Figure 1 This is a schematic diagram of a hydrogel microsphere preparation system provided in Embodiment 1 of the present invention;

[0047] Figure 2 This is a schematic diagram of the hydrogel microsphere system provided in Experimental Example 2 of the present invention for wound repair in mice;

[0048] 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 oxidized inflammatory exudate in vitro and gradually cross-links to form hydrogel-encapsulated ordinary microspheres.

[0049] Figure 4 This is a scanning electron microscope image of the hydrogel microsphere system provided in Experiment Example 4 of the present invention, which simulates the absorption and cross-linking of oxidized inflammatory exudate in vitro.

[0050] Figure 5 The results of the experimental test obtained by using a rotational rheometer to perform alternating "high strain-low strain" rheological tests on the hydrogel microsphere system provided in Experimental Example 5 of the present invention to simulate the absorption and cross-linking of inflammatory exudate in vitro. Detailed Implementation

[0051] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] Example 1

[0053] This embodiment prepares a hydrogel microsphere system comprising inflammatory-responsive hydrogel microspheres a with diselenyl bonds as inflammatory-responsive groups and ordinary microspheres b with hexamethylenediamine as crosslinking molecules. Microsphere a has a molecular weight of 200 kDa of hyaluronic acid, delivers berberine as the drug, and has a diameter of 120 micrometers. Microsphere b has a molecular weight of 250 kDa of hyaluronic acid, delivers asiaticoside as the drug, and has a diameter of 180 micrometers.

[0054] The specific steps are as follows, and the preparation diagram is shown below. Figure 1 As shown:

[0055] S1. Prepare a 1.5 wt% hyaluronic acid solution, and then mix and dissolve it in an aqueous phase W1 for the preparation of microspheres a according to the molar ratio of hyaluronic acid (CAS No. 9004-61-9): selenocysteine ​​(CAS No. 2697-61-2): 1-ethyl-(3-dimethylaminopropyl)carbodiimide (CAS No.: 1892-57-5) = 1:0.6:1.

[0056] S2. Prepare a 3 wt% hyaluronic acid solution, and then mix and dissolve it in an aqueous phase W2 for the preparation of microspheres b according to the molar ratio of hyaluronic acid: hexamethylenediamine (CAS No.: 124-09-4): 1-ethyl-(3-dimethylaminopropyl)carbodiimide (CAS No.: 1892-57-5) = 1:0.6:1.

[0057] S3. Mineral oil (CAS No.: 8042-47-5) and amphoteric Span 80 (CAS No.: 1338-43-8) are mixed evenly at a volume ratio of 1:80 to obtain oil phase O;

[0058] S4. Aqueous phases W1 and W2 and oil phase O were mixed and stirred at a volume ratio of 20:80 to obtain mixtures C1 and C2. The stirring speed was 300 rpm and the reaction time was 12 h.

[0059] S5. After the reaction is complete, wash the mixtures C1 and C2 with isopropanol (CAS No.: 67-63-0), then transfer them to 50mL centrifuge tubes and centrifuge at 1500 rpm for 3 min. Discard the liquid to obtain microspheres B1 and B2, respectively.

[0060] S6. Microspheres B1 obtained by centrifugation were soaked in a berberine solution (CAS No.: 2086-83-1) at a mass ratio of 1:50, wherein the berberine solution concentration was 2 wt%, to obtain a microsphere B1 suspension; microspheres B2 were soaked in a asiaticoside solution (CAS No.: 16830-15-2) at a mass ratio of 1:25, wherein the asiaticoside solution concentration was 0.5 wt%, to obtain a microsphere B2 suspension; then the microsphere B1 suspension and the microsphere B2 suspension were transferred to a freeze dryer for complete freeze-drying to obtain inflammatory-responsive hydrogel microspheres a and ordinary microspheres b. The freeze dryer temperature was -55℃.

[0061] S7. The freeze-dried microspheres are screened using a sieve, wherein the upper limit of the sieve hole size is 200 micrometers and the lower limit is 100 micrometers;

[0062] S8. By mixing hydrogel microspheres a and ordinary microspheres b at a mass ratio of 1:1, a hydrogel microsphere system for time-delivered drugs can be obtained.

[0063] Example 2

[0064] This embodiment prepares a hydrogel microsphere system comprising inflammatory-responsive hydrogel microspheres a with phenylboronic acid ester bonds as inflammatory-responsive groups and ordinary microspheres b with ethylenediamine as crosslinking molecules. Microsphere a has a molecular weight of 350 kDa of hyaluronic acid, delivers myricetin as the drug, and has a diameter of 80 micrometers. Microsphere b has a molecular weight of 400 kDa of hyaluronic acid, delivers asiaticoside as the drug, and has a diameter of 120 micrometers.

[0065] The specific steps are as follows:

[0066] S1. Prepare a 1 wt% hyaluronic acid solution, and then mix and dissolve it in an aqueous phase W1 for preparing microspheres a according to the molar ratio of oxidized hyaluronic acid: myricetin-crosslinked aminophenylboronic acid (CAS No.: 80460-73-7) = 1:1.

[0067] S2. Prepare a 1 wt% hyaluronic acid solution, and then mix and dissolve it in an aqueous phase W2 for the preparation of microspheres b according to the molar ratio of hyaluronic acid: ethylenediamine: 1-ethyl-(3-dimethylaminopropyl)carbodiimide = 1:0.5:1.

[0068] S3. Mineral oil and amphoteric Span 80 are mixed evenly at a volume ratio of 1:75 to obtain oil phase O;

[0069] S4. Aqueous phases W1 and W2 and oil phase O were mixed and stirred at a volume ratio of 10:90 to obtain mixtures C1 and C2. The stirring speed was 400 rpm and the reaction time was 24 h.

[0070] S5. After the reaction is complete, wash the mixtures C1 and C2 with isopropanol, then transfer them to 50 mL centrifuge tubes and centrifuge at 1000 rpm for 3 min. Discard the liquid to obtain microspheres B1 and B2, respectively.

[0071] S6. Microspheres B1 obtained by centrifugation were further soaked in a myricetin (CAS No.: 529-44-2) solution at a mass ratio of 1:45, wherein the myricetin solution concentration was 0.5 wt%, to obtain a microsphere B1 suspension; microspheres B2 were soaked in a asiaticoside (CAS No.: 34540-22-2) solution at a mass ratio of 1:20, wherein the asiaticoside solution concentration was 1 wt%, to obtain a microsphere B2 suspension. Subsequently, the microsphere B1 suspension and the microsphere B2 suspension were transferred to a freeze dryer for complete freeze-drying to obtain inflammatory-responsive hydrogel microspheres a and ordinary microspheres b. The freeze dryer temperature was -55℃.

[0072] S7. The freeze-dried microspheres are screened using a sieve, wherein the upper limit of the sieve hole size is 150 micrometers and the lower limit is 50 micrometers;

[0073] S8. Mix hydrogel microspheres a and ordinary microspheres b at a mass ratio of 1:1 to obtain a time-delivery drug hydrogel microsphere system.

[0074] Example 3

[0075] This embodiment prepares a hydrogel microsphere system comprising inflammatory-responsive hydrogel microspheres a with Schiff base bonds as inflammatory-responsive groups and ordinary microspheres b with ethylenediamine as crosslinking molecules. Microsphere a has a molecular weight of 150 kDa of hyaluronic acid, delivers dihydromyricetin (CAS No.: 27200-12-0) as the drug, and has a diameter of 150 micrometers. Microsphere b has a molecular weight of 300 kDa of hyaluronic acid, delivers minoxidil (CAS No.: 38304-91-5) as the drug, and has a diameter of 180 micrometers.

[0076] The specific steps are as follows:

[0077] S1. Prepare a 0.5 wt% oxidized hyaluronic acid solution, and then mix and dissolve it in an aqueous phase W1 for the preparation of microspheres a according to the molar ratio of oxidized hyaluronic acid to dihydromyricetin crosslinked aminophenylboronic acid (CAS No.: 3945-69-5) = 1:1.

[0078] S2. Prepare a 1.5 wt% hyaluronic acid solution, and then mix and dissolve it in an aqueous phase W2 for the preparation of microspheres b according to the molar ratio of hyaluronic acid: ethylenediamine: 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (CAS No.: 80460-73-7) = 1:0.5:1.

[0079] S3. Mineral oil and amphoteric Span 80 are mixed evenly at a volume ratio of 1:70 to obtain oil phase O;

[0080] S4. Aqueous phases W1 and W2 and oil phase O were mixed and stirred at a volume ratio of 10:90 to obtain mixtures C1 and C2. The stirring speed was 300 rpm and the reaction time was 12 h.

[0081] S5. After the reaction is complete, wash the mixtures C1 and C2 with isopropanol, then transfer them to 50 mL centrifuge tubes and centrifuge at 1200 rpm for 5 min. Discard the liquid to obtain microspheres B1 and B2, respectively.

[0082] S6. Microspheres B1 obtained by centrifugation were further soaked in a dihydromyricetin solution at a mass ratio of 1:30, wherein the concentration of the dihydromyricetin solution was 0.5 wt%, to obtain a microsphere B1 suspension; microspheres B2 were soaked in a minoxidil solution at a mass ratio of 1:25, wherein the concentration of the minoxidil solution was 1.5 wt%, to obtain a microsphere B2 suspension. Subsequently, the microsphere B1 suspension and the microsphere B2 suspension were transferred to a freeze dryer for complete freeze-drying to obtain inflammatory-responsive hydrogel microspheres a and ordinary microspheres b. The freeze dryer temperature was -55℃.

[0083] S7. The freeze-dried microspheres are screened using a sieve, wherein the upper limit of the sieve hole size is 200 micrometers and the lower limit is 100 micrometers;

[0084] S8. Mix hydrogel microspheres a and ordinary microspheres b at a mass ratio of 1:1 to obtain a time-delivery drug hydrogel microsphere system.

[0085] Example 4

[0086] This embodiment prepares a hydrogel microsphere system comprising inflammatory-responsive hydrogel microspheres a with matrix metalloproteinase-responsive peptide chains as inflammatory-responsive groups and ordinary microspheres b with hexamethylenediamine as crosslinking molecules. Microsphere a has a molecular weight of 200 kDa of hyaluronic acid, delivers curcumin (CAS No.: 458-37-7) as the drug, and has a diameter of 120 micrometers. Microsphere b has a molecular weight of 300 kDa of hyaluronic acid, delivers astragaloside A (CAS No.: 83207-58-3) as the drug, and has a diameter of 180 micrometers.

[0087] The specific steps are as follows:

[0088] S1. Prepare a 1.5 wt% hyaluronic acid solution, and then mix and dissolve the hyaluronic acid, matrix metalloproteinase responsive peptide chain (CAS No.: 9000-70-8), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in a molar ratio of 1:1:1 to prepare an aqueous phase W1 for the preparation of microspheres a;

[0089] S2. Prepare a 3 wt% hyaluronic acid solution, and then mix and dissolve it in an aqueous phase W2 according to the molar ratio of hyaluronic acid: hexamethylenediamine: activator = 1:0.5:1 for the preparation of microspheres b;

[0090] S3. Mineral oil and amphoteric Span 80 are mixed evenly at a volume ratio of 1:80 to obtain oil phase O;

[0091] S4. Aqueous phases W1 and W2 and oil phase O were mixed and stirred at a volume ratio of 15:85 to obtain mixtures C1 and C2. The stirring speed was 250 rpm and the reaction time was 12 h.

[0092] S5. After the reaction is complete, wash the mixtures C1 and C2 with isopropanol, then transfer them to 50 mL centrifuge tubes and centrifuge at 1500 rpm for 3 min. Discard the liquid to obtain microspheres B1 and B2, respectively.

[0093] S6. Microspheres B1 obtained by centrifugation were further soaked in a curcumin solution at a mass ratio of 1:50, wherein the concentration of dihydromyricetin solution was 0.5 wt%, to obtain a microsphere B1 suspension; microspheres B2 were soaked in an astragaloside A solution at a mass ratio of 1:30, wherein the concentration of astragaloside A solution was 1 wt%, to obtain a microsphere B2 suspension. Subsequently, the microsphere B1 suspension and the microsphere B2 suspension were transferred to a freeze dryer for complete freeze-drying to obtain inflammatory-responsive hydrogel microspheres a and ordinary microspheres b. The freeze dryer temperature was -55℃.

[0094] S7. The freeze-dried microspheres are screened using a sieve, wherein the upper limit of the sieve hole size is 200 micrometers and the lower limit is 100 micrometers;

[0095] S8. Mix hydrogel microspheres a and ordinary microspheres b at a mass ratio of 1:1 to obtain a time-delivery drug hydrogel microsphere system.

[0096] Example 5

[0097] This embodiment prepares a hydrogel microsphere system comprising inflammatory-responsive hydrogel microspheres a with disulfide bonds as inflammatory-responsive groups and ordinary microspheres b with hexamethylenediamine as crosslinking molecules. Microsphere a has a molecular weight of 150 kDa of hyaluronic acid, delivers shikonin (CAS No.: 517-89-5) as the drug, and has a diameter of 80 micrometers. Microsphere b has a molecular weight of 200 kDa of hyaluronic acid, delivers lidocaine (CAS No.: 137-58-6) as the drug, and has a diameter of 85 micrometers.

[0098] The specific steps are as follows:

[0099] S1. Prepare a 1.5 wt% hyaluronic acid solution, and then mix and dissolve it in an aqueous phase W1 for the preparation of microspheres a according to the molar ratio of hyaluronic acid: cystamine (CAS No.: 51-85-4): 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride = 1:0.6:1.

[0100] S2. Prepare a 2 wt% hyaluronic acid solution, and then mix and dissolve it in an aqueous phase W2 for the preparation of microspheres b according to the molar ratio of hyaluronic acid: hexamethylenediamine: 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride = 1:0.6:1.

[0101] S3. Mineral oil and amphoteric Span 80 are mixed evenly at a volume ratio of 1:80 to obtain oil phase O;

[0102] S4. Aqueous phases W1 and W2 and oil phase O were mixed and stirred at a volume ratio of 20:80 to obtain mixtures C1 and C2. The stirring speed was 250 rpm and the reaction time was 12 h.

[0103] S5. After the reaction is complete, wash the mixtures C1 and C2 with isopropanol, then transfer them to 50 mL centrifuge tubes and centrifuge at 1000 rpm for 3 min. Discard the liquid to obtain microspheres B1 and B2, respectively.

[0104] S6. Microspheres B1 obtained by centrifugation were soaked in a shikonin solution at a mass ratio of 1:50, wherein the shikonin solution concentration was 1.5 wt%, to obtain a microsphere B1 suspension; microspheres B2 were soaked in a lidocaine solution at a mass ratio of 1:20, wherein the lidocaine solution concentration was 2 wt%, to obtain a microsphere B2 suspension. Subsequently, the microsphere B1 suspension and the microsphere B2 suspension were transferred to a freeze dryer for complete freeze-drying to obtain inflammatory-responsive hydrogel microspheres a and ordinary microspheres b. The freeze dryer temperature was -55℃.

[0105] S7. The freeze-dried microspheres are screened using a sieve, wherein the upper limit of the sieve hole size is 100 micrometers and the lower limit is 50 micrometers;

[0106] S8. Mix hydrogel microspheres a and ordinary microspheres b at a mass ratio of 1:1 to obtain a time-delivery drug hydrogel microsphere system.

[0107] Example 6

[0108] This embodiment prepares an inflammatory-responsive hydrogel microsphere system comprising inflammatory-responsive microspheres a with ferrocene-cyclodextrin self-assembled structures as inflammatory-responsive groups and ordinary microspheres b with ethylenediamine as crosslinking molecules. Microsphere a has a molecular weight of 250 kDa of hyaluronic acid, delivers triamcinolone acetonide (CAS No.: 124-94-7) as the drug, and has a diameter of 110 μm. Microsphere b has a molecular weight of 450 kDa of hyaluronic acid, delivers fibroblast exosomes as the drug, and has a diameter of 150 μm.

[0109] The specific steps are as follows:

[0110] S1. Prepare a 1 wt% oxidized hyaluronic acid solution, and then mix and dissolve the oxidized hyaluronic acid, ferrocene-cyclodextrin self-assembled molecules (CAS No.: 12176-38-4; CAS No.: 12619-70-4), and N,N'-carbonyldiimidazole (CAS No.: 530-62-1) in a molar ratio of 1:1:1 to prepare an aqueous phase W1 for the preparation of microspheres a;

[0111] S2. Prepare a 1.5 wt% hyaluronic acid solution, and then mix and dissolve it in an aqueous phase W2 for the preparation of microspheres b according to the molar ratio of hyaluronic acid: hexamethylenediamine: (4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride = 1:0.5:1.

[0112] S3. Mineral oil and amphoteric Span 80 are mixed evenly at a volume ratio of 1:80 to obtain oil phase O;

[0113] S4. Aqueous phases W1 and W2 and oil phase O were mixed and stirred at a volume ratio of 10:90 to obtain mixtures C1 and C2. The stirring speed was 350 rpm and the reaction time was 12 h.

[0114] S5. After the reaction is complete, wash the mixtures C1 and C2 with isopropanol, then transfer them to 50 mL centrifuge tubes and centrifuge at 1200 rpm for 5 min. Discard the liquid to obtain microspheres B1 and B2, respectively.

[0115] S6. Microspheres B1 obtained by centrifugation were further soaked in a triamcinolone acetonide solution at a mass ratio of 1:30, where the triamcinolone acetonide solution concentration was 1 wt%, to obtain a microsphere B1 suspension; microspheres B2 were soaked in a fibroblast exosome solution at a mass ratio of 1:30, where the fibroblast exosome solution concentration was 0.1 wt%, to obtain a microsphere B1 suspension. Subsequently, the microsphere B1 suspension and the microsphere B2 suspension were transferred to a freeze dryer for complete freeze-drying to obtain inflammatory-responsive hydrogel microspheres a and ordinary microspheres b. The freeze dryer temperature was -55℃.

[0116] S7. The freeze-dried microspheres are screened using a sieve, wherein the upper limit of the sieve hole size is 200 micrometers and the lower limit is 100 micrometers;

[0117] S8. Mix hydrogel microspheres a and ordinary microspheres b at a mass ratio of 1:1 to obtain a time-delivery drug hydrogel microsphere system.

[0118] Comparative Example 1

[0119] This comparative example prepared an inflammatory-responsive hydrogel microsphere system comprising inflammatory-responsive microspheres a with diselenyl bonds as inflammatory-responsive groups and ordinary microspheres b with hexamethylenediamine as crosslinking molecules. Microsphere a has a molecular weight of 20 kDa of hyaluronic acid, delivers berberine as the drug, and has a diameter of 110 μm. Microsphere b has a molecular weight of 50 kDa of hyaluronic acid, delivers asiaticoside as the drug, and has a diameter of 160 μm.

[0120] The specific steps are as follows: Steps S1-S8 are the same as in Example 1.

[0121] Comparative Example 2

[0122] This comparative example prepared an inflammatory-responsive hydrogel microsphere system comprising inflammatory-responsive microspheres a with diselenyl bonds as inflammatory-responsive groups and ordinary microspheres b with hexamethylenediamine as crosslinking molecules. Microsphere a has a molecular weight of 1000 kDa of hyaluronic acid, delivers berberine as the drug, and has a diameter of 140 μm. Microsphere b has a molecular weight of 900 kDa of hyaluronic acid, delivers asiaticoside as the drug, and has a diameter of 160 μm.

[0123] The specific steps are as follows: Steps S1-S8 are the same as in Example 1.

[0124] Comparative Example 3

[0125] The comparative example is the hydrogel microsphere a prepared in Example 1.

[0126] Comparative Example 4

[0127] This comparative example is the ordinary microsphere b prepared in Example 1.

[0128] Comparative Example 5

[0129] This embodiment prepares an inflammatory-responsive hydrogel patch a with diselenyl bonds as inflammatory-responsive groups and ordinary microspheres b with hexamethylenediamine as crosslinking molecules. The hyaluronic acid of microsphere a has a molecular weight of 200 kDa, delivers berberine as the drug, and has a diameter of 120 micrometers. The hyaluronic acid of microsphere b has a molecular weight of 250 kDa, delivers asiaticoside as the drug, and has a diameter of 180 micrometers.

[0130] The preparation steps for microsphere b are the same as in Example 1. The specific preparation steps for hydrogel patch a are as follows:

[0131] S1. Prepare a 1.5 wt% hyaluronic acid solution, and then mix and dissolve it in an aqueous phase W1 for preparing patch a according to the molar ratio of hyaluronic acid: selenocysteine: 1-ethyl-(3-dimethylaminopropyl)carbodiimide (CAS No.: 1892-57-5): berberine = 1:0.6:1:0.1.

[0132] S2. Microspheres b are added to aqueous phase W1 at a hyaluronic acid mass ratio of 1:1 and mixed evenly to obtain W2;

[0133] S3. After transferring the aqueous phase W2 into the mold and reacting for 12 hours, the hydrogel microsphere system can be obtained.

[0134] Comparative Example 6

[0135] This comparative example prepared an inflammatory-responsive hydrogel microsphere system comprising diselenyl bonds as inflammatory-responsive groups and pure asiaticoside drug powder, wherein the exponential parameters of microsphere a are the same as in Example 1.

[0136] The specific steps are as follows: The preparation steps of the inflammatory responsive microspheres a are the same as S1-S7 in Example 1;

[0137] S8. Mix hydrogel microspheres a and asiaticoside powder of the same drug weight as in Example 1 according to the relevant proportions in Example 1.

[0138] Comparative Example 7

[0139] This comparative example prepared a hydrogel microsphere system comprising ordinary microspheres b with hexamethylenediamine as the crosslinking molecule and pure berberine drug powder, wherein the exponential parameters of microspheres b are the same as in Example 1.

[0140] The specific steps are as follows: The preparation steps of ordinary microspheres b are the same as S1-S7 in Example 1;

[0141] S8. Mix hydrogel microspheres b with berberine drug powder of the same drug mass as in Example 1 according to the relevant proportions in Example 1.

[0142] Experimental Example 1

[0143] The hydrogel microsphere system obtained in Example 1 simulated the process of absorbing and gradually cross-linking oxidized inflammatory exudate to form a hydrogel that encapsulates ordinary microspheres in vitro:

[0144] For the hydrogel system obtained in Example 1, a 1 mM hydrogen peroxide solution was used to simulate reactive oxygen species at the site of inflammation, such as... Figure 3 As shown, 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 morphology, while the ordinary microspheres remain stable, forming a jelly-like structure similar to encapsulated fruit particles. The surface hydrogel microsphere system exhibits responsive differentiation in the inflammatory exudate environment, which is helpful for subsequent staged wound treatment.

[0145] Experiment Example 2

[0146] Scanning electron microscope (SEM) images of the hydrogel microsphere system from Example 1, showing the in vitro absorption and cross-linking of oxidized inflammatory exudate. Specifically, the hydrogel system obtained in Example 1, after being lyophilized with reactive oxygen species, was quenched in liquid nitrogen and broken into pieces. The microscopic structure of the hydrogel was then observed using a scanning electron microscope. Figure 4 As can be seen, a structure of hydrogel encapsulating microspheres has been formed. The hydrogel is loose and porous, while the microspheres have a relatively dense morphology, indicating that no significant changes have occurred.

[0147] Experimental Example 3

[0148] The hydrogel microsphere system of Example 1 was subjected to alternating high-strain and low-strain rheological tests using a rotational rheometer to simulate the absorption and cross-linking of inflammatory exudate in vitro, and the self-healing properties of the hydrogel were observed. The results are as follows: Figure 5 As shown, when high strain occurs, the hydrogel is disrupted, with its elastic modulus lower than its viscous modulus, and it exists in a sol state. However, upon returning to a low strain state, the hydrogel rapidly recovers to a gel state, with its elastic modulus greater than its viscous modulus. These results indicate that the hydrogel possesses excellent self-healing properties, capable of spontaneously recovering to a gel state after damage, which is beneficial for its use in dynamic wound environments.

[0149] Experiment Example 4

[0150] Biocompatibility experiment:

[0151] The hydrogel microsphere system of Example 1 was co-cultured with mouse fibroblast L929 cells in a Transwell culture chamber for 24 hours, and then the cytotoxicity of the hydrogel microsphere system was determined by thiazolyl blue. The results showed that the cell viability after co-culturing with the hydrogel microsphere system of Example 1 was above 95%, which met the biosafety standards.

[0152] Experimental Example 5

[0153] The hydrogel microsphere system of Example 1 was immersed in PBS solution and an inflammatory wound environment simulation solution for 24 hours, respectively. The mass before and after immersion was measured to determine the liquid absorption rate of the hydrogel microsphere system. The results showed that the liquid absorption rate of the hydrogel microsphere system was 152% in PBS solution and 541% in the inflammatory wound environment simulation solution. These results indicate that in an inflammatory environment, the hydrogel microsphere system can more effectively absorb exudate and transform it into hydrogel, which is helpful for the treatment of wounds in highly inflammatory environments.

[0154] Experimental Example 6

[0155] Different drugs require different release rates. The inflammatory phase of a wound mainly lasts for 4-6 days, so anti-inflammatory drugs need to be released preferentially in the inflammatory environment. On the other hand, healing-promoting drugs mainly target the proliferative and remodeling phases of the wound, so their release needs to be as slow as possible in the early stages or the release time of the fastest rate needs to be delayed. This differentiated release of drugs can ensure that a single dose meets the needs of the entire healing process.

[0156] The microspheres from Examples 1-6 and Comparative Examples 1-7 were placed in solutions simulating a wound environment. The mass of the microspheres was weighed at different time points, and the degradation time of the microspheres was determined. The release amount of the drug was then tested using an ELISA reader after placing the microspheres in solutions simulating a wound environment. The results are shown in Table 1.

[0157] Table 1

[0158]

[0159] Table 1 shows that Comparative Example 1 and Comparative Example 2 respectively illustrate the effects of low (less than 74 kDa) and high (greater than 800 kDa) molecular weights of hyaluronic acid on hydrogel degradation time and drug release. As shown in Comparative Example 1, when the molecular weight is too low, the microspheres degrade within 2 days, failing to function during the inflammatory phase. Simultaneously, due to structural damage, drug release is too rapid; the healing-promoting drug is completely released within 48 hours, failing to function during the proliferation and healing-promoting phases. Conversely, when the molecular weight is too high, anti-inflammatory drugs cannot be effectively released. In Comparative Example 2, only 31% of the anti-inflammatory drug was released within 48 hours, failing to effectively treat the inflammation.

[0160] The hyaluronic acid in the hydrogel microsphere framework constructed in Comparative Examples 3-5 and Examples 1-6 of this invention has a suitable molecular weight (74-800 kDa), avoiding excessively rapid microsphere degradation or excessively slow drug release. Simultaneously, the inflammatory-responsive microspheres in the hydrogel microsphere system can responsively release drugs in simulated wound environment liquid, reaching 40%-52% within 12 hours, effectively curbing excessive inflammation in the wound. The drug release rate then slows down, but is still significantly faster than in Comparative Example 2. Meanwhile, the healing-promoting drug is released slowly, reaching 28%-30% within 48 hours. During the proliferative phase, the release rate accelerates, increasing by more than 40% within 96 hours, effectively delaying release and achieving targeted treatment. More importantly, the degradation time is 10-14 days, meeting the requirements for single-dose treatment. It should be noted that Comparative Examples 3 and 4 show that the degradation rate of the inflammatory-responsive microspheres is faster than that of ordinary microspheres, which meets the design requirements of this patented material. However, a single microsphere cannot meet the requirements for targeted drug release and the synergistic effect of multiple drugs. For example, Comparative Examples 3 and 4 could only load a single drug, failing to achieve the desired synergistic effect of multiple drugs. Comparative Examples 6 / 7 used non-microsphered berberine or non-microsphered asiaticoside, which, although possessing anti-inflammatory / healing-promoting effects, resulted in excessively rapid drug release due to the non-microsphered treatment, failing to achieve the expected release and healing times.

[0161] Traditional wound dressings (gauze, sponges, etc.) often fail quickly due to rapid degradation or drug release, rendering them ineffective for subsequent treatment and even causing physiological toxicity due to drug bursts. Conversely, slow degradation or drug release (e.g., in 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 the degradation rate and drug release within a suitable range. This invention effectively achieves this requirement by adjusting the molecular weight of hyaluronic acid. When the molecular weight of hyaluronic acid is kept within a suitable range, its physical entanglement can effectively regulate the drug release rate, while simultaneously allowing for stable degradation by hyaluronidase in the body.

[0162] Experimental Example 7

[0163] To investigate the impact of different drug releases at different stages of wound healing in Examples 1-6 and Comparative Examples 1-7 on wound healing, a full-thickness dorsal wound model of diabetic patients was constructed in C57BL / 6J mice. Diabetic wounds, due to the persistent high-glucose environment, lead to severe inflammation, further damaging cells and blood vessels and hindering wound healing. Therefore, it is necessary to specifically release anti-inflammatory drugs during the inflammatory phase to alleviate inflammation, followed by the release of healing-promoting drugs during the wound's proliferation and remodeling phases to ensure comprehensive wound healing.

[0164] Materials from Comparative Examples 1-7 and Examples 1-6 were placed at the wound site and wrapped externally with 3M film to prevent material detachment from the tissue. Tissue was collected on day 6, homogenized, and the ratio of CD86 positivity (M1 macrophages) to CD206 positivity (M2 macrophages) was calculated by flow cytometry. The wound healing rate was recorded and analyzed on day 10; the results are shown in Table 2.

[0165] Table 2

[0166]

[0167] CD86 is a marker of M1 macrophages and an indicator of pro-inflammatory activity; CD206 is a marker of M2 macrophages and an indicator of anti-inflammatory activity. A higher CD86 / CD206 ratio indicates that M1 macrophages predominate at the wound site, resulting in a strong inflammatory atmosphere that is not conducive to wound healing. Conversely, a lower ratio indicates that the wound is in a state of anti-inflammatory remission, with the inflammatory phase about to end and transitioning to the proliferative and remodeling phases, which is conducive to wound healing.

[0168] Comparative Example 1 shows that when the hydrogel microspheres degrade too quickly, the drug cannot maintain its effect. The CD86 / CD206 ratio is 129%, higher than 100%, indicating that inflammation is suppressed, but the overall environment remains pro-inflammatory. Ultimately, the wound healing rate on day 10 is only 55%. Comparative Example 2 shows that when the hydrogel degrades too slowly, although inflammation is effectively relieved on day 7, with a CD86 / CD206 ratio of 44%, the lack of timely release of the healing-promoting drug prevents rapid healing of the wound tissue during the proliferative and remodeling phases. Ultimately, the wound healing rate on day 10 is only 66%. Comparative Examples 3 and 4 show that simply controlling inflammation or promoting the repair of tissues related to the proliferative and remodeling phases is not effective in promoting wound healing; both methods result in healing rates below 50%, and the wound healing process is significantly slowed. Comparative Example 5 shows that hydrogel patch technology can also effectively deliver drugs differentially to promote wound healing, but the neglect of wound exudate absorption during the inflammatory phase leads to a poorer effect on inflammation control, resulting in a final healing rate of only 71%. Comparative Examples 6 and 7 were treated without microspheres, which prevented the anti-inflammatory drug (Comparative Example 6) and the healing-promoting drug (Comparative Example 7) from being released at the expected rate, resulting in a final healing rate of only 47% and 42% for the mice, respectively.

[0169] As shown in Examples 1-6, when the differentiated drug delivery rate of the microspheres is appropriate, it can effectively control inflammation. The CD86 / CD206 ratio is 29%-38%, which is at a very low level, ensuring a smooth transition during the inflammatory phase. Furthermore, during the proliferation and remodeling phases, the healing-promoting drugs can act precisely, effectively accelerating the wound healing rate. By day 10, the wound healing rate is 83%-88%, meaning the wound is about to close. A schematic diagram of the specific healing process is shown below. Figure 2 As shown.

[0170] The above results demonstrate that wound healing in diabetic patients is the result of the combined effects of various stages. Simply controlling or acting on the remodeling of tissues in the proliferative and remodeling phases cannot effectively promote wound healing. Furthermore, slow drug release can also occur when the drug is released too quickly or too slowly, hindering its effectiveness. The time-sequential drug delivery hydrogel microsphere system designed in this invention effectively addresses the different treatment needs at different stages of wound healing, releasing drugs specifically to promote healing.

[0171] The hydrogel system of Embodiment 1 of the present invention can absorb inflammatory exudate in the early stage of the wound area and release berberine drug in response, clearing reactive oxygen species and inhibiting inflammation at the wound site; at the same time, ordinary microspheres can release asiaticoside to promote cell migration, angiogenesis and promote wound healing, thereby improving the quality of healing.

[0172] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

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

2. The hydrogel microsphere system for time-sequential drug delivery according to claim 1, characterized in that, The inflammatory responsive groups include one or more of the following: diselenyl groups, disulfide groups, phenylboronic acid ester bonds, thioether bonds, oxalate polyesters, acylpyrrolidine-2-carboxamide groups, ferrocene-cyclodextrin self-assembled structural groups, ketthiols, concanavalin A, metalloproteinase-responsive peptide chains, Schiff base bonds, and pH-responsive peptide chains.

3. The hydrogel microsphere system for time-sequential 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 time-sequential drug delivery according to claim 1, characterized in that, The anti-inflammatory drugs include one or more of gallic acid, epigallocatechin gallate, triamcinolone, 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 the following: astragaloside A, asiaticoside, hydroxyasiaticoside, lidocaine, vascular endothelial growth factor, fibroblast exosomes, adipose-derived stem cell exosomes, minoxidil, and verteporphyrin.

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

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

8. A method for preparing a time-delivery drug hydrogel microsphere system according to any one of claims 1 to 7, characterized in that, The method includes: Step 1: Preparation of inflammatory-responsive hydrogel microspheres and ordinary microspheres Hyaluronic acid and inflammatory-responsive molecules were mixed and dissolved in a certain proportion to prepare an inflammatory-responsive hydrogel aqueous phase W1; hyaluronic acid and non-responsive molecules were mixed and dissolved in a certain proportion to prepare a normal hydrogel aqueous phase W2. Mineral oil and Span 80 were mixed evenly in a certain proportion to obtain oil phase O; The aqueous phases W1 and W2 of the inflammatory-responsive hydrogel and the oil phase O were mixed and stirred in a certain proportion to obtain the inflammatory-responsive hydrogel emulsion C1 and the ordinary hydrogel emulsion C2. The inflammatory-responsive hydrogel emulsion and the ordinary hydrogel emulsion were washed with isopropanol and centrifuged to obtain drug-free inflammatory-responsive hydrogel microspheres B1 and drug-free ordinary hydrogel microspheres B2. Step 2, drug loading Unloaded inflammatory-responsive hydrogel microspheres were soaked in an anti-inflammatory drug solution to obtain a mixture of drug-loaded inflammatory-responsive hydrogel microspheres; unloaded ordinary hydrogel microspheres were soaked in a healing-promoting drug solution to obtain a mixture of drug-loaded ordinary hydrogel microspheres; the mixtures of drug-loaded inflammatory-responsive hydrogel microspheres and drug-loaded ordinary hydrogel microspheres were freeze-dried and then screened to obtain drug-loaded inflammatory-responsive hydrogel microspheres a and drug-loaded ordinary hydrogel microspheres b, respectively. An inflammatory-responsive hydrogel microsphere system was obtained by mixing drug-loaded inflammatory-responsive hydrogel microspheres a and drug-loaded ordinary hydrogel microspheres b in a certain proportion.

9. The method for preparing a time-delivery drug hydrogel microsphere system according to claim 8, characterized in that, In step one, the concentration of hyaluronic acid is 1-10 wt%, the molar ratio of hyaluronic acid to inflammatory 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. The use of a time-delivery hydrogel microsphere system according to any one of claims 1 to 7 in the preparation of a drug for treating skin trauma.

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